Irving Weissman

All Publications

• SIRPα controls CD47-dependent platelet clearance in mice and humans. bioRxiv : the preprint server for biology Shoham, M., Yiu, Y. Y., Hansen, P. S., Subramaniam, A., Broberg, M., Gars, E., Raveh, T., Weissman, I. L., Sinnott-Armstrong, N., Krishnan, A., Ollila, H. M., Tal, M. C. 2023

  Abstract

  Over the last decade, more data has revealed that increased surface expression of the "don't eat me" CD47 protein on cancer cells plays a role in immune evasion and tumor progression, with CD47 blockade emerging as a new therapy in immuno-oncology. CD47 is critical in regulating cell homeostasis and clearance, as binding of CD47 to the inhibitory receptor SIRPα can prevent phagocytosis and macrophage-mediated cell clearance. The purpose of this study was to examine the role of the CD47-SIRPα signal in platelet homeostasis and clearance. Therapeutic reagents targeting the CD47-SIRPα axis are very promising for treatment of hematologic malignancies and solid tumors, but lead to transient anemia or thrombocytopenia in a subset of patients. We found that platelet homeostatic clearance is regulated through the CD47-SIRPα axis and that therapeutic blockade to disrupt this interaction in mice and in humans has a significant impact on platelet levels. Furthermore, we identified genetic variations at the SIRPA locus that impact platelet levels in humans such that higher SIRPA gene expression is associated with higher platelet levels. SIRPA expression at either end of the normal range may affect clinical outcomes of treatment with anti-CD47 therapy.

  View details for DOI 10.1101/2023.12.09.570874

  View details for PubMedID 38106070

  View details for PubMedCentralID PMC10723388

• Prospective isolation of neural stem and progenitor cells from the developing human brain. STAR protocols Liu, D. D., He, J. Q., Uchida, N., Weissman, I. L., Sinha, R. 2023; 4 (4): 102674

  Abstract

  Prospective isolation of defined cell types is critical for the functional study of stem cells, especially in primary human tissues. Here, we present a protocol for purifying 10 transcriptomically and functionally distinct neural stem and progenitor cell types from the developing human brain using fluorescence-activated cell sorting. We describe steps for tissue dissociation, staining, and cell sorting as well as downstream functional experiments for measuring clonogenicity, differentiation, and engraftment potential of purified populations. For complete details on the use and execution of this protocol, please refer to Liu etal. (2023).1.

  View details for DOI 10.1016/j.xpro.2023.102674

  View details for PubMedID 37897731

• Immune Surveillance of Acute Myeloid Leukemia Is Mediated by HLA-Presented Antigens on Leukemia Progenitor Cells. Blood cancer discovery Nelde, A., Schuster, H., Heitmann, J. S., Bauer, J., Maringer, Y., Zwick, M., Volkmer, J. P., Chen, J. Y., Stanger, A. M., Lehmann, A., Appiah, B., Märklin, M., Rücker-Braun, E., Salih, H. R., Roerden, M., Schroeder, S. M., Häring, M. F., Schlosser, A., Schetelig, J., Schmitz, M., Boerries, M., Köhler, N., Lengerke, C., Majeti, R., Weissman, I. L., Rammensee, H. G., Walz, J. S. 2023: OF1-OF22

  Abstract

  Therapy-resistant leukemia stem and progenitor cells (LSC) are a main cause of acute myeloid leukemia (AML) relapse. LSC-targeting therapies may thus improve outcome of patients with AML. Here we demonstrate that LSCs present HLA-restricted antigens that induce T-cell responses allowing for immune surveillance of AML. Using a mass spectrometry-based immunopeptidomics approach, we characterized the antigenic landscape of patient LSCs and identified AML- and AML/LSC-associated HLA-presented antigens absent from normal tissues comprising nonmutated peptides, cryptic neoepitopes, and neoepitopes of common AML driver mutations of NPM1 and IDH2. Functional relevance of shared AML/LSC antigens is illustrated by presence of their cognizant memory T cells in patients. Antigen-specific T-cell recognition and HLA class II immunopeptidome diversity correlated with clinical outcome. Together, these antigens shared among AML and LSCs represent prime targets for T cell-based therapies with potential of eliminating residual LSCs in patients with AML.The elimination of therapy-resistant leukemia stem and progenitor cells (LSC) remains a major challenge in the treatment of AML. This study identifies and functionally validates LSC-associated HLA class I and HLA class II-presented antigens, paving the way to the development of LSC-directed T cell-based immunotherapeutic approaches for patients with AML. See related commentary by Ritz, p. 437 .

  View details for DOI 10.1158/2643-3230.BCD-23-0020

  View details for PubMedID 37847741

• Stem-Cell Aging and Pathways to Precancer Evolution. The New England journal of medicine Jamieson, C. H., Weissman, I. L. 2023; 389 (14): 1310-1319

  View details for DOI 10.1056/NEJMra2304431

  View details for PubMedID 37792614

• Combination of Distinct Vascular Stem/Progenitor Cells for Neovascularization and Ischemic Rescue. Arteriosclerosis, thrombosis, and vascular biology Zhao, L., Lee, A. S., Sasagawa, K., Sokol, J., Wang, Y., Ransom, R. C., Zhao, X., Ma, C., Steininger, H. M., Koepke, L. S., Borrelli, M. R., Brewer, R. E., Lee, L. L., Huang, X., Ambrosi, T. H., Sinha, R., Hoover, M. Y., Seita, J., Weissman, I. L., Wu, J. C., Wan, D. C., Xiao, J., Longaker, M. T., Nguyen, P. K., Chan, C. K. 2023

  Abstract

  Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia.The ActinCreER/R26VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions.Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice.These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

  View details for DOI 10.1161/ATVBAHA.122.317943

  View details for PubMedID 37051932

• Multiple Forms of Neural Cell Death in the Cyclical Brain Degeneration of A Colonial Chordate. Cells Anselmi, C., Caicci, F., Bocci, T., Guidetti, M., Priori, A., Giusti, V., Levy, T., Raveh, T., Voskoboynik, A., Weissman, I. L., Manni, L. 2023; 12 (7)

  Abstract

  Human neuronal loss occurs through different cellular mechanisms, mainly studied in vitro. Here, we characterized neuronal death in B. schlosseri, a marine colonial tunicate that shares substantial genomic homology with mammals and has a life history in which controlled neurodegeneration happens simultaneously in the brains of adult zooids during a cyclical phase named takeover. Using an ultrastructural and transcriptomic approach, we described neuronal death forms in adult zooids before and during the takeover phase while comparing adult zooids in takeover with their buds where brains are refining their structure. At takeover, we found in neurons clear morphologic signs of apoptosis (i.e., chromatin condensation, lobed nuclei), necrosis (swollen cytoplasm) and autophagy (autophagosomes, autolysosomes and degradative multilamellar bodies). These results were confirmed by transcriptomic analyses that highlighted the specific genes involved in these cell death pathways. Moreover, the presence of tubulovesicular structures in the brain medulla alongside the over-expression of prion disease genes in late cycle suggested a cell-to-cell, prion-like propagation recalling the conformational disorders typical of some human neurodegenerative diseases. We suggest that improved understanding of how neuronal alterations are regulated in the repeated degeneration-regeneration program of B. schlosseri may yield mechanistic insights relevant to the study of human neurodegenerative diseases.

  View details for DOI 10.3390/cells12071041

  View details for PubMedID 37048113

• Purification and characterization of human neural stem and progenitor cells. Cell Liu, D. D., He, J. Q., Sinha, R., Eastman, A. E., Toland, A. M., Morri, M., Neff, N. F., Vogel, H., Uchida, N., Weissman, I. L. 2023; 186 (6): 1179

  Abstract

  The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

  View details for DOI 10.1016/j.cell.2023.02.017

  View details for PubMedID 36931245

• Innate immune cell activation causes lung fibrosis in a humanized model of long COVID. Proceedings of the National Academy of Sciences of the United States of America Cui, L., Fang, Z., De Souza, C. M., Lerbs, T., Guan, Y., Li, I., Charu, V., Chen, S. Y., Weissman, I., Wernig, G. 2023; 120 (10): e2217199120

  Abstract

  COVID-19 remains a global pandemic of an unprecedented magnitude with millions of people now developing "COVID lung fibrosis." Single-cell transcriptomics of lungs of patients with long COVID revealed a unique immune signature demonstrating the upregulation of key proinflammatory and innate immune effector genes CD47, IL-6, and JUN. We modeled the transition to lung fibrosis after COVID and profiled the immune response with single-cell mass cytometry in JUN mice. These studies revealed that COVID mediated chronic immune activation reminiscent to long COVID in humans. It was characterized by increased CD47, IL-6, and phospho-JUN (pJUN) expression which correlated with disease severity and pathogenic fibroblast populations. When we subsequently treated a humanized COVID lung fibrosis model by combined blockade of inflammation and fibrosis, we not only ameliorated fibrosis but also restored innate immune equilibrium indicating possible implications for clinical management of COVID lung fibrosis in patients.

  View details for DOI 10.1073/pnas.2217199120

  View details for PubMedID 36848564

• Cell-type-specific aging clocks to quantify aging and rejuvenation in neurogenic regions of the brain NATURE AGING Buckley, M. T., Sun, E. D., George, B. M., Liu, L., Schaum, N., Xu, L., Reyes, J. M., Goodell, M. A., Weissman, I. L., Wyss-Coray, T., Rando, T. A., Brunet, A. 2023; 3 (1): 121-+

  View details for DOI 10.1038/s43587-022-00335-4

  View details for Web of Science ID 000922722500016

• Radiotherapy in combination with CD47 blockade elicits a macrophage-mediated abscopal effect. Nature cancer Nishiga, Y., Drainas, A. P., Baron, M., Bhattacharya, D., Barkal, A. A., Ahrari, Y., Mancusi, R., Ross, J. B., Takahashi, N., Thomas, A., Diehn, M., Weissman, I. L., Graves, E. E., Sage, J. 2022

  Abstract

  Radiation therapy is a mainstay of cancer treatment but does not always lead to complete tumor regression. Here we combine radiotherapy with blockade of the 'don't-eat-me' cell-surface molecule CD47 in small cell lung cancer (SCLC), a highly metastatic form of lung cancer. CD47 blockade potently enhances the local antitumor effects of radiotherapy in preclinical models of SCLC. Notably, CD47 blockade also stimulates off-target 'abscopal' effects inhibiting non-irradiated SCLC tumors in mice receiving radiation. These abscopal effects are independent of T cells but require macrophages that migrate into non-irradiated tumor sites in response to inflammatory signals produced by radiation and are locally activated by CD47 blockade to phagocytose cancer cells. Similar abscopal antitumor effects were observed in other cancer models treated with radiation and CD47 blockade. The systemic activation of antitumor macrophages following radiotherapy and CD47 blockade may be particularly important in patients with cancer who suffer from metastatic disease.

  View details for DOI 10.1038/s43018-022-00456-0

  View details for PubMedID 36411318

• Increased macrophage phagocytic activity with TLR9 agonist conjugation of an anti- Borrelia burgdorferi monoclonal antibody. Clinical immunology (Orlando, Fla.) Jahanbani, S., Hansen, P. S., Blum, L. K., Bastounis, E. E., Ramadoss, N. S., Pandrala, M., Kirschmann, J. M., Blacker, G. S., Love, Z. Z., Weissman, I. L., Nemati, F., Tal, M. C., Robinson, W. H. 2022: 109180

  Abstract

  Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12-24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.

  View details for DOI 10.1016/j.clim.2022.109180

  View details for PubMedID 36396013

• MDS-482 Impact Of Magrolimab in Combination With Azacitidine on Red Blood Cells (RBCs) in Patients With Higher-Risk Myelodysplastic Syndromes (HR MDS). Clinical lymphoma, myeloma & leukemia Chen, J., Johnson, L., McKenna, K., Choi, T., Duan, J., Feng, D., Tsai, J., Garcia-Martin, N., Sompalli, K., Maute, R., Vyas, P., Majeti, R., Takimoto, C., Liu, J., Ramsingh, G., Chao, M., Volkmer, J., Weissman, I. 2022; 22 Suppl 2: S317-S318

  Abstract

  CONTEXT: Magrolimab is an antibody blocking CD47, a "don't eat me" signal expressed on cancer cells, to escape immune surveillance and macrophage-mediated clearance. Preclinical studies found that CD47 is critical to RBC homeostasis, with CD47 deficiency decreasing RBC half-life. Fc-mediated opsonization also depletes RBCs, raising concerns that potential on-target anemia could result from the use of anti-CD47 agents. Several clinical trials demonstrated that magrolimab can be safely administered as monotherapy, with an initial lower "priming" dose yielding transient anemia with compensatory reticulocytosis and no anemia observed at higher maintenance doses. However, the underlying mechanism has not been fully defined.OBJECTIVE: To describe manageable anemia in magrolimab-treated patients and further investigate the underlying mechanisms in preclinical models.DESIGN: Prospective analysis from a ph1 trial of magrolimab+azacitidine (NCT03248479). Complete blood counts (CBCs), peripheral blood, and bone marrow (BM) were collected from patients at prespecified time points. CBCs were measured, and blood and BM samples were analyzed by flow cytometry for CD47 expression on RBCs and white blood cells (WBCs). Preclinical modeling studies were conducted with intact and Fc-deficient anti-mouse CD47 (MIAP410) and anti-human CD47 (magrolimab) antibodies in murine models, including C57BL/6J B-hSIRPA/hCD47 mice.PATIENTS: 57 patients with HR MDS.INTERVENTIONS: Magrolimab IV 1 mg/kg (priming) then 30 mg/kg QW, then Q2W (maintenance). Azacitidine 75 mg/m2 days 1-7 (each 28-day cycle).RESULTS: Treatment with magrolimab+azacitidine resulted in tolerable anemia that correlated with rapid, near-complete loss of CD47 in RBCs but not WBCs. The initial 1-mg/kg priming dose was sufficient for CD47 loss, which persisted with subsequent 30-mg/kg maintenance doses. Both findings are consistent with prior clinical observations of magrolimab monotherapy in patients with solid tumors and magrolimab+rituximab in patients with lymphoma. Our preclinical studies with mouse models revealed that CD47 removal is mechanistically independent of previously described RBC antigen modulation mechanisms and cellular compartments. Instead, this CD47 loss requires anti-CD47 cross-linking between RBCs and non-RBCs.CONCLUSIONS: These results support the idea that on-target magrolimab-mediated anemia is mitigated by a near-complete loss of RBC CD47. Patients with HR MDS treated with magrolimab+azacitidine had tolerable anemia with priming and maintenance doses.

  View details for DOI 10.1016/S2152-2650(22)01421-5

  View details for PubMedID 36163968

• Impact Of Magrolimab in Combination With Azacitidine on Red Blood Cells (RBCs) in Patients With Higher-Risk Myelodysplastic Syndromes (HR MDS) Chen, J., Johnson, L., McKenna, K., Choi, T., Duan, J., Feng, D., Tsai, J., Garcia-Martin, N., Sompalli, K., Maute, R., Vyas, P., Majeti, R., Takimoto, C., Liu, J., Ramsingh, G., Chao, M., Volkmer, J., Weissman, I. CIG MEDIA GROUP, LP. 2022: S317-S318

  View details for Web of Science ID 000897948100300

• Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos. Cell reports Roodgar, M., Suchy, F. P., Nguyen, L. H., Bajpai, V. K., Sinha, R., Vilches-Moure, J. G., Van Bortle, K., Bhadury, J., Metwally, A., Jiang, L., Jian, R., Chiang, R., Oikonomopoulos, A., Wu, J. C., Weissman, I. L., Mankowski, J. L., Holmes, S., Loh, K. M., Nakauchi, H., VandeVoort, C. A., Snyder, M. P. 2022; 40 (9): 111264

  Abstract

  As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.

  View details for DOI 10.1016/j.celrep.2022.111264

  View details for PubMedID 36044843

• Two distinct evolutionary conserved neural degeneration pathways characterized in a colonial chordate. Proceedings of the National Academy of Sciences of the United States of America Anselmi, C., Kowarsky, M., Gasparini, F., Caicci, F., Ishizuka, K. J., Palmeri, K. J., Raveh, T., Sinha, R., Neff, N., Quake, S. R., Weissman, I. L., Voskoboynik, A., Manni, L. 2022; 119 (29): e2203032119

  Abstract

  Colonial tunicates are marine organisms that possess multiple brains simultaneously during their colonial phase. While the cyclical processes of neurogenesis and neurodegeneration characterizing their life cycle have been documented previously, the cellular and molecular changes associated with such processes and their relationship with variation in brain morphology and individual (zooid) behavior throughout adult life remains unknown. Here, we introduce Botryllus schlosseri as an invertebrate model for neurogenesis, neural degeneration, and evolutionary neuroscience. Our analysis reveals that during the weekly colony budding (i.e., asexual reproduction), prior to programmed cell death and removal by phagocytes, decreases in the number of neurons in the adult brain are associated with reduced behavioral response and significant change in the expression of 73 mammalian homologous genes associated with neurodegenerative disease. Similarly, when comparing young colonies (1 to 2 y of age) to those reared in a laboratory for 20 y, we found that older colonies contained significantly fewer neurons and exhibited reduced behavioral response alongside changes in the expression of 148 such genes (35 of which were differentially expressed across both timescales). The existence of two distinct yet apparently related neurodegenerative pathways represents a novel platform to study the gene products governing the relationship between aging, neural regeneration and degeneration, and loss of nervous system function. Indeed, as a member of an evolutionary clade considered to be a sister group of vertebrates, this organism may be a fundamental resource in understanding how evolution has shaped these processes across phylogeny and obtaining mechanistic insight.

  View details for DOI 10.1073/pnas.2203032119

  View details for PubMedID 35858312

• ISSCR Presidents look back on their presidency, the evolution of the field, and the Society STEM CELL REPORTS Zon, L., Keller, G., Daley, G. Q., Watt, F. M., Weissman, I. L., Fuchs, E., Gage, F. H., Yamanaka, S., Rossant, J., Morrison, S., Temple, S., Clevers, H. C., Srivastava, D., Mummery, C. L., Little, M. 2022; 17 (6): 1237-1244

  Abstract

  In celebration of the ISSCR's 20th anniversary we asked past ISSCR presidents the question, "During your presidential year, what key achievements or issue(s) in the field stood out to you?" The collection of responses provides a glimpse of the evolution of the field and the ISSCR over the past 20 years.

  View details for Web of Science ID 000825548800003

  View details for PubMedID 35705012

  View details for PubMedCentralID PMC9214063

• Impact of magrolimab treatment in combination with azacitidine on red blood cells in patients with higher-risk myelodysplastic syndrome (HR-MDS). Chen, J., Johnson, L., McKenna, K., Choi, T. S., Duan, J., Feng, D., Tsai, J. M., Garcia-Martin, N., Sompalli, K., Maute, R., Vyas, P., Majeti, R., Takimoto, C. M., Liu, J., Ramsingh, G., Chao, M., Volkmer, J., Weissman, I. L. LIPPINCOTT WILLIAMS & WILKINS. 2022

  View details for Web of Science ID 000863680301955

• Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection Sheikh-Mohamed, S., Chao, G., Isho, B., Zuo, M., Cohen, C., Lustig, Y., Nahass, G., Salomon, R. E., Blacker, G., Fazel-Zarandi, M., Rathod, B., Colwill, K., Jamal, A. J., Li, Z., Delaunay, K., Takaoka, A., Garnham-Takaoka, J., Patel, A., Fahim, C., Patterson, A., Liu, A., Haq, N., Barati, S., Gilbert, L., Green, K., Mozafarihashjin, M., Samaan, P., Budylowski, P., Siqueira, W., Mubareka, S., Ostrowski, M., Rini, J., Rojas, O., Weissman, I. L., Tal, M., McGeer, A., Regev, G., Straus, S., Gingras, A., Gommerman, J. L. AMER ASSOC IMMUNOLOGISTS. 2022

  View details for DOI 10.4049/jimmunol.208.Supp.59.14

  View details for Web of Science ID 000892531600313

• Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection. Mucosal immunology Sheikh-Mohamed, S., Isho, B., Chao, G. Y., Zuo, M., Cohen, C., Lustig, Y., Nahass, G. R., Salomon-Shulman, R. E., Blacker, G., Fazel-Zarandi, M., Rathod, B., Colwill, K., Jamal, A., Li, Z., de Launay, K. Q., Takaoka, A., Garnham-Takaoka, J., Patel, A., Fahim, C., Paterson, A., Li, A. X., Haq, N., Barati, S., Gilbert, L., Green, K., Mozafarihashjin, M., Samaan, P., Budylowski, P., Siqueira, W. L., Mubareka, S., Ostrowski, M., Rini, J. M., Rojas, O. L., Weissman, I. L., Tal, M. C., McGeer, A., Regev-Yochay, G., Straus, S., Gingras, A., Gommerman, J. L. 2022

  Abstract

  Although SARS-CoV-2 infects the upper respiratory tract, we know little about the amount, type, and kinetics of antibodies (Ab) generated in the oral cavity in response to COVID-19 vaccination. We collected serum and saliva samples from participants receiving two doses of mRNA COVID-19 vaccines and measured the level of anti-SARS-CoV-2 Ab. We detected anti-Spike and anti-Receptor Binding Domain (RBD) IgG and IgA, as well as anti-Spike/RBD associated secretory component in the saliva of most participants after dose 1. Administration of a second dose of mRNA boosted the IgG but not the IgA response, with only 30% of participants remaining positive for IgA at this timepoint. At 6 months post-dose 2, these participants exhibited diminished anti-Spike/RBD IgG levels, although secretory component-associated anti-Spike Ab were more stable. Examining two prospective cohorts we found that participants who experienced breakthrough infections with SARS-CoV-2 variants had lower levels of vaccine-induced serum anti-Spike/RBD IgA at 2-4 weeks post-dose 2 compared to participants who did not experience an infection, whereas IgG levels were comparable between groups. These data suggest that COVID-19 vaccines that elicit a durable IgA response may have utility in preventing infection.

  View details for DOI 10.1038/s41385-022-00511-0

  View details for PubMedID 35468942

• 2021 Jeffrey M. Hoeg Award Lecture: Defining the Role of Efferocytosis in Cardiovascular Disease: A Focus on the CD47 (Cluster of Differentiation 47) Axis. Arteriosclerosis, thrombosis, and vascular biology Jarr, K., Kojima, Y., Weissman, I. L., Leeper, N. J. 2022: 101161ATVBAHA122317049

  Abstract

  A key feature of atherogenesis is the accumulation of diseased and dying cells within the lesional necrotic core. While the burden of intraplaque apoptotic cells may be driven in part by an increase in programmed cell death, mounting evidence suggests that their presence may primarily be dictated by a defect in programmed cell removal, or efferocytosis. In this brief review, we will summarize the evidence suggesting that inflammation-dependent changes within the plaque render target cells inedible and reduce the appetite of lesional phagocytes. We will present the genetic causation studies, which indicate these phenomena promote lesion expansion and plaque vulnerability, and the interventional data which suggest that these processes can be reversed. Particular emphasis is provided related to the antiphagocytic CD47 (cluster of differentiation 47) do not eat me axis, which has emerged as a novel antiatherosclerotic translational target that is predicted to provide benefit independent of traditional cardiovascular risk factors.

  View details for DOI 10.1161/ATVBAHA.122.317049

  View details for PubMedID 35387480

• Molecular hallmarks of heterochronic parabiosis at single-cell resolution. Nature Palovics, R., Keller, A., Schaum, N., Tan, W., Fehlmann, T., Borja, M., Kern, F., Bonanno, L., Calcuttawala, K., Webber, J., McGeever, A., Tabula Muris Consortium, Luo, J., Pisco, A. O., Karkanias, J., Neff, N. F., Darmanis, S., Quake, S. R., Wyss-Coray, T., Almanzar, N., Antony, J., Baghel, A. S., Bakerman, I., Bansal, I., Barres, B. A., Beachy, P. A., Berdnik, D., Bilen, B., Brownfield, D., Cain, C., Chan, C. K., Chen, M. B., Clarke, M. F., Conley, S. D., Demers, A., Demir, K., de Morree, A., Divita, T., du Bois, H., Ebadi, H., Espinoza, F. H., Fish, M., Gan, Q., George, B. M., Gillich, A., Gomez-Sjoberg, R., Green, F., Genetiano, G., Gu, X., Gulati, G. S., Hahn, O., Haney, M. S., Hang, Y., Harris, L., He, M., Hosseinzadeh, S., Huang, A., Huang, K. C., Iram, T., Isobe, T., Ives, F., Jones, R. C., Kao, K. S., Karnam, G., Kershner, A. M., Khoury, N., Kim, S. K., Kiss, B. M., Kong, W., Krasnow, M. A., Kumar, M. E., Kuo, C. S., Lam, J., Lee, D. P., Lee, S. E., Lehallier, B., Leventhal, O., Li, G., Li, Q., Liu, L., Lo, A., Lu, W., Lugo-Fagundo, M. F., Manjunath, A., May, A. P., Maynard, A., McKay, M., McNerney, M. W., Merrill, B., Metzger, R. J., Mignardi, M., Min, D., Nabhan, A. N., Ng, K. M., Nguyen, P. K., Noh, J., Nusse, R., Patkar, R., Peng, W. C., Penland, L., Pollard, K., Puccinelli, R., Qi, Z., Rando, T. A., Rulifson, E. J., Segal, J. M., Sikandar, S. S., Sinha, R., Sit, R. V., Sonnenburg, J., Staehli, D., Szade, K., Tan, M., Tato, C., Tellez, K., Torrez Dulgeroff, L. B., Travaglini, K. J., Tropini, C., Tsui, M., Waldburger, L., Wang, B. M., van Weele, L. J., Weinberg, K., Weissman, I. L., Wosczyna, M. N., Wu, S. M., Xiang, J., Xue, S., Yamauchi, K. A., Yang, A. C., Yerra, L. P., Youngyunpipatkul, J., Yu, B., Zanini, F., Zardeneta, M. E., Zee, A., Zhao, C., Zhang, F., Zhang, H., Zhang, M. J., Zhou, L., Zou, J. 2022

  Abstract

  The ability to slow or reverse biological ageing would have major implications for mitigating disease risk and maintaining vitality1. Although an increasing number of interventions show promise for rejuvenation2, their effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. Here we performed single-cell RNA sequencing on 20 organs to reveal cell-type-specific responses to young and aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, haematopoietic stem cells and hepatocytes are among those cell types that are especially responsive. On the pathway level, young blood invokes new gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. We observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it in select cell types. Together, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

  View details for DOI 10.1038/s41586-022-04461-2

  View details for PubMedID 35236985

• CD47 Blockade Leads to Chemokine-Dependent Monocyte Infiltration and Loss of B Cells from the Splenic Marginal Zone. Journal of immunology (Baltimore, Md. : 1950) Yiu, Y. Y., Hansen, P. S., Torrez Dulgeroff, L. B., Blacker, G., Myers, L., Galloway, S., Gars, E., Colace, O., Mansfield, P., Hasenkrug, K. J., Weissman, I. L., Tal, M. C. 2022

  Abstract

  CD47 is an important innate immune checkpoint through its interaction with its inhibitory receptor on macrophages, signal-regulatory protein alpha (SIRPalpha). Therapeutic blockade of CD47-SIRPalpha interactions is a promising immuno-oncology treatment that promotes clearance of cancer cells. However, CD47-SIRPalpha interactions also maintain homeostatic lymphocyte levels. In this study, we report that the mouse splenic marginal zone B cell population is dependent on intact CD47-SIRPalpha interactions and blockade of CD47 leads to the loss of these cells. This depletion is accompanied by elevated levels of monocyte-recruiting chemokines CCL2 and CCL7 and infiltration of CCR2+Ly6Chi monocytes into the mouse spleen. In the absence of CCR2 signaling, there is no infiltration and reduced marginal zone B cell depletion. These data suggest that CD47 blockade leads to clearance of splenic marginal zone B cells.

  View details for DOI 10.4049/jimmunol.2100352

  View details for PubMedID 35236754

• The pleiotropic benefits of statins include the ability to reduce CD47 and amplify the effect of pro-efferocytic therapies in atherosclerosis. Nature cardiovascular research Jarr, K., Ye, J., Kojima, Y., Ye, Z., Gao, H., Schmid, S., Luo, L., Baylis, R. A., Lotfi, M., Lopez, N., Eberhard, A. V., Smith, B. R., Weissman, I. L., Maegdefessel, L., Leeper, N. J. 2022; 1 (3): 253-262

  Abstract

  The pleiotropic benefits of statins may result from their impact on vascular inflammation. The molecular process underlying this phenomenon is not fully elucidated. Here, RNA sequencing designed to investigate gene expression patterns following CD47-SIRPalpha inhibition identifies a link between statins, efferocytosis, and vascular inflammation. In vivo and in vitro studies provide evidence that statins augment programmed cell removal by inhibiting the nuclear translocation of NFkappaB1 p50 and suppressing the expression of the critical 'don't eat me' molecule, CD47. Statins amplify the phagocytic capacity of macrophages, and thus the anti-atherosclerotic effects of CD47-SIRPalpha blockade, in an additive manner. Analyses of clinical biobank specimens suggest a similar link between statins and CD47 expression in humans, highlighting the potential translational implications. Taken together, our findings identify efferocytosis and CD47 as pivotal mediators of statin pleiotropy. In turn, statins amplify the anti-atherosclerotic effects of pro-phagocytic therapies independently of any lipid-lowering effect.

  View details for DOI 10.1038/s44161-022-00023-x

  View details for PubMedID 35990913

• PNP Hydrogel Prevents Formation of Symblephara in Mice After Ocular Alkali Injury. Translational vision science & technology Swarup, A., Grosskopf, A. K., Stapleton, L. M., Subramaniam, V. R., Li, B., Weissman, I. L., Appel, E. A., Wu, A. Y. 2022; 11 (2): 31

  Abstract

  Purpose: To create an alkali injury symblephara mouse model to study conjunctival fibrosis pathophysiology and test polymer nanoparticle (PNP) hydrogel as a preventative therapeutic.Methods: Mice were injured using NaOH-soaked filter paper to determine the optimal NaOH concentration to induce the formation of symblephara. Injured mice were observed for 7 days to detect the formation of symblephara. Forniceal shortening observed on hematoxylin and eosin (H&E)-stained tissue sections was used as a symblephara marker. Alpha-smooth muscle actin (alpha-SMA) expression, Masson's trichrome assay, and periodic acid-Schiff (PAS) staining were used to determine myofibroblast expression, collagen deposition, and goblet cell integrity. PNP hydrogel, with multivalent, noncovalent interactions between modified biopolymers and nanoparticles, was applied immediately after alkali injury to determine its ability to prevent the formation of symblephara.Results: Forniceal shortening was observed in H&E images with 1N NaOH for 2 minutes after 7 days without globe destruction. PNP hydrogel prevented forniceal shortening after alkali injury as observed by H&E histology. alpha-SMA expression and collagen deposition in eye tissue sections were increased in the fornix after injury with 1N NaOH compared with uninjured controls. PNP hydrogel treatment immediately after injury reduced alpha-SMA expression and collagen deposition in the forniceal region. Mucin-secreting goblet cells stained with PAS were significantly lower in alkali-injured and PNP hydrogel-treated conjunctivas than in uninjured control conjunctivas.Conclusions: We observed that 1N NaOH for 2 minutes induced maximal forniceal shortening and symblephara in mice. PNP hydrogel prevented forniceal shortening and conjunctival fibrosis after injury. This first murine model for symblephara will be useful to study fibrosis pathophysiology after conjunctival injury and to determine therapeutic targets for cicatrizing diseases.Translational Relevance: This mouse model of symblephara can be useful for studying conjunctival scarring disease pathophysiology and preventative therapeutics. We tested PNP hydrogel, which prevented the formation of symblephara after injury.

  View details for DOI 10.1167/tvst.11.2.31

  View details for PubMedID 35191963

• Tractable Human Skeletal Stem Cell Diversity Shapes Bone Development and Regeneration Ambrosi, T., Taheri, S., Sinha, R., Goodnough, L., Steininger, H., Weissman, I., Longaker, M., Sahoo, D., Chan, C. WILEY. 2022: 266-267

  View details for Web of Science ID 000778074501234

• Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication. Nature medicine Theruvath, J., Menard, M., Smith, B. A., Linde, M. H., Coles, G. L., Dalton, G. N., Wu, W., Kiru, L., Delaidelli, A., Sotillo, E., Silberstein, J. L., Geraghty, A. C., Banuelos, A., Radosevich, M. T., Dhingra, S., Heitzeneder, S., Tousley, A., Lattin, J., Xu, P., Huang, J., Nasholm, N., He, A., Kuo, T. C., Sangalang, E. R., Pons, J., Barkal, A., Brewer, R. E., Marjon, K. D., Vilches-Moure, J. G., Marshall, P. L., Fernandes, R., Monje, M., Cochran, J. R., Sorensen, P. H., Daldrup-Link, H. E., Weissman, I. L., Sage, J., Majeti, R., Bertozzi, C. R., Weiss, W. A., Mackall, C. L., Majzner, R. G. 1800

  Abstract

  The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

  View details for DOI 10.1038/s41591-021-01625-x

  View details for PubMedID 35027753

• CD47 expression attenuates Ebola virus-induced immunopathology in mice. Antiviral research Rao, D., O'Donnell, K. L., Carmody, A., Weissman, I. L., Hasenkrug, K. J., Marzi, A. 1800: 105226

  Abstract

  It has been shown that a very early cell-intrinsic response to infection is the upregulation of CD47 cell surface expression, a molecule known for delivering a "don't eat me signal" that inhibits macrophage-mediated phagocytosis and antigen presentation. Thus, blockade of CD47 signaling during lymphocytic choriomenigitis virus infections of mice has been shown to enhance the kinetics and potency of immune responses, thereby producing faster recovery. It seems counterintuitive that one of the earliest responses to infection would be immunoinhibitory, but it has been hypothesized that CD47 induction acts as an innate immune system checkpoint to prevent immune overactivation and immunopathogenic responses during certain infections. In the current study we examined the effect of CD47 blockade on lethal Ebola virus infection of mice. At 6 days post-infection, CD47 blockade was associated with significantly increased activation of B cells along with increases in recently cytolytic CD8+ T cells. However, the anti-CD47-treated mice exhibited increased weight loss, higher virus titers, and succumbed more rapidly. The anti-CD47-treated mice also had increased inflammatory cytokines in the plasma indicative of a "cytokine storm". Thus, in the context of this rapid hemorrhagic disease, CD47 blockade indeed exacerbated immunopathology and disease severity.

  View details for DOI 10.1016/j.antiviral.2021.105226

  View details for PubMedID 34923028

• Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development. eLife Phansalkar, R., Krieger, J., Zhao, M., Kolluru, S. S., Jones, R. C., Quake, S. R., Weissman, I., Bernstein, D., Winn, V. D., D'Amato, G., Red-Horse, K. 1800; 10

  Abstract

  Most cell fate trajectories during development follow a diverging, tree-like branching pattern, but the opposite can occur when distinct progenitors contribute to the same cell type. During this convergent differentiation, it is unknown if cells 'remember' their origins transcriptionally or whether this influences cell behavior. Most coronary blood vessels of the heart develop from two different progenitor sources-the endocardium (Endo) and sinus venosus (SV)-but whether transcriptional or functional differences related to origin are retained is unknown. We addressed this by combining lineage tracing with single-cell RNA sequencing (scRNAseq) in embryonic and adult mouse hearts. Shortly after coronary development begins, capillary endothelial cells (ECs) transcriptionally segregated into two states that retained progenitor-specific gene expression. Later in development, when the coronary vasculature is well established but still remodeling, capillary ECs again segregated into two populations, but transcriptional differences were primarily related to tissue localization rather than lineage. Specifically, ECs in the heart septum expressed genes indicative of increased local hypoxia and decreased blood flow. Adult capillary ECs were more homogeneous with respect to both lineage and location. In agreement, SV- and Endo-derived ECs in adult hearts displayed similar responses to injury. Finally, scRNAseq of developing human coronary vessels indicated that the human heart followed similar principles. Thus, over the course of development, transcriptional heterogeneity in coronary ECs is first influenced by lineage, then by location, until heterogeneity declines in the homeostatic adult heart. These results highlight the plasticity of ECs during development, and the validity of the mouse as a model for human coronary development.

  View details for DOI 10.7554/eLife.70246

  View details for PubMedID 34910626

• Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report. Annals of the New York Academy of Sciences Cable, J., Pei, D., Reid, L. M., Wang, X. W., Bhatia, S., Karras, P., Melenhorst, J. J., Grompe, M., Lathia, J. D., Song, E., Kuo, C. J., Zhang, N., White, R. M., Ma, S. K., Ma, L., Chin, Y. R., Shen, M. M., Ng, I. O., Kaestner, K. H., Zhou, L., Sikandar, S., Schmitt, C. A., Guo, W., Wong, C. C., Ji, J., Tang, D. G., Dubrovska, A., Yang, C., Wiedemeyer, W. R., Weissman, I. L. 2021

  Abstract

  The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability.On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

  View details for DOI 10.1111/nyas.14719

  View details for PubMedID 34850398

• JSP191 As a Single-Agent Conditioning Regimen Results in Successful Engraftment, Donor Myeloid Chimerism, and Production of Donor Derived Naive Lymphocytes in Patients with Severe Combined Immunodeficiency (SCID) Agarwa, R., Dvorak, C. C., Prockop, S., Kwon, H., Long-Boyle, J. R., Le, A., Brown, J. W., Merkel, E., Truong, K., Velasco, B., Arulprakasam, K., Harada, N., Dougall, K. A., Prohaska, S. S., Pang, W. W., Heller, K. N., Weissman, I. L., Cowan, M. J., Logan, A. C., O'Reilly, R. J., Parkman, R., Weinberg, K. I., Roncarolo, M., Shizuru, J. A. AMER SOC HEMATOLOGY. 2021

  View details for DOI 10.1182/blood-2021-153074

  View details for Web of Science ID 000736398802095

• CD47 REGULATES PARASITE BURDEN AND PROMOTES PATHOGENESIS IN MURINE MALARIA MODELS Oakley, M. S., Malla, P., Dulgeroff, L., Majam, V., Chorazeczewski, J. K., Okoth, W. A., Meredith, S., Rotstein, D. S., Weissman, I. L., Kumar, S. AMER SOC TROP MED & HYGIENE. 2021: 334-335

  View details for Web of Science ID 000778105603140

• A FUNCTIONAL GENETIC SCREEN UNCOVERS REGULATORS OF INTRATUMORAL MACROPHAGE FUNCTION AND REVEALS CD24 AS A NOVEL TARGET FOR CANCER IMMUNOTHERAPY BY MACROPHAGES Barkal, A., Brewer, R., Weissman, I. BMJ PUBLISHING GROUP. 2021: A283

  View details for DOI 10.1136/jitc-2021-SITC2021.261

  View details for Web of Science ID 000774877500253

• Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis. Nature Kamber, R. A., Nishiga, Y., Morton, B., Banuelos, A. M., Barkal, A. A., Vences-Catalan, F., Gu, M., Fernandez, D., Seoane, J. A., Yao, D., Liu, K., Lin, S., Spees, K., Curtis, C., Jerby-Arnon, L., Weissman, I. L., Sage, J., Bassik, M. C. 2021

  Abstract

  Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

  View details for DOI 10.1038/s41586-021-03879-4

  View details for PubMedID 34497417

• A Clinical PET Imaging Tracer ([18F]DASA-23) to Monitor Pyruvate Kinase M2 Induced Glycolytic Reprogramming in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research Beinat, C., Patel, C. B., Haywood, T., Murty, S., Naya, L., Castillo, J. B., Reyes, S. T., Phillips, M., Buccino, P., Shen, B., Park, J. H., Koran, M. E., Alam, I. S., James, M. L., Holley, D., Halbert, K., Gandhi, H., He, J. Q., Granucci, M., Johnson, E., Liu, D. D., Uchida, N., Sinha, R., Chu, P., Born, D. E., Warnock, G. I., Weissman, I., Hayden Gephart, M., Khalighi, M. M., Massoud, T. F., Iagaru, A., Davidzon, G., Thomas, R., Nagpal, S., Recht, L. D., Gambhir, S. S. 2021

  Abstract

  PURPOSE: Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain. We describe the development, validation, and translation of a novel positron emission tomography (PET) tracer to study PKM2 in GBM. We evaluated 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and GBM patients.EXPERIMENTAL DESIGN: [18F]DASA-23 was synthesized with a molar activity of 100.47 {plus minus} 29.58 GBq/mol and radiochemical purity >95%. We performed initial testing of [18F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next we produced [18F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers, and a pilot cohort of glioma patients.RESULTS: In mouse imaging studies, [18F]DASA-23 clearly delineated the U87 GBM from surrounding healthy brain tissue and had a tumor-to-brain ratio (TBR) of 3.6 {plus minus} 0.5. In human volunteers, [18F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In GBM patients, [18F]DASA-23 successfully outlined tumors visible on contrast-enhanced magnetic resonance imaging (MRI). The uptake of [18F]DASA-23 was markedly elevated in GBMs compared to normal brain, and it identified a metabolic non-responder within 1-week of treatment initiation.CONCLUSIONS: We developed and translated [18F]DASA-23 as a new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These results warrant further clinical evaluation of [18F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism.

  View details for DOI 10.1158/1078-0432.CCR-21-0544

  View details for PubMedID 34475101

• Combining CD47 blockade with trastuzumab eliminates HER2-positive breast cancer cells and overcomes trastuzumab tolerance. Proceedings of the National Academy of Sciences of the United States of America Upton, R., Banuelos, A., Feng, D., Biswas, T., Kao, K., McKenna, K., Willingham, S., Ho, P. Y., Rosental, B., Tal, M. C., Raveh, T., Volkmer, J., Pegram, M. D., Weissman, I. L. 2021; 118 (29)

  Abstract

  Trastuzumab, a targeted anti-human epidermal-growth-factor receptor-2 (HER2) monoclonal antibody, represents a mainstay in the treatment of HER2-positive (HER2+) breast cancer. Although trastuzumab treatment is highly efficacious for early-stage HER2+ breast cancer, the majority of advanced-stage HER2+ breast cancer patients who initially respond to trastuzumab acquire resistance to treatment and relapse, despite persistence of HER2 gene amplification/overexpression. Here, we sought to leverage HER2 overexpression to engage antibody-dependent cellular phagocytosis (ADCP) through a combination of trastuzumab and anti-CD47 macrophage checkpoint immunotherapy. We have previously shown that blockade of CD47, a surface protein expressed by many malignancies (including HER2+ breast cancer), is an effective anticancer therapy. CD47 functions as a "don't eat me" signal through its interaction with signal regulatory protein-alpha (SIRPalpha) on macrophages to inhibit phagocytosis. Hu5F9-G4 (magrolimab), a humanized monoclonal antibody against CD47, blocks CD47's "don't eat me" signal, thereby facilitating macrophage-mediated phagocytosis. Preclinical studies have shown that combining Hu5F9-G4 with tumor-targeting antibodies, such as rituximab, further enhances Hu5F9-G4's anticancer effects via ADCP. Clinical trials have additionally demonstrated that Hu5F9-G4, in combination with rituximab, produced objective responses in patients whose diffuse large B cell lymphomas had developed resistance to rituximab and chemotherapy. These studies led us to hypothesize that combining Hu5F9-G4 with trastuzumab would produce an anticancer effect in antibody-dependent cellular cytotoxicity (ADCC)-tolerant HER2+ breast cancer. This combination significantly suppressed the growth of ADCC-tolerant HER2+ breast cancers via Fc-dependent ADCP. Our study demonstrates that combining trastuzumab and Hu5F9-G4 represents a potential new treatment option for HER2+ breast cancer patients, even for patients whose tumors have progressed after trastuzumab.

  View details for DOI 10.1073/pnas.2026849118

  View details for PubMedID 34257155

• Distinct skeletal stem cell types orchestrate long bone skeletogenesis. eLife Ambrosi, T. H., Sinha, R., Steininger, H. M., Hoover, M. Y., Murphy, M. P., Koepke, L. S., Wang, Y., Lu, W., Morri, M., Neff, N. F., Weissman, I. L., Longaker, M. T., Chan, C. K. 2021; 10

  Abstract

  Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

  View details for DOI 10.7554/eLife.66063

  View details for PubMedID 34280086

• Overexpression of CD47 is associated with brain overgrowth and 16p11.2 deletion syndrome. Proceedings of the National Academy of Sciences of the United States of America Li, J., Brickler, T., Banuelos, A., Marjon, K., Shcherbina, A., Banerjee, S., Bian, J., Narayanan, C., Weissman, I. L., Chetty, S. 2021; 118 (15)

  Abstract

  Copy number variation (CNV) at the 16p11.2 locus is associated with neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia. CNVs of the 16p gene can manifest in opposing head sizes. Carriers of 16p11.2 deletion tend to have macrocephaly (or brain enlargement), while those with 16p11.2 duplication frequently have microcephaly. Increases in both gray and white matter volume have been observed in brain imaging studies in 16p11.2 deletion carriers with macrocephaly. Here, we use human induced pluripotent stem cells (hiPSCs) derived from controls and subjects with 16p11.2 deletion and 16p11.2 duplication to understand the underlying mechanisms regulating brain overgrowth. To model both gray and white matter, we differentiated patient-derived iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). In both NPCs and OPCs, we show that CD47 (a "don't eat me" signal) is overexpressed in the 16p11.2 deletion carriers contributing to reduced phagocytosis both in vitro and in vivo. Furthermore, 16p11.2 deletion NPCs and OPCs up-regulate cell surface expression of calreticulin (a prophagocytic "eat me" signal) and its binding sites, indicating that these cells should be phagocytosed but fail to be eliminated due to elevations in CD47. Treatment of 16p11.2 deletion NPCs and OPCs with an anti-CD47 antibody to block CD47 restores phagocytosis to control levels. While the CD47 pathway is commonly implicated in cancer progression, we document a role for CD47 in psychiatric disorders associated with brain overgrowth.

  View details for DOI 10.1073/pnas.2005483118

  View details for PubMedID 33833053

• CD47 blockade reduces the pathologic features of experimental cerebral malaria and promotes survival of hosts with Plasmodium infection. Proceedings of the National Academy of Sciences of the United States of America Torrez Dulgeroff, L. B., Oakley, M. S., Tal, M. C., Yiu, Y. Y., He, J. Q., Shoham, M., Majam, V., Okoth, W. A., Malla, P., Kumar, S., Weissman, I. L. 2021; 118 (11)

  Abstract

  CD47 is an antiphagocytic "don't eat me" signal that inhibits programmed cell removal of self. As red blood cells (RBCs) age they lose CD47 expression and become susceptible to programmed cell removal by macrophages. CD47-/- mice infected with Plasmodium yoelii, which exhibits an age-based preference for young RBCs, were previously demonstrated to be highly resistant to malaria infection. Our study sought to test the therapeutic benefit of CD47 blockade on ameliorating the clinical syndromes of experimental cerebral malaria (ECM), using the Plasmodium berghei ANKA (Pb-A) murine model. In vitro we tested the effect of anti-CD47 mAb on Plasmodium-infected RBC phagocytosis and found that anti-CD47 treatment significantly increased clearance of Plasmodium-infected RBCs. Infection of C57BL/6 mice with Pb-A is lethal and mice succumb to the clinical syndromes of CM between days 6 and 10 postinfection. Strikingly, treatment with anti-CD47 resulted in increased survival during the cerebral phase of Pb-A infection. Anti-CD47-treated mice had increased lymphocyte counts in the peripheral blood and increased circulating levels of IFN-γ, TNF-α, and IL-22. Despite increased circulating levels of inflammatory cytokines, anti-CD47-treated mice had reduced pathological features in the brain. Survival of ECM in anti-CD47-treated mice was correlated with reduced cellular accumulation in the cerebral vasculature, improved blood-brain barrier integrity, and reduced cytotoxic activity of infiltrating CD8+ T cells. These results demonstrate the therapeutic benefit of anti-CD47 to reduce morbidity in a lethal model of ECM, which may have implications for preventing mortality in young African children who are the highest casualties of CM.

  View details for DOI 10.1073/pnas.1907653118

  View details for PubMedID 33836556

  View details for PubMedCentralID PMC7980459

• Humanized anti-CD47 monoclonal antibody magrolimab (Hu5F9-G4) plus trastuzumab potentiates antibody-dependent cellular phagocytosis (ADCP), and cooperate to inhibit human HER2+breast cancer (BC) xenografts growth in vivo Upton, R., Feng, D., Banuelos, A. M., Biswas, T., Willingham, S., Kao, K. S., McKenna, K., Rosenthal, B., Tal, M. C., Volkmer, J., Pegram, M. D., Weissman, I. L. AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000618737700575

• Restoring metabolism of myeloid cells reverses cognitive decline in ageing. Nature Minhas, P. S., Latif-Hernandez, A., McReynolds, M. R., Durairaj, A. S., Wang, Q., Rubin, A., Joshi, A. U., He, J. Q., Gauba, E., Liu, L., Wang, C., Linde, M., Sugiura, Y., Moon, P. K., Majeti, R., Suematsu, M., Mochly-Rosen, D., Weissman, I. L., Longo, F. M., Rabinowitz, J. D., Andreasson, K. I. 2021

  Abstract

  Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty1-3. The ageing brain is also vulnerable to inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer's disease4-6. Systemically, circulating pro-inflammatory factors can promote cognitive decline7,8, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration9,10. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E2 (PGE2), a major modulator of inflammation11. In ageing macrophages and microglia, PGE2 signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.

  View details for DOI 10.1038/s41586-020-03160-0

  View details for PubMedID 33473210

• Safe and Effective In Vivo Targeting and Gene Editing in Hematopoietic Stem Cells: Strategies for Accelerating Development National Institutes of Health/Bill & Melinda Gates Foundation Expert Scientific Roundtable Webinar Meeting. Human gene therapy Cannon, P., Asokan, A., Czechowicz, A., Hammond, P., Kohn, D. B., Lieber, A., Malik, P., Marks, P., Porteus, M., Verhoeyen, E., Weissman, D., Weissman, I., Kiem, H. 2021

  Abstract

  Introduction On May 11, 2020, the National Institutes of Health (NIH) and the Bill & Melinda Gates Foundation (Gates Foundation) held an exploratory expert scientific roundtable to inform an NIH-Gates Foundation collaboration on the development of scalable, sustainable, and accessible HIV and sickle cell disease (SCD) therapies based on in vivo gene editing of hematopoietic stem cells (HSC). A particular emphasis was on how such therapies could be developed for low-resource settings in sub-Saharan Africa. Paula Cannon, Ph.D., of the University of Southern California and Hans-Peter Kiem, M.D., Ph.D., of the Fred Hutchinson Cancer Research Center served as roundtable co-chairs. Welcoming remarks were provided by the leadership of NIH, NHLBI, and BMGF, who cited the importance of assessing the state of the science and charting a path toward finding safe, effective, and durable gene-based therapies for HIV and sickle cell disease. These remarks were followed by three sessions in which participants heard presentations on and discussed the therapeutic potential of modified HSCs, leveraging HSC biology and differentiation, and in vivo HSC targeting approaches. This roundtable serves as the beginning of an ongoing discussion among NIH, the Gates Foundation, research and patient communities, and the public at large. As this collaboration progresses, these communities will be engaged as we collectively navigate the complex scientific and ethical issues surrounding in vivo HSC targeting and editing. Summarized excerpts from each of the presentations are below, reflecting the individual views and perspectives of each presenter.

  View details for DOI 10.1089/hum.2020.263

  View details for PubMedID 33427035

• Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery. Immunity Chiou, S. H., Tseng, D. n., Reuben, A. n., Mallajosyula, V. n., Molina, I. S., Conley, S. n., Wilhelmy, J. n., McSween, A. M., Yang, X. n., Nishimiya, D. n., Sinha, R. n., Nabet, B. Y., Wang, C. n., Shrager, J. B., Berry, M. F., Backhus, L. n., Lui, N. S., Wakelee, H. A., Neal, J. W., Padda, S. K., Berry, G. J., Delaidelli, A. n., Sorensen, P. H., Sotillo, E. n., Tran, P. n., Benson, J. A., Richards, R. n., Labanieh, L. n., Klysz, D. D., Louis, D. M., Feldman, S. A., Diehn, M. n., Weissman, I. L., Zhang, J. n., Wistuba, I. I., Futreal, P. A., Heymach, J. V., Garcia, K. C., Mackall, C. L., Davis, M. M. 2021; 54 (3): 586–602.e8

  Abstract

  To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRβ chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.

  View details for DOI 10.1016/j.immuni.2021.02.014

  View details for PubMedID 33691136

• Absence of CD11a Expression Identifies Embryonic Hematopoietic Stem Cell Precursors via Competitive Neonatal Transplantation Assay. Frontiers in cell and developmental biology Karimzadeh, A., Varady, E. S., Scarfone, V. M., Chao, C., Grathwohl, K., Nguyen, P. U., Ghorbanian, Y., Weissman, I. L., Serwold, T., Inlay, M. A. 2021; 9: 734176

  Abstract

  Hematopoietic stem cells (HSCs) are defined by their self-renewal, multipotency, and bone marrow (BM) engraftment abilities. How HSCs emerge during embryonic development remains unclear, but are thought to arise from hemogenic endothelium through an intermediate precursor called "pre-HSCs." Pre-HSCs have self-renewal and multipotent activity, but lack BM engraftability. They can be identified functionally by transplantation into neonatal recipients, or by in vitro co-culture with cytokines and stroma followed by transplantation into adult recipients. While pre-HSCs express markers such as Kit and CD144, a precise surface marker identity for pre-HSCs has remained elusive due to the fluctuating expression of common HSC markers during embryonic development. We have previously determined that the lack of CD11a expression distinguishes HSCs in adults as well as multipotent progenitors in the embryo. Here, we use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed "11a- eKLS" (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all in vivo long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs.

  View details for DOI 10.3389/fcell.2021.734176

  View details for PubMedID 34513848

• Reactivation of the pluripotency program precedes formation of the cranial neural crest. Science (New York, N.Y.) Zalc, A., Sinha, R., Gulati, G. S., Wesche, D. J., Daszczuk, P., Swigut, T., Weissman, I. L., Wysocka, J. 2021; 371 (6529)

  Abstract

  During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor Oct4 is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4+ CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.

  View details for DOI 10.1126/science.abb4776

  View details for PubMedID 33542111

• Sexual and asexual development: two distinct programs producing the same tunicate. Cell reports Kowarsky, M. n., Anselmi, C. n., Hotta, K. n., Burighel, P. n., Zaniolo, G. n., Caicci, F. n., Rosental, B. n., Neff, N. F., Ishizuka, K. J., Palmeri, K. J., Okamoto, J. n., Gordon, T. n., Weissman, I. L., Quake, S. R., Manni, L. n., Voskoboynik, A. n. 2021; 34 (4): 108681

  Abstract

  Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri, we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways.

  View details for DOI 10.1016/j.celrep.2020.108681

  View details for PubMedID 33503429

• Hoxb5 defines the heterogeneity of self-renewal capacity in the hematopoietic stem cell compartment. Biochemical and biophysical research communications Sakamaki, T. n., Kao, K. S., Nishi, K. n., Chen, J. Y., Sadaoka, K. n., Fujii, M. n., Takaori-Kondo, A. n., Weissman, I. L., Miyanishi, M. n. 2021; 539: 34–41

  Abstract

  Self-renewal and multipotency are essential functions of hematopoietic stem cells (HSCs). To maintain homeostatic hematopoiesis, functionally uniform HSCs have been thought to be an ideal cell-of-origin. Recent technological advances in the field have allowed us to analyze HSCs with single cell resolution and implicate that functional heterogeneity may exist even within the highly purified HSC compartment. However, due in part to the technical limitations of analyzing extremely rare populations and our incomplete understanding of HSC biology, neither the biological meaning of why heterogeneity exists nor the precise mechanism of how heterogeneity is determined within the HSC compartment is entirely known. Here we show the first evidence that self-renewal capacity varies with the degree of replication stress dose and results in heterogeneity within the HSC compartment. Using the Hoxb5-reporter mouse line which enables us to distinguish between long-term (LT)-HSCs and short-term (ST)-HSCs, we have found that ST-HSCs quickly lose self-renewal capacity under high stress environments but can maintain self-renewal under low stress environments for long periods of time. Critically, exogeneous Hoxb5 expression confers protection against loss of self-renewal to Hoxb5-negative HSCs and can partially alter the cell fate of ST-HSCs to that of LT-HSCs. Our results demonstrate that Hoxb5 imparts functional heterogeneity in the HSC compartment by regulating self-renewal capacity. Additionally, Hoxb5-positive HSCs may exist as fail-safe system to protect from the exhaustion of HSCs throughout an organism's lifespan.

  View details for DOI 10.1016/j.bbrc.2020.12.077

  View details for PubMedID 33418191

• Aged skeletal stem cells generate an inflammatory degenerative niche. Nature Ambrosi, T. H., Marecic, O., McArdle, A., Sinha, R., Gulati, G. S., Tong, X., Wang, Y., Steininger, H. M., Hoover, M. Y., Koepke, L. S., Murphy, M. P., Sokol, J., Seo, E. Y., Tevlin, R., Lopez, M., Brewer, R. E., Mascharak, S., Lu, L., Ajanaku, O., Conley, S. D., Seita, J., Morri, M., Neff, N. F., Sahoo, D., Yang, F., Weissman, I. L., Longaker, M. T., Chan, C. K. 2021

  Abstract

  Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.

  View details for DOI 10.1038/s41586-021-03795-7

  View details for PubMedID 34381212

• Epidermal-Derived Hedgehog Signaling Drives Mesenchymal Proliferation during Digit Tip Regeneration. Journal of clinical medicine Maan, Z. N., Rinkevich, Y., Barrera, J., Chen, K., Henn, D., Foster, D., Bonham, C. A., Padmanabhan, J., Sivaraj, D., Duscher, D., Hu, M., Yan, K., Januszyk, M., Longaker, M. T., Weissman, I. L., Gurtner, G. C. 2021; 10 (18)

  Abstract

  Hand injuries often result in significant functional impairments and are rarely completely restored. The spontaneous regeneration of injured appendages, which occurs in salamanders and newts, for example, has been reported in human fingertips after distal amputation, but this type of regeneration is rare in mammals and is incompletely understood. Here, we study fingertip regeneration by amputating murine digit tips, either distally to initiate regeneration, or proximally, causing fibrosis. Using an unbiased microarray analysis, we found that digit tip regeneration is significantly associated with hair follicle differentiation, Wnt, and sonic hedgehog (SHH) signaling pathways. Viral over-expression and genetic knockouts showed the functional significance of these pathways during regeneration. Using transgenic reporter mice, we demonstrated that, while both canonical Wnt and HH signaling were limited to epidermal tissues, downstream hedgehog signaling (through Gli) occurred in mesenchymal tissues. These findings reveal a mechanism for epidermal/mesenchyme interactions, governed by canonical hedgehog signaling, during digit regeneration. Further research into these pathways could lead to improved therapeutic outcomes after hand injuries in humans.

  View details for DOI 10.3390/jcm10184261

  View details for PubMedID 34575372

• Effect of CD47 Blockade on Vascular Inflammation. The New England journal of medicine Jarr, K. U., Nakamoto, R. n., Doan, B. H., Kojima, Y. n., Weissman, I. L., Advani, R. H., Iagaru, A. n., Leeper, N. J. 2021; 384 (4): 382–83

  View details for DOI 10.1056/NEJMc2029834

  View details for PubMedID 33503349

• Wounds Inhibit Tumor Growth In Vivo ANNALS OF SURGERY Hu, M. S., Maan, Z. N., Leavitt, T., Hong, W., Rennert, R. C., Marshall, C. D., Borrelli, M. R., Zhu, T. N., Esquivel, M., Zimmermann, A., McArdle, A., Chung, M. T., Foster, D. S., Jones, R., Gurtner, G. C., Giaccia, A. J., Lorenz, H., Weissman, I. L., Longaker, M. T. 2021; 273 (1): 173–80

  View details for DOI 10.1097/SLA.0000000000003255

  View details for Web of Science ID 000613348700039

• Proteomic analysis of young and old mouse hematopoietic stem cells and their progenitors reveals post-transcriptional regulation in stem cells. eLife Zaro, B. W., Noh, J. J., Mascetti, V. L., Demeter, J., George, B., Zukowska, M., Gulati, G. S., Sinha, R., Flynn, R. A., Banuelos, A., Zhang, A., Wilkinson, A. C., Jackson, P., Weissman, I. L. 2020; 9

  Abstract

  The balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for a healthy blood supply; imbalances underlie hematological diseases. The importance of HSCs and their progenitors have led to their extensive characterization at genomic and transcriptomic levels. However, the proteomics of hematopoiesis remains incompletely understood. Here we report a proteomics resource from mass spectrometry of mouse young adult and old adult mouse HSCs, multipotent progenitors and oligopotent progenitors; 12 cell types in total. We validated differential protein levels, including confirmation that Dnmt3a protein levels are undetected in young adult mouse HSCs until forced into cycle. Additionally, through integrating proteomics and RNA-sequencing datasets, we identified a subset of genes with apparent post-transcriptional repression in young adult mouse HSCs. In summary, we report proteomic coverage of young and old mouse HSCs and progenitors, with broader implications for understanding mechanisms for stem cell maintenance, niche interactions and fate determination.

  View details for DOI 10.7554/eLife.62210

  View details for PubMedID 33236985

• A molecular cell atlas of the human lung from single-cell RNA sequencing. Nature Travaglini, K. J., Nabhan, A. N., Penland, L., Sinha, R., Gillich, A., Sit, R. V., Chang, S., Conley, S. D., Mori, Y., Seita, J., Berry, G. J., Shrager, J. B., Metzger, R. J., Kuo, C. S., Neff, N., Weissman, I. L., Quake, S. R., Krasnow, M. A. 2020

  Abstract

  Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles1-9, it has been difficult to systematically identify and localize all molecularcell types in individual organs to create a full molecular cell atlas. Here, using droplet- and plate-based single-cell RNA sequencing of approximately 75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, we create an extensive cell atlas of the human lung. We define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 out of 45 previously known cell types and 14 previously unknown ones. This comprehensive molecular atlas identifies the biochemical functions of lung cells and the transcription factors and markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signalling interactions and immune cell homing; and identifies cell types that are directly affected by lung disease genes and respiratory viruses. By comparing human and mouse data, we identified 17 molecular cell types that have been gained or lost during lung evolution and others with substantially altered expression profiles, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions and interactions are achieved in development and tissue engineering and altered in disease and evolution.

  View details for DOI 10.1038/s41586-020-2922-4

  View details for PubMedID 33208946

• Effects of ultra-high dose rate FLASH irradiation on the tumor microenvironment in Lewis lung carcinoma: role of myosin light chain. International journal of radiation oncology, biology, physics Kim, Y., Gwak, S., Hong, B., Oh, J., Choi, H., Kim, M. S., Oh, D., Lartey, F., Rafat, M., Schuler, E., Kim, H., von Eyben, R., Weissman, I. L., Koch, C. J., Maxim, P. G., Loo, B. W., Ahn, G. 2020

  Abstract

  PURPOSE: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR.METHODS AND MATERIALS: Lewis lung carcinoma (LLC) were subcutaneously implanted in mice followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hr post-IR and stained for CD31, phosphorylated myosin-light chain (p-MLC), gammaH2AX, intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8alpha T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days prior to 15 Gy CONV IR. Tumors were similarly harvested and analyzed as above.RESULTS: By immunostaining, we observed that CONV IR at 6 hr post-IR resulted in constricted vessel morphology, increased expression of phosphorylated myosin light chain (p-MLC), and much higher numbers of gammaH2AX (surrogate marker for DNA double strand break)-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, we found that MLC activation by reactive oxygen species (ROS) is unlikely since FLASH IR produced significantly higher ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase abrogated IR-induced gammaH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR including the reduction in the vasculature collapse, fewer gammaH2AX-positive cells, and increased immune cell influx to the tumors.CONCLUSIONS: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of those microenvironmental changes differentially regulated between CONV and FLASH IR.

  View details for DOI 10.1016/j.ijrobp.2020.11.012

  View details for PubMedID 33186615

• Articular cartilage regeneration by activated skeletal stem cells. Nature medicine Murphy, M. P., Koepke, L. S., Lopez, M. T., Tong, X., Ambrosi, T. H., Gulati, G. S., Marecic, O., Wang, Y., Ransom, R. C., Hoover, M. Y., Steininger, H., Zhao, L., Walkiewicz, M. P., Quarto, N., Levi, B., Wan, D. C., Weissman, I. L., Goodman, S. B., Yang, F., Longaker, M. T., Chan, C. K. 2020

  Abstract

  Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2-4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5-7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

  View details for DOI 10.1038/s41591-020-1013-2

  View details for PubMedID 32807933

• Digit tip regeneration relies on germ layer restricted Wnt and Hedgehog signaling Barrera, J., Maan, Z., Rinkevich, Y., Henn, D., Chen, K., Bonham, C. A., Padmanabhan, J., Januszyk, M., Weissman, I. L., Gurtner, G. C. WILEY. 2020: S5

  View details for Web of Science ID 000548418300006

• James L. Gowans 1924-2020. Nature immunology Howard, J. C., Weissman, I. L. 2020; 21 (6): 595

  View details for DOI 10.1038/s41590-020-0696-3

  View details for PubMedID 32457533

• Magrolimab and gemcitabine-cisplatin combination enhance phagocytic elimination of bladder cancer. Kiss, B., Volkmer, A., Feng, D., McKenna, K., Mihardja, S., Chao, M., Takimoto, C. M., Liao, J. C., Volkmer, J., Weissman, I. L. LIPPINCOTT WILLIAMS & WILKINS. 2020

  View details for Web of Science ID 000560368306191

• Functional cytotoxic T cells exhibit tissue-specific phenotypic differences during chronic Friend virus infection Myers, L., Tal, M., Messer, R. J., Carmody, A. B., Weissman, I. L., Hasenkrug, K. J. AMER ASSOC IMMUNOLOGISTS. 2020

  View details for Web of Science ID 000589972401051

• Polymorphisms in SIRPA impact macrophage phagocytosis in response to therapeutic antibody blockade. Yiu, Y. Y., Barkal, A. A., Tal, M. C., Ollila, H. M., Hansen, P. S., Mansfield, P., Weissman, I. L. AMER ASSOC CANCER RESEARCH. 2020: 49

  View details for Web of Science ID 000518188200069

• Evolutionary perspective on the hematopoietic system through a colonial chordate: allogeneic immunity and hematopoiesis. Current opinion in immunology Rosental, B., Raveh, T., Voskoboynik, A., Weissman, I. L. 2020; 62: 91–98

  Abstract

  Evolution and selection have shaped diverse immune systems throughout phylogeny, the vast majority of which remain unexplored. Botryllus schlosseri is a colonial tunicate, a sister group to vertebrates, that develops as a chordate, then metamorphoses to an asexually reproductive invertebrate that every week makes the same body plan from budded stem cells. Genetically distinct B. schlosseri colonies can fuse to form a chimera, or reject each other based on allogeneic recognition. In chimeras, circulating germline and somatic stem cells participate in development; stem cells compete in all individuals in the fused colonies, with rejection preventing germline parasitism. Here we review the isolation and characterization of B. schlosseri hematopoietic stem cells (HSC) and their niches, and the role of the immune effector cells in allorecognition.

  View details for DOI 10.1016/j.coi.2019.12.006

  View details for PubMedID 31954962

• A single-cell transcriptomic atlas characterizes ageing tissues in the mouse. Nature 2020

  Abstract

  Ageing is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death1. Despite rapid advances over recent years, many of the molecular and cellular processes that underlie the progressive loss of healthy physiology are poorly understood2. To gain a better insight into these processes, here we generate a single-cell transcriptomic atlas across the lifespan of Mus musculus that includes data from 23 tissues and organs. We found cell-specific changes occurring across multiple cell types and organs, as well as age-related changes in the cellular composition of different organs. Using single-cell transcriptomic data, we assessed cell-type-specific manifestations of different hallmarks of ageing-such as senescence3, genomic instability4 and changes in the immune system2. This transcriptomic atlas-which we denote Tabula Muris Senis, or 'Mouse Ageing Cell Atlas'-provides molecular information about how the most important hallmarks of ageing are reflected in a broad range of tissues and cell types.

  View details for DOI 10.1038/s41586-020-2496-1

  View details for PubMedID 32669714

• Clonally expanding smooth muscle cells promote atherosclerosis by escaping efferocytosis and activating the complement cascade. Proceedings of the National Academy of Sciences of the United States of America Wang, Y. n., Nanda, V. n., Direnzo, D. n., Ye, J. n., Xiao, S. n., Kojima, Y. n., Howe, K. L., Jarr, K. U., Flores, A. M., Tsantilas, P. n., Tsao, N. n., Rao, A. n., Newman, A. A., Eberhard, A. V., Priest, J. R., Ruusalepp, A. n., Pasterkamp, G. n., Maegdefessel, L. n., Miller, C. L., Lind, L. n., Koplev, S. n., Björkegren, J. L., Owens, G. K., Ingelsson, E. n., Weissman, I. L., Leeper, N. J. 2020

  Abstract

  Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

  View details for DOI 10.1073/pnas.2006348117

  View details for PubMedID 32541024

• Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection. Cell reports Cham, L. B., Torrez Dulgeroff, L. B., Tal, M. C., Adomati, T. n., Li, F. n., Bhat, H. n., Huang, A. n., Lang, P. A., Moreno, M. E., Rivera, J. M., Galkina, S. A., Kosikova, G. n., Stoddart, C. A., McCune, J. M., Myers, L. M., Weissman, I. L., Lang, K. S., Hasenkrug, K. J. 2020; 31 (2): 107494

  Abstract

  Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases.

  View details for DOI 10.1016/j.celrep.2020.03.058

  View details for PubMedID 32294445

• Semaphorin 3A mediated brain tumor stem cell proliferation and invasion in EGFRviii mutant gliomas. BMC cancer Higgins, D. M., Caliva, M. n., Schroeder, M. n., Carlson, B. n., Upadhyayula, P. S., Milligan, B. D., Cheshier, S. H., Weissman, I. L., Sarkaria, J. N., Meyer, F. B., Henley, J. R. 2020; 20 (1): 1213

  Abstract

  Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors.GBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7.Immunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD.These findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

  View details for DOI 10.1186/s12885-020-07694-4

  View details for PubMedID 33302912

• Pro-efferocytic nanoparticles are specifically taken up by lesional macrophages and prevent atherosclerosis. Nature nanotechnology Flores, A. M., Hosseini-Nassab, N. n., Jarr, K. U., Ye, J. n., Zhu, X. n., Wirka, R. n., Koh, A. L., Tsantilas, P. n., Wang, Y. n., Nanda, V. n., Kojima, Y. n., Zeng, Y. n., Lotfi, M. n., Sinclair, R. n., Weissman, I. L., Ingelsson, E. n., Smith, B. R., Leeper, N. J. 2020

  Abstract

  Atherosclerosis is the process that underlies heart attack and stroke. A characteristic feature of the atherosclerotic plaque is the accumulation of apoptotic cells in the necrotic core. Prophagocytic antibody-based therapies are currently being explored to stimulate the phagocytic clearance of apoptotic cells; however, these therapies can cause off-target clearance of healthy tissues, which leads to toxicities such as anaemia. Here we developed a macrophage-specific nanotherapy based on single-walled carbon nanotubes loaded with a chemical inhibitor of the antiphagocytic CD47-SIRPα signalling axis. We demonstrate that these single-walled carbon nanotubes accumulate within the atherosclerotic plaque, reactivate lesional phagocytosis and reduce the plaque burden in atheroprone apolipoprotein-E-deficient mice without compromising safety, and thereby overcome a key translational barrier for this class of drugs. Single-cell RNA sequencing analysis reveals that prophagocytic single-walled carbon nanotubes decrease the expression of inflammatory genes linked to cytokine and chemokine pathways in lesional macrophages, which demonstrates the potential of 'Trojan horse' nanoparticles to prevent atherosclerotic cardiovascular disease.

  View details for DOI 10.1038/s41565-019-0619-3

  View details for PubMedID 31988506

• Ageing hallmarks exhibit organ-specific temporal signatures. Nature Schaum, N. n., Lehallier, B. n., Hahn, O. n., Pálovics, R. n., Hosseinzadeh, S. n., Lee, S. E., Sit, R. n., Lee, D. P., Losada, P. M., Zardeneta, M. E., Fehlmann, T. n., Webber, J. T., McGeever, A. n., Calcuttawala, K. n., Zhang, H. n., Berdnik, D. n., Mathur, V. n., Tan, W. n., Zee, A. n., Tan, M. n., Pisco, A. O., Karkanias, J. n., Neff, N. F., Keller, A. n., Darmanis, S. n., Quake, S. R., Wyss-Coray, T. n. 2020

  Abstract

  Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.

  View details for DOI 10.1038/s41586-020-2499-y

  View details for PubMedID 32669715

• Targeting macrophage checkpoint inhibitor SIRPa for anticancer therapy. JCI insight Liu, J. n., Xavy, S. n., Mihardja, S. n., Chen, S. n., Sompalli, K. n., Feng, D. n., Choi, T. S., Agoram, B. n., Majeti, R. n., Weissman, I. L., Volkmer, J. P. 2020

  Abstract

  The SIRPα-CD47 interaction provides a macrophage immune checkpoint pathway that plays a critical role in cancer immune evasion across multiple cancers. Here, we report the engineering of a humanized anti-SIRPα monoclonal antibody (1H9) for antibody target cancer therapy. 1H9 has broad activity across a wide range of SIRPα variants. Binding of 1H9 to SIRPα blocks its interaction with CD47, thereby promoting macrophage-mediated phagocytosis of cancer cells. Pre-clinical studies in vitro and in vivo demonstrate that 1H9 synergizes with other therapeutic antibodies to promote phagocytosis of tumor cells and inhibit tumor growth in both syngeneic and xenograft tumor models, leading to survival benefit. Thus, 1H9 can potentially act as a universal agent to enhance therapeutic efficacy when used in combination with most tumor-targeting antibodies. We report for the first time, a comparison of anti-SIRPα and anti-CD47 antibodies in CD47/SIRPα double humanized mice, and found that 1H9 exhibits a substantially reduced antigen-sink effect due to the limited tissue distribution of SIRPα expression. Toxicokinetic studies in non-human primates show that 1H9 is well tolerated with no treatment-related adverse effects noted. These data highlight the clinical potential of 1H9 as a pan-therapeutic with the desired properties when used in combination with tumor-targeting antibodies.

  View details for DOI 10.1172/jci.insight.134728

  View details for PubMedID 32427583

• Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition. mBio Tal, M. C., Torrez Dulgeroff, L. B., Myers, L. n., Cham, L. B., Mayer-Barber, K. D., Bohrer, A. C., Castro, E. n., Yiu, Y. Y., Lopez Angel, C. n., Pham, E. n., Carmody, A. B., Messer, R. J., Gars, E. n., Kortmann, J. n., Markovic, M. n., Hasenkrug, M. n., Peterson, K. E., Winkler, C. W., Woods, T. A., Hansen, P. n., Galloway, S. n., Wagh, D. n., Fram, B. J., Nguyen, T. n., Corey, D. n., Kalluru, R. S., Banaei, N. n., Rajadas, J. n., Monack, D. M., Ahmed, A. n., Sahoo, D. n., Davis, M. M., Glenn, J. S., Adomati, T. n., Lang, K. S., Weissman, I. L., Hasenkrug, K. J. 2020; 11 (3)

  Abstract

  It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

  View details for DOI 10.1128/mBio.01293-20

  View details for PubMedID 32576678

• Heme oxygenase-1 deficiency triggers exhaustion of hematopoietic stem cells. EMBO reports Szade, K., Zukowska, M., Szade, A., Nowak, W., Skulimowska, I., Ciesla, M., Bukowska-Strakova, K., Gulati, G. S., Kachamakova-Trojanowska, N., Kusienicka, A., Einwallner, E., Kijowski, J., Czauderna, S., Esterbauer, H., Benes, V., L Weissman, I., Dulak, J., Jozkowicz, A. 2019: e47895

  Abstract

  While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1-/- ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured invitro with HO-1-/- mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1-/- animals have reduced quiescence and regenerative potential. Young HO-1-/- HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1+/+ HSCs transplanted into HO-1-/- recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1-/- HSCs to the HO-1+/+ recipients recovers the regenerative potential of HO-1-/- HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.

  View details for DOI 10.15252/embr.201947895

  View details for PubMedID 31885181

• Neogenin-1 distinguishes between myeloid-biased and balanced Hoxb5+ mouse long-term hematopoietic stem cells. Proceedings of the National Academy of Sciences of the United States of America Gulati, G. S., Zukowska, M., Noh, J. J., Zhang, A., Wesche, D. J., Sinha, R., George, B. M., Weissman, I. L., Szade, K. 2019

  Abstract

  Hematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single cell transplants and lineage tracing suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled with Hoxb5, a specific reporter of long-term HSCs (LT-HSCs). We show that NEO1+ Hoxb5 + LT-HSCs expand with age and respond to myeloablative stress in young mice while NEO1- Hoxb5 + LT-HSCs exhibit no significant change in number. Furthermore, NEO1+ Hoxb5 + LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1- Hoxb5 + LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+ Hoxb5 + LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution while NEO1- Hoxb5 + LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression analysis reveals erythroid and myeloid priming in the NEO1+ fraction and association of quiescence and self-renewal-related transcription factors with NEO1- LT-HSCs. Finally, transplanted NEO1+ Hoxb5 + LT-HSCs rarely generate NEO1- Hoxb5 + LT-HSCs while NEO1- Hoxb5 + LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced NEO1- Hoxb5 + LT-HSCs can hierarchically precede active, myeloid-biased NEO1+ Hoxb5 + LT-HSCs.

  View details for DOI 10.1073/pnas.1911024116

  View details for PubMedID 31754028

• The Ban on US Government Funding Research Using Human Fetal Tissues: How Does This Fit with the NIH Mission to Advance Medical Science for the Benefit of the Citizenry? Stem cell reports McCune, J. M., Weissman, I. L. 2019; 13 (5): 777–86

  Abstract

  Some have argued that human fetal tissue research is unnecessary and/or immoral. Recently, the Trump administration has taken the drastic--and we believe misguided--step to effectively ban government-funded research on fetal tissue altogether. In this article, we show that entire lines of research and their clinical outcomes would not have progressed had fetal tissue been unavailable. We argue that this research has been carried out in a manner that is ethical and legal, and that it has provided knowledge that has saved lives, particularly those of pregnant women, their unborn fetuses, and newborns. We believe that those who support a ban on the use of fetal tissue are halting medical progress and therefore endangering the health and lives of many, and for this they should accept responsibility. At the very least, we challenge them to be true to their beliefs: if they wish to short-circuit a scientific process that has led to medical advances, they should pledge to not accept for themselves the health benefits that such advances provide.

  View details for DOI 10.1016/j.stemcr.2019.10.003

  View details for PubMedID 31722191

• Adult stem cells and regenerative medicine-a symposium report. Annals of the New York Academy of Sciences Cable, J., Fuchs, E., Weissman, I., Jasper, H., Glass, D., Rando, T. A., Blau, H., Debnath, S., Oliva, A., Park, S., Passegue, E., Kim, C., Krasnow, M. A. 2019

  Abstract

  Adult stem cells are rare, undifferentiated cells found in all tissues of the body. Although normally kept in a quiescent, nondividing state, these cells can proliferate and differentiate to replace naturally dying cells within their tissue and to repair its wounds in response to injury. Due to their proliferative nature and ability to regenerate tissue, adult stem cells have the potential to treat a variety of degenerative diseases as well as aging. In addition, since stem cells are often thought to be the source of malignant tumors, understanding the mechanisms that keep their proliferative abilities in check can pave the way for new cancer therapies. While adult stem cells have had limited practical and clinical applications to date, several clinical trials of stem cell-based therapies are underway. This report details recent research presented at the New York Academy of Sciences on March 14, 2019 on understanding the factors that regulate stem cell activity and differentiation, with the hope of translating these findings into the clinic.

  View details for DOI 10.1111/nyas.14243

  View details for PubMedID 31655007

• Digit Tip Regeneration Relies on Germ Layer Restricted Wnt and Hedgehog Signaling Maan, Z. N., Januszyk, M., Rinkevich, Y., Weissman, I., Gurtner, G. ELSEVIER SCIENCE INC. 2019: S220–S221

  View details for Web of Science ID 000492740900422

• Neutrophil and monocyte kinetics play critical roles in mouse peritoneal adhesion formation. Blood advances Tsai, J. M., Shoham, M., Fernhoff, N. B., George, B. M., Marjon, K. D., McCracken, M. N., Kao, K. S., Sinha, R., Volkmer, A. K., Miyanishi, M., Seita, J., Rinkevich, Y., Weissman, I. L. 2019; 3 (18): 2713–21

  Abstract

  Peritoneal adhesions are pathological fibroses that ensnare organs after abdominal surgery. This dense connective tissue can cause small bowel obstruction, female infertility, and chronic abdominal pain. The pathogenesis of adhesions is a fibrotic response to tissue damage coordinated between mesothelial cells, fibroblasts, and immune cells. We have previously demonstrated that peritoneal adhesions are a consequence of mechanical injury to the mesothelial layer sustained during surgery. Neutrophils are among the first leukocytes involved in the early response to tissue damage. Here, we show that when subjected to mechanical stress, activated mesothelial cells directly recruit neutrophils and monocytes through upregulation of chemokines such as CXCL1 and monocyte chemoattractant protein 1 (MCP-1). We find that neutrophils within the adhesion sites undergo cell death and form neutrophil extracellular traps (NETosis) that contribute to pathogenesis. Conversely, tissue-resident macrophages were profoundly depleted throughout the disease time course. We show that this is distinct from traditional inflammatory kinetics such as after sham surgery or chemically induced peritonitis, and suggest that adhesions result from a primary difference in inflammatory kinetics. We find that transient depletion of circulating neutrophils significantly decreases adhesion burden, and further recruitment of monocytes with thioglycolate or MCP-1 also improves outcomes. Our findings suggest that the combination of neutrophil depletion and monocyte recruitment is sufficient to prevent adhesion formation, thus providing insight for potential clinical interventions.

  View details for DOI 10.1182/bloodadvances.2018024026

  View details for PubMedID 31519647

• Phagocytosis checkpoints as new targets for cancer immunotherapy. Nature reviews. Cancer Feng, M., Jiang, W., Kim, B. Y., Zhang, C. C., Fu, Y., Weissman, I. L. 2019

  Abstract

  Cancer immunotherapies targeting adaptive immune checkpoints have substantially improved patient outcomes across multiple metastatic and treatment-refractory cancer types. However, emerging studies have demonstrated that innate immune checkpoints, which interfere with the detection and clearance of malignant cells through phagocytosis and suppress innate immune sensing, also have a key role in tumour-mediated immune escape and might, therefore, be potential targets for cancer immunotherapy. Indeed, preclinical studies and early clinical data have established the promise of targeting phagocytosis checkpoints, such as the CD47-signal-regulatory protein alpha (SIRPalpha) axis, either alone or in combination with other cancer therapies. In this Review, we highlight the current understanding of how cancer cells evade the immune system by disrupting phagocytic clearance and the effect of phagocytosis checkpoint blockade on induction of antitumour immune responses. Given the role of innate immune cells in priming adaptive immune responses, an improved understanding of the tumour-intrinsic processes that inhibit essential immune surveillance processes, such as phagocytosis and innate immune sensing, could pave the way for the development of highly effective combination immunotherapy strategies that modulate both innate and adaptive antitumour immune responses.

  View details for DOI 10.1038/s41568-019-0183-z

  View details for PubMedID 31462760

• The GABA receptor GABRR1 is expressed on and functional in hematopoietic stem cells and megakaryocyte progenitors. Proceedings of the National Academy of Sciences of the United States of America Zhu, F., Feng, M., Sinha, R., Murphy, M. P., Luo, F., Kao, K. S., Szade, K., Seita, J., Weissman, I. L. 2019

  Abstract

  GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR-) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR- HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.

  View details for DOI 10.1073/pnas.1906251116

  View details for PubMedID 31451629

• CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature Barkal, A. A., Brewer, R. E., Markovic, M., Kowarsky, M., Barkal, S. A., Zaro, B. W., Krishnan, V., Hatakeyama, J., Dorigo, O., Barkal, L. J., Weissman, I. L. 2019

  Abstract

  Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.

  View details for DOI 10.1038/s41586-019-1456-0

  View details for PubMedID 31367043

• CD47-Targeted Near-Infrared Photoimmunotherapy for Human Bladder Cancer CLINICAL CANCER RESEARCH Kiss, B., van den Berg, N. S., Ertsey, R., McKenna, K., Mach, K. E., Zhang, C., Volkmer, J., Weissman, I. L., Rosenthal, E. L., Liao, J. C. 2019; 25 (12): 3561–71

  View details for DOI 10.1158/1078-0432.CCR-18-3267

  View details for Web of Science ID 000472077200011

• Evolutionarily conserved resistance to phagocytosis observed in melanoma cells is insensitive to upregulation of pro-phagocytic signals and to CD47 blockade. Melanoma research Anderson, K. L., Snyder, K. M., Ito, D., Lins, D. C., Mills, L. J., Weiskopf, K., Ring, N. G., Ring, A. M., Shimizu, Y., Mescher, M. F., Weissman, I. L., Modiano, J. F. 2019

  Abstract

  Therapeutic activation of macrophage phagocytosis has the ability to restrain tumour growth through phagocytic clearance of tumour cells and activation of the adaptive immune response. Our objective for this study was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. In order to identify evolutionarily conserved mechanisms of resistance that may be important for melanoma cell survival, we utilized a multi-species approach and examined the phagocytosis of human, mouse, and dog melanoma cells. We observed that melanoma cells from all three species displayed unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the 'don't eat me' signal CD47 or by chemotherapeutic enhancement of known 'eat me' signals. Additionally, CD47 blockade failed to promote anti-melanoma immune responses or tumour regression in vivo. This melanoma resistance to phagocytosis was not mediated by soluble factors, and it was unaffected by siRNA-mediated knockdown of 47 prospective 'don't eat me' signals or by CRISPR-Cas-mediated CD47 knockout. Unexpectedly, CD47 knockout also did not enhance phagocytosis of lymphoma cells, but it eliminated the pro-phagocytic effect of CD47 blockade, suggesting that the pro-phagocytic effects of CD47 blockade are due in part to Fc receptor engagement. From this study, we conclude that melanoma cells possess an evolutionarily conserved resistance to macrophage phagocytosis. Further investigation will be needed to overcome the mechanisms that mediate melanoma cell resistance to innate immunity.This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

  View details for DOI 10.1097/CMR.0000000000000629

  View details for PubMedID 31205227

• Antibody Conditioning Enables MHC-Mismatched Hematopoietic Stem Cell Transplants and Organ Graft Tolerance. Cell stem cell George, B. M., Kao, K. S., Kwon, H., Velasco, B. J., Poyser, J., Chen, A., Le, A. C., Chhabra, A., Burnett, C. E., Cajuste, D., Hoover, M., Loh, K. M., Shizuru, J. A., Weissman, I. L. 2019

  Abstract

  Hematopoietic cell transplantation can correct hematological and immunological disorders by replacing a diseased blood system with a healthy one, but this currently requires depleting a patient's existing hematopoietic system with toxic and non-specific chemotherapy, radiation, or both. Here we report an antibody-based conditioning protocol with reduced toxicity and enhanced specificity for robust hematopoietic stem cell (HSC) transplantation and engraftment in recipient mice. Host pre-treatment with six monoclonal antibodies targeting CD47, Tcells, NK cells, and HSCs followed by donor HSC transplantation enabled stable hematopoietic system reconstitution in recipients with mismatches at half (haploidentical) or all major histocompatibility complex (MHC) genes. This approach allowed tolerance to heart tissue from HSC donor strains in haploidentical recipients, showing potential applications for solid organ transplantation without immune suppression. Fully mismatched chimeric mice developed antibody responses to nominal antigens, showing preserved functional immunity. These findings suggest approaches for transplanting immunologically mismatched HSCs and solid organs with limited toxicity.

  View details for DOI 10.1016/j.stem.2019.05.018

  View details for PubMedID 31204177

• Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice BLOOD Pang, W. W., Czechowicz, A., Logan, A. C., Bhardwaj, R., Poyser, J., Park, C. Y., Weissman, I. L., Shizuru, J. A. 2019; 133 (19): 2069–78

  View details for DOI 10.1182/blood-2018-06-858159

  View details for Web of Science ID 000467385500012

• Anti-human CD117 antibody-mediated bone marrow niche clearance in nonhuman primates and humanized NSG mice BLOOD Kwon, H., Logan, A. C., Chhabra, A., Pang, W. W., Czechowicz, A., Tate, K., Le, A., Poyser, J., Hollis, R., Kelly, B. V., Kohn, D. B., Weissman, I. L., Prohaska, S. S., Shizuru, J. A. 2019; 133 (19): 2104–8

  View details for DOI 10.1182/blood-2018-06-853879

  View details for Web of Science ID 000467385500016

• Voices: The Importance of Fetal Tissue Research CELL STEM CELL Weissman, I., Rawlins, E., Jensen, K., Barker, R., Beltrao-Braga, P., Dekel, B., Frisen, J., Surani, A., Fehlings, M. 2019; 24 (3): 357–59

  Abstract

  The value of fetal tissue research cannot be understated and has led to remarkable advances in understanding human development, therapeutic discovery, and stem cell research across many organ systems. We asked 9 researchers to reflect on the impact and key contributions of fetal tissue in their field of expertise.

  View details for DOI 10.1016/j.stem.2019.01.012

  View details for Web of Science ID 000460672700008

  View details for PubMedID 30770301

• First-in-Human, First-in-Class Phase I Trial of the Anti-CD47 Antibody Hu5F9-G4 in Patients With Advanced Cancers. Journal of clinical oncology : official journal of the American Society of Clinical Oncology Sikic, B. I., Lakhani, N., Patnaik, A., Shah, S. A., Chandana, S. R., Rasco, D., Colevas, A. D., O'Rourke, T., Narayanan, S., Papadopoulos, K., Fisher, G. A., Villalobos, V., Prohaska, S. S., Howard, M., Beeram, M., Chao, M. P., Agoram, B., Chen, J. Y., Huang, J., Axt, M., Liu, J., Volkmer, J., Majeti, R., Weissman, I. L., Takimoto, C. H., Supan, D., Wakelee, H. A., Aoki, R., Pegram, M. D., Padda, S. K. 2019: JCO1802018

  Abstract

  PURPOSE: To evaluate the safety, pharmacokinetics, and pharmacodynamics of Hu5F9-G4 (5F9), a humanized IgG4 antibody that targets CD47 to enable phagocytosis.PATIENTS AND METHODS: Adult patients with solid tumors were treated in four cohorts: part A, to determine a priming dose; part B, to determine a weekly maintenance dose; part C, to study a loading dose in week 2; and a tumor biopsy cohort.RESULTS: Sixty-two patients were treated: 11 in part A, 14 in B, 22 in C, and 15 in the biopsy cohort. Part A used doses that ranged from 0.1 to 3 mg/kg. On the basis of tolerability and receptor occupancy studies that showed 100% CD47 saturation on RBCs, 1 mg/kg was selected as the priming dose. In subsequent groups, patients were treated with maintenance doses that ranged from 3 to 45 mg/kg, and most toxicities were mild to moderate. These included transient anemia (57% of patients), hemagglutination on peripheral blood smear (36%), fatigue (64%), headaches (50%), fever (45%), chills (45%), hyperbilirubinemia (34%), lymphopenia (34%), infusion-related reactions (34%), and arthralgias (18%). No maximum tolerated dose was reached with maintenance doses up to 45 mg/kg. At doses of 10 mg/kg or more, the CD47 antigen sink was saturated by 5F9, and a 5F9 half-life of approximately 13 days was observed. Strong antibody staining of tumor tissue was observed in a patient at 30 mg/kg. Two patients with ovarian/fallopian tube cancers had partial remissions for 5.2 and 9.2 months.CONCLUSION: 5F9 is well tolerated using a priming dose at 1 mg/kg on day 1 followed by maintenance doses of up to 45 mg/kg weekly.

  View details for PubMedID 30811285

• Wounds Inhibit Tumor Growth In Vivo. Annals of surgery Hu, M. S., Maan, Z. N., Leavitt, T., Hong, W. X., Rennert, R. C., Marshall, C. D., Borrelli, M. R., Zhu, T. N., Esquivel, M., Zimmermann, A., McArdle, A., Chung, M. T., Foster, D. S., Jones, R. E., Gurtner, G. C., Giaccia, A. J., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2019

  Abstract

  OBJECTIVE: The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo.SUMMARY OF BACKGROUND DATA: Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo.METHODS: To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells.RESULTS: Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds.CONCLUSION: Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.

  View details for PubMedID 30829705

• Regenerating the field of cardiovascular cell therapy. Nature biotechnology Chien, K. R., Frisen, J., Fritsche-Danielson, R., Melton, D. A., Murry, C. E., Weissman, I. L. 2019

  Abstract

  The retraction of >30 falsified studies by Anversa et al. has had a disheartening impact on the cardiac cell therapeutics field. The premise of heart muscle regeneration by the transdifferentiation of bone marrow cells or putative adult resident cardiac progenitors has been largely disproven. Over the past 18 years, a generation of physicians and scientists has lost years chasing these studies, and patients have been placed at risk with little scientific grounding. Funding agencies invested hundreds of millions of dollars in irreproducible work, and both academic institutions and the scientific community ignored troubling signals over a decade of questionable work. Our collective retrospective analysis identifies preventable problems at the level of the editorial and peer-review process, funding agencies and academic institutions. This Perspective provides a chronology of the forces that led to this scientific debacle, integrating direct knowledge of the process. We suggest a science-driven path forward that includes multiple novel approaches to the problem of heart muscle regeneration.

  View details for PubMedID 30778231

• A functional subset of CD8+ T cells during chronic exhaustion is defined by SIRPalpha expression. Nature communications Myers, L. M., Tal, M. C., Torrez Dulgeroff, L. B., Carmody, A. B., Messer, R. J., Gulati, G., Yiu, Y. Y., Staron, M. M., Angel, C. L., Sinha, R., Markovic, M., Pham, E. A., Fram, B., Ahmed, A., Newman, A. M., Glenn, J. S., Davis, M. M., Kaech, S. M., Weissman, I. L., Hasenkrug, K. J. 2019; 10 (1): 794

  Abstract

  Prolonged exposure of CD8+ T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8+ T cells defined by surface expression of SIRPalpha, a protein not previously reported on lymphocytes. On SIRPalpha+ CD8+ T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPalpha+ cells that actively proliferate, transcribe IFNgamma and show cytolytic activity. Furthermore, target cells that express the ligand for SIRPalpha, CD47, are more susceptible to CD8+ T cell-killing in vivo. SIRPalpha+ CD8+ T cells are evident in mice infected with Friend retrovirus, LCMV Clone 13, and in patients with chronic HCV infections. Furthermore, therapeutic blockade of PD-L1 to reinvigorate CD8+ T cells during chronic infection expands the cytotoxic subset of SIRPalpha+ CD8+ T cells.

  View details for PubMedID 30770827

• Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice. Blood Pang, W. W., Czechowicz, A., Logan, A. C., Bhardwaj, R., Poyser, J., Park, C. Y., Weissman, I. L., Shizuru, J. A. 2019

  Abstract

  The myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Further, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

  View details for PubMedID 30745302

• Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo. Proceedings of the National Academy of Sciences of the United States of America Lu, R., Czechowicz, A., Seita, J., Jiang, D., Weissman, I. L. 2019

  Abstract

  While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

  View details for PubMedID 30622181

• Tumor-associated macrophages enhance tumor hypoxia and aerobic glycolysis. Cancer research Jeong, H., Kim, S., Hong, B., Lee, C., Kim, Y., Bok, S., Oh, J., Gwak, S., Yoo, M. Y., Lee, M. S., Chung, S., Defrene, J., Tessier, P., Pelletier, M., Jeon, H., Roh, T., Kim, B., Kim, K. H., Ju, J. H., Kim, S., Lee, Y., Kim, D., Kim, I. H., Kim, H. J., Park, J., Lee, Y., Lee, J. S., Cheon, G. J., Weissman, I. L., Chung, D. H., Jeon, Y. K., Ahn, G. 2019

  Abstract

  Tumor hypoxia and aerobic glycolysis are well-known resistance factors for anticancer therapies. Here we demonstrate that tumor-associated macrophages (TAM) enhance tumor hypoxia and aerobic glycolysis in mice subcutaneous tumors and in non-small cell lung cancer (NSCLC) patients. We found a strong correlation between CD68 TAM immunostaining and positron emission tomography (PET) 18fluoro-deoxyglucose (FDG) uptake in 98 matched tumors of NSCLC patients. We also observed a significant correlation between CD68 and glycolytic gene signatures in 513 NSCLC patients from the TCGA database. TAM secreted tumor necrosis factor-alpha (TNF-alpha) to promote tumor cell glycolysis while increased AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha) in TAM facilitated tumor hypoxia. Depletion of TAM by clodronate was sufficient to abrogate aerobic glycolysis and tumor hypoxia, thereby improving tumor response to anticancer therapies. TAM depletion led to a significant increase in programmed death-ligand 1 (PD-L1) expression in aerobic cancer cells as well as T cell infiltration in tumors, resulting in antitumor efficacy by PD-L1 antibodies which were otherwise completely ineffective. These data suggest that TAM can significantly alter tumor metabolism, further complicating tumor response to anticancer therapies including immunotherapy.

  View details for PubMedID 30610087

• Microglia are effector cells of CD47-SIRPalpha antiphagocytic axis disruption against glioblastoma. Proceedings of the National Academy of Sciences of the United States of America Hutter, G., Theruvath, J., Graef, C. M., Zhang, M., Schoen, M. K., Manz, E. M., Bennett, M. L., Olson, A., Azad, T. D., Sinha, R., Chan, C., Assad Kahn, S., Gholamin, S., Wilson, C., Grant, G., He, J., Weissman, I. L., Mitra, S. S., Cheshier, S. H. 2019

  Abstract

  Glioblastoma multiforme (GBM) is a highly aggressive malignant brain tumor with fatal outcome. Tumor-associated macrophages and microglia (TAMs) have been found to be major tumor-promoting immune cells in the tumor microenvironment. Hence, modulation and reeducation of tumor-associated macrophages and microglia in GBM is considered a promising antitumor strategy. Resident microglia and invading macrophages have been shown to have distinct origin and function. Whereas yolk sac-derived microglia reside in the brain, blood-derived monocytes invade the central nervous system only under pathological conditions like tumor formation. We recently showed that disruption of the SIRPalpha-CD47 signaling axis is efficacious against various brain tumors including GBM primarily by inducing tumor phagocytosis. However, most effects are attributed to macrophages recruited from the periphery but the role of the brain resident microglia is unknown. Here, we sought to utilize a model to distinguish resident microglia and peripheral macrophages within the GBM-TAM pool, using orthotopically xenografted, immunodeficient, and syngeneic mouse models with genetically color-coded macrophages (Ccr2 RFP) and microglia (Cx3cr1 GFP). We show that even in the absence of phagocytizing macrophages (Ccr2 RFP/RFP), microglia are effector cells of tumor cell phagocytosis in response to anti-CD47 blockade. Additionally, macrophages and microglia show distinct morphological and transcriptional changes. Importantly, the transcriptional profile of microglia shows less of an inflammatory response which makes them a promising target for clinical applications.

  View details for PubMedID 30602457

• Single-cell analysis reveals T cell infiltration in old neurogenic niches. Nature Dulken, B. W., Buckley, M. T., Navarro Negredo, P. n., Saligrama, N. n., Cayrol, R. n., Leeman, D. S., George, B. M., Boutet, S. C., Hebestreit, K. n., Pluvinage, J. V., Wyss-Coray, T. n., Weissman, I. L., Vogel, H. n., Davis, M. M., Brunet, A. n. 2019

  Abstract

  The mammalian brain contains neurogenic niches that comprise neural stem cells and other cell types. Neurogenic niches become less functional with age, but how they change during ageing remains unclear. Here we perform single-cell RNA sequencing of young and old neurogenic niches in mice. The analysis of 14,685 single-cell transcriptomes reveals a decrease in activated neural stem cells, changes in endothelial cells and microglia, and an infiltration of T cells in old neurogenic niches. T cells in old brains are clonally expanded and are generally distinct from those in old blood, which suggests that they may experience specific antigens. T cells in old brains also express interferon-γ, and the subset of neural stem cells that has a high interferon response shows decreased proliferation in vivo. We find that T cells can inhibit the proliferation of neural stem cells in co-cultures and in vivo, in part by secreting interferon-γ. Our study reveals an interaction between T cells and neural stem cells in old brains, opening potential avenues through which to counteract age-related decline in brain function.

  View details for DOI 10.1038/s41586-019-1362-5

  View details for PubMedID 31270459

• Dysregulated integrin αVβ3 and CD47 signaling promotes joint inflammation, cartilage breakdown, and progression of osteoarthritis. JCI insight Wang, Q. n., Onuma, K. n., Liu, C. n., Wong, H. n., Bloom, M. S., Elliott, E. E., Cao, R. R., Hu, N. n., Lingampalli, N. n., Sharpe, O. n., Zhao, X. n., Sohn, D. H., Lepus, C. M., Sokolove, J. n., Mao, R. n., Cisar, C. T., Raghu, H. n., Chu, C. R., Giori, N. J., Willingham, S. B., Prohaska, S. S., Cheng, Z. n., Weissman, I. L., Robinson, W. H. 2019; 4 (18)

  Abstract

  Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVβ3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVβ3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVβ3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVβ3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVβ3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVβ3 and CD47 signaling in OA pathogenesis and suggest that activation of αVβ3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVβ3, CD47, and their signaling pathways as a disease-modifying therapy.

  View details for DOI 10.1172/jci.insight.128616

  View details for PubMedID 31534047

• RUNX3 levels in human hematopoietic progenitors are regulated by aging and dictate erythroid-myeloid balance. Haematologica Balogh, P. n., Adelman, E. R., Pluvinage, J. V., Capaldo, B. J., Freeman, K. C., Singh, S. n., Elagib, K. E., Nakamura, Y. n., Kurita, R. n., Sashida, G. n., Zunder, E. R., Li, H. n., Gru, A. A., Price, E. A., Schrier, S. L., Weissman, I. L., Figueroa, M. E., Pang, W. W., Goldfarb, A. N. 2019

  Abstract

  Healthy bone marrow progenitors yield a coordinated balance of hematopoietic lineages. This balance shifts with aging toward enhanced granulopoiesis with diminished erythropoiesis and lymphopoiesis, changes which likely contribute to the development of bone marrow disorders in the elderly. In this study, RUNX3 was identified as a hematopoietic stem and progenitor cell factor whose levels decline with aging in humans and mice. This decline is exaggerated in hematopoietic stem and progenitor cells from subjects diagnosed with unexplained anemia of the elderly. Hematopoietic stem cells from elderly unexplained anemia patients had diminished erythroid but unaffected granulocytic colony forming potential. Knockdown studies revealed human hematopoietic stem and progenitor cells to be strongly influenced by RUNX3 levels, with modest deficiencies abrogating erythroid differentiation at multiple steps while retaining capacity for granulopoiesis. Transcriptome profiling indicated control by RUNX3 of key erythroid transcription factors, including KLF1 and GATA1. These findings thus implicate RUNX3 as a participant in HSPC aging, and a key determinant of erythroid-myeloid lineage balance.

  View details for DOI 10.3324/haematol.2018.208918

  View details for PubMedID 31171641

• Hematopoietic stem cell-independent hematopoiesis and the origins of innate-like B lymphocytes. Development (Cambridge, England) Ghosn, E. n., Yoshimoto, M. n., Nakauchi, H. n., Weissman, I. L., Herzenberg, L. A. 2019; 146 (15)

  Abstract

  The current paradigm that a single long-term hematopoietic stem cell can regenerate all components of the mammalian immune system has been challenged by recent findings in mice. These findings show that adult tissue-resident macrophages and innate-like lymphocytes develop early in fetal hematopoiesis from progenitors that emerge prior to, and apparently independently of, conventional long-term hematopoietic stem cells. Here, we discuss these recent findings, which show that an early and distinct wave of hematopoiesis occurs for all major hematopoietic lineages. These data provide evidence that fetal hematopoietic progenitors not derived from the bona fide long-term hematopoietic stem cells give rise to tissue-resident immune cells that persist throughout adulthood. We also discuss recent insights into B lymphocyte development and attempt to synthesize seemingly contradictory recent findings on the origins of innate-like B-1a lymphocytes during fetal hematopoiesis.

  View details for DOI 10.1242/dev.170571

  View details for PubMedID 31371526

• Therapeutic Targeting of the Macrophage Immune Checkpoint CD47 in Myeloid Malignancies. Frontiers in oncology Chao, M. P., Takimoto, C. H., Feng, D. D., McKenna, K., Gip, P., Liu, J., Volkmer, J., Weissman, I. L., Majeti, R. 2019; 9: 1380

  Abstract

  In recent years, immunotherapies have been clinically investigated in AML and other myeloid malignancies. While most of these are focused on stimulating the adaptive immune system (including T cell checkpoint inhibitors), several key approaches targeting the innate immune system have been identified. Macrophages are a key cell type in the innate immune response with CD47 being identified as a dominant macrophage checkpoint. CD47 is a "do not eat me" signal, overexpressed in myeloid malignancies that leads to tumor evasion of phagocytosis by macrophages. Blockade of CD47 leads to engulfment of leukemic cells and therapeutic elimination. Pre-clinical data has demonstrated robust anti-cancer activity in multiple hematologic malignancies including AML and myelodysplastic syndrome (MDS). In addition, clinical studies have been underway with CD47 targeting agents in both AML and MDS as monotherapy and in combination. This review will describe the role of CD47 in myeloid malignancies and pre-clinical data supporting CD47 targeting. In addition, initial clinical data of CD47 targeting in AML/MDS will be reviewed, and including the first-in-class anti-CD47 antibody magrolimab.

  View details for DOI 10.3389/fonc.2019.01380

  View details for PubMedID 32038992

• De novo mutations in mitochondrial DNA of iPSCs produce immunogenic neoepitopes in mice and humans. Nature biotechnology Deuse, T. n., Hu, X. n., Agbor-Enoh, S. n., Koch, M. n., Spitzer, M. H., Gravina, A. n., Alawi, M. n., Marishta, A. n., Peters, B. n., Kosaloglu-Yalcin, Z. n., Yang, Y. n., Rajalingam, R. n., Wang, D. n., Nashan, B. n., Kiefmann, R. n., Reichenspurner, H. n., Valantine, H. n., Weissman, I. L., Schrepfer, S. n. 2019

  Abstract

  The utility of autologous induced pluripotent stem cell (iPSC) therapies for tissue regeneration depends on reliable production of immunologically silent functional iPSC derivatives. However, rejection of autologous iPSC-derived cells has been reported, although the mechanism underlying rejection is largely unknown. We hypothesized that de novo mutations in mitochondrial DNA (mtDNA), which has far less reliable repair mechanisms than chromosomal DNA, might produce neoantigens capable of eliciting immune recognition and rejection. Here we present evidence in mice and humans that nonsynonymous mtDNA mutations can arise and become enriched during reprogramming to the iPSC stage, long-term culture and differentiation into target cells. These mtDNA mutations encode neoantigens that provoke an immune response that is highly specific and dependent on the host major histocompatibility complex genotype. Our results reveal that autologous iPSCs and their derivatives are not inherently immunologically inert for autologous transplantation and suggest that iPSC-derived products should be screened for mtDNA mutations.

  View details for DOI 10.1038/s41587-019-0227-7

  View details for PubMedID 31427818

• Irradiation or temozolomide chemotherapy enhances anti-CD47 treatment of glioblastoma. Innate immunity Gholamin, S. n., Youssef, O. A., Rafat, M. n., Esparza, R. n., Kahn, S. n., Shahin, M. n., Giaccia, A. J., Graves, E. E., Weissman, I. n., Mitra, S. n., Cheshier, S. H. 2019: 1753425919876690

  View details for DOI 10.1177/1753425919876690

  View details for PubMedID 31547758

• RBC-Specific CD47 Pruning Confers Protection and Underlies the Transient Anemia in Patients Treated with Anti-CD47 Antibody 5F9 Chen, J. Y., McKenna, K., Choi, T. S., Duan, J., Brown, L., Stewart, J. J., Sompalli, K., Vyas, P., Schrier, S., Majeti, R., Weissman, I. L., Elrod, K., Chao, M., Takimoto, C. H., Liu, J., Volkmer, J. AMER SOC HEMATOLOGY. 2018

  View details for DOI 10.1182/blood-2018-99-115674

  View details for Web of Science ID 000454837606298

• Combination Treatment with 5F9 and Azacitidine Enhances Phagocytic Elimination of Acute Myeloid Leukemia Feng, D., Gip, P., McKenna, K., Zhao, F., Mata, O., Choi, T. S., Duan, J., Sompalli, K., Majeti, R., Weissman, I. L., Takimoto, C. H., Chao, M., Chen, J. Y., Liu, J., Volkmer, J. AMER SOC HEMATOLOGY. 2018

  View details for DOI 10.1182/blood-2018-99-120170

  View details for Web of Science ID 000454837607335

• Surgical adhesions in mice are derived from mesothelial cells and can be targeted by antibodies against mesothelial markers. Science translational medicine Tsai, J. M., Sinha, R., Seita, J., Fernhoff, N., Christ, S., Koopmans, T., Krampitz, G. W., McKenna, K. M., Xing, L., Sandholzer, M., Sales, J. H., Shoham, M., McCracken, M., Joubert, L., Gordon, S. R., Poux, N., Wernig, G., Norton, J. A., Weissman, I. L., Rinkevich, Y. 2018; 10 (469)

  Abstract

  Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical and infectious morbidity. The primary molecular chain of events leading to the initiation of adhesions has been elusive, chiefly due to the lack of an identifiable cell of origin. Using clonal analysis and lineage tracing, we have identified injured surface mesothelium expressing podoplanin (PDPN) and mesothelin (MSLN) as a primary instigator of peritoneal adhesions after surgery in mice. We demonstrate that an anti-MSLN antibody diminished adhesion formation in a mouse model where adhesions were induced by surgical ligation to form ischemic buttons and subsequent surgical abrasion of the peritoneum. RNA sequencing and bioinformatics analyses of mouse mesothelial cells from injured mesothelium revealed aspects of the pathological mechanism of adhesion development and yielded several potential regulators of this process. Specifically, we show that PDPN+MSLN+ mesothelium responded to hypoxia by early up-regulation of hypoxia-inducible factor 1 alpha (HIF1alpha) that preceded adhesion development. Inhibition of HIF1alpha with small molecules ameliorated the injury program in damaged mesothelium and was sufficient to diminish adhesion severity in a mouse model. Analyses of human adhesion tissue suggested that similar surface markers and signaling pathways may contribute to surgical adhesions in human patients.

  View details for PubMedID 30487249

• Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens. Nature genetics Haney, M. S., Bohlen, C. J., Morgens, D. W., Ousey, J. A., Barkal, A. A., Tsui, C. K., Ego, B. K., Levin, R., Kamber, R. A., Collins, H., Tucker, A., Li, A., Vorselen, D., Labitigan, L., Crane, E., Boyle, E., Jiang, L., Chan, J., Rincon, E., Greenleaf, W. J., Li, B., Snyder, M. P., Weissman, I. L., Theriot, J. A., Collins, S. R., Barres, B. A., Bassik, M. C. 2018

  Abstract

  Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-beta aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

  View details for PubMedID 30397336

• Publisher Correction: Notch1 regulates the initiation of metastasis and self-renewal of Group 3 medulloblastoma. Nature communications Kahn, S. A., Wang, X., Nitta, R. T., Gholamin, S., Theruvath, J., Hutter, G., Azad, T. D., Wadi, L., Bolin, S., Ramaswamy, V., Esparza, R., Liu, K., Edwards, M., Swartling, F. J., Sahoo, D., Li, G., Wechsler-Reya, R. J., Reimand, J., Cho, Y., Taylor, M. D., Weissman, I. L., Mitra, S. S., Cheshier, S. H. 2018; 9 (1): 4651

  Abstract

  The original version of this Article omitted Suzana A. Kahn, Siddhartha S. Mitra & Samuel H. Cheshier as jointly supervising authors. This has now been corrected in both the PDF and HTML versions of the Article.

  View details for PubMedID 30389946

• Method of Isolating and Transplanting the Hematopoietic Stem Cell with Its Microenvironment Which Improves Functional Hematopoietic Engraftment Borrelli, M. R., Lopez, M., Gulati, G., Murphy, M. P., Sinha, R., Longaker, M. T., Weissman, I. L., Newman, A. M., Chan, C. K., Sokol, J. ELSEVIER SCIENCE INC. 2018: E224

  View details for DOI 10.1016/j.jamcollsurg.2018.08.605

  View details for Web of Science ID 000447772500541

• Identification of the Human Skeletal Stem Cell. Cell Chan, C. K., Gulati, G. S., Sinha, R., Tompkins, J. V., Lopez, M., Carter, A. C., Ransom, R. C., Reinisch, A., Wearda, T., Murphy, M., Brewer, R. E., Koepke, L. S., Marecic, O., Manjunath, A., Seo, E. Y., Leavitt, T., Lu, W., Nguyen, A., Conley, S. D., Salhotra, A., Ambrosi, T. H., Borrelli, M. R., Siebel, T., Chan, K., Schallmoser, K., Seita, J., Sahoo, D., Goodnough, H., Bishop, J., Gardner, M., Majeti, R., Wan, D. C., Goodman, S., Weissman, I. L., Chang, H. Y., Longaker, M. T. 2018; 175 (1): 43

  Abstract

  Stem cell regulation and hierarchical organization ofhuman skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

  View details for PubMedID 30241615

• Screening for genes that regulate the differentiation of human megakaryocytic lineage cells. Proceedings of the National Academy of Sciences of the United States of America Zhu, F., Feng, M., Sinha, R., Seita, J., Mori, Y., Weissman, I. L. 2018

  Abstract

  Different combinations of transcription factors (TFs) function at each stage of hematopoiesis, leading to distinct expression patterns of lineage-specific genes. The identification of such regulators and their functions in hematopoiesis remain largely unresolved. In this study, we utilized screening approaches to study the transcriptional regulators of megakaryocyte progenitor (MkP) generation, a key step before platelet production. Promising candidate genes were generated from a microarray platform gene expression commons and individually manipulated in human hematopoietic stem and progenitor cells (HSPCs). Deletion of some of the candidate genes (the hit genes) by CRISPR/Cas9 led to decreased MkP generation during HSPC differentiation, while more MkPs were produced when some hit genes were overexpressed in HSPCs. We then demonstrated that overexpression of these genes can increase the frequency of mature megakaryocytic colonies by functional colony forming unit-megakaryocyte (CFU-Mk) assay and the release of platelets after in vitro maturation. Finally, we showed that the histone deacetylase inhibitors could also increase MkP differentiation, possibly by regulating some of the newly identified TFs. Therefore, identification of such regulators will advance the understanding of basic mechanisms of HSPC differentiation and conceivably enable the generation and maturation of megakaryocytes and platelets in vitro.

  View details for PubMedID 30150396

• Stem cell competition is central to leukemogenesis Weissman, I. L. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-SY39-03

  View details for Web of Science ID 000468818901073

• HIF-1 alpha activation in myeloid cells accelerates dextran sodium sulfate-induced colitis progression in mice DISEASE MODELS & MECHANISMS Kim, Y., Lee, M., Gu, H., Kim, J., Jeong, S., Yeo, S., Lee, Y., Im, S., Sung, Y., Kim, H., Weissman, I. L., Ahn, G. 2018; 11 (7)

  Abstract

  Inflammatory bowel disease (IBD) is a chronic inflammatory disease, in which the intestinal epithelium loses its barrier function. Given the existence of the oxygen gradient in the intestinal epithelium and that inflammation further contributes to the tissue hypoxia, we investigated the role of hypoxia-inducible factor (HIF), a transcription factor activated under hypoxic conditions in myeloid cells, in the progression of IBD. To do this, we utilized myeloid-specific knockout (KO) mice targeting HIF pathways, created by a Cre-loxP system with human MRP8 (hMRP8), an intracellular calcium-binding protein, as the myeloid promoter. By feeding 5% dextran sodium sulfate (DSS) to hMRP8 von Hippel Lindau (Vhl) KO mice, in which HIF-1α and HIF-2α are constitutively activated in myeloid cells, we found that these mice were highly susceptible to DSS-induced colitis, demonstrating greater body weight loss, increased mortality, faster onset of rectal bleeding, shortened colon length, and increased CD11b- or Gr-1-positive myeloid cells in the colon compared with wild-type (WT) mice. These parameters were restored to, if not better than, the WT levels when we examined hMRP8 Hif-1a KO mice upon 5% DSS feeding. hMRP8 Hif-2a KO mice, on the other hand, exhibited a similar degree of DSS-induced colitis to that of WT mice. Lastly, when DSS was given together with azoxymethane to induce tumorigenesis in the colon, we found that hMRP8 Hif-1a KO mice exhibited comparable levels of colorectal tumors to those of WT mice, indicating that HIF-1α in myeloid cells is dispensable for tumorigenesis. Collectively, our results suggest that HIF-1α activation in myeloid cells critically regulates IBD progression.

  View details for PubMedID 29967068

  View details for PubMedCentralID PMC6078398

• Isolation and functional assessment of mouse skeletal stem cell lineage NATURE PROTOCOLS Gulati, G. S., Murphy, M. P., Marecic, O., Lopez, M., Brewer, R. E., Koepke, L. S., Manjunath, A., Ransom, R. C., Salhotra, A., Weissman, I. L., Longaker, M. T., Chan, C. F. 2018; 13 (6): 1294–1309

  Abstract

  There are limited methods available to study skeletal stem, progenitor, and progeny cell activity in normal and diseased contexts. Most protocols for skeletal stem cell isolation are based on the extent to which cells adhere to plastic or whether they express a limited repertoire of surface markers. Here, we describe a flow cytometry-based approach that does not require in vitro selection and that uses eight surface markers to distinguish and isolate mouse skeletal stem cells (mSSCs); bone, cartilage, and stromal progenitors (mBCSPs); and five downstream differentiated subtypes, including chondroprogenitors, two types of osteoprogenitors, and two types of hematopoiesis-supportive stroma. We provide instructions for the optimal mechanical and chemical digestion of bone and bone marrow, as well as the subsequent flow-cytometry-activated cell sorting (FACS) gating schemes required to maximally yield viable skeletal-lineage cells. We also describe a methodology for renal subcapsular transplantation and in vitro colony-formation assays on the isolated mSSCs. The isolation of mSSCs can be completed in 9 h, with at least 1 h more required for transplantation. Experience with flow cytometry and mouse surgical procedures is recommended before attempting the protocol. Our system has wide applications and has already been used to study skeletal response to fracture, diabetes, and osteoarthritis, as well as hematopoietic stem cell-niche interactions in the bone marrow.

  View details for PubMedID 29748647

• Computational correction of index switching in multiplexed sequencing libraries NATURE METHODS Larsson, A. M., Stanley, G., Sinha, R., Weissman, I. L., Sandberg, R. 2018; 15 (5): 305–7

  View details for PubMedID 29702636

• HUMANIZED ANTI-CD47 ANTIBODY (HU-5F9-G4) FOR METASTATIC BLADDER CANCER IS SUPERIOR TO CONVENTIONAL CHEMOTHERAPY WITH CISPLATIN AND GEMCITABINE IN A MURINE BLADDER CANCER MODEL. Kiss, B., Volkmer, A., Liao, J., Volkmer, J., Weissman, I. ELSEVIER SCIENCE INC. 2018: E864

  View details for Web of Science ID 000429166602468

• Improving immune-vascular crosstalk for cancer immunotherapy NATURE REVIEWS IMMUNOLOGY Huang, Y., Kim, B. S., Chan, C. K., Hahn, S. M., Weissman, I. L., Jiang, W. 2018; 18 (3): 195–203

  Abstract

  The vasculature of tumours is highly abnormal and dysfunctional. Consequently, immune effector cells have an impaired ability to penetrate solid tumours and often exhibit compromised functions. Normalization of the tumour vasculature can enhance tissue perfusion and improve immune effector cell infiltration, leading to immunotherapy potentiation. However, recent studies have demonstrated that the stimulation of immune cell functions can also help to normalize tumour vessels. In this Opinion article, we propose that the reciprocal regulation between tumour vascular normalization and immune reprogramming forms a reinforcing loop that reconditions the tumour immune microenvironment to induce durable antitumour immunity. A deeper understanding of these pathways could pave the way for identifying new biomarkers and developing more effective combination treatment strategies for patients with cancer.

  View details for PubMedID 29332937

• Anti-Human CD117 Antibodies Mediate Clearance of Myelodysplastic Syndrome Hematopoietic Stem Cells and Facilitate Establishment of Normal Hematopoiesis in Transplantation Pang, W. W., Czechowicz, A., Poyser, J., Park, C. Y., Weissman, I. L., Shizuru, J. A. ELSEVIER SCIENCE INC. 2018: S230–S231

  View details for Web of Science ID 000425476000317

• A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells CELL REPORTS Ang, L., Tan, A., Autio, M. I., Goh, S., Choo, S., Lee, K., Tan, J., Pan, B., Lee, J., Lum, J., Lim, C., Yeo, I., Wong, C., Liu, M., Oh, J., Chia, C., Loh, C., Chen, A., Chen, Q., Weissman, I. L., Loh, K. M., Lim, B. 2018; 22 (8): 2190–2205

  Abstract

  How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah-/-Rag2-/-Il2rg-/- mouse model of liver failure.

  View details for PubMedID 29466743

• Proefferocytic Therapy Promotes Transforming Growth Factor-beta Signaling and Prevents Aneurysm Formation CIRCULATION Kojima, Y., Werner, N., Ye, J., Nanda, V., Tsao, N., Wang, Y., Flores, A. M., Miller, C. L., Weissman, I., Deng, H., Xu, B., Dalman, R. L., Eken, S. M., Pelisek, J., Li, Y., Maegdefessel, L., Leeper, N. J. 2018; 137 (7): 750–53

  View details for PubMedID 29440201

  View details for PubMedCentralID PMC5819616

• Mentoring the Next Generation: Irving Weissman CELL STEM CELL Weissman, I. 2018; 22 (2): 151–52

  Abstract

  Mentor-mentee relationships are essential for professional development, but developing these interpersonal skills is not often highlighted as a priority in scientific endeavors. In a yearlong series, Cell Stem Cell interviews prominent scientists who have prioritized mentorship over the years. Here, we chat with Dr. Irving Weissman about his views.

  View details for DOI 10.1016/j.stem.2018.01.006

  View details for Web of Science ID 000423840400006

  View details for PubMedID 29413391

• Where Hematopoietic Stem Cells Live: The Bone Marrow Niche ANTIOXIDANTS & REDOX SIGNALING Szade, K., Gulati, G. S., Chan, C. F., Kao, K. S., Miyanishi, M., Marjon, K. D., Sinha, R., George, B. M., Chen, J. Y., Weissman, I. L. 2018

  Abstract

  Hematopoietic stem cells (HSCs) can sustain the production of blood throughout one's lifetime. However, for proper self-renewal of its own population and differentiation to blood, the HSC requires a specialized microenvironment called the "niche." Recent Advances: Recent studies using novel mouse models have shed new light on the cellular architecture and function of the HSC niche. Here, we review the different cells that constitute the HSC niche and the molecular mechanisms that underlie HSC and niche interaction. We discuss the evidence and potential features that distinguish the HSC niche from other microenvironments in the bone marrow. The relevance of the niche in malignant transformation of the HSCs and harboring cancer metastasis to the bone is also outlined. In addition, we address how the niche may regulate reactive oxygen species levels surrounding the HSCs. Critical Issues and Future Directions: We propose future directions and remaining challenges in investigating the niche of HSCs. We discuss how a better understanding of the HSC niche may help in restoring an aged hematopoietic system, fighting against malignancies, and transplanting purified HSCs safely and effectively into patients. Antioxid. Redox Signal. 00, 000-000.

  View details for PubMedID 29113449

• Single-cell analysis of early progenitor cells that build coronary arteries. Nature Su, T. n., Stanley, G. n., Sinha, R. n., D'Amato, G. n., Das, S. n., Rhee, S. n., Chang, A. H., Poduri, A. n., Raftrey, B. n., Dinh, T. T., Roper, W. A., Li, G. n., Quinn, K. E., Caron, K. M., Wu, S. n., Miquerol, L. n., Butcher, E. C., Weissman, I. n., Quake, S. n., Red-Horse, K. n. 2018

  Abstract

  Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

  View details for PubMedID 29973725

• Notch1 regulates the initiation of metastasis and self-renewal of Group 3 medulloblastoma. Nature communications Kahn, S. A., Wang, X. n., Nitta, R. T., Gholamin, S. n., Theruvath, J. n., Hutter, G. n., Azad, T. D., Wadi, L. n., Bolin, S. n., Ramaswamy, V. n., Esparza, R. n., Liu, K. W., Edwards, M. n., Swartling, F. J., Sahoo, D. n., Li, G. n., Wechsler-Reya, R. J., Reimand, J. n., Cho, Y. J., Taylor, M. D., Weissman, I. L., Mitra, S. S., Cheshier, S. H. 2018; 9 (1): 4121

  Abstract

  Medulloblastoma is the most common malignant brain tumor of childhood. Group 3 medulloblastoma, the most aggressive molecular subtype, frequently disseminates through the leptomeningeal cerebral spinal fluid (CSF) spaces in the brain and spinal cord. The mechanism of dissemination through the CSF remains poorly understood, and the molecular pathways involved in medulloblastoma metastasis and self-renewal are largely unknown. Here we show that NOTCH1 signaling pathway regulates both the initiation of metastasis and the self-renewal of medulloblastoma. We identify a mechanism in which NOTCH1 activates BMI1 through the activation of TWIST1. NOTCH1 expression and activity are directly related to medulloblastoma metastasis and decreased survival rate of tumor-bearing mice. Finally, medulloblastoma-bearing mice intrathecally treated with anti-NRR1, a NOTCH1 blocking antibody, present lower frequency of spinal metastasis and higher survival rate. These findings identify NOTCH1 as a pivotal driver of Group 3 medulloblastoma metastasis and self-renewal, supporting the development of therapies targeting this pathway.

  View details for PubMedID 30297829

• Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration. Journal of visualized experiments : JoVE Tsai, J. M., Weissman, I. L., Rinkevich, Y. 2018

  Abstract

  Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe's morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

  View details for PubMedID 29912198

• Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature 2018; 562 (7727): 367–72

  Abstract

  Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3'-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

  View details for DOI 10.1038/s41586-018-0590-4

  View details for PubMedID 30283141

• CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin's Lymphoma. The New England journal of medicine Advani, R., Flinn, I., Popplewell, L., Forero, A., Bartlett, N. L., Ghosh, N., Kline, J., Roschewski, M., LaCasce, A., Collins, G. P., Tran, T., Lynn, J., Chen, J. Y., Volkmer, J., Agoram, B., Huang, J., Majeti, R., Weissman, I. L., Takimoto, C. H., Chao, M. P., Smith, S. M. 2018; 379 (18): 1711–21

  Abstract

  BACKGROUND: The Hu5F9-G4 (hereafter, 5F9) antibody is a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis. 5F9 synergizes with rituximab to eliminate B-cell non-Hodgkin's lymphoma cells by enhancing macrophage-mediated antibody-dependent cellular phagocytosis. This combination was evaluated clinically.METHODS: We conducted a phase 1b study involving patients with relapsed or refractory non-Hodgkin's lymphoma. Patients may have had diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma. 5F9 (at a priming dose of 1 mg per kilogram of body weight, administered intravenously, with weekly maintenance doses of 10 to 30 mg per kilogram) was given with rituximab to determine safety and efficacy and to suggest a phase 2 dose.RESULTS: A total of 22 patients (15 with DLBCL and 7 with follicular lymphoma) were enrolled. Patients had received a median of 4 (range, 2 to 10) previous therapies, and 95% of the patients had disease that was refractory to rituximab. Adverse events were predominantly of grade 1 or 2. The most common adverse events were anemia and infusion-related reactions. Anemia (an expected on-target effect) was mitigated by the strategy of 5F9 prime and maintenance dosing. Dose-limiting side effects were rare. A selected phase 2 dose of 30 mg of 5F9 per kilogram led to an approximate 100% CD47-receptor occupancy on circulating white and red cells. A total of 50% of the patients had an objective (i.e., complete or partial) response, with 36% having a complete response. The rates of objective response and complete response were 40% and 33%, respectively, among patients with DLBCL and 71% and 43%, respectively, among those with follicular lymphoma. At a median follow-up of 6.2 months among patients with DLBCL and 8.1 months among those with follicular lymphoma, 91% of the responses were ongoing.CONCLUSIONS: The macrophage checkpoint inhibitor 5F9 combined with rituximab showed promising activity in patients with aggressive and indolent lymphoma. No clinically significant safety events were observed in this initial study. (Funded by Forty Seven and the Leukemia and Lymphoma Society; ClinicalTrials.gov number, NCT02953509 .).

  View details for PubMedID 30380386

• Complex mammalian-like haematopoietic system found in a colonial chordate. Nature Rosental, B. n., Kowarsky, M. n., Seita, J. n., Corey, D. M., Ishizuka, K. J., Palmeri, K. J., Chen, S. Y., Sinha, R. n., Okamoto, J. n., Mantalas, G. n., Manni, L. n., Raveh, T. n., Clarke, D. N., Tsai, J. M., Newman, A. M., Neff, N. F., Nolan, G. P., Quake, S. R., Weissman, I. L., Voskoboynik, A. n. 2018

  Abstract

  Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life1. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

  View details for PubMedID 30518860

• Programmed cell removal by calreticulin in tissue homeostasis and cancer. Nature communications Feng, M. n., Marjon, K. D., Zhu, F. n., Weissman-Tsukamoto, R. n., Levett, A. n., Sullivan, K. n., Kao, K. S., Markovic, M. n., Bump, P. A., Jackson, H. M., Choi, T. S., Chen, J. n., Banuelos, A. M., Liu, J. n., Gip, P. n., Cheng, L. n., Wang, D. n., Weissman, I. L. 2018; 9 (1): 3194

  Abstract

  Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to "labeling" by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes.

  View details for PubMedID 30097573

• Hypoxia-inducible factor-1α regulates microglial functions affecting neuronal survival in the acute phase of ischemic stroke in mice. Oncotarget Bok, S., Kim, Y. E., Woo, Y., Kim, S., Kang, S. J., Lee, Y., Park, S. K., Weissman, I. L., Ahn, G. O. 2017; 8 (67): 111508-111521

  Abstract

  Cells universally adapt to ischemic conditions by turning on a transcription factor hypoxia-inducible factor (HIF), in which its role is known to differ widely across many different types of cells. Given that microglia have been reported as an essential mediator of neuroinflammation in many brain diseases, we examined the role of HIF in microglia in the progression of an acute phase of ischemic stroke by challenging our novel strains of myeloid-specific Hif-1α or Hif-2α knockout (KO) mice created by Cre-loxP system via middle cerebral artery occlusion (MCAO). We observed that Hif-1α but not Hif-2α KO mice exhibited an improved recovery compared to wild-type (WT) mice determined by behavioral tests. Immunostaining analyses revealed that there were increased numbers of both mature and immature neurons while microglia and apoptotic cells were significantly decreased in the dentate gyrus of Hif-1α KO mice following MCAO. By isolating microglia with fluorescence-activated cell sorter, we found that HIF-1α-deficient microglia were impaired in phagocytosis, reactive oxygen species (ROS) production, and tumor necrosis factor-α (TNF-α) secretion. We further observed a significant decrease in the expression of Cd36 and milk fat globule-epidermal growth factor 8 (Mfg-e8) genes, both of which contain hypoxia-responsive element (HRE). Knocking down either of these genes in BV2 microglial cells was sufficient to abrogate HIF-mediated increase in phagocytosis, production of intracellular ROS, or TNF-α secretion. Our results therefore suggest that HIF-1α in microglia is a novel therapeutic target to protect neuronal survival following an acute phase of ischemic stroke.

  View details for DOI 10.18632/oncotarget.22851

  View details for PubMedID 29340071

  View details for PubMedCentralID PMC5762339

• Anti-SIRP alpha antibody immunotherapy enhances neutrophil and macrophage antitumor activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ring, N., Herndler-Brandstetter, D., Weiskopf, K., Shan, L., Volkmer, J., George, B. M., Lietzenmayer, M., McKenna, K. M., Naik, T. J., McCarty, A., Zheng, Y., Ring, A. M., Flavell, R. A., Weissman, I. L. 2017; 114 (49): E10578–E10585

  Abstract

  Cancer immunotherapy has emerged as a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. A key factor that limits therapeutic success is the infiltration of tumors by cells of the myeloid lineage. The inhibitory receptor signal regulatory protein-α (SIRPα) is a myeloid-specific immune checkpoint that engages the "don't eat me" signal CD47 expressed on tumors and normal tissues. We therefore developed the monoclonal antibody KWAR23, which binds human SIRPα with high affinity and disrupts its binding to CD47. Administered by itself, KWAR23 is inert, but given in combination with tumor-opsonizing monoclonal antibodies, KWAR23 greatly augments myeloid cell-dependent killing of a collection of hematopoietic and nonhematopoietic human tumor-derived cell lines. Following KWAR23 antibody treatment in a human SIRPA knockin mouse model, both neutrophils and macrophages infiltrate a human Burkitt's lymphoma xenograft and inhibit tumor growth, generating complete responses in the majority of treated animals. We further demonstrate that a bispecific anti-CD70/SIRPα antibody outperforms individually delivered antibodies in specific types of cancers. These studies demonstrate that SIRPα blockade induces potent antitumor activity by targeting multiple myeloid cell subsets that frequently infiltrate tumors. Thus, KWAR23 represents a promising candidate for combination therapy.

  View details for PubMedID 29158380

• Lessons from immuno-oncology: a new era for cancer nanomedicine? NATURE REVIEWS DRUG DISCOVERY Jiang, W., Yuan, H., Chan, C. K., von Roemeling, C. A., Yan, Z., Weissman, I. L., Kim, B. S. 2017; 16 (6): 369–70

  Abstract

  Despite a decade of intensive preclinical research, the translation of cancer nanomedicine to the clinic has been slow. Here, we discuss how recent lessons learned from the successes with immuno-oncology therapies could be applied to cancer nanomedicine and how this may help to overcome some of the key technical challenges in this field.

  View details for PubMedID 28303024

• PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity. Nature Gordon, S. R., Maute, R. L., Dulken, B. W., Hutter, G., George, B. M., McCracken, M. N., Gupta, R., Tsai, J. M., Sinha, R., Corey, D., Ring, A. M., Connolly, A. J., Weissman, I. L. 2017; 545 (7655): 495-499

  Abstract

  Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin's lymphoma. Although it is well established that PD-1-PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1-PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1-PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.

  View details for DOI 10.1038/nature22396

  View details for PubMedID 28514441

• intestinal stem-cell self-renewal. Nature Yan, K. S., Janda, C. Y., Chang, J., Zheng, G. X., Larkin, K. A., Luca, V. C., Chia, L. A., Mah, A. T., Han, A., Terry, J. M., Ootani, A., Roelf, K., Lee, M., Yuan, J., Li, X., Bolen, C. R., Wilhelmy, J., Davies, P. S., Ueno, H., von Furstenberg, R. J., Belgrader, P., Ziraldo, S. B., Ordonez, H., Henning, S. J., Wong, M. H., Snyder, M. P., Weissman, I. L., Hsueh, A. J., Mikkelsen, T. S., Garcia, K. C., Kuo, C. J. 2017; 545 (7653): 238-242

  Abstract

  The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5(+) intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5(+) ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5(+) ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5(+) ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

  View details for DOI 10.1038/nature22313

  View details for PubMedID 28467820

• Identification of mouse cochlear progenitors that develop hair and supporting cells in the organ of Corti NATURE COMMUNICATIONS Xu, J., Ueno, H., Xu, C. Y., Chen, B., Weissman, I. L., Xu, P. 2017; 8

  Abstract

  The adult mammalian cochlear sensory epithelium houses two major types of cells, mechanosensory hair cells and underlying supporting cells, and lacks regenerative capacity. Recent evidence indicates that a subset of supporting cells can spontaneously regenerate hair cells after ablation only within the first week postparturition. Here in vivo clonal analysis of mouse inner ear cells during development demonstrates clonal relationship between hair and supporting cells in sensory organs. We report the identification in mouse of a previously unknown population of multipotent stem/progenitor cells that are capable of not only contributing to the hair and supporting cells but also to other cell types, including glia, in cochlea undergoing development, maturation and repair in response to damage. These multipotent progenitors originate from Eya1-expressing otic progenitors. Our findings also provide evidence for detectable regenerative potential in the postnatal cochlea beyond 1 week of age.

  View details for DOI 10.1038/ncomms15046

  View details for Web of Science ID 000400960800001

  View details for PubMedID 28492243

• Non-equivalence of Wnt and R-spondin ligands during Lgr5(+) intestinal stem-cell self-renewal NATURE Yan, K. S., Janda, C. Y., Chang, J., Zheng, G. X., Larkin, K. A., Luca, V. C., Chia, L. A., Mah, A. T., Han, A., Terry, J. M., Ootani, A., Roelf, K., Lee, M., Yuan, J., Li, X., Bolen, C. R., Wilhelmy, J., Davies, P. S., Ueno, H., von Furstenberg, R. J., Belgrader, P., Ziraldo, S. B., Ordonez, H., Henning, S. J., Wong, M. H., Snyder, M. P., Weissman, I. L., Hsueh, A. J., Mikkelsen, T. S., Garcia, K. C., Kuo, C. J. 2017; 545 (7653): 238-?

  Abstract

  The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5(+) intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5(+) ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5(+) ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5(+) ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

  View details for DOI 10.1038/nature22313

  View details for Web of Science ID 000400963800037

• Unifying mechanism for different fibrotic diseases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wernig, G., Chen, S., Cui, L., Van Neste, C., Tsai, J. M., Kambham, N., Vogel, H., Natkunam, Y., Gilliland, D. G., Nolan, G., Weissman, I. L. 2017; 114 (18): 4757-4762

  Abstract

  Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun-mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

  View details for DOI 10.1073/pnas.1621375114

  View details for PubMedID 28424250

• Normal and Neoplastic Stem Cells. Cold Spring Harbor symposia on quantitative biology McCracken, M. N., George, B. M., Kao, K. S., Marjon, K. D., Raveh, T., Weissman, I. L. 2017

  Abstract

  A stem cell is broadly defined as a cell that retains the capacity to self-renew, a feature that confers the ability to continuously make identical daughter cells or additional cells that will differentiate into downstream progeny. This highly regulated genetic program to retain "stemness" is under active investigation. Research in our laboratory has explored similarities and differences in embryonic, tissue-specific, and neoplastic stem cells and their terminally differentiated counterparts. In this review, we will focus on the contributions of our laboratory, in particular on the studies that identified the mouse hematopoietic stem cell (HSC) and the human leukemic stem cell. These studies have led to significant improvements in both preclinical and clinical research, including improved clinical bone marrow transplantation protocols, isolation of nonleukemic HSCs, a cancer immunotherapy currently in clinical trials, and development of a HSC reporter mouse. These studies and the current follow-up research by us and others will continue to identify the properties, function, and regulation of both normal and neoplastic stem cells.

  View details for DOI 10.1101/sqb.2016.81.030965

  View details for PubMedID 28416577

• A CD47-associated super-enhancer links pro-inflammatory signalling to CD47 upregulation in breast cancer NATURE COMMUNICATIONS Betancur, P. A., Abraham, B. J., Yiu, Y. Y., Willingham, S. B., Khameneh, F., Zarnegar, M., Kuo, A. H., McKenna, K., Kojima, Y., Leeper, N. J., Ho, P., Gip, P., Swigut, T., Sherwood, R. I., Clarke, M. F., Somlo, G., Young, R. A., Weissman, I. L. 2017; 8

  Abstract

  CD47 is a cell surface molecule that inhibits phagocytosis of cells that express it by binding to its receptor, SIRPα, on macrophages and other immune cells. CD47 is expressed at different levels by neoplastic and normal cells. Here, to reveal mechanisms by which different neoplastic cells generate this dominant 'don't eat me' signal, we analyse the CD47 regulatory genomic landscape. We identify two distinct super-enhancers (SEs) associated with CD47 in certain cancer cell types. We show that a set of active constituent enhancers, located within the two CD47 SEs, regulate CD47 expression in different cancer cell types and that disruption of CD47 SEs reduces CD47 gene expression. Finally we report that the TNF-NFKB1 signalling pathway directly regulates CD47 by interacting with a constituent enhancer located within a CD47-associated SE specific to breast cancer. These results suggest that cancers can evolve SE to drive CD47 overexpression to escape immune surveillance.

  View details for DOI 10.1038/ncomms14802

  View details for Web of Science ID 000398343600001

  View details for PubMedID 28378740

• Localized hepatic lobular regeneration by central-vein-associated lineage-restricted progenitors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tsai, J. M., Koh, P. W., Stefanska, A., Xing, L., Walmsley, G. G., Poux, N., Weissman, I. L., Rinkevich, Y. 2017; 114 (14): 3654-3659

  Abstract

  The regeneration of organ morphology and function following tissue loss is critical to restore normal physiology, yet few cases are documented in mammalian postnatal life. Partial hepatectomy of the adult mammalian liver activates compensatory hepatocyte hypertrophy and cell division across remaining lobes, resulting in restitution of organ mass but with permanent alteration of architecture. Here, we identify a time window in early postnatal life wherein partial amputation culminates in a localized regeneration instead of global hypertrophy and proliferation. Quantifications of liver mass, enzymatic activity, and immunohistochemistry demonstrate that damaged lobes underwent multilineage regeneration, reforming a lobe often indistinguishable from undamaged ones. Clonal analysis during regeneration reveals local clonal expansions of hepatocyte stem/progenitors at injured sites that are lineage but not fate restricted. Tetrachimeric mice show clonal selection occurs during development with further selections following injury. Surviving progenitors associate mainly with central veins, in a pattern of selection different from that of normal development. These results illuminate a previously unknown program of liver regeneration after acute injury and allow for exploration of latent regenerative programs with potential applications to adult liver regeneration.

  View details for DOI 10.1073/pnas.1621361114

  View details for Web of Science ID 000398159000047

  View details for PubMedID 28330992

• Practical Immuno-PET Radiotracer Design Considerations for Human Immune Checkpoint Imaging JOURNAL OF NUCLEAR MEDICINE Mayer, A. T., Natarajan, A., Gordon, S. R., Maute, R. L., McCracken, M. N., Ring, A. M., Weissman, I. L., Gambhir, S. S. 2017; 58 (4): 538-546

  Abstract

  Immune checkpoint blockade has emerged as a promising cancer treatment paradigm. Unfortunately, there are still a large number of patients and malignancies that do not respond to therapy. A major barrier to validating biomarkers for the prediction and monitoring of responders to clinical checkpoint blockade has been the lack of imaging tools to accurately assess dynamic immune checkpoint expression. Here, we sought to optimize noninvasive immuno-PET imaging of human programmed death-ligand 1 (PD-L1) expression, in a preclinical model, using a small high-affinity engineered protein scaffold (HAC-PD1). Six HAC-PD1 radiotracer variants were developed and used in preclinical imaging and biodistribution studies to assess their ability to detect human PD-L1 expression in vivo. Radiotracer design modifications included chelate, glycosylation, and radiometal. HACA-PD1 was adopted as the naming convention for aglycosylated tracer variants. NOD scid γ-(NSG) mice were inoculated with subcutaneous tumors engineered to either be constitutively positive (CT26 hPD-L1) or be negative (ΔmPD-L1 CT26) for human PD-L1 expression. When the tumors had grown to an average size of 1 cm in diameter, mice were injected with 0.75-2.25 MBq (∼10 μg) of an engineered radiotracer variant and imaged. At 1 h after injection, organs were harvested for biodistribution. Of the practical immuno-PET tracer modifications considered, glycosylation was the most prominent design factor affecting tracer uptake, specificity, and clearance. In imaging studies, aglycosylated (64)Cu-NOTA-HACA-PD1 most accurately visualized human PD-L1 expression in vivo. We reasoned that because of the scaffold's small size (14 kDa), its pharmacokinetics may be suitable for labeling with the short-lived and widely clinically available radiometal (68)Ga. At 1 h after injection, (68)Ga-NOTA-HACA-PD1 and (68)Ga-DOTA-HACA-PD1 exhibited promising target-to-background ratios in ex vivo biodistribution studies (12.3 and 15.2 tumor-to-muscle ratios, respectively). Notably, all HAC-PD1 radiotracer variants enabled much earlier detection of human PD-L1 expression (1 h after injection) than previously reported radiolabeled antibodies (>24 h after injection). This work provides a template for assessing immuno-PET tracer design parameters and supports the translation of small engineered protein radiotracers for imaging human immune checkpoints.

  View details for DOI 10.2967/jnumed.116.177659

  View details for Web of Science ID 000398249600012

  View details for PubMedCentralID PMC5373501

• Disrupting the CD47-SIRP alpha anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors SCIENCE TRANSLATIONAL MEDICINE Gholamin, S., Mitra, S. S., Feroze, A. H., Liu, J., Kahn, S. A., Zhang, M., Esparza, R., Richard, C., Ramaswamy, V., Remke, M., Volkmer, A. K., Willingham, S., Ponnuswami, A., McCarty, A., Lovelace, P., Storm, T. A., Schubert, S., Hutter, G., Narayanan, C., Chu, P., Raabe, E. H., Harsh, G., Taylor, M. D., Monje, M., Cho, Y., Majeti, R., Volkmer, J. P., Fisher, P. G., Grant, G., Steinberg, G. K., Vogel, H., Edwards, M., Weissman, I. L., Cheshier, S. H. 2017; 9 (381)

  Abstract

  Morbidity and mortality associated with pediatric malignant primary brain tumors remain high in the absence of effective therapies. Macrophage-mediated phagocytosis of tumor cells via blockade of the anti-phagocytic CD47-SIRPα interaction using anti-CD47 antibodies has shown promise in preclinical xenografts of various human malignancies. We demonstrate the effect of a humanized anti-CD47 antibody, Hu5F9-G4, on five aggressive and etiologically distinct pediatric brain tumors: group 3 medulloblastoma (primary and metastatic), atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Hu5F9-G4 demonstrated therapeutic efficacy in vitro and in vivo in patient-derived orthotopic xenograft models. Intraventricular administration of Hu5F9-G4 further enhanced its activity against disseminated medulloblastoma leptomeningeal disease. Notably, Hu5F9-G4 showed minimal activity against normal human neural cells in vitro and in vivo, a phenomenon reiterated in an immunocompetent allograft glioma model. Thus, Hu5F9-G4 is a potentially safe and effective therapeutic agent for managing multiple pediatric central nervous system malignancies.

  View details for DOI 10.1126/scitranslmed.aaf2968

  View details for PubMedID 28298418

• Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling CELL Ho, C. C., Chhabra, A., Starkl, P., Schnorr, P., Wilmes, S., Moraga, I., Kwon, H., Gaudenzio, N., Sibilano, R., Wehrman, T. S., Gakovic, M., Sockolosky, J. T., Tiffany, M. R., Ring, A. M., Piehler, J., Weissman, I. L., Galli, S. J., Shizuru, J. A., Garcia, K. C. 2017; 168 (6): 1041-?

  Abstract

  Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

  View details for DOI 10.1016/j.cell.2017.02.011

  View details for Web of Science ID 000396287900012

  View details for PubMedID 28283060

• Mechanisms of melanoma cell resistance to phagocytosis Anderson, K. L., Snyder, K. M., Lins, D., Weiskopf, K., Shimizu, Y., Weissman, I., Mescher, M., Modiano, J. AMER ASSOC CANCER RESEARCH. 2017

  View details for DOI 10.1158/2326-6074.TUMIMM16-A65

  View details for Web of Science ID 000419245100049

• Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate. Nature communications Wahlestedt, M., Erlandsson, E., Kristiansen, T., Lu, R., Brakebusch, C., Weissman, I. L., Yuan, J., Martin-Gonzalez, J., Bryder, D. 2017; 8: 14533

  Abstract

  Ageing associates with significant alterations in somatic/adult stem cells and therapies to counteract these might have profound benefits for health. In the blood, haematopoietic stem cell (HSC) ageing is linked to several functional shortcomings. However, besides the recent realization that individual HSCs might be preset differentially already from young age, HSCs might also age asynchronously. Evaluating the prospects for HSC rejuvenation therefore ultimately requires approaching those HSCs that are functionally affected by age. Here we combine genetic barcoding of aged murine HSCs with the generation of induced pluripotent stem (iPS) cells. This allows us to specifically focus on aged HSCs presenting with a pronounced lineage skewing, a hallmark of HSC ageing. Functional and molecular evaluations reveal haematopoiesis from these iPS clones to be indistinguishable from that associating with young mice. Our data thereby provide direct support to the notion that several key functional attributes of HSC ageing can be reversed.

  View details for DOI 10.1038/ncomms14533

  View details for PubMedID 28224997

  View details for PubMedCentralID PMC5322498

• Breaking Down the Barriers to Precision Cancer Nanomedicine TRENDS IN BIOTECHNOLOGY von Roemeling, C., Jian, W., Chan, C. K., Weissman, I. L., Kim, B. Y. 2017; 35 (2): 159-171

  Abstract

  Nanomedicine offers unique advantages in treating human cancers. However, physiological and pathological barriers within normal and disease tissues, which are highly variable among individuals, often hinder its effectiveness. The body possesses specific innate responses to nanoparticles (NPs), which when combined with unique pathophysiological signatures in the tumor microenvironment, can severely limit the utility of nanomedicine in the oncological setting. Furthermore, with the successes of cancer immunotherapies, understanding nanoimmune interactions and developing immune-smart cancer nanomedicine that can take advantage of the body's immune functions will increasingly become clinically relevant. Therefore, a better understanding of the important native and acquired biological processes that dictate the fate of nanomedicine is integral to developing more effective individualized platforms for treating cancer patients.

  View details for DOI 10.1016/j.tibtech.2016.07.006

  View details for Web of Science ID 000393261300009

• Breaking Down the Barriers to Precision Cancer Nanomedicine. Trends in biotechnology von Roemeling, C., Jiang, W., Chan, C. K., Weissman, I. L., Kim, B. Y. 2017; 35 (2): 159-171

  Abstract

  Nanomedicine offers unique advantages in treating human cancers. However, physiological and pathological barriers within normal and disease tissues, which are highly variable among individuals, often hinder its effectiveness. The body possesses specific innate responses to nanoparticles (NPs), which when combined with unique pathophysiological signatures in the tumor microenvironment, can severely limit the utility of nanomedicine in the oncological setting. Furthermore, with the successes of cancer immunotherapies, understanding nanoimmune interactions and developing immune-smart cancer nanomedicine that can take advantage of the body's immune functions will increasingly become clinically relevant. Therefore, a better understanding of the important native and acquired biological processes that dictate the fate of nanomedicine is integral to developing more effective individualized platforms for treating cancer patients.

  View details for DOI 10.1016/j.tibtech.2016.07.006

  View details for PubMedID 27492049

• The Role of Efferocytosis in Atherosclerosis CIRCULATION Kojima, Y., Weissman, I. L., Leeper, N. J. 2017; 135 (5): 476-489

  Abstract

  The necrotic core has long been a hallmark of the vulnerable atherosclerotic plaque. Although apoptotic cells are cleared quickly in almost all other tissue beds, their removal appears to be significantly impaired in the diseased blood vessel. Emerging evidence indicates that this phenomenon is caused by a defect in efferocytosis, the process by which apoptotic tissue is recognized for engulfment by phagocytic cells such as macrophages. Genetic and experimental data suggest that efferocytosis is impaired during atherogenesis caused by dysregulation of so-called eat me ligands, which govern the edibility of cells undergoing programmed cell death. The following is a summary of recent data indicating that efferocytosis is a major unappreciated driver of lesion expansion but also a reversible defect that can potentially be targeted as a means to prevent plaque progression.

  View details for DOI 10.1161/CIRCULATIONAHA.116.025684

  View details for Web of Science ID 000393716200009

  View details for PubMedID 28137963

  View details for PubMedCentralID PMC5302553

• Pharmacological rescue of diabetic skeletal stem cell niches. Science translational medicine Tevlin, R., Seo, E. Y., Marecic, O., McArdle, A., Tong, X., Zimdahl, B., Malkovskiy, A., Sinha, R., Gulati, G., Li, X., Wearda, T., Morganti, R., Lopez, M., Ransom, R. C., Duldulao, C. R., Rodrigues, M., Nguyen, A., Januszyk, M., Maan, Z., Paik, K., Yapa, K., Rajadas, J., Wan, D. C., Gurtner, G. C., Snyder, M., Beachy, P. A., Yang, F., Goodman, S. B., Weissman, I. L., Chan, C. K., Longaker, M. T. 2017; 9 (372)

  Abstract

  Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.

  View details for DOI 10.1126/scitranslmed.aag2809

  View details for PubMedID 28077677

• Surgical debulking promotes recruitment of macrophages and triggers glioblastoma phagocytosis in combination with CD47 blocking immunotherapy. Oncotarget Zhu, H., Leiss, L., Yang, N., Rygh, C. B., Mitra, S. S., Cheshier, S. H., Weissman, I. L., Huang, B., Miletic, H., Bjerkvig, R., Enger, P. Ø., Li, X., Wang, J. 2017

  Abstract

  Surgical resection is a standard component of treatment in the clinical management of patients with glioblastoma multiforme (GBM). However, experimental therapies are rarely investigated in the context of tumor debulking in preclinical models. Here, a surgical debulking GBM xenograft model was developed in nude rats, and was used in combination with CD47 blocking immunotherapy, a novel treatment strategy that triggers phagocytosis of tumor cells by macrophages in diverse cancer types including GBM. Orthotopic patient-derived xenograft tumors expressing CD47 were resected at 4 weeks after implantation and immediately thereafter treated with anti-CD47 or control antibodies injected into the cavity. Debulking prolonged survival (median survival, 68.5 vs 42.5 days, debulking and non-debulking survival times, respectively; n = 6 animals/group; P = 0.0005). Survival was further improved in animals that underwent combination treatment with anti-CD47 mAbs (median survival, 81.5 days vs 69 days, debulking + anti-CD47 vs debulking + control IgG, respectively; P = 0.0007). Immunohistochemistical staining of tumor sections revealed an increase in recruitment of cells positive for CD68, a marker for macrophages/immune cell types, to the surgical site (50% vs 10%, debulking vs non-debulking, respectively). Finally, analysis of tumor protein lysates on antibody microarrays demonstrated an increase in pro-inflammatory cytokines, such as CXCL10, and a decrease in angiogenic proteins in debulking + anti-CD47 vs non-debulking + IgG tumors. The results indicated that surgical resection combined with anti-CD47 blocking immunotherapy promoted an inflammatory response and prolonged survival in animals, and is therefore an attractive strategy for clinical translation.

  View details for DOI 10.18632/oncotarget.14553

  View details for PubMedID 28076333

• Delivery of monocyte lineage cells in a biomimetic scaffold enhances tissue repair. JCI insight Hu, M. S., Walmsley, G. G., Barnes, L. A., Weiskopf, K. n., Rennert, R. C., Duscher, D. n., Januszyk, M. n., Maan, Z. N., Hong, W. X., Cheung, A. T., Leavitt, T. n., Marshall, C. D., Ransom, R. C., Malhotra, S. n., Moore, A. L., Rajadas, J. n., Lorenz, H. P., Weissman, I. L., Gurtner, G. C., Longaker, M. T. 2017; 2 (19)

  Abstract

  The monocyte lineage is essential to normal wound healing. Macrophage inhibition or knockout in mice results in impaired wound healing through reduced neovascularization, granulation tissue formation, and reepithelialization. Numerous studies have either depleted macrophages or reduced their activity in the context of wound healing. Here, we demonstrate that by increasing the number of macrophages or monocytes in the wound site above physiologic levels via pullulan-collagen composite dermal hydrogel scaffold delivery, the rate of wound healing can be significantly accelerated in both wild-type and diabetic mice, with no adverse effect on the quality of repair. Macrophages transplanted onto wounds differentiate into M1 and M2 phenotypes of different proportions at various time points, ultimately increasing angiogenesis. Given that monocytes can be readily isolated from peripheral blood without in vitro manipulation, these findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.

  View details for PubMedID 28978794

• Age-associated changes in human hematopoietic stem cells. Seminars in hematology Pang, W. W., Schrier, S. L., Weissman, I. L. 2017; 54 (1): 39-42

  Abstract

  Aging has a broad impact on the function of the human hematopoietic system. This review will focus primarily on the effect of aging on the human hematopoietic stem cell (HSC) population. With age, even though human HSCs increase in number, they have decreased self-renewal capacity and reconstitution potential upon transplantation. As a population, human HSCs become more myeloid-biased in their differentiation potential. This is likely due to the human HSC population becoming more clonal with age, selecting for myeloid-biased HSC clones. The HSC clones that come to predominate with age may also contain disease-causing genetic and epigenetic changes that confer an increased risk of developing into an age-associated clonal hematopoietic disease, such as myelodysplastic syndrome, myeloproliferative disorders, or leukemia. The selection of these aged human HSC clones may be in part due to changes in the aging bone marrow microenvironment. While there have been significant advances in the understanding of the effect of aging on mouse hematopoiesis and mouse HSCs, we have comparatively less detailed analyses of the effect of aging on human HSCs. Continued evaluation of human HSCs in the context of aging will be important to determine how applicable the findings in mice and other model organisms are to the human clinical setting.

  View details for DOI 10.1053/j.seminhematol.2016.10.004

  View details for PubMedID 28088986

• Age-associated changes in human hematopoietic stem cells SEMINARS IN HEMATOLOGY Pang, W. W., Schrier, S. L., Weissmann, I. L. 2017; 54 (1): 39-42

  Abstract

  Aging has a broad impact on the function of the human hematopoietic system. This review will focus primarily on the effect of aging on the human hematopoietic stem cell (HSC) population. With age, even though human HSCs increase in number, they have decreased self-renewal capacity and reconstitution potential upon transplantation. As a population, human HSCs become more myeloid-biased in their differentiation potential. This is likely due to the human HSC population becoming more clonal with age, selecting for myeloid-biased HSC clones. The HSC clones that come to predominate with age may also contain disease-causing genetic and epigenetic changes that confer an increased risk of developing into an age-associated clonal hematopoietic disease, such as myelodysplastic syndrome, myeloproliferative disorders, or leukemia. The selection of these aged human HSC clones may be in part due to changes in the aging bone marrow microenvironment. While there have been significant advances in the understanding of the effect of aging on mouse hematopoiesis and mouse HSCs, we have comparatively less detailed analyses of the effect of aging on human HSCs. Continued evaluation of human HSCs in the context of aging will be important to determine how applicable the findings in mice and other model organisms are to the human clinical setting.

  View details for DOI 10.1053/j.seminhematol.2016.10.004

  View details for Web of Science ID 000393445800007

• External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas. Stem cells (Dayton, Ohio) Lee, A. S., Tang, C. n., Hong, W. X., Park, S. n., Bazalova, M. n., Nelson, G. n., Sanchez-Freire, V. n., Bakerman, I. n., Zhang, W. n., Neofytou, E. n., Connolly, A. J., Chan, C. K., Graves, E. E., Weissman, I. L., Nguyen, P. K., Wu, J. C. 2017

  Abstract

  Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present the evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by utilizing a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced re-seeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1x10(3) higher doses to form new teratomas. We demonstrate that radiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. This article is protected by copyright. All rights reserved.

  View details for PubMedID 28600830

• Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy. Nature immunology Barkal, A. A., Weiskopf, K. n., Kao, K. S., Gordon, S. R., Rosental, B. n., Yiu, Y. Y., George, B. M., Markovic, M. n., Ring, N. G., Tsai, J. M., McKenna, K. M., Ho, P. Y., Cheng, R. Z., Chen, J. Y., Barkal, L. J., Ring, A. M., Weissman, I. L., Maute, R. L. 2017

  Abstract

  Exciting progress in the field of cancer immunotherapy has renewed the urgency of the need for basic studies of immunoregulation in both adaptive cell lineages and innate cell lineages. Here we found a central role for major histocompatibility complex (MHC) class I in controlling the phagocytic function of macrophages. Our results demonstrated that expression of the common MHC class I component β2-microglobulin (β2M) by cancer cells directly protected them from phagocytosis. We further showed that this protection was mediated by the inhibitory receptor LILRB1, whose expression was upregulated on the surface of macrophages, including tumor-associated macrophages. Disruption of either MHC class I or LILRB1 potentiated phagocytosis of tumor cells both in vitro and in vivo, which defines the MHC class I-LILRB1 signaling axis as an important regulator of the effector function of innate immune cells, a potential biomarker for therapeutic response to agents directed against the signal-regulatory protein CD47 and a potential target of anti-cancer immunotherapy.

  View details for PubMedID 29180808

• An atlas of transcriptional, chromatin accessibility, and surface marker changes in human mesoderm development SCIENTIFIC DATA Koh, P. W., Sinha, R., Barkal, A. A., Morganti, R. M., Chen, A., Weissman, I. L., Ang, L. T., Kundaje, A., Loh, K. M. 2016; 3

  Abstract

  Mesoderm is the developmental precursor to myriad human tissues including bone, heart, and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end, we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites, sclerotome, dermomyotome) and separately, into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq, ATAC-seq, and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level, knowledge that might one day be exploited for regenerative medicine.

  View details for DOI 10.1038/sdata.2016.109

  View details for PubMedID 27996962

• Practical ImmunoPET radiotracer design considerations for human immune checkpoint imaging. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Mayer, A. T., Natarajan, A., Gordon, S., Maute, R., McCracken, M., Ring, A., Weissman, I., Gambhir, S. S. 2016

  Abstract

  Immune checkpoint blockade has emerged as a promising cancer treatment paradigm. Unfortunately, there are still a large number of patients and malignancies that do not respond to therapy. A major barrier to validating biomarkers for the prediction and monitoring of responders to clinical checkpoint blockade has been the lack of imaging tools to accurately assess dynamic immune checkpoint expression. Here, we sought to optimize noninvasive immuno-PET imaging of human programmed death-ligand 1 (PD-L1) expression, in a preclinical model, using a small high-affinity engineered protein scaffold (HAC-PD1). Six HAC-PD1 radiotracer variants were developed and used in preclinical imaging and biodistribution studies to assess their ability to detect human PD-L1 expression in vivo. Radiotracer design modifications included chelate, glycosylation, and radiometal. HACA-PD1 was adopted as the naming convention for aglycosylated tracer variants. NOD scid γ-(NSG) mice were inoculated with subcutaneous tumors engineered to either be constitutively positive (CT26 hPD-L1) or be negative (ΔmPD-L1 CT26) for human PD-L1 expression. When the tumors had grown to an average size of 1 cm in diameter, mice were injected with 0.75-2.25 MBq (∼10 μg) of an engineered radiotracer variant and imaged. At 1 h after injection, organs were harvested for biodistribution. Of the practical immuno-PET tracer modifications considered, glycosylation was the most prominent design factor affecting tracer uptake, specificity, and clearance. In imaging studies, aglycosylated (64)Cu-NOTA-HACA-PD1 most accurately visualized human PD-L1 expression in vivo. We reasoned that because of the scaffold's small size (14 kDa), its pharmacokinetics may be suitable for labeling with the short-lived and widely clinically available radiometal (68)Ga. At 1 h after injection, (68)Ga-NOTA-HACA-PD1 and (68)Ga-DOTA-HACA-PD1 exhibited promising target-to-background ratios in ex vivo biodistribution studies (12.3 and 15.2 tumor-to-muscle ratios, respectively). Notably, all HAC-PD1 radiotracer variants enabled much earlier detection of human PD-L1 expression (1 h after injection) than previously reported radiolabeled antibodies (>24 h after injection). This work provides a template for assessing immuno-PET tracer design parameters and supports the translation of small engineered protein radiotracers for imaging human immune checkpoints.

  View details for PubMedID 27980047

• Evidence that beta 7 Integrin Regulates Hematopoietic Stem Cell Homing and Engraftment Through Interaction with MAdCAM-1 STEM CELLS AND DEVELOPMENT Murakami, J. L., Xu, B., Franco, C. B., Hu, X., Galli, S. J., Weissman, I. L., Chen, C. 2016; 25 (1): 18-26

  Abstract

  α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis is controversial. We identified a subset of hematopoietic stem cells (HSCs) in the bone marrow (BM) that expressed β7 integrin. These β7(+) HSCs were capable of multilineage, long-term reconstitution and had an inherent competitive advantage over β7(-) HSCs. On the other hand, HSCs that lacked β7 integrin (β7KO) had reduced engraftment potential. Interestingly, quantitative RT-PCR and flow cytometry revealed that β7KO HSCs expressed lower levels of the chemokine receptor CXCR4. Accordingly, β7KO HSCs exhibited impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on BM endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with its endothelial ligand MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment.

  View details for DOI 10.1089/scd.2014.0551

  View details for PubMedID 26422691

  View details for PubMedCentralID PMC4692116

• Eradication of Canine Diffuse Large B-Cell Lymphoma in a Murine Xenograft Model with CD47 Blockade and Anti-CD20 CANCER IMMUNOLOGY RESEARCH Weiskopf, K., Anderson, K. L., Ito, D., Schnorr, P. J., Tomiyasu, H., Ring, A. M., Bloink, K., Efe, J., Rue, S., Lowery, D., Barkal, A., Prohaska, S., McKenna, K. M., Cornax, I., O'Brien, T. D., O'Sullivan, M. G., Weissman, I. L., Modiano, J. F. 2016; 4 (12): 1072-1087

  Abstract

  Cancer immunotherapies hold much promise, but their potential in veterinary settings has not yet been fully appreciated. Canine lymphomas are among the most common tumors of dogs and bear remarkable similarity to human disease. In this study, we examined the combination of CD47 blockade with anti-CD20 passive immunotherapy for canine lymphoma. The CD47/SIRPα axis is an immune checkpoint that regulates macrophage activation. In humans, CD47 is expressed on cancer cells and enables evasion from phagocytosis. CD47-blocking therapies are now under investigation in clinical trials for a variety of human cancers. We found the canine CD47/SIRPα axis to be conserved biochemically and functionally. We identified high-affinity SIRPα variants that antagonize canine CD47 and stimulate phagocytosis of canine cancer cells in vitro When tested as Fc fusion proteins, these therapeutic agents exhibited single-agent efficacy in a mouse xenograft model of canine lymphoma. As robust synergy between CD47 blockade and tumor-specific antibodies has been demonstrated for human cancer, we evaluated the combination of CD47 blockade with 1E4-cIgGB, a canine-specific antibody to CD20. 1E4-cIgGB could elicit a therapeutic response against canine lymphoma in vivo as a single agent. However, augmented responses were observed when combined with CD47-blocking therapies, resulting in synergy in vitro and in vivo and eliciting cures in 100% of mice bearing canine lymphoma. Our findings support further testing of CD47-blocking therapies alone and in combination with CD20 antibodies in the veterinary setting. Cancer Immunol Res; 4(12); 1072-87. ©2016 AACR.

  View details for DOI 10.1158/2326-6066.CIR-16-0105

  View details for Web of Science ID 000389702900009

  View details for PubMedID 27856424

• LYVE1 Marks the Divergence of Yolk Sac Definitive Hemogenic Endothelium from the Primitive Erythroid Lineage CELL REPORTS Lee, L. K., Ghorbanian, Y., Wang, W., Wang, Y., Kim, Y. J., Weissman, I. L., Inlay, M. A., Mikkola, H. K. 2016; 17 (9): 2286-2298

  Abstract

  The contribution of the different waves and sites of developmental hematopoiesis to fetal and adult blood production remains unclear. Here, we identify lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) as a marker of yolk sac (YS) endothelium and definitive hematopoietic stem and progenitor cells (HSPCs). Endothelium in mid-gestation YS and vitelline vessels, but not the dorsal aorta and placenta, were labeled by Lyve1-Cre. Most YS HSPCs and erythro-myeloid progenitors were Lyve1-Cre lineage traced, but primitive erythroid cells were not, suggesting that they represent distinct lineages. Fetal liver (FL) and adult HSPCs showed 35%-40% Lyve1-Cre marking. Analysis of circulation-deficient Ncx1(-/-) concepti identified the YS as a major source of Lyve1-Cre labeled HSPCs. FL proerythroblast marking was extensive at embryonic day (E) 11.5-13.5, but decreased to hematopoietic stem cell (HSC) levels by E16.5, suggesting that HSCs from multiple sources became responsible for erythropoiesis. Lyve1-Cre thus marks the divergence between YS primitive and definitive hematopoiesis and provides a tool for targeting YS definitive hematopoiesis and FL colonization.

  View details for DOI 10.1016/j.celrep.2016.10.080

  View details for Web of Science ID 000390893600011

  View details for PubMedID 27880904

• Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos. Cell stem cell Masaki, H., Kato-Itoh, M., Takahashi, Y., Umino, A., Sato, H., Ito, K., Yanagida, A., Nishimura, T., Yamaguchi, T., Hirabayashi, M., Era, T., Loh, K. M., Wu, S. M., Weissman, I. L., Nakauchi, H. 2016; 19 (5): 587-592

  Abstract

  Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17(+) endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17(+) endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.

  View details for DOI 10.1016/j.stem.2016.10.013

  View details for PubMedID 27814480

• Immune Priming of the Tumor Microenvironment by Radiation TRENDS IN CANCER Jiang, W., Chan, C. K., Weissman, I. L., Kim, B. S., Hahn, S. M. 2016; 2 (11): 638–45

  Abstract

  Ionizing irradiation can induce a multitude of alterations within the tumor microenvironment. Unlike targeted therapies, radiation delivered to the tumor bed can prompt phenotypic changes in both normal stromal and cancer cells, leading to molecular and physiological alterations within the tumor microenvironment. These environmental modulations directly influence the degree of immunogenicity of the tumor microenvironment and may ultimately affect tumor responsiveness to cancer immunotherapies. Here we review the preclinical evidence for tumor microenvironment-mediated immune suppression and how radiation can modulate immune properties within a tumor. We then discuss the therapeutic opportunities for combining radiation with molecular agents to enhance tumor immunogenicity and how this represents a potential exciting strategy to complement immunotherapies including immune checkpoint blockers in cancer treatment.

  View details for PubMedID 28741502

• Myeloid Cell Origins, Differentiation, and Clinical Implications. Microbiology spectrum Weiskopf, K., Schnorr, P. J., Pang, W. W., Chao, M. P., Chhabra, A., Seita, J., Feng, M., Weissman, I. L. 2016; 4 (5)

  Abstract

  The hematopoietic stem cell (HSC) is a multipotent stem cell that resides in the bone marrow and has the ability to form all of the cells of the blood and immune system. Since its first purification in 1988, additional studies have refined the phenotype and functionality of HSCs and characterized all of their downstream progeny. The hematopoietic lineage is divided into two main branches: the myeloid and lymphoid arms. The myeloid arm is characterized by the common myeloid progenitor and all of its resulting cell types. The stages of hematopoiesis have been defined in both mice and humans. During embryological development, the earliest hematopoiesis takes place in yolk sac blood islands and then migrates to the fetal liver and hematopoietic organs. Some adult myeloid populations develop directly from yolk sac progenitors without apparent bone marrow intermediates, such as tissue-resident macrophages. Hematopoiesis also changes over time, with a bias of the dominating HSCs toward myeloid development as animals age. Defects in myelopoiesis contribute to many hematologic disorders, and some of these can be overcome with therapies that target the aberrant stage of development. Furthermore, insights into myeloid development have informed us of mechanisms of programmed cell removal. The CD47/SIRPα axis, a myeloid-specific immune checkpoint, limits macrophage removal of HSCs but can be exploited by hematologic and solid malignancies. Therapeutics targeting CD47 represent a new strategy for treating cancer. Overall, an understanding of hematopoiesis and myeloid cell development has implications for regenerative medicine, hematopoietic cell transplantation, malignancy, and many other diseases.

  View details for DOI 10.1128/microbiolspec.MCHD-0031-2016

  View details for PubMedID 27763252

• Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy. Science translational medicine Chhabra, A., Ring, A. M., Weiskopf, K., Schnorr, P. J., Gordon, S., Le, A. C., Kwon, H., Ring, N. G., Volkmer, J., Ho, P. Y., Tseng, S., Weissman, I. L., Shizuru, J. A. 2016; 8 (351): 351ra105-?

  Abstract

  Hematopoietic stem cell (HSC) transplantation can cure diverse diseases of the blood system, including hematologic malignancies, anemias, and autoimmune disorders. However, patients must undergo toxic conditioning regimens that use chemotherapy and/or radiation to eliminate host HSCs and enable donor HSC engraftment. Previous studies have shown that anti-c-Kit monoclonal antibodies deplete HSCs from bone marrow niches, allowing donor HSC engraftment in immunodeficient mice. We show that host HSC clearance is dependent on Fc-mediated antibody effector functions, and enhancing effector activity through blockade of CD47, a myeloid-specific immune checkpoint, extends anti-c-Kit conditioning to fully immunocompetent mice. The combined treatment leads to elimination of >99% of host HSCs and robust multilineage blood reconstitution after HSC transplantation. This targeted conditioning regimen that uses only biologic agents has the potential to transform the practice of HSC transplantation and enable its use in a wider spectrum of patients.

  View details for DOI 10.1126/scitranslmed.aae0501

  View details for PubMedID 27510901

• Antibody Therapy Targeting CD47 and CD271 Effectively Suppresses Melanoma Metastasis in Patient-Derived Xenografts. Cell reports Ngo, M., Han, A., Lakatos, A., Sahoo, D., Hachey, S. J., Weiskopf, K., Beck, A. H., Weissman, I. L., Boiko, A. D. 2016; 16 (6): 1701-1716

  Abstract

  The high rate of metastasis and recurrence among melanoma patients indicates the existence of cells within melanoma that have the ability to both initiate metastatic programs and bypass immune recognition. Here, we identify CD47 as a regulator of melanoma tumor metastasis and immune evasion. Protein and gene expression analysis of clinical melanoma samples reveals that CD47, an anti-phagocytic signal, correlates with melanoma metastasis. Antibody-mediated blockade of CD47 coupled with targeting of CD271(+) melanoma cells strongly inhibits tumor metastasis in patient-derived xenografts. This therapeutic effect is mediated by drastic changes in the tumor and metastatic site immune microenvironments, both of whichwhich exhibit greatly increased density of differentiated macrophages and significantly fewer inflammatory monocytes, pro-metastatic macrophages (CCR2(+)/VEGFR1(+)), and neutrophils, all of which are associated with disease progression. Thus, antibody therapy that activates the innate immune response in combination with selective targeting of CD271(+) melanoma cells represents a powerful therapeutic approach against metastatic melanoma.

  View details for DOI 10.1016/j.celrep.2016.07.004

  View details for PubMedID 27477289

• CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature Kojima, Y., Volkmer, J., McKenna, K., Civelek, M., Lusis, A. J., Miller, C. L., DiRenzo, D., Nanda, V., Ye, J., Connolly, A. J., Schadt, E. E., Quertermous, T., Betancur, P., Maegdefessel, L., Matic, L. P., Hedin, U., Weissman, I. L., Leeper, N. J. 2016; 536 (7614): 86-90

  Abstract

  Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or 'efferocytosis'. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-α as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.

  View details for PubMedID 27437576

• Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types CELL Loh, K. M., Chen, A., Koh, P. W., Deng, T. Z., Sinha, R., Tsai, J. M., Barkal, A. A., Shen, K. Y., Jain, R., Morganti, R. M., Shyh-Chang, N., Fernhoff, N. B., George, B. M., Wernig, G., Salomon, R. E., Chen, Z., Vogel, H., Epstein, J. A., Kundaje, A., Talbot, W. S., Beachy, P. A., Ang, L. T., Weissman, I. L. 2016; 166 (2): 451-467

  Abstract

  Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

  View details for DOI 10.1016/j.cell.2016.06.011

  View details for PubMedID 27419872

• CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer JOURNAL OF CLINICAL INVESTIGATION Weiskopf, K., Jahchan, N. S., Schnorr, P. J., Cristea, S., Ring, A. M., Maute, R. L., Volkmer, A. K., Volkmer, J., Liu, J., Lim, J. S., Yang, D., Seitz, G., Thuyen Nguyen, T., Wu, D., Jude, K., Guerston, H., Barkal, A., Trapani, F., George, J., Poirier, J. T., Gardner, E. E., Miles, L. A., de Stanchina, E., Lofgren, S. M., Vogel, H., Winslow, M. M., Dive, C., Thomas, R. K., Rudin, C. M., van de Rijn, M., Majeti, R., Garcia, K. C., Weissman, I. L., Sage, J. 2016; 126 (7): 2610-2620

  Abstract

  Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

  View details for DOI 10.1172/JCI81603

  View details for Web of Science ID 000379094800024

  View details for PubMedID 27294525

  View details for PubMedCentralID PMC4922696

• Developmental cell death programs license cytotoxic cells to eliminate histocompatible partners PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Corey, D. M., rosental, B., Kowarsky, M., Sinha, R., Ishizuka, K. J., Palmeri, K. J., Quake, S. R., Voskoboynik, A., Weissman, I. L. 2016; 113 (23): 6520-6525

  Abstract

  In a primitive chordate model of natural chimerism, one chimeric partner is often eliminated in a process of allogeneic resorption. Here, we identify the cellular framework underlying loss of tolerance to one partner within a natural Botryllus schlosseri chimera. We show that the principal cell type mediating chimeric partner elimination is a cytotoxic morula cell (MC). Proinflammatory, developmental cell death programs render MCs cytotoxic and, in collaboration with activated phagocytes, eliminate chimeric partners during the "takeover" phase of blastogenic development. Among these genes, the proinflammatory cytokine IL-17 enhances cytotoxicity in allorecognition assays. Cellular transfer of FACS-purified MCs from allogeneic donors into recipients shows that the resorption response can be adoptively acquired. Transfer of 1 × 10(5) allogeneic MCs eliminated 33 of 78 (42%) recipient primary buds and 20 of 76 (20.5%) adult parental adult organisms (zooids) by 14 d whereas transfer of allogeneic cell populations lacking MCs had only minimal effects on recipient colonies. Furthermore, reactivity of transferred cells coincided with the onset of developmental-regulated cell death programs and disproportionately affected developing tissues within a chimera. Among chimeric partner "losers," severe developmental defects were observed in asexually propagating tissues, reflecting a pathologic switch in gene expression in developmental programs. These studies provide evidence that elimination of one partner in a chimera is an immune cell-based rejection that operates within histocompatible pairs and that maximal allogeneic responses involve the coordination of both phagocytic programs and the "arming" of cytotoxic cells.

  View details for DOI 10.1073/pnas.1606276113

  View details for Web of Science ID 000377155400052

  View details for PubMedID 27217570

• How One Thing Led to Another. Annual review of immunology Weissman, I. 2016; 34: 1-30

  Abstract

  I started research in high school, experimenting on immunological tolerance to transplantation antigens. This led to studies of the thymus as the site of maturation of T cells, which led to the discovery, isolation, and clinical transplantation of purified hematopoietic stem cells (HSCs). The induction of immune tolerance with HSCs has led to isolation of other tissue-specific stem cells for regenerative medicine. Our studies of circulating competing germline stem cells in colonial protochordates led us to document competing HSCs. In human acute myelogenous leukemia we showed that all preleukemic mutations occur in HSCs, and determined their order; the final mutations occur in a multipotent progenitor derived from the preleukemic HSC clone. With these, we discovered that CD47 is an upregulated gene in all human cancers and is a "don't eat me" signal; blocking it with antibodies leads to cancer cell phagocytosis. CD47 is the first known gene common to all cancers and is a target for cancer immunotherapy.

  View details for DOI 10.1146/annurev-immunol-032414-112003

  View details for PubMedID 27168238

• Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Krampitz, G. W., George, B. M., Willingham, S. B., Volkmer, J., Weiskopf, K., Jahchan, N., Newman, A. M., Sahoo, D., Zemek, A. J., Yanovsky, R. L., Nguyen, J. K., Schnorr, P. J., Mazur, P. K., Sage, J., Longacre, T. A., Visser, B. C., Poultsides, G. A., Norton, J. A., Weissman, I. L. 2016; 113 (16): 4464-4469

  Abstract

  Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

  View details for DOI 10.1073/pnas.1600007113

  View details for PubMedID 27035983

• Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo PLOS ONE Zhang, M., Hutter, G., Kahn, S. A., Azad, T. D., Gholamin, S., Xu, C. Y., Liu, J., Achrol, A. S., Richard, C., Sommerkamp, P., Schoen, M. K., McCracken, M. N., Majeti, R., Weissman, I., Mitra, S. S., Cheshier, S. H. 2016; 11 (4)

  Abstract

  Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

  View details for DOI 10.1371/journal.pone.0153550

  View details for Web of Science ID 000374541200027

  View details for PubMedID 27092773

  View details for PubMedCentralID PMC4836698

• New tools for studying microglia in the mouse and human CNS. Proceedings of the National Academy of Sciences of the United States of America Bennett, M. L., Bennett, F. C., Liddelow, S. A., Ajami, B., Zamanian, J. L., Fernhoff, N. B., Mulinyawe, S. B., Bohlen, C. J., Adil, A., Tucker, A., Weissman, I. L., Chang, E. F., Li, G., Grant, G. A., Hayden Gephart, M. G., Barres, B. A. 2016; 113 (12): E1738-46

  Abstract

  The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

  View details for DOI 10.1073/pnas.1525528113

  View details for PubMedID 26884166

  View details for PubMedCentralID PMC4812770

• New tools for studying microglia in the mouse and human CNS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bennett, M. L., Bennett, F. C., Liddelow, S. A., Ajami, B., Zamanian, J. L., Fernhoff, N. B., Mulinyawe, S. B., Bohlen, C. J., Adil, A., Tucker, A., Weissman, I. L., Chang, E. F., Li, G., Grant, G. A., Gephart, M. G., Barres, B. A. 2016; 113 (12): E1738-E1746

  Abstract

  The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

  View details for DOI 10.1073/pnas.1525528113

  View details for Web of Science ID 000372488200020

  View details for PubMedCentralID PMC4812770

• Dynamic Patterns of Clonal Evolution in Tumor Vasculature Underlie Alterations in Lymphocyte-Endothelial Recognition to Foster Tumor Immune Escape. Cancer research Corey, D. M., Rinkevich, Y., Weissman, I. L. 2016; 76 (6): 1348-1353

  Abstract

  Although tumor blood vessels have been a major therapeutic target for cancer chemotherapy, little is known regarding the stepwise development of the tumor microenvironment. Here, we use a multicolor Cre-dependent marker system to trace clonality within the tumor microenvironment to show that tumor blood vessels follow a pattern of dynamic clonal evolution. In an advanced melanoma tumor microenvironment, the vast majority of tumor vasculature clones are derived from a common precursor. Quantitative lineage analysis reveals founder clones diminish in frequency and are replaced by subclones as tumors evolve. These tumor-specific blood vessels are characterized by a developmental switch to a more invasive and immunologically silent phenotype. Gene expression profiling and pathway analysis reveals selection for traits promoting upregulation of alternative angiogenic programs such as unregulated HGF-MET signaling and enhanced autocrine signaling through VEGF and PDGF. Furthermore, we show a developmental switch in the expression of functionally significant primary lymphocyte adhesion molecules on tumor endothelium, such as the loss in expression of the mucosal addressin MAdCAM-1, whose counter receptor a4β7 on lymphocytes controls lymphocyte homing. Changes in adhesive properties on tumor endothelial subclones are accompanied by decreases in expression of lymphocyte chemokines CXCL16, CXCL13, CXCL12, CXCL9, CXCL10, and CCL19. These evolutionary patterns in the expressed genetic program within tumor endothelium will have both a quantitative and functional impact on lymphocyte distribution that may well influence tumor immune function and underlie escape mechanisms from current antiangiogenic pharmacotherapies. Cancer Res; 76(6); 1348-53. ©2015 AACR.

  View details for DOI 10.1158/0008-5472.CAN-15-1150

  View details for PubMedID 26719541

  View details for PubMedCentralID PMC4794394

• A Mechanism for Somatic Brain Mosaicism CELL Weissman, I. L., Gage, F. H. 2016; 164 (4): 593-595

  Abstract

  Double-strand break repair is required for neural development, and brain cells contain somatic genomic variations. Now, Wei et al. demonstrate that neural stem and progenitor cells undergo very frequent DNA breaks in a very restricted set of genes involved in neural cell adhesion and synapse function.

  View details for DOI 10.1016/j.cell.2016.01.048

  View details for Web of Science ID 000369998300003

  View details for PubMedID 26871622

• Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche NATURE Chen, J. Y., Miyanishi, M., Wang, S. K., Yamazaki, S., Sinha, R., Kao, K. S., Seita, J., Sahoo, D., Nakauchi, H., Weissman, I. L. 2016; 530 (7589): 223-?

  Abstract

  Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5(+) HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5(+)) are directly attached to VE-cadherin(+) cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.

  View details for DOI 10.1038/nature16943

  View details for Web of Science ID 000369916700040

  View details for PubMedID 26863982

  View details for PubMedCentralID PMC4854608

• Training the next generation of biomedical investigators in glycosciences JOURNAL OF CLINICAL INVESTIGATION Agre, P., Bertozzi, C., Bissell, M., Campbell, K. P., Cummings, R. D., Desai, U. R., Estes, M., Flotte, T., Fogleman, G., Gage, F., Ginsburg, D., Gordon, J. I., Hart, G., Hascall, V., Kiessling, L., Kornfeld, S., Lowe, J., Magnani, J., Mahal, L. K., Medzhitov, R., Roberts, R. J., Sackstein, R., Sarkar, R., Schnaar, R., Schwartz, N., Varki, A., Walt, D., Weissman, I. 2016; 126 (2): 405-408

  Abstract

  This position statement originated from a working group meeting convened on April 15, 2015, by the NHLBI and incorporates follow-up contributions by the participants as well as other thought leaders subsequently consulted, who together represent research fields relevant to all branches of the NIH. The group was deliberately composed not only of individuals with a current research emphasis in the glycosciences, but also of many experts from other fields, who evinced a strong interest in being involved in the discussions. The original goal was to discuss the value of creating centers of excellence for training the next generation of biomedical investigators in the glycosciences. A broader theme that emerged was the urgent need to bring the glycosciences back into the mainstream of biology by integrating relevant education into the curricula of medical, graduate, and postgraduate training programs, thus generating a critical sustainable workforce that can advance the much-needed translation of glycosciences into a more complete understanding of biology and the enhanced practice of medicine.

  View details for DOI 10.1172/JCI85905

  View details for Web of Science ID 000370677300001

  View details for PubMedID 26829621

  View details for PubMedCentralID PMC4731185

• Surveillance of Stem Cell Fate and Function: A System for Assessing Cell Survival and Collagen Expression In Situ TISSUE ENGINEERING PART A Walmsley, G. G., Senarath-Yapa, K., Wearda, T. L., Menon, S., Hu, M. S., Duscher, D., Maan, Z. N., Tsai, J. M., Zielins, E. R., Weissman, I. L., Gurtner, G. C., Lorenz, H. P., Longaker, M. T. 2016; 22 (1-2): 31-40

  Abstract

  Cell-based therapy is an emerging paradigm in skeletal regenerative medicine. However, the primary means by which transplanted cells contribute to bone repair and regeneration remain controversial. To gain an insight into the mechanisms of how both transplanted and endogenous cells mediate skeletal healing, we used a transgenic mouse strain expressing both the topaz variant of green fluorescent protein under the control of the collagen, type I, alpha 1 promoter/enhancer sequence (Col1a1(GFP)) and membrane-bound tomato red fluorescent protein constitutively in all cell types (R26(mTmG)). A comparison of healing in parietal versus frontal calvarial defects in these mice revealed that frontal osteoblasts express Col1a1 to a greater degree than parietal osteoblasts. Furthermore, the scaffold-based application of adipose-derived stromal cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and osteoblasts derived from these mice to critical-sized calvarial defects allowed for investigation of cell survival and function following transplantation. We found that ASCs led to significantly faster rates of bone healing in comparison to BM-MSCs and osteoblasts. ASCs displayed both increased survival and increased Col1a1 expression compared to BM-MSCs and osteoblasts following calvarial defect transplantation, which may explain their superior regenerative capacity in the context of bone healing. Using this novel reporter system, we were able to elucidate how cell-based therapies impact bone healing and identify ASCs as an attractive candidate for cell-based skeletal regenerative therapy. These insights potentially influence stem cell selection in translational clinical trials evaluating cell-based therapeutics for osseous repair and regeneration.

  View details for DOI 10.1089/ten.tea.2015.0221

  View details for Web of Science ID 000368520300005

  View details for PubMedCentralID PMC4741228

• Surveillance of Stem Cell Fate and Function: A System for Assessing Cell Survival and Collagen Expression In Situ. Tissue engineering. Part A Walmsley, G. G., Senarath-Yapa, K., Wearda, T. L., Menon, S., Hu, M. S., Duscher, D., Maan, Z. N., Tsai, J. M., Zielins, E. R., Weissman, I. L., Gurtner, G. C., Lorenz, H. P., Longaker, M. T. 2016; 22 (1-2): 31-40

  Abstract

  Cell-based therapy is an emerging paradigm in skeletal regenerative medicine. However, the primary means by which transplanted cells contribute to bone repair and regeneration remain controversial. To gain an insight into the mechanisms of how both transplanted and endogenous cells mediate skeletal healing, we used a transgenic mouse strain expressing both the topaz variant of green fluorescent protein under the control of the collagen, type I, alpha 1 promoter/enhancer sequence (Col1a1(GFP)) and membrane-bound tomato red fluorescent protein constitutively in all cell types (R26(mTmG)). A comparison of healing in parietal versus frontal calvarial defects in these mice revealed that frontal osteoblasts express Col1a1 to a greater degree than parietal osteoblasts. Furthermore, the scaffold-based application of adipose-derived stromal cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and osteoblasts derived from these mice to critical-sized calvarial defects allowed for investigation of cell survival and function following transplantation. We found that ASCs led to significantly faster rates of bone healing in comparison to BM-MSCs and osteoblasts. ASCs displayed both increased survival and increased Col1a1 expression compared to BM-MSCs and osteoblasts following calvarial defect transplantation, which may explain their superior regenerative capacity in the context of bone healing. Using this novel reporter system, we were able to elucidate how cell-based therapies impact bone healing and identify ASCs as an attractive candidate for cell-based skeletal regenerative therapy. These insights potentially influence stem cell selection in translational clinical trials evaluating cell-based therapeutics for osseous repair and regeneration.

  View details for DOI 10.1089/ten.TEA.2015.0221

  View details for PubMedID 26486617

  View details for PubMedCentralID PMC4741228

• Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maute, R. L., Gordon, S. R., Mayer, A. T., McCracken, M. N., Natarajan, A., Ring, N. G., Kimura, R., Tsai, J. M., Manglik, A., Kruse, A. C., Gambhir, S. S., Weissman, I. L., Ring, A. M. 2015; 112 (47): E6506-E6514

  View details for DOI 10.1073/pnas.1519623112

  View details for Web of Science ID 000365173100015

  View details for PubMedID 26604307

• Neural Placode Tissue Derived From Myelomeningocele Repair Serves as a Viable Source of Oligodendrocyte Progenitor Cells. Neurosurgery Mitra, S. S., Feroze, A. H., Gholamin, S., Richard, C., Esparza, R., Zhang, M., Azad, T. D., Alrfaei, B., Kahn, S. A., Hutter, G., Guzman, R., Creasey, G. H., Plant, G. W., Weissman, I. L., Edwards, M. S., Cheshier, S. 2015; 77 (5): 794-802

  Abstract

  The presence, characteristics, and potential clinical relevance of neural progenitor populations within the neural placodes of myelomeningocele patients remain to be studied. Neural stem cells are known to reside adjacent to ependyma-lined surfaces along the central nervous system axis.Given such neuroanatomic correlation and regenerative capacity in fetal development, we assessed myelomeningocele-derived neural placode tissue as a potentially novel source of neural stem and progenitor cells.Nonfunctional neural placode tissue was harvested from infants during the surgical repair of myelomeningocele and subsequently further analyzed by in vitro studies, flow cytometry, and immunofluorescence. To assess lineage potential, neural placode-derived neurospheres were subjected to differential media conditions. Through assessment of platelet-derived growth factor receptor α (PDGFRα) and CD15 cell marker expression, Sox2+Olig2+ putative oligodendrocyte progenitor cells were successfully isolated.PDGFRαCD15 cell populations demonstrated the highest rate of self-renewal capacity and multipotency of cell progeny. Immunofluorescence of neural placode-derived neurospheres demonstrated preferential expression of the oligodendrocyte progenitor marker, CNPase, whereas differentiation to neurons and astrocytes was also noted, albeit to a limited degree.Neural placode tissue contains multipotent progenitors that are preferentially biased toward oligodendrocyte progenitor cell differentiation and presents a novel source of such cells for use in the treatment of a variety of pediatric and adult neurological disease, including spinal cord injury, multiple sclerosis, and metabolic leukoencephalopathies.

  View details for DOI 10.1227/NEU.0000000000000918

  View details for PubMedID 26225855

• Evolution of normal and neoplastic tissue stem cells: progress after Robert Hooke PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Weissman, I. 2015; 370 (1680)

  View details for DOI 10.1098/rstb.2014.0364

  View details for PubMedID 26416675

• Skeletal Stem Cell Niche Aberrancies Underlie Impaired Fracture Healing in a Mouse Model of Type 2 Diabetes. Plastic and reconstructive surgery Tevlin, R., Young Seo, E., Marecic, O., Wearda, T., Mc Ardle, A., Januszyk, M., Gulati, G., Maan, Z., Hu, M. S., Walmsley, G. G., Gurtner, G. C., Chan, C. K., Weissman, I. L., Longaker, M. T. 2015; 136 (4): 73-?

  View details for DOI 10.1097/01.prs.0000472372.96995.3e

  View details for PubMedID 26397581

• Sleep disruption impairs haematopoietic stem cell transplantation in mice NATURE COMMUNICATIONS Rolls, A., Pang, W. W., Ibarra, I., Colas, D., Bonnavion, P., Korin, B., Heller, H. C., Weissman, I. L., de Lecea, L. 2015; 6

  Abstract

  Many of the factors affecting the success of haematopoietic cell transplantation are still unknown. Here we show in mice that donor sleep deprivation reduces the ability of its haematopoietic stem cells (HSCs) to engraft and reconstitute the blood and bone marrow of an irradiated recipient by more than 50%. We demonstrate that sleep deprivation downregulates the expression of microRNA (miR)-19b, a negative regulator of the suppressor of cytokine signalling (SOCS) genes, which inhibit HSC migration and homing. Accordingly, HSCs from sleep-deprived mice have higher levels of SOCS genes expression, lower migration capacity in vitro and reduced homing to the bone marrow in vivo. Recovery of sleep after sleep deprivation restored the reconstitution potential of the HSCs. Taken together, this study provides insights into cellular and molecular mechanisms underlying the effects of sleep deprivation on HSCs, emphasizing the potentially critical role of donor sleep in the success of bone marrow transplantation.

  View details for DOI 10.1038/ncomms9516

  View details for Web of Science ID 000364930800001

  View details for PubMedID 26465715

  View details for PubMedCentralID PMC4621781

• Molecular Pathways: Activating T Cells after Cancer Cell Phagocytosis from Blockade of CD47 "Don't Eat Me" Signals. Clinical cancer research McCracken, M. N., Cha, A. C., Weissman, I. L. 2015; 21 (16): 3597-3601

  Abstract

  Recent advances with immunotherapy agents for the treatment of cancer have provided remarkable, and in some cases, curative results. Our laboratory has identified CD47 as an important "don't eat me" signal expressed on malignant cells. Blockade of the CD47:SIRP-α axis between tumor cells and innate immune cells (monocytes, macrophages, and dendritic cells) increases tumor cell phagocytosis in both solid tumors (including, but not limited to, bladder, breast, colon, lung, and pancreatic) and hematologic malignancies. These phagocytic innate cells are also professional antigen-presenting cells (APC), providing a link from innate to adaptive antitumor immunity. Preliminary studies have demonstrated that APCs present antigens from phagocytosed tumor cells, causing T-cell activation. Therefore, agents that block the CD47:SIRP-α engagement are attractive therapeutic targets as a monotherapy or in combination with additional immune-modulating agents for activating antitumor T cells in vivo. Clin Cancer Res; 21(16); 3597-601. ©2015 AACR.

  View details for DOI 10.1158/1078-0432.CCR-14-2520

  View details for PubMedID 26116271

• Identification and characterization of an injury-induced skeletal progenitor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Marecic, O., Tevlin, R., McArdle, A., Seo, E. Y., Wearda, T., Duldulao, C., Walmsley, G. G., Nguyen, A., Weissman, I. L., Chan, C. K., Longaker, M. T. 2015; 112 (32): 9920-9925

  Abstract

  The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.

  View details for DOI 10.1073/pnas.1513066112

  View details for PubMedID 26216955

• Prospective isolation of human erythroid lineage-committed progenitors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mori, Y., Chen, J. Y., Pluvinage, J. V., Seita, J., Weissman, I. L. 2015; 112 (31): 9638-9643

  Abstract

  Determining the developmental pathway leading to erythrocytes and being able to isolate their progenitors are crucial to understanding and treating disorders of red cell imbalance such as anemia, myelodysplastic syndrome, and polycythemia vera. Here we show that the human erythrocyte progenitor (hEP) can be prospectively isolated from adult bone marrow. We found three subfractions that possessed different expression patterns of CD105 and CD71 within the previously defined human megakaryocyte/erythrocyte progenitor (hMEP; Lineage(-) CD34(+) CD38(+) IL-3Rα(-) CD45RA(-)) population. Both CD71(-) CD105(-) and CD71(+) CD105(-) MEPs, at least in vitro, still retained bipotency for the megakaryocyte (MegK) and erythrocyte (E) lineages, although the latter subpopulation is skewed in differentiation toward the erythroid lineage. Notably, the proliferative and differentiation output of the CD71(intermediate(int)/+) CD105(+) subset of cells within the MEP population was completely restricted to the erythroid lineage with the loss of MegK potential. CD71(+) CD105(-) MEPs are erythrocyte-biased MEPs (E-MEPs) and CD71(int/+) CD105(+) cells are EPs. These previously unclassified populations may facilitate further understanding of the molecular mechanisms governing human erythroid development and serve as potential therapeutic targets in disorders of the erythroid lineage.

  View details for DOI 10.1073/pnas.1512076112

  View details for PubMedID 26195758

• Expression of TCR-V peptides by murine bone marrow cells does not identify T-cell progenitors JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Abbey, J. L., Karsunky, H., Serwold, T., Papathanasiou, P., Weissman, I. L., O'Neill, H. C. 2015; 19 (8): 1956-1964

  Abstract

  Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2(+) Cβ(-) are found in several lymphoid sites, and among the lineage-negative (Lin(-) ) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin(-) Vβ8.2(+) Cβ(-) BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin(-) Vβ8.2(+) Cβ(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

  View details for DOI 10.1111/jcmm.12572

  View details for Web of Science ID 000358925100020

  View details for PubMedID 25754612

  View details for PubMedCentralID PMC4549046

• En1 fibroblasts and melanoma. Melanoma management Walmsley, G. G., Hu, M. S., Maan, Z. N., Rinkevich, Y., Weissman, I. L., Longaker, M. T. 2015; 2 (3): 191-192

  View details for DOI 10.2217/mmt.15.23

  View details for PubMedID 30190847

  View details for PubMedCentralID PMC6094637

• Stem cells are units of natural selection for tissue formation, for germline development, and in cancer development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Weissman, I. L. 2015; 112 (29): 8922-8928

  Abstract

  It is obvious that natural selection operates at the level of individuals and collections of individuals. Nearly two decades ago we showed that in multi-individual colonies of protochordate colonial tunicates sharing a blood circulation, there exists an exchange of somatic stem cells and germline stem cells, resulting in somatic chimeras and stem cell competitions for gonadal niches. Stem cells are unlike other cells in the body in that they alone self-renew, so that they form clones that are perpetuated for the life of the organism. Stem cell competitions have allowed the emergence of competitive somatic and germline stem cell clones. Highly successful germline stem cells usually outcompete less successful competitors both in the gonads of the genotype partner from which they arise and in the gonads of the natural parabiotic partners. Therefore, natural selection also operates at the level of germline stem cell clones. In the colonial tunicate Botryllus schlosseri the formation of natural parabionts is prevented by a single-locus highly polymorphic histocompatibility gene called Botryllus histocompatibility factor. This limits germline stem cell predation to kin, as the locus has hundreds of alleles. We show that in mice germline stem cells compete for gonad niches, and in mice and humans, blood-forming stem cells also compete for bone marrow niches. We show that the clonal progression from blood-forming stem cells to acute leukemias by successive genetic and epigenetic events in blood stem cells also involves competition and selection between clones and propose that this is a general theme in cancer.

  View details for DOI 10.1073/pnas.1505464112

  View details for Web of Science ID 000358225100052

  View details for PubMedID 26195745

  View details for PubMedCentralID PMC4517284

• "Velcro" Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein alpha(SIRP alpha) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis JOURNAL OF BIOLOGICAL CHEMISTRY Ho, C. C., Guo, N., Sockolosky, J. T., Ring, A. M., Weiskopf, K., Oezkan, E., Mori, Y., Weissman, I. L., Garcia, K. C. 2015; 290 (20): 12650-12663

  Abstract

  CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

  View details for DOI 10.1074/jbc.M115.648220

  View details for Web of Science ID 000354569000019

  View details for PubMedCentralID PMC4432284

• "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein a (SIRPa) antagonists that enhance antibody-dependent cellular phagocytosis. journal of biological chemistry Ho, C. C., Guo, N., Sockolosky, J. T., Ring, A. M., Weiskopf, K., Özkan, E., Mori, Y., Weissman, I. L., Garcia, K. C. 2015; 290 (20): 12650-12663

  Abstract

  CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

  View details for DOI 10.1074/jbc.M115.648220

  View details for PubMedID 25837251

  View details for PubMedCentralID PMC4432284

• Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science Rinkevich, Y., Walmsley, G. G., Hu, M. S., Maan, Z. N., Newman, A. M., Drukker, M., Januszyk, M., Krampitz, G. W., Gurtner, G. C., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2015; 348 (6232)

  Abstract

  Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

  View details for DOI 10.1126/science.aaa2151

  View details for PubMedID 25883361

  View details for PubMedCentralID PMC5088503

• CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer. Proceedings of the National Academy of Sciences of the United States of America Cheah, M. T., Chen, J. Y., Sahoo, D., Contreras-Trujillo, H., Volkmer, A. K., Scheeren, F. A., Volkmer, J., Weissman, I. L. 2015; 112 (15): 4725-4730

  Abstract

  Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.

  View details for DOI 10.1073/pnas.1424795112

  View details for PubMedID 25825750

• Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands CELL Moraga, I., Wernig, G., Wilmes, S., Gryshkova, V., Richter, C. P., Hong, W., Sinha, R., Guo, F., Fabionar, H., Wehrman, T. S., Krutzik, P., Demharter, S., Plo, I., Weissman, I. L., Minary, P., Majeti, R., Constantinescu, S. N., Piehler, J., Garcia, K. C. 2015; 160 (6): 1196-1208

  Abstract

  Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

  View details for DOI 10.1016/j.cell.2015.02.011

  View details for PubMedID 25728669

• Macrophages are critical effectors of antibody therapies for cancer. mAbs Weiskopf, K., Weissman, I. L. 2015; 7 (2): 303-310

  Abstract

  Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

  View details for DOI 10.1080/19420862.2015.1011450

  View details for PubMedID 25667985

• Live Fibroblast Harvest Reveals Surface Marker Shift In Vitro TISSUE ENGINEERING PART C-METHODS Walmsley, G. G., Rinkevich, Y., Hu, M. S., Montoro, D. T., Lo, D. D., McArdle, A., Maan, Z. N., Morrison, S. D., Duscher, D., Whittam, A. J., Wong, V. W., Weissman, I. L., Gurtner, G. C., Longaker, M. T. 2015; 21 (3): 314-321

  Abstract

  Current methods for the isolation of fibroblasts require extended ex vivo manipulation in cell culture. As a consequence, prior studies investigating fibroblast biology may fail to adequately represent cellular phenotypes in vivo. To overcome this problem, we describe a detailed protocol for the isolation of fibroblasts from the dorsal dermis of adult mice that bypasses the need for cell culture, thereby preserving the physiological, transcriptional, and proteomic profiles of each cell. Using the described protocol we characterized the transcriptional programs and the surface expression of 176 CD markers in cultured versus uncultured fibroblasts. The differential expression patterns we observed highlight the importance of a live harvest for investigations of fibroblast biology.

  View details for DOI 10.1089/ten.tec.2014.0118

  View details for Web of Science ID 000350043400009

  View details for PubMedID 25275778

  View details for PubMedCentralID PMC4346232

• Macrophages eat cancer cells using their own calreticulin as a guide: Roles of TLR and Btk. Proceedings of the National Academy of Sciences of the United States of America Feng, M., Chen, J. Y., Weissman-Tsukamoto, R., Volkmer, J., Ho, P. Y., McKenna, K. M., Cheshier, S., Zhang, M., Guo, N., Gip, P., Mitra, S. S., Weissman, I. L. 2015; 112 (7): 2145-2150

  Abstract

  Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

  View details for DOI 10.1073/pnas.1424907112

  View details for PubMedID 25646432

• Botryllus schlosseri, an emerging model for the study of aging, stem cells, and mechanisms of regeneration INVERTEBRATE REPRODUCTION & DEVELOPMENT Voskoboynik, A., Weissman, I. L. 2015; 59: 33-38

  View details for DOI 10.1080/07924259.2014.944673

  View details for Web of Science ID 000346071100007

• Botryllus schlosseri, an emerging model for the study of aging, stem cells, and mechanisms of regeneration. Invertebrate reproduction & development Voskoboynik, A., Weissman, I. L. 2015; 59 (sup1): 33-38

  Abstract

  The decline of tissue regenerative potential with the loss of stem cell function is a hallmark of mammalian aging. We study Botryllus schlosseri, a colonial chordate which exhibits robust stem cell-mediated regeneration capacities throughout life. Larvae, derived by sexual reproduction and chordate development, metamorphose to clonal founders that undergo weekly formation of new individuals by budding from stem cells. Individuals are transient structures which die through massive apoptosis, and successive buds mature to replicate an entire new body. As a result, their stem cells, which are the only self-renewing cells in a tissue, are the only cells which remain through the entire life of the genotype and retain the effects of time. During aging, a significant decrease in the colonies' regenerative potential is observed and both sexual and asexual reproductions will eventually halt. When a parent colony is experimentally separated into a number of clonal replicates, they frequently undergo senescence simultaneously, suggesting a heritable factor that determines lifespan in these colonies. The availability of the recently published B. schlosseri genome coupled with its unique life cycle features promotes the use of this model organism for the study of the evolution of aging, stem cells, and mechanisms of regeneration.

  View details for DOI 10.1080/07924259.2014.944673

  View details for PubMedID 26136618

  View details for PubMedCentralID PMC4464096

• Identification and specification of the mouse skeletal stem cell. Cell Chan, C. K., Seo, E. Y., Chen, J. Y., Lo, D., McArdle, A., Sinha, R., Tevlin, R., Seita, J., Vincent-Tompkins, J., Wearda, T., Lu, W., Senarath-Yapa, K., Chung, M. T., Marecic, O., Tran, M., Yan, K. S., Upton, R., Walmsley, G. G., Lee, A. S., Sahoo, D., Kuo, C. J., Weissman, I. L., Longaker, M. T. 2015; 160 (1-2): 285-298

  Abstract

  How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

  View details for DOI 10.1016/j.cell.2014.12.002

  View details for PubMedID 25594184

• Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation. Blood Reinisch, A., Etchart, N., Thomas, D., Hofmann, N. A., Fruehwirth, M., Sinha, S., Chan, C. K., Senarath-Yapa, K., Seo, E., Wearda, T., Hartwig, U. F., Beham-Schmid, C., Trajanoski, S., Lin, Q., Wagner, W., Dullin, C., Alves, F., Andreeff, M., Weissman, I. L., Longaker, M. T., Schallmoser, K., Majeti, R., Strunk, D. 2015; 125 (2): 249-260

  Abstract

  In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a bone marrow cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13 and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs) as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

  View details for DOI 10.1182/blood-2014-04-572255

  View details for PubMedID 25406351

• SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts. Cell stem cell Deuse, T., Wang, D., Stubbendorff, M., Itagaki, R., Grabosch, A., Greaves, L. C., Alawi, M., Grünewald, A., Hu, X., Hua, X., Velden, J., Reichenspurner, H., Robbins, R. C., Jaenisch, R., Weissman, I. L., Schrepfer, S. 2015; 16 (1): 33-38

  Abstract

  The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies.

  View details for DOI 10.1016/j.stem.2014.11.003

  View details for PubMedID 25465116

• Lift NIH restrictions on chimera research. Science (New York, N.Y.) Sharma, A. n., Sebastiano, V. n., Scott, C. T., Magnus, D. n., Koyano-Nakagawa, N. n., Garry, D. J., Witte, O. N., Nakauchi, H. n., Wu, J. C., Weissman, I. L., Wu, S. M. 2015; 350 (6261): 640

  View details for PubMedID 26542560

• Hematopoietic stem cells, regenerative medicine, and leukemogenesis Thomas' Hematopoietic Cell Transplantation Weissman, I. 2015; 5: 25–57
• Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential. PloS one Liu, J., Wang, L., Zhao, F., Tseng, S., Narayanan, C., Shura, L., Willingham, S., Howard, M., Prohaska, S., Volkmer, J., Chao, M., Weissman, I. L., Majeti, R. 2015; 10 (9)

  View details for DOI 10.1371/journal.pone.0137345

  View details for PubMedID 26390038

• Pericytes are progenitors for coronary artery smooth muscle. eLife Volz, K. S., Jacobs, A. H., Chen, H. I., Poduri, A., McKay, A. S., Riordan, D. P., Kofler, N., Kitajewski, J., Weissman, I., Red-Horse, K. 2015; 4

  Abstract

  Epicardial cells on the heart's surface give rise to coronary artery smooth muscle cells (caSMCs) located deep in the myocardium. However, the differentiation steps between epicardial cells and caSMCs are unknown as are the final maturation signals at coronary arteries. Here, we use clonal analysis and lineage tracing to show that caSMCs derive from pericytes, mural cells associated with microvessels, and that these cells are present in adults. During development following the onset of blood flow, pericytes at arterial remodeling sites upregulate Notch3 while endothelial cells express Jagged-1. Deletion of Notch3 disrupts caSMC differentiation. Our data support a model wherein epicardial-derived pericytes populate the entire coronary microvasculature, but differentiate into caSMCs at arterial remodeling zones in response to Notch signaling. Our data are the first demonstration that pericytes are progenitors for smooth muscle, and their presence in adult hearts reveals a new potential cell type for targeting during cardiovascular disease.

  View details for DOI 10.7554/eLife.10036

  View details for PubMedID 26479710

  View details for PubMedCentralID PMC4728130

• Upregulation of CD11A on Hematopoietic Stem Cells Denotes the Loss of Long-Term Reconstitution Potential STEM CELL REPORTS Fathman, J. W., Fernhoff, N. B., Seita, J., Chao, C., Scarfone, V. M., Weissman, I. L., Inlay, M. A. 2014; 3 (5): 707-715

  View details for DOI 10.1016/j.stemcr.2014.09.007

  View details for Web of Science ID 000345118600003

• Upregulation of CD11A on hematopoietic stem cells denotes the loss of long-term reconstitution potential. Stem cell reports Fathman, J. W., Fernhoff, N. B., Seita, J., Chao, C., Scarfone, V. M., Weissman, I. L., Inlay, M. A. 2014; 3 (5): 707-715

  Abstract

  Small numbers of hematopoietic stem cells (HSCs) generate large numbers of mature effector cells through the successive amplification of transiently proliferating progenitor cells. HSCs and their downstream progenitors have been extensively characterized based on their cell-surface phenotype and functional activities during transplantation assays. These cells dynamically lose and acquire specific sets of surface markers during differentiation, leading to the identification of markers that allow for more refined separation of HSCs from early hematopoietic progenitors. Here, we describe a marker, CD11A, which allows for the enhanced purification of mouse HSCs. We show through in vivo transplantations that upregulation of CD11A on HSCs denotes the loss of their long-term reconstitution potential. Surprisingly, nearly half of phenotypic HSCs (defined as Lin-KIT(+)SCA-1(+)CD150(+)CD34-) are CD11A(+) and lack long-term self-renewal potential. We propose that CD11A(+)Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are multipotent progenitors and CD11A-Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are true HSCs.

  View details for DOI 10.1016/j.stemcr.2014.09.007

  View details for PubMedID 25418718

• Endoscopic molecular imaging of human bladder cancer using a CD47 antibody SCIENCE TRANSLATIONAL MEDICINE Pan, Y., Volkmer, J., Mach, K. E., Rouse, R. V., Liu, J., Sahoo, D., Chang, T. C., Metzner, T. J., Kang, L., van de Rijn, M., Skinner, E. C., Gambhir, S. S., Weissman, I. L., Liao, J. C. 2014; 6 (260)

  Abstract

  A combination of optical imaging technologies with cancer-specific molecular imaging agents is a potentially powerful strategy to improve cancer detection and enable image-guided surgery. Bladder cancer is primarily managed endoscopically by white light cystoscopy with suboptimal diagnostic accuracy. Emerging optical imaging technologies hold great potential for improved diagnostic accuracy but lack imaging agents for molecular specificity. Using fluorescently labeled CD47 antibody (anti-CD47) as molecular imaging agent, we demonstrated consistent identification of bladder cancer with clinical grade fluorescence imaging systems, confocal endomicroscopy, and blue light cystoscopy in fresh surgically removed human bladders. With blue light cystoscopy, the sensitivity and specificity for CD47-targeted imaging were 82.9 and 90.5%, respectively. We detected variants of bladder cancers, which are diagnostic challenges, including carcinoma in situ, residual carcinoma in tumor resection bed, recurrent carcinoma following prior intravesical immunotherapy with Bacillus Calmette-Guérin (BCG), and excluded cancer from benign but suspicious-appearing mucosa. CD47-targeted molecular imaging could improve diagnosis and resection thoroughness for bladder cancer.

  View details for DOI 10.1126/scitranslmed.3009457

  View details for Web of Science ID 000343920500006

• Endoscopic molecular imaging of human bladder cancer using a CD47 antibody. Science translational medicine Pan, Y., Volkmer, J., Mach, K. E., Rouse, R. V., Liu, J., Sahoo, D., Chang, T. C., Metzner, T. J., Kang, L., van de Rijn, M., Skinner, E. C., Gambhir, S. S., Weissman, I. L., Liao, J. C. 2014; 6 (260): 260ra148-?

  Abstract

  A combination of optical imaging technologies with cancer-specific molecular imaging agents is a potentially powerful strategy to improve cancer detection and enable image-guided surgery. Bladder cancer is primarily managed endoscopically by white light cystoscopy with suboptimal diagnostic accuracy. Emerging optical imaging technologies hold great potential for improved diagnostic accuracy but lack imaging agents for molecular specificity. Using fluorescently labeled CD47 antibody (anti-CD47) as molecular imaging agent, we demonstrated consistent identification of bladder cancer with clinical grade fluorescence imaging systems, confocal endomicroscopy, and blue light cystoscopy in fresh surgically removed human bladders. With blue light cystoscopy, the sensitivity and specificity for CD47-targeted imaging were 82.9 and 90.5%, respectively. We detected variants of bladder cancers, which are diagnostic challenges, including carcinoma in situ, residual carcinoma in tumor resection bed, recurrent carcinoma following prior intravesical immunotherapy with Bacillus Calmette-Guérin (BCG), and excluded cancer from benign but suspicious-appearing mucosa. CD47-targeted molecular imaging could improve diagnosis and resection thoroughness for bladder cancer.

  View details for DOI 10.1126/scitranslmed.3009457

  View details for PubMedID 25355698

• In utero depletion of fetal hematopoietic stem cells improves engraftment after neonatal transplantation in mice. Blood Derderian, S. C., Togarrati, P. P., King, C., Moradi, P. W., Reynaud, D., Czechowicz, A., Weissman, I. L., MacKenzie, T. C. 2014; 124 (6): 973-980

  Abstract

  Although in utero hematopoietic cell transplantation is a promising strategy to treat congenital hematopoietic disorders, levels of engraftment have not been therapeutic for diseases in which donor cells have no survival advantage. We used an antibody against the murine c-Kit receptor (ACK2) to deplete fetal host hematopoietic stem cells (HSCs) and increase space within the hematopoietic niche for donor cell engraftment. Fetal mice were injected with ACK2 on embryonic days 13.5 to 14.5 and surviving pups were transplanted with congenic hematopoietic cells on day of life 1. Low-dose ACK2 treatment effectively depleted HSCs within the bone marrow with minimal toxicity and the antibody was cleared from the serum before the neonatal transplantation. Chimerism levels were significantly higher in treated pups than in controls; both myeloid and lymphoid cell chimerism increased because of higher engraftment of HSCs in the bone marrow. To test the strategy of repeated HSC depletion and transplantation, some mice were treated with ACK2 postnatally, but the increase in engraftment was lower than that seen with prenatal treatment. We demonstrate a successful fetal conditioning strategy associated with minimal toxicity. Such strategies could be used to achieve clinically relevant levels of engraftment to treat congenital stem cell disorders.

  View details for DOI 10.1182/blood-2014-02-550327

  View details for PubMedID 24879814

• Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L., Longaker, M. T. 2014; 111 (27): 9846-9851

  Abstract

  The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

  View details for DOI 10.1073/pnas.1410097111

  View details for Web of Science ID 000338514800040

  View details for PubMedCentralID PMC4103362

• Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration. Proceedings of the National Academy of Sciences of the United States of America Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L., Longaker, M. T. 2014; 111 (27): 9846-9851

  Abstract

  The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

  View details for DOI 10.1073/pnas.1410097111

  View details for PubMedID 24958860

• Quiescent Hematopoietic Stem Cells Accumulate DNA Damage during Aging that Is Repaired upon Entry into Cell Cycle. Cell stem cell Beerman, I., Seita, J., Inlay, M. A., Weissman, I. L., Rossi, D. J. 2014; 15 (1): 37-50

  Abstract

  Hematopoietic stem cells (HSCs) maintain homeostasis and regenerate the blood system throughout life. It has been postulated that HSCs may be uniquely capable of preserving their genomic integrity in order to ensure lifelong function. To directly test this, we quantified DNA damage in HSCs and downstream progenitors from young and old mice, revealing that strand breaks significantly accrue in HSCs during aging. DNA damage accumulation in HSCs was associated with broad attenuation of DNA repair and response pathways that was dependent upon HSC quiescence. Accordingly, cycling fetal HSCs and adult HSCs driven into cycle upregulated these pathways leading to repair of strand breaks. Our results demonstrate that HSCs are not comprehensively geno-protected during aging. Rather, HSC quiescence and concomitant attenuation of DNA repair and response pathways underlies DNA damage accumulation in HSCs during aging. These results provide a potential mechanism through which premalignant mutations accrue in HSCs.

  View details for DOI 10.1016/j.stem.2014.04.016

  View details for PubMedID 24813857

  View details for PubMedCentralID PMC4082747

• Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ali, S. R., Hippenmeyer, S., Saadat, L. V., Luo, L., Weissman, I. L., Ardehali, R. 2014; 111 (24): 8850-8855

  Abstract

  The mammalian heart has long been considered a postmitotic organ, implying that the total number of cardiomyocytes is set at birth. Analysis of cell division in the mammalian heart is complicated by cardiomyocyte binucleation shortly after birth, which makes it challenging to interpret traditional assays of cell turnover [Laflamme MA, Murray CE (2011) Nature 473(7347):326-335; Bergmann O, et al. (2009) Science 324(5923):98-102]. An elegant multi-isotope imaging-mass spectrometry technique recently calculated the low, discrete rate of cardiomyocyte generation in mice [Senyo SE, et al. (2013) Nature 493(7432):433-436], yet our cellular-level understanding of postnatal cardiomyogenesis remains limited. Herein, we provide a new line of evidence for the differentiated α-myosin heavy chain-expressing cardiomyocyte as the cell of origin of postnatal cardiomyogenesis using the "mosaic analysis with double markers" mouse model. We show limited, life-long, symmetric division of cardiomyocytes as a rare event that is evident in utero but significantly diminishes after the first month of life in mice; daughter cardiomyocytes divide very seldom, which this study is the first to demonstrate, to our knowledge. Furthermore, ligation of the left anterior descending coronary artery, which causes a myocardial infarction in the mosaic analysis with double-marker mice, did not increase the rate of cardiomyocyte division above the basal level for up to 4 wk after the injury. The clonal analysis described here provides direct evidence of postnatal mammalian cardiomyogenesis.

  View details for DOI 10.1073/pnas.1408233111

  View details for Web of Science ID 000337300100041

  View details for PubMedCentralID PMC4066522

• Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice. Proceedings of the National Academy of Sciences of the United States of America Ali, S. R., Hippenmeyer, S., Saadat, L. V., Luo, L., Weissman, I. L., Ardehali, R. 2014; 111 (24): 8850-8855

  Abstract

  The mammalian heart has long been considered a postmitotic organ, implying that the total number of cardiomyocytes is set at birth. Analysis of cell division in the mammalian heart is complicated by cardiomyocyte binucleation shortly after birth, which makes it challenging to interpret traditional assays of cell turnover [Laflamme MA, Murray CE (2011) Nature 473(7347):326-335; Bergmann O, et al. (2009) Science 324(5923):98-102]. An elegant multi-isotope imaging-mass spectrometry technique recently calculated the low, discrete rate of cardiomyocyte generation in mice [Senyo SE, et al. (2013) Nature 493(7432):433-436], yet our cellular-level understanding of postnatal cardiomyogenesis remains limited. Herein, we provide a new line of evidence for the differentiated α-myosin heavy chain-expressing cardiomyocyte as the cell of origin of postnatal cardiomyogenesis using the "mosaic analysis with double markers" mouse model. We show limited, life-long, symmetric division of cardiomyocytes as a rare event that is evident in utero but significantly diminishes after the first month of life in mice; daughter cardiomyocytes divide very seldom, which this study is the first to demonstrate, to our knowledge. Furthermore, ligation of the left anterior descending coronary artery, which causes a myocardial infarction in the mosaic analysis with double-marker mice, did not increase the rate of cardiomyocyte division above the basal level for up to 4 wk after the injury. The clonal analysis described here provides direct evidence of postnatal mammalian cardiomyogenesis.

  View details for DOI 10.1073/pnas.1408233111

  View details for PubMedID 24876275

• Clonal Origins of the Hematopoietic System: The Single Most Elegant Experiment JOURNAL OF IMMUNOLOGY Weissman, I. L. 2014; 192 (11): 4943-4944

  View details for DOI 10.4049/jimmunol.1400902

  View details for Web of Science ID 000337171800003

  View details for PubMedID 24837150

• In Vivo clonal analysis reveals lineage-restricted progenitor characteristics in Mammalian kidney development, maintenance, and regeneration. Cell reports Rinkevich, Y., Montoro, D. T., Contreras-Trujillo, H., Harari-Steinberg, O., Newman, A. M., Tsai, J. M., Lim, X., Van-Amerongen, R., Bowman, A., Januszyk, M., Pleniceanu, O., Nusse, R., Longaker, M. T., Weissman, I. L., Dekel, B. 2014; 7 (4): 1270-1283

  Abstract

  The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

  View details for DOI 10.1016/j.celrep.2014.04.018

  View details for PubMedID 24835991

  View details for PubMedCentralID PMC4425291

• Identification of Multipotent Progenitors that Emerge Prior to Hematopoietic Stem Cells in Embryonic Development. Stem cell reports Inlay, M. A., Serwold, T., Mosley, A., Fathman, J. W., Dimov, I. K., Seita, J., Weissman, I. L. 2014; 2 (4): 457-472

  Abstract

  Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability) gradually arise. Whereas self-renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryogenesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS) than the aorta-gonad-mesonephros (AGM) or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis.

  View details for DOI 10.1016/j.stemcr.2014.02.001

  View details for PubMedID 24749071

  View details for PubMedCentralID PMC3986503

• Clonal Tracking of Rhesus Macaque Hematopoiesis Highlights a Distinct Lineage Origin for Natural Killer Cells CELL STEM CELL Wu, C., Li, B., Lu, R., Koelle, S. J., Yang, Y., Jares, A., Krouse, A. E., Metzger, M., Liang, F., Lore, K., Wu, C. O., Donahue, R. E., Chen, I. S., Weissman, I., Dunbar, C. E. 2014; 14 (4): 486-499

  Abstract

  Analysis of hematopoietic stem cell function in nonhuman primates provides insights that are relevant for human biology and therapeutic strategies. In this study, we applied quantitative genetic barcoding to track the clonal output of transplanted autologous rhesus macaque hematopoietic stem and progenitor cells over a time period of up to 9.5 months. We found that unilineage short-term progenitors reconstituted myeloid and lymphoid lineages at 1 month but were supplanted over time by multilineage clones, initially myeloid restricted, then myeloid-B clones, and then stable myeloid-B-T multilineage, long-term repopulating clones. Surprisingly, reconstitution of the natural killer (NK) cell lineage, and particularly the major CD16(+)/CD56(-) peripheral blood NK compartment, showed limited clonal overlap with T, B, or myeloid lineages, and therefore appears to be ontologically distinct. Thus, in addition to providing insights into clonal behavior over time, our analysis suggests an unexpected paradigm for the relationship between NK cells and other hematopoietic lineages in primates.

  View details for DOI 10.1016/j.stem.2014.01.020

  View details for Web of Science ID 000334766400012

  View details for PubMedID 24702997

  View details for PubMedCentralID PMC3979461

• Abstract 140: identification, characterization, and prospective isolation of a fibroblast lineage contributing to dermal development, cutaneous scarring, radiation fibrosis, and cancer stroma. Plastic and reconstructive surgery Walmsley, G. G., Rinkevich, Y., Hu, M. S., McArdle, A., Maan, Z. N., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2014; 133 (3): 157-?

  View details for DOI 10.1097/01.prs.0000444968.20280.4d

  View details for PubMedID 25942251

• Abstract 161: identification of cell-intrinsic mechanisms and differentially regulated genetic pathways responsible for the age-related functional decline in aged skeletal stem cells. Plastic and reconstructive surgery McArdle, A., Chan, C., Seita, J., Senarath-Yapa, K., Hu, M., Walmsley, G. G., Zielins, E., Atashroo, D., Tevlin, R., Weissman, I., Longaker, M. T. 2014; 133 (3): 178-?

  View details for DOI 10.1097/01.prs.0000444990.75431.f1

  View details for PubMedID 25942271

• Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission. Proceedings of the National Academy of Sciences of the United States of America Corces-Zimmerman, M. R., Hong, W., Weissman, I. L., Medeiros, B. C., Majeti, R. 2014; 111 (7): 2548-2553

  Abstract

  Cancer is widely characterized by the sequential acquisition of genetic lesions in a single lineage of cells. Our previous studies have shown that, in acute myeloid leukemia (AML), mutation acquisition occurs in functionally normal hematopoietic stem cells (HSCs). These preleukemic HSCs harbor some, but not all, of the mutations found in the leukemic cells. We report here the identification of patterns of mutation acquisition in human AML. Our findings support a model in which mutations in "landscaping" genes, involved in global chromatin changes such as DNA methylation, histone modification, and chromatin looping, occur early in the evolution of AML, whereas mutations in "proliferative" genes occur late. Additionally, we analyze the persistence of preleukemic mutations in patients in remission and find CD34+ progenitor cells and various mature cells that harbor preleukemic mutations. These findings indicate that preleukemic HSCs can survive induction chemotherapy, identifying these cells as a reservoir for the reevolution of relapsed disease. Finally, through the study of several cases of relapsed AML, we demonstrate various evolutionary patterns for the generation of relapsed disease and show that some of these patterns are consistent with involvement of preleukemic HSCs. These findings provide key insights into the monitoring of minimal residual disease and the identification of therapeutic targets in human AML.

  View details for DOI 10.1073/pnas.1324297111

  View details for PubMedID 24550281

• Transcriptional activation of hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis through VEGF and S100A8 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ahn, G., Seita, J., Hong, B., Kim, Y., Bok, S., Lee, C., Kim, K. S., Lee, J. C., Leeper, N. J., Cooke, J. P., Kim, H. J., Kim, I. H., Weissman, I. L., Brown, J. M. 2014; 111 (7): 2698-2703

  Abstract

  Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.

  View details for DOI 10.1073/pnas.1320243111

  View details for Web of Science ID 000331396500062

  View details for PubMedID 24497508

  View details for PubMedCentralID PMC3932909

• Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell stem cell Loh, K. M., Ang, L. T., Zhang, J., Kumar, V., Ang, J., Auyeong, J. Q., Lee, K. L., Choo, S. H., Lim, C. Y., Nichane, M., Tan, J., Noghabi, M. S., Azzola, L., Ng, E. S., Durruthy-Durruthy, J., Sebastiano, V., Poellinger, L., Elefanty, A. G., Stanley, E. G., Chen, Q., Prabhakar, S., Weissman, I. L., Lim, B. 2014; 14 (2): 237-252

  Abstract

  Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.

  View details for DOI 10.1016/j.stem.2013.12.007

  View details for PubMedID 24412311

• Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips LAB ON A CHIP Earhart, C. M., Hughes, C. E., Gaster, R. S., Ooi, C. C., Wilson, R. J., Zhou, L. Y., Humke, E. W., Xu, L., Wong, D. J., Willingham, S. B., Schwartz, E. J., Weissman, I. L., Jeffrey, S. S., Neal, J. W., Rohatgi, R., Wakeleebe, H. A., Wang, S. X. 2014; 14 (1): 78-88

  View details for DOI 10.1039/c3lc50580d

  View details for Web of Science ID 000327669000008

• Discriminating cellular heterogeneity using microwell-based RNA cytometry. Nature communications Dimov, I. K., Lu, R., Lee, E. P., Seita, J., Sahoo, D., Park, S., Weissman, I. L., Lee, L. P. 2014; 5: 3451-?

  Abstract

  Discriminating cellular heterogeneity is important for understanding cellular physiology. However, it is limited by the technical difficulties of single-cell measurements. Here we develop a two-stage system to determine cellular heterogeneity. In the first stage, we perform multiplex single-cell RNA cytometry in a microwell array containing over 60,000 reaction chambers. In the second stage, we use the RNA cytometry data to determine cellular heterogeneity by providing a heterogeneity likelihood score (HLS). Moreover, we use Monte-Carlo simulation and RNA cytometry data to calculate the minimum number of cells required for detecting heterogeneity. We apply this system to characterize the RNA distributions of ageing-related genes in a highly purified mouse haematopoietic stem cell population. We identify genes that reveal novel heterogeneity of these cells. We also show that changes in expression of genes such as Birc6 during ageing can be attributed to the shift of relative portions of cells in the high-expressing subgroup versus low-expressing subgroup.

  View details for DOI 10.1038/ncomms4451

  View details for PubMedID 24667995

• Discriminating cellular heterogeneity using microwell-based RNA cytometry. Nature communications Dimov, I. K., Lu, R., Lee, E. P., Seita, J., Sahoo, D., Park, S., Weissman, I. L., Lee, L. P. 2014; 5: 3451-?

  View details for DOI 10.1038/ncomms4451

  View details for PubMedID 24667995

• Learning from host-defense peptides: cationic, amphipathic peptoids with potent anticancer activity. PloS one Huang, W., Seo, J., Willingham, S. B., Czyzewski, A. M., Gonzalgo, M. L., Weissman, I. L., Barron, A. E. 2014; 9 (2)

  Abstract

  Cationic, amphipathic host defense peptides represent a promising group of agents to be developed for anticancer applications. Poly-N-substituted glycines, or peptoids, are a class of biostable, peptidomimetic scaffold that can display a great diversity of side chains in highly tunable sequences via facile solid-phase synthesis. Herein, we present a library of anti-proliferative peptoids that mimics the cationic, amphipathic structural feature of the host defense peptides and explore the relationships between the structure, anticancer activity and selectivity of these peptoids. Several peptoids are found to be potent against a broad range of cancer cell lines at low-micromolar concentrations including cancer cells with multidrug resistance (MDR), causing cytotoxicity in a concentration-dependent manner. They can penetrate into cells, but their cytotoxicity primarily involves plasma membrane perturbations. Furthermore, peptoid 1, the most potent peptoid synthesized, significantly inhibited tumor growth in a human breast cancer xenotransplantation model without any noticeable acute adverse effects in mice. Taken together, our work provided important structural information for designing host defense peptides or their mimics for anticancer applications. Several cationic, amphipathic peptoids are very attractive for further development due to their high solubility, stability against protease degradation, their broad, potent cytotoxicity against cancer cells and their ability to overcome multidrug resistance.

  View details for DOI 10.1371/journal.pone.0090397

  View details for PubMedID 24587350

  View details for PubMedCentralID PMC3938723

• Osteoclast derivation from mouse bone marrow. Journal of visualized experiments : JoVE Tevlin, R., McArdle, A., Chan, C. K., Pluvinage, J., Walmsley, G. G., Wearda, T., Marecic, O., Hu, M. S., Paik, K. J., Senarath-Yapa, K., Atashroo, D. A., Zielins, E. R., Wan, D. C., Weissman, I. L., Longaker, M. T. 2014

  Abstract

  Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease. As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation. An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

  View details for DOI 10.3791/52056

  View details for PubMedID 25407120

• BLT-humanized C57BL/6 Rag2(-/-)gamma(-/-)(c)CD47(-/-) mice are resistant to GVHD and develop B- and T-cell immunity to HIV infection BLOOD Lavender, K. J., Pang, W. W., Messer, R. J., Duley, A. K., Race, B., Phillips, K., Scott, D., Peterson, K. E., Chan, C. K., Dittmer, U., Dudek, T., Allen, T. M., Weissman, I. L., Hasenkrug, K. J. 2013; 122 (25): 4013-4020

  Abstract

  The use of C57BL/6 Rag2(-/-)γc(-/-) mice as recipients for xenotransplantation with human immune systems (humanization) has been problematic because C57BL/6 SIRPα does not recognize human CD47, and such recognition is required to suppress macrophage-mediated phagocytosis of transplanted human hematopoietic stem cells (HSCs). We show that genetic inactivation of CD47 on the C57BL/6 Rag2(-/-)γc(-/-) background negates the requirement for CD47-signal recognition protein α (SIRPα) signaling and induces tolerance to transplanted human HSCs. These triple-knockout, bone marrow, liver, thymus (TKO-BLT) humanized mice develop organized lymphoid tissues including mesenteric lymph nodes, splenic follicles and gut-associated lymphoid tissue that demonstrate high levels of multilineage hematopoiesis. Importantly, these mice have an intact complement system and showed no signs of graft-versus-host disease (GVHD) out to 29 weeks after transplantation. Sustained, high-level HIV-1 infection was observed via either intrarectal or intraperitoneal inoculation. TKO-BLT mice exhibited hallmarks of human HIV infection including CD4(+) T-cell depletion, immune activation, and development of HIV-specific B- and T-cell responses. The lack of GVHD makes the TKO-BLT mouse a significantly improved model for long-term studies of pathogenesis, immune responses, therapeutics, and vaccines to human pathogens.

  View details for DOI 10.1182/blood-2013-06-506949

  View details for Web of Science ID 000329739100009

  View details for PubMedID 24021673

  View details for PubMedCentralID PMC3862274

• Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips. Lab on a chip Earhart, C. M., Hughes, C. E., Gaster, R. S., Ooi, C. C., Wilson, R. J., Zhou, L. Y., Humke, E. W., Xu, L., Wong, D. J., Willingham, S. B., Schwartz, E. J., Weissman, I. L., Jeffrey, S. S., Neal, J. W., Rohatgi, R., Wakelee, H. A., Wang, S. X. 2013; 14 (1): 78-88

  Abstract

  Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of "liquid biopsies" from cancer patients.

  View details for DOI 10.1039/c3lc50580d

  View details for PubMedID 23969419

• Parabiosis in Mice: A Detailed Protocol JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Kamran, P., Sereti, K., Zhao, P., Ali, S. R., Weissman, I. L., Ardehali, R. 2013

  Abstract

  Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 1864(1), the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival(2). In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

  View details for DOI 10.3791/50556

  View details for Web of Science ID 000209228800009

  View details for PubMedCentralID PMC3938334

• Improving macrophage responses to therapeutic antibodies by molecular engineering of SIRPα variants. Oncoimmunology Weiskopf, K., Ring, A. M., Schnorr, P. J., Volkmer, J. P., Volkmer, A. K., Weissman, I. L., Garcia, K. C. 2013; 2 (9): e25773

  Abstract

  CD47 transduces inhibitory signals through signal-regulatory protein α (SIRPα), a plasma membrane receptor expressed by macrophages. Many cancers upregulate CD47 to evade immunosurveillance. We have recently engineered SIRPα variants that potently antagonize CD47 for use as anticancer immunotherapeutics. These high-affinity SIRPα variants synergize with antineoplastic antibodies by lowering the threshold for macrophage-mediated destruction of malignant cells.

  View details for DOI 10.4161/onci.25773

  View details for PubMedID 24319639

  View details for PubMedCentralID PMC3850276

• Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nature medicine Lee, A. S., Tang, C., Rao, M. S., Weissman, I. L., Wu, J. C. 2013; 19 (8): 998-1004

  Abstract

  Human pluripotent stem cells (PSCs) are a leading candidate for cell-based therapies because of their capacity for unlimited self renewal and pluripotent differentiation. These advances have recently culminated in the first-in-human PSC clinical trials by Geron, Advanced Cell Technology and the Kobe Center for Developmental Biology for the treatment of spinal cord injury and macular degeneration. Despite their therapeutic promise, a crucial hurdle for the clinical implementation of human PSCs is their potential to form tumors in vivo. In this Perspective, we present an overview of the mechanisms underlying the tumorigenic risk of human PSC-based therapies and discuss current advances in addressing these challenges.

  View details for DOI 10.1038/nm.3267

  View details for PubMedID 23921754

  View details for PubMedCentralID PMC3967018

• Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, C. K., Lindau, P., Jiang, W., Chen, J. Y., Zhang, L. F., Chen, C., Seita, J., Sahoo, D., Kim, J., Lee, A., Park, S., Nag, D., Gong, Y., Kulkarni, S., Luppen, C. A., Theologis, A. A., Wan, D. C., DeBoer, A., Seo, E. Y., Vincent-Tompkins, J. D., Loh, K., Walmsley, G. G., Kraft, D. L., Wu, J. C., Longaker, M. T., Weissman, I. L. 2013; 110 (31): 12643-12648

  Abstract

  Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

  View details for DOI 10.1073/pnas.1310212110

  View details for PubMedID 23858471

• Identification of a colonial chordate histocompatibility gene. Science Voskoboynik, A., Newman, A. M., Corey, D. M., Sahoo, D., Pushkarev, D., Neff, N. F., Passarelli, B., Koh, W., Ishizuka, K. J., Palmeri, K. J., Dimov, I. K., Keasar, C., Fan, H. C., Mantalas, G. L., Sinha, R., Penland, L., Quake, S. R., Weissman, I. L. 2013; 341 (6144): 384-387

  Abstract

  Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

  View details for DOI 10.1126/science.1238036

  View details for PubMedID 23888037

• Engineered SIRPa variants as immunotherapeutic adjuvants to anticancer antibodies. Science Weiskopf, K., Ring, A. M., Ho, C. C., Volkmer, J., Levin, A. M., Volkmer, A. K., Ozkan, E., Fernhoff, N. B., van de Rijn, M., Weissman, I. L., Garcia, K. C. 2013; 341 (6141): 88-91

  Abstract

  CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction. Here, we modified the binding domain of human SIRPα, the receptor for CD47, for use as a CD47 antagonist. We engineered high-affinity SIRPα variants with approximately 50,000-fold increased affinity for human CD47 relative to wild-type SIRPα. As high-affinity SIRPα monomers, they potently antagonized CD47 on cancer cells but did not induce macrophage phagocytosis on their own. Instead, they exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosis in vitro and enhancing antitumor responses in vivo. This "one-two punch" directs immune responses against tumor cells while lowering the threshold for macrophage activation, thereby providing a universal method for augmenting the efficacy of therapeutic anticancer antibodies.

  View details for DOI 10.1126/science.1238856

  View details for PubMedID 23722425

• Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tseng, D., Volkmer, J., Willingham, S. B., Contreras-Trujillo, H., Fathman, J. W., Fernhoff, N. B., Seita, J., Inlay, M. A., Weiskopf, K., Miyanishi, M., Weissman, I. L. 2013; 110 (27): 11103-11108

  Abstract

  Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8(+))] but decreased priming of OT-II T cells (CD4(+)). The CD4(+) T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3(+)) regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8(+) T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.

  View details for DOI 10.1073/pnas.1305569110

  View details for Web of Science ID 000321978000057

  View details for PubMedID 23690610

• Use of a KIT-specific monoclonal antibody to bypass imatinib resistance in gastrointestinal stromal tumors. Oncoimmunology Edris, B., Willingham, S., Weiskopf, K., Volkmer, A. K., Volkmer, J. P., Mühlenberg, T., Weissman, I. L., van de Rijn, M. 2013; 2 (6): e24452

  Abstract

  Acquired resistance to imatinib is a significant problem for the clinical management of gastrointestinal stromal tumor (GIST) patients, and second-line small molecules have shown limited efficacy in this setting. We have recently demonstrated that a monoclonal antibody targeting KIT could potentially bypass imatinib resistance in preclinical models of GIST.

  View details for DOI 10.4161/onci.24452

  View details for PubMedID 23894705

  View details for PubMedCentralID PMC3716740

• Use of a KIT-specific monoclonal antibody to bypass imatinib resistance in gastrointestinal stromal tumors ONCOIMMUNOLOGY Edris, B., Willingham, S., Weiskopf, K., Volkmer, A. K., Volkmer, J., Muehlenberg, T., Weissman, I. L., van de Rijn, M. 2013; 2 (6)

  Abstract

  Acquired resistance to imatinib is a significant problem for the clinical management of gastrointestinal stromal tumor (GIST) patients, and second-line small molecules have shown limited efficacy in this setting. We have recently demonstrated that a monoclonal antibody targeting KIT could potentially bypass imatinib resistance in preclinical models of GIST.

  View details for DOI 10.4161/onci.24452

  View details for Web of Science ID 000322060900007

  View details for PubMedCentralID PMC3716740

• Brain Tumor Stem Cell Multipotency Correlates with Nanog Expression and Extent of Passaging in Human Glioblastoma Xenografts ONCOTARGET Higgins, D. M., Wang, R., Milligan, B., Schroeder, M., Carlson, B., Pokorny, J., Cheshier, S. H., Meyer, F. B., Weissman, I. L., Sarkaria, J. N., Henley, J. R. 2013; 4 (5): 792-801

  Abstract

  Glioblastoma multiforme (GBM) is the most common primary brain tumor, with a median survival of only 15 months. A subpopulation of cells, the brain tumor stem cells (BTSCs), may be responsible for the malignancy of this disease. Xenografts have proven to be a robust model of human BTSCs, but the effects of long-term passaging have yet to be determined. Here we present a study detailing changes in BTSC multipotency, invasive migration, and proliferation after serial passaging of human GBM xenografts. Immunocytochemistry and tumorsphere formation assays demonstrated the presence of BTSCs in both early generation (EG-BTSCs; less than 15 passages) and late generation (LG-BTSCs; more than 24 passages) xenografts. The EG-BTSCs upregulated expression of lineage markers for neurons and oligodendrocytes upon differentiation, indicating multipotency. In contrast, the LG-BTSCs were restricted to an astrocytic differentiation. Quantitative migration and proliferation assays showed that EG-BTSCs are more migratory and proliferative than LG-BTSCs. However, both populations respond similarly to the chemokine SDF-1 by increasing invasive migration. These differences between the EG- and LG-BTSCs were correlated with a significant decrease in nanog expression as determined by qRT-PCR. Mice implanted intracranially with EG-BTSCs showed shorter survival when compared to LG-BTSCs. Moreover, differentiation prior to implantation of EG-BTSCs, but not LG-BTSCs, led to increased survival. Thus, nanog may identify multipotent BTSCs. Furthermore, limited passaging of xenografts preserves these multipotent BTSCs, which may be an essential underlying feature of GBM lethality.

  View details for Web of Science ID 000322580000015

  View details for PubMedID 23801022

• Azacitidine fails to eradicate leukemic stem/progenitor cell populations in patients with acute myeloid leukemia and myelodysplasia LEUKEMIA Craddock, C., Quek, L., Goardon, N., Freeman, S., Siddique, S., Raghavan, M., Aztberger, A., Schuh, A., Grimwade, D., IVEY, A., Virgo, P., Hills, R., McSkeane, T., Arrazi, J., Knapper, S., Brookes, C., Davies, B., Price, A., Wall, K., Griffiths, M., Cavenagh, J., Majeti, R., Weissman, I., Burnett, A., Vyas, P. 2013; 27 (5): 1028-1036

  Abstract

  Epigenetic therapies demonstrate significant clinical activity in acute myeloid leukemia (AML) and myelodysplasia (MDS) and constitute an important new class of therapeutic agents. However hematological responses are not durable and disease relapse appears inevitable. Experimentally, leukemic stem/progenitor cells (LSC) propagate disease in animal models of AML and it has been postulated that their relative chemo-resistance contributes to disease relapse. We serially measured LSC numbers in patients with high-risk AML and MDS treated with 5'-azacitidine and sodium valproate (VAL-AZA). Fifteen out of seventy-nine patients achieved a complete remission (CR) or complete remission with incomplete blood count recovery (CRi) with VAL-AZA therapy. There was no significant reduction in the size of the LSC-containing population in non-responders. While the LSC-containing population was substantially reduced in all patients achieving a CR/CRi it was never eradicated and expansion of this population antedated morphological relapse. Similar studies were performed in seven patients with newly diagnosed AML treated with induction chemotherapy. Eradication of the LSC-containing population was observed in three patients all of whom achieved a durable CR in contrast to patients with resistant disease where LSC persistence was observed. LSC quantitation provides a novel biomarker of disease response and relapse in patients with AML treated with epigenetic therapies. New drugs that target this cellular population in vivo are required.

  View details for DOI 10.1038/leu.2012.312

  View details for Web of Science ID 000318698300005

  View details for PubMedID 23223186

• Anti-KIT monoclonal antibody inhibits imatinib-resistant gastrointestinal stromal tumor growth PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Edris, B., Willingham, S. B., Weiskopf, K., Volkmer, A. K., Volkmer, J., Muehlenberg, T., Montgomery, K. D., Contreras-Trujillo, H., Czechowicz, A., Fletcher, J. A., West, R. B., Weissman, I. L., van de Rijn, M. 2013; 110 (9): 3501-3506

  Abstract

  Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract and arises from the interstitial cells of Cajal. It is characterized by expression of the receptor tyrosine kinase CD117 (KIT). In 70-80% of GIST cases, oncogenic mutations in KIT are present, leading to constitutive activation of the receptor, which drives the proliferation of these tumors. Treatment of GIST with imatinib, a small-molecule tyrosine kinase inhibitor, inhibits KIT-mediated signaling and initially results in disease control in 70-85% of patients with KIT-positive GIST. However, the vast majority of patients eventually develop resistance to imatinib treatment, leading to disease progression and posing a significant challenge in the clinical management of these tumors. Here, we show that an anti-KIT monoclonal antibody (mAb), SR1, is able to slow the growth of three human GIST cell lines in vitro. Importantly, these reductions in cell growth were equivalent between imatinib-resistant and imatinib-sensitive GIST cell lines. Treatment of GIST cell lines with SR1 reduces cell-surface KIT expression, suggesting that mAb-induced KIT down-regulation may be a mechanism by which SR1 inhibits GIST growth. Furthermore, we also show that SR1 treatment enhances phagocytosis of GIST cells by macrophages, indicating that treatment with SR1 may enhance immune cell-mediated tumor clearance. Finally, using two xenotransplantation models of imatinib-sensitive and imatinib-resistant GIST, we demonstrate that SR1 is able to strongly inhibit tumor growth in vivo. These results suggest that treatment with mAbs targeting KIT may represent an alternative, or complementary, approach for treating GIST.

  View details for DOI 10.1073/pnas.1222893110

  View details for PubMedID 23382202

• Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ardehali, R., Ali, S. R., Inlay, M. A., Abilez, O. J., Chen, M. Q., Blauwkamp, T. A., Yazawa, M., Gong, Y., Nusse, R., Drukker, M., Weissman, I. L. 2013; 110 (9): 3405-3410

  Abstract

  A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.

  View details for DOI 10.1073/pnas.1220832110

  View details for Web of Science ID 000315841900046

  View details for PubMedID 23391730

  View details for PubMedCentralID PMC3587189

• Hematopoietic stem cell and progenitor cell mechanisms in myelodysplastic syndromes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pang, W. W., Pluvinage, J. V., Price, E. A., Sridhar, K., Arber, D. A., Greenberg, P. L., Schrier, S. L., Park, C. Y., Weissman, I. L. 2013; 110 (8): 3011-3016

  Abstract

  Myelodysplastic syndromes (MDS) are a group of disorders characterized by variable cytopenias and ineffective hematopoiesis. Hematopoietic stem cells (HSCs) and myeloid progenitors in MDS have not been extensively characterized. We transplanted purified human HSCs from MDS samples into immunodeficient mice and show that HSCs are the disease-initiating cells in MDS. We identify a recurrent loss of granulocyte-macrophage progenitors (GMPs) in the bone marrow of low risk MDS patients that can distinguish low risk MDS from clinical mimics, thus providing a simple diagnostic tool. The loss of GMPs is likely due to increased apoptosis and increased phagocytosis, the latter due to the up-regulation of cell surface calreticulin, a prophagocytic marker. Blocking calreticulin on low risk MDS myeloid progenitors rescues them from phagocytosis in vitro. However, in the high-risk refractory anemia with excess blasts (RAEB) stages of MDS, the GMP population is increased in frequency compared with normal, and myeloid progenitors evade phagocytosis due to up-regulation of CD47, an antiphagocytic marker. Blocking CD47 leads to the selective phagocytosis of this population. We propose that MDS HSCs compete with normal HSCs in the patients by increasing their frequency at the expense of normal hematopoiesis, that the loss of MDS myeloid progenitors by programmed cell death and programmed cell removal are, in part, responsible for the cytopenias, and that up-regulation of the "don't eat me" signal CD47 on MDS myeloid progenitors is an important transition step leading from low risk MDS to high risk MDS and, possibly, to acute myeloid leukemia.

  View details for DOI 10.1073/pnas.1222861110

  View details for PubMedID 23388639

• Repeated, Long-Term Cycling of Putative Stem Cells between Niches in a Basal Chordate DEVELOPMENTAL CELL Rinkevich, Y., Voskoboynik, A., Rosner, A., Rabinowitz, C., Paz, G., Oren, M., Douek, J., Alfassi, G., Moiseeva, E., Ishizuka, K. J., Palmeri, K. J., Weissman, I. L., Rinkevich, B. 2013; 24 (1): 76-88

  Abstract

  The mechanisms that sustain stem cells are fundamental to tissue maintenance. Here, we identify "cell islands" (CIs) as a niche for putative germ and somatic stem cells in Botryllus schlosseri, a colonial chordate that undergoes weekly cycles of death and regeneration. Cells within CIs express markers associated with germ and somatic stem cells and gene products that implicate CIs as signaling centers for stem cells. Transplantation of CIs induced long-term germline and somatic chimerism, demonstrating self-renewal and pluripotency of CI cells. Cell labeling and in vivo time-lapse imaging of CI cells reveal waves of migrations from degrading CIs into developing buds, contributing to soma and germline development. Knockdown of cadherin, which is highly expressed within CIs, elicited the migration of CI cells to circulation. Piwi knockdown resulted in regeneration arrest. We suggest that repeated trafficking of stem cells allows them to escape constraints imposed by the niche, enabling self-preservation throughout life.

  View details for DOI 10.1016/j.devcel.2012.11.010

  View details for Web of Science ID 000316305200007

  View details for PubMedID 23260626

• Immunogenicity of in vitro maintained and matured populations: potential barriers to engraftment of human pluripotent stem cell derivatives. Methods in molecular biology (Clifton, N.J.) Tang, C., Weissman, I. L., Drukker, M. 2013; 1029: 17-31

  Abstract

  The potential to develop into any cell type makes human pluripotent stem cells (hPSCs) one of the most promising sources for regenerative treatments. Hurdles to their clinical applications include (1) formation of heterogeneously differentiated cultures, (2) the risk of teratoma formation from residual undifferentiated cells, and (3) immune rejection of engrafted cells. The recent production of human isogenic (genetically identical) induced PSCs (hiPSCs) has been proposed as a "solution" to the histocompatibility barrier. In theory, differentiated cells derived from patient-specific hiPSC lines should be histocompatible to their donor/recipient. However, propagation, maintenance, and non-physiologic differentiation of hPSCs in vitro may produce other, likely less powerful, immune responses. In light of recent progress towards the clinical application of hPSCs, this review focuses on two antigen presentation phenomena that may lead to rejection of isogenic hPSC derivates: namely, the expression of aberrant antigens as a result of long-term in vitro maintenance conditions or incomplete somatic cell reprogramming, and the unbalanced presentation of receptors and ligands involved in immune recognition due to accelerated differentiation. Finally, we discuss immunosuppressive approaches that could potentially address these immunological concerns.

  View details for DOI 10.1007/978-1-62703-478-4_2

  View details for PubMedID 23756939

• Do pluripotent stem cells exist in adult mice as very small embryonic stem cells? Stem cell reports Miyanishi, M., Mori, Y., Seita, J., Chen, J. Y., Karten, S., Chan, C. K., Nakauchi, H., Weissman, I. L. 2013; 1 (2): 198-208

  Abstract

  Very small embryonic-like stem cells (VSELs) isolated from bone marrow (BM) have been reported to be pluripotent. Given their nonembryonic source, they could replace blastocyst-derived embryonic stem cells in research and medicine. However, their multiple-germ-layer potential has been incompletely studied. Here, we show that we cannot find VSELs in mouse BM with any of the reported stem cell potentials, specifically for hematopoiesis. We found that: (1) most events within the "VSEL" flow-cytometry gate had little DNA and the cells corresponding to these events (2) could not form spheres, (3) did not express Oct4, and (4) could not differentiate into blood cells. These results provide a failure to confirm the existence of pluripotent VSELs.

  View details for DOI 10.1016/j.stemcr.2013.07.001

  View details for PubMedID 24052953

• The genome sequence of the colonial chordate, Botryllus schlosseri. eLife Voskoboynik, A., Neff, N. F., Sahoo, D., Newman, A. M., Pushkarev, D., Koh, W., Passarelli, B., Fan, H. C., Mantalas, G. L., Palmeri, K. J., Ishizuka, K. J., Gissi, C., Griggio, F., Ben-Shlomo, R., Corey, D. M., Penland, L., White, R. A., Weissman, I. L., Quake, S. R. 2013; 2

  Abstract

  Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI:http://dx.doi.org/10.7554/eLife.00569.001.

  View details for DOI 10.7554/eLife.00569

  View details for PubMedID 23840927

  View details for PubMedCentralID PMC3699833

• Do pluripotent stem cells exist in adult mice as very small embryonic stem cells? Stem cell reports Miyanishi, M., Mori, Y., Seita, J., Chen, J. Y., Karten, S., Chan, C. K., Nakauchi, H., Weissman, I. L. 2013; 1 (2): 198-208

  Abstract

  Very small embryonic-like stem cells (VSELs) isolated from bone marrow (BM) have been reported to be pluripotent. Given their nonembryonic source, they could replace blastocyst-derived embryonic stem cells in research and medicine. However, their multiple-germ-layer potential has been incompletely studied. Here, we show that we cannot find VSELs in mouse BM with any of the reported stem cell potentials, specifically for hematopoiesis. We found that: (1) most events within the "VSEL" flow-cytometry gate had little DNA and the cells corresponding to these events (2) could not form spheres, (3) did not express Oct4, and (4) could not differentiate into blood cells. These results provide a failure to confirm the existence of pluripotent VSELs.

  View details for DOI 10.1016/j.stemcr.2013.07.001

  View details for PubMedID 24052953

  View details for PubMedCentralID PMC3757755

• E. Donnall Thomas (1920-2012) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Blume, K. G., Weissman, I. L. 2012; 109 (51): 20777-20778

  View details for DOI 10.1073/pnas.1218913109

  View details for Web of Science ID 000313123700017

  View details for PubMedID 23197829

  View details for PubMedCentralID PMC3529056

• In vivo directed differentiation of pluripotent stem cells for skeletal regeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Levi, B., Hyun, J. S., Montoro, D. T., Lo, D. D., Chan, C. K., Hu, S., Sun, N., Lee, M., Grova, M., Connolly, A. J., Wu, J. C., Gurtner, G. C., Weissman, I. L., Wan, D. C., Longaker, M. T. 2012; 109 (50): 20379-20384

  Abstract

  Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

  View details for DOI 10.1073/pnas.1218052109

  View details for PubMedID 23169671

• Identification and prospective isolation of a mesothelial precursor lineage giving rise to smooth muscle cells and fibroblasts for mammalian internal organs, and their vasculature NATURE CELL BIOLOGY Rinkevich, Y., Mori, T., Sahoo, D., Xu, P., Bermingham, J. R., Weissman, I. L. 2012; 14 (12): 1251-?

  Abstract

  Fibroblasts and smooth muscle cells (FSMCs) are principal cell types of connective and adventitial tissues that participate in the development, physiology and pathology of internal organs, with incompletely defined cellular origins. Here, we identify and prospectively isolate from the mesothelium a mouse cell lineage that is committed to FSMCs. The mesothelium is an epithelial monolayer covering the vertebrate thoracic and abdominal cavities and internal organs. Time-lapse imaging and transplantation experiments reveal robust generation of FSMCs from the mesothelium. By targeting mesothelin (MSLN), a surface marker expressed on mesothelial cells, we identify and isolate precursors capable of clonally generating FSMCs. Using a genetic lineage tracing approach, we show that embryonic and adult mesothelium represents a common lineage to trunk FSMCs, and trunk vasculature, with minimal contributions from neural crest, or circulating cells. The isolation of FSMC precursors enables the examination of multiple aspects of smooth muscle and fibroblast biology as well as the prospective isolation of these precursors for potential regenerative medicine purposes.

  View details for DOI 10.1038/ncb2610

  View details for Web of Science ID 000311890300007

  View details for PubMedID 23143399

• CD19(-)CD45(low/-)CD38(high)/CD138(+) plasma cells enrich for human tumorigenic myeloma cells LEUKEMIA Kim, D., Park, C. Y., Medeiros, B. C., Weissman, I. L. 2012; 26 (12): 2530-2537

  Abstract

  Multiple myeloma is a hematological neoplasm characterized by the accumulation of clonal plasma cells in the bone marrow. Its frequent relapse following achievement of clinical remissions implicates the existence of therapy-resistant myeloma-initiating cells. To date, results on the identity of myeloma-initiating cells have differed. Here, we prospectively identified a myeloma-initiating population by fractionating and transplanting patient bone marrow cells into human bone-bearing immunocompromised mice. Xenotransplantation of fractionated CD138(+)/CD38(high) cells from 40% of patients (8/20) led to a repopulation of CD19(+)CD38(low) or CD138(+)CD38(+) B-lineage cells in human bone grafts; and these grafts were clonally derived from patient myeloma cells. Meanwhile, CD19(+)CD38(low) xenografts were detected in human bone-bearing mice transplanted with CD19(+)CD38(low/-) B cells from 8 of 22 samples but were not clonally related to patient myeloma cells. Further fractionation and xenotransplantation of CD138(+)CD38(high) cells demonstrated that (CD45(low/-) or CD19(-)) CD38(high)/CD138(+) plasma cells, but not (CD45(high) or CD19(+)) CD38(high)/CD138(+) plasmablasts enrich for myeloma-initiating cells. Quantitative reverse transcription-PCR of two serially transplantable xenografts, which were CD19(-)CD138(+), revealed that they were Pax5 (a B-cell-specific transactivator)-negative. These results suggest that CD19(-)CD45(low/-) fully differentiated plasma cells enrich for long-lived and tumor-initiating cells whereas B cells or plasmablasts do not.

  View details for DOI 10.1038/leu.2012.140

  View details for Web of Science ID 000312186000012

  View details for PubMedID 22733078

• Anti-CD47 antibodies promote phagocytosis and inhibit the growth of human myeloma cells LEUKEMIA Kim, D., Wang, J., Willingham, S. B., Martin, R., Wernig, G., Weissman, I. L. 2012; 26 (12): 2538-2545

  Abstract

  Multiple myeloma is a plasma cell neoplasm residing in bone marrow. Despite advances in myeloma therapies, novel therapies are required to improve patient outcomes. CD47 is highly expressed on myeloma cells and a potential therapeutic candidate for myeloma therapies. Flow cytometric analysis of patient bone marrow cells revealed that myeloma cells overexpress CD47 when compared with non-myeloma cells in 73% of patients (27/37). CD47 expression protects cells from phagocytosis by transmitting an inhibitory signal to macrophages. Here we show that blocking CD47 with an anti-CD47 monoclonal antibody increased phagocytosis of myeloma cells in vitro. In xenotransplantation models, anti-CD47 antibodies inhibited the growth of RPMI 8226 myeloma cells and led to tumor regression (42/57 mice), implicating the eradication of myeloma-initiating cells. Moreover, anti-CD47 antibodies retarded the growth of patient myeloma cells and alleviated bone resorption in human bone-bearing mice. Irradiation of mice before myeloma cell xenotransplantation abolished the therapeutic efficacy of anti-CD47 antibodies delivered 2 weeks after radiation, and coincided with a reduction of myelomonocytic cells in spleen, bone marrow and liver. These results are consistent with the hypothesis that anti-CD47 blocking antibodies inhibit myeloma growth, in part, by increasing phagocytosis of myeloma cells.

  View details for DOI 10.1038/leu.2012.141

  View details for Web of Science ID 000312186000013

  View details for PubMedID 22648449

• Clonal Level Lineage Commitment of Mouse Hematopoietic Stem Cells in Vivo 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Lu, R., Czechowicz, A., Seita, J., Weissman, I. L. AMER SOC HEMATOLOGY. 2012

  View details for Web of Science ID 000313838900137

• Reply to Soto-Pantoja et al. and Zhao et al.: Targeting CD47 on human solid tumors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Willingham, S. B., Volkmer, J., Weiskopf, K., Ring, A. M., Weissman, I. L. 2012; 109 (42): E2844-E2845

  View details for DOI 10.1073/pnas.1209743109

  View details for Web of Science ID 000310515800003

• Human Neural Stem Cells Induce Functional Myelination in Mice with Severe Dysmyelination SCIENCE TRANSLATIONAL MEDICINE Uchida, N., Chen, K., Dohse, M., Hansen, K. D., Dean, J., Buser, J. R., Riddle, A., Beardsley, D. J., Wan, Y., Gong, X., Thuan Nguyen, T., Cummings, B. J., Anderson, A. J., Tamaki, S. J., Tsukamoto, A., Weissman, I. L., Matsumoto, S. G., Sherman, L. S., Kroenke, C. D., Back, S. A. 2012; 4 (155)

  Abstract

  Shiverer-immunodeficient (Shi-id) mice demonstrate defective myelination in the central nervous system (CNS) and significant ataxia by 2 to 3 weeks of life. Expanded, banked human neural stem cells (HuCNS-SCs) were transplanted into three sites in the brains of neonatal or juvenile Shi-id mice, which were asymptomatic or showed advanced hypomyelination, respectively. In both groups of mice, HuCNS-SCs engrafted and underwent preferential differentiation into oligodendrocytes. These oligodendrocytes generated compact myelin with normalized nodal organization, ultrastructure, and axon conduction velocities. Myelination was equivalent in neonatal and juvenile mice by quantitative histopathology and high-field ex vivo magnetic resonance imaging, which, through fractional anisotropy, revealed CNS myelination 5 to 7 weeks after HuCNS-SC transplantation. Transplanted HuCNS-SCs generated functional myelin in the CNS, even in animals with severe symptomatic hypomyelination, suggesting that this strategy may be useful for treating dysmyelinating diseases.

  View details for DOI 10.1126/scitranslmed.3004371

  View details for Web of Science ID 000309966800003

  View details for PubMedID 23052293

• Remodeling of Endogenous Mammary Epithelium by Breast Cancer Stem Cells STEM CELLS Parashurama, N., Lobo, N. A., Ito, K., Mosley, A. R., Habte, F. G., Zabala, M., Smith, B. R., Lam, J., Weissman, I. L., Clarke, M. F., Gambhir, S. S. 2012; 30 (10): 2114-2127

  Abstract

  Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC.

  View details for DOI 10.1002/stem.1205

  View details for Web of Science ID 000308928300005

  View details for PubMedID 22899386

• Flipping the script on macrophages in leiomyosarcoma. Oncoimmunology Edris, B., Weiskopf, K., Weissman, I. L., van de Rijn, M. 2012; 1 (7): 1202-1204

  Abstract

  Macrophages promote the growth of leiomyosarcoma (LMS), a malignant soft-tissue tumor. CD47 on tumor cells binds to the macrophagic receptor signal regulatory protein α (SIRPα) and prevents phagocytosis. We showed that anti-CD47 monoclonal antibodies (mAbs) allow macrophages to engulf LMS cells and prevent tumor growth and metastases. Therefore, anti-CD47 mAbs represent a promising targeted immunotherapy for LMS.

  View details for DOI 10.4161/onci.20799

  View details for PubMedID 23170280

  View details for PubMedCentralID PMC3494646

• The road to purified hematopoietic stem cell transplants is paved with antibodies. Current opinion in immunology Logan, A. C., Weissman, I. L., Shizuru, J. A. 2012; 24 (5): 640-648

  Abstract

  Hematopoietic progenitor cell replacement therapy remains a surprisingly unrefined process. In general, unmanipulated bone marrow or mobilized peripheral blood (MPB) grafts which carry potentially harmful passenger cells are administered after treating recipients with high-dose chemotherapy and/or radiotherapy to eradicate malignant disease, eliminate immunologic barriers to allogeneic cell engraftment, and to 'make space' for rare donor stem cells within the stem cell niche. The sequalae of such treatments are substantial, including direct organ toxicity and nonspecific inflammation that contribute to the development of graft-versus-host disease (GVHD) and poor immune reconstitution. Passenger tumor cells that contaminate autologous hematopoietic grafts may contribute to relapse post-transplant. Use of antibodies to rid grafts of unwanted cell populations, and to eliminate or minimize the need for nonspecifically cytotoxic therapies used to condition transplant recipients, will dramatically improve the safety profile of allogeneic and gene-modified autologous hematopoietic stem cell therapies.

  View details for DOI 10.1016/j.coi.2012.08.002

  View details for PubMedID 22939368

• Flipping the script on macrophages in leiomyosarcoma ONCOIMMUNOLOGY Edris, B., Weiskopf, K., Weissman, I. L., van de Rijn, M. 2012; 1 (7): 1202-1204

  Abstract

  Macrophages promote the growth of leiomyosarcoma (LMS), a malignant soft-tissue tumor. CD47 on tumor cells binds to the macrophagic receptor signal regulatory protein α (SIRPα) and prevents phagocytosis. We showed that anti-CD47 monoclonal antibodies (mAbs) allow macrophages to engulf LMS cells and prevent tumor growth and metastases. Therefore, anti-CD47 mAbs represent a promising targeted immunotherapy for LMS.

  View details for DOI 10.4161/onci.20799

  View details for Web of Science ID 000316279900033

  View details for PubMedCentralID PMC3494646

• Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors NATURE COMMUNICATIONS Blauwkamp, T. A., Nigam, S., Ardehali, R., Weissman, I. L., Nusse, R. 2012; 3

  Abstract

  The pluripotent nature of human embryonic stem cells (hESCs) makes them convenient for deriving therapeutically relevant cells. Here we show using Wnt reporter hESC lines that the cells are heterogeneous with respect to endogenous Wnt signalling activity. Moreover, the level of Wnt signalling activity in individual cells correlates with differences in clonogenic potential and lineage-specific differentiation propensity. The addition of Wnt protein or, conversely, a small-molecule Wnt inhibitor (IWP2) reduces heterogeneity, allowing stable expansion of Wnt(high) or Wnt(low) hESC populations, respectively. On differentiation, the Wnt(high) hESCs predominantly form endodermal and cardiac cells, whereas the Wnt(low) hESCs generate primarily neuroectodermal cells. Thus, heterogeneity with respect to endogenous Wnt signalling underlies much of the inefficiency in directing hESCs towards specific cell types. The relatively uniform differentiation potential of the Wnt(high) and Wnt(low) hESCs leads to faster and more efficient derivation of targeted cell types from these populations.

  View details for DOI 10.1038/ncomms2064

  View details for Web of Science ID 000309338100037

  View details for PubMedID 22990866

  View details for PubMedCentralID PMC3657997

• Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia SCIENCE TRANSLATIONAL MEDICINE Jan, M., Snyder, T. M., Corces-Zimmerman, M. R., Vyas, P., Weissman, I. L., Quake, S. R., Majeti, R. 2012; 4 (149)

  Abstract

  Given that most bone marrow cells are short-lived, the accumulation of multiple leukemogenic mutations in a single clonal lineage has been difficult to explain. We propose that serial acquisition of mutations occurs in self-renewing hematopoietic stem cells (HSCs). We investigated this model through genomic analysis of HSCs from six patients with de novo acute myeloid leukemia (AML). Using exome sequencing, we identified mutations present in individual AML patients harboring the FLT3-ITD (internal tandem duplication) mutation. We then screened the residual HSCs and detected some of these mutations including mutations in the NPM1, TET2, and SMC1A genes. Finally, through single-cell analysis, we determined that a clonal progression of multiple mutations occurred in the HSCs of some AML patients. These preleukemic HSCs suggest the clonal evolution of AML genomes from founder mutations, revealing a potential mechanism contributing to relapse. Such preleukemic HSCs may constitute a cellular reservoir that should be targeted therapeutically for more durable remissions.

  View details for DOI 10.1126/scitranslmed.3004315

  View details for Web of Science ID 000308491600005

  View details for PubMedID 22932223

• Cardiomyocytes Undergo Division Postnatally to Generate New Cardiomyocytes in Mouse Models of Aging and Cardiac Injury Basic Cardiovascular Sciences Scientific Session Ali, S. R., Saadat, L. S., Hippenmeyer, S., Luo, L., Weissman, I. L., Ardehali, R. LIPPINCOTT WILLIAMS & WILKINS. 2012

  View details for Web of Science ID 000312506400043

• Gene Expression Commons: An Open Platform for Absolute Gene Expression Profiling PLOS ONE Seita, J., Sahoo, D., Rossi, D. J., Bhattacharya, D., Serwold, T., Inlay, M. A., Ehrlich, L. I., Fathman, J. W., Dill, D. L., Weissman, I. L. 2012; 7 (7)

  Abstract

  Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000) of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/) which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  View details for DOI 10.1371/journal.pone.0040321

  View details for Web of Science ID 000306548900020

  View details for PubMedID 22815738

  View details for PubMedCentralID PMC3399844

• Janus-like opposing roles of CD47 in autoimmune brain inflammation in humans and mice JOURNAL OF EXPERIMENTAL MEDICINE Han, M. H., Lundgren, D. H., Jaiswal, S., Chao, M., Graham, K. L., Garris, C. S., Axtell, R. C., Ho, P. P., Lock, C. B., Woodard, J. I., Brownell, S. E., Zoudilova, M., Hunt, J. F., Baranzini, S. E., Butcher, E. C., Raine, C. S., Sobel, R. A., Han, D. K., Weissman, I., Steinman, L. 2012; 209 (7): 1325-1334

  Abstract

  Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

  View details for DOI 10.1084/jem.20101974

  View details for Web of Science ID 000306174300008

  View details for PubMedID 22734047

  View details for PubMedCentralID PMC3405500

• Stem Cell Therapies Could Change Medicine ... If They Get the Chance CELL STEM CELL Weissman, I. 2012; 10 (6): 663-665

  Abstract

  Stem cell therapies have the potential to revolutionize the way we practice medicine. However, in the current climate several barriers and false assumptions stand in the way of achieving that goal.

  View details for DOI 10.1016/j.stem.2012.05.014

  View details for Web of Science ID 000305768400011

  View details for PubMedID 22704505

• Cyclin-A1 represents a new immunogenic targetable antigen expressed in acute myeloid leukemia stem cells with characteristics of a cancer-testis antigen BLOOD Ochsenreither, S., Majeti, R., Schmitt, T., Stirewalt, D., Keilholz, U., Loeb, K. R., Wood, B., Choi, Y. E., Bleakley, M., Warren, E. H., Hudecek, M., Akatsuka, Y., Weissman, I. L., Greenberg, P. D. 2012; 119 (23): 5492-5501

  Abstract

  Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1-specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell-based therapy approaches.

  View details for DOI 10.1182/blood-2011-07-365890

  View details for Web of Science ID 000307391400023

  View details for PubMedID 22529286

  View details for PubMedCentralID PMC3369684

• Isolation of primitive endoderm, mesoderm, vascular endothelial and trophoblast progenitors from human pluripotent stem cells NATURE BIOTECHNOLOGY Drukker, M., Tang, C., Ardehali, R., Rinkevich, Y., Seita, J., Lee, A. S., Mosley, A. R., Weissman, I. L., Soen, Y. 2012; 30 (6): 531-?

  Abstract

  To identify early populations of committed progenitors derived from human embryonic stem cells (hESCs), we screened self-renewing, BMP4-treated and retinoic acid-treated cultures with >400 antibodies recognizing cell-surface antigens. Sorting of >30 subpopulations followed by transcriptional analysis of developmental genes identified four distinct candidate progenitor groups. Subsets detected in self-renewing cultures, including CXCR4(+) cells, expressed primitive endoderm genes. Expression of Cxcr4 in primitive endoderm was confirmed in visceral endoderm of mouse embryos. BMP4-induced progenitors exhibited gene signatures of mesoderm, trophoblast and vascular endothelium, suggesting correspondence to gastrulation-stage primitive streak, chorion and allantois precursors, respectively. Functional studies in vitro and in vivo confirmed that ROR2(+) cells produce mesoderm progeny, APA(+) cells generate syncytiotrophoblasts and CD87(+) cells give rise to vasculature. The same progenitor classes emerged during the differentiation of human induced pluripotent stem cells (hiPSCs). These markers and progenitors provide tools for purifying human tissue-regenerating progenitors and for studying the commitment of pluripotent stem cells to lineage progenitors.

  View details for DOI 10.1038/nbt.2239

  View details for Web of Science ID 000305158600023

  View details for PubMedID 22634564

  View details for PubMedCentralID PMC3672406

• Cyclin-A1 expression in acute myeloid leukemia stem cells and its representation as an immunogenic antigen that can be targeted by cytotoxic T cells. 48th Annual Meeting of the American-Society-of-Clinical-Oncology (ASCO) Ochsenreither, S., Majeti, R., Loeb, K., Stirewalt, D. L., Keilholz, U., Weissman, I. L., Greenberg, P. D. AMER SOC CLINICAL ONCOLOGY. 2012

  View details for Web of Science ID 000318009801719

• Mechanisms of targeting CD47-SIRP alpha in hematologic malignancies Response BLOOD Chao, M. P., Majeti, R., Weissman, I. 2012; 119 (18): 4334-4335

  View details for DOI 10.1182/blood-2012-02-411249

  View details for Web of Science ID 000305284600032

• The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Willingham, S. B., Volkmer, J., Gentles, A. J., Sahoo, D., Dalerba, P., Mitra, S. S., Wang, J., Contreras-Trujillo, H., Martin, R., Cohen, J. D., Lovelace, P., Scheeren, F. A., Chao, M. P., Weiskopf, K., Tang, C., Volkmer, A. K., Naik, T. J., Storm, T. A., Mosley, A. R., Edris, B., Schmid, S. M., Sun, C. K., Chua, M., Murillo, O., Rajendran, P., Cha, A. C., Chin, R. K., Kim, D., Adorno, M., Raveh, T., Tseng, D., Jaiswal, S., Enger, P. O., Steinberg, G. K., Li, G., So, S. K., Majeti, R., Harsh, G. R., van de Rijn, M., Teng, N. N., Sunwoo, J. B., Alizadeh, A. A., Clarke, M. F., Weissman, I. L. 2012; 109 (17): 6662-6667

  Abstract

  CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

  View details for DOI 10.1073/pnas.1121623109

  View details for PubMedID 22451913

• Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Edris, B., Weiskopf, K., Volkmer, A. K., Volkmer, J., Willingham, S. B., Contreras-Trujillo, H., Liu, J., Majeti, R., West, R. B., Fletcher, J. A., Beck, A. H., Weissman, I. L., van de Rijn, M. 2012; 109 (17): 6656-6661

  Abstract

  Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

  View details for DOI 10.1073/pnas.1121629109

  View details for PubMedID 22451919

• Effect of nucleophosmin1 haploinsufficiency on hematopoietic stem cells LEUKEMIA Raval, A., Kusler, B., Pang, W. W., Weissman, I. L., Mitchell, B. S., Park, C. Y. 2012; 26 (4): 853-855

  View details for DOI 10.1038/leu.2011.270

  View details for Web of Science ID 000302788300040

  View details for PubMedID 21979879

• Regenerative Therapy after Myocardial Infarction: Role of mtDNA in SCNT Derived Embryonic Stem Cells 32nd Annual Meeting and Scientific Sessions of the International-Society-for-Heart-and-Lung-Transplantation / Meeting of the ISHLT Academy - Core Competencies in Mechanical Circulatory Support Hua, X., Deuse, T., Velden, J., Kirak, O., Jaenisch, R., Weissman, I., Ricklefs, F., Reichenspurner, H., Robbins, R. C., Schrepfer, S. ELSEVIER SCIENCE INC. 2012: S117–S117

  View details for Web of Science ID 000302207900330

• The CD47-SIRP alpha pathway in cancer immune evasion and potential therapeutic implications CURRENT OPINION IN IMMUNOLOGY Chao, M. P., Weissman, I. L., Majeti, R. 2012; 24 (2): 225-232

  Abstract

  Multiple lines of investigation have demonstrated that the immune system plays an important role in preventing tumor initiation and controlling tumor growth. Accordingly, many cancers have evolved diverse mechanisms to evade such monitoring. While multiple immune cell types mediate tumor surveillance, recent evidence demonstrates that macrophages, and other phagocytic cells, play a key role in regulating tumor growth through phagocytic clearance. In this review we highlight the role of tumor immune evasion through the inhibition of phagocytosis, specifically through the CD47-signal-regulatory protein-α pathway, and discuss how targeting this pathway might lead to more effective cancer immunotherapies.

  View details for DOI 10.1016/j.coi.2012.01.010

  View details for Web of Science ID 000303187600017

  View details for PubMedID 22310103

  View details for PubMedCentralID PMC3319521

• Perturbation of the Hematopoietic System during Embryonic Liver Development Due to Disruption of Polyubiquitin Gene Ubc in Mice PLOS ONE Ryu, K., Park, H., Rossi, D. J., Weissman, I. L., Kopito, R. R. 2012; 7 (2)

  Abstract

  Disruption of the polyubiquitin gene Ubc leads to a defect in fetal liver development, which can be partially rescued by increasing the amount of ubiquitin. However, it is still not known why Ubc is required for fetal liver development and the nature of the defective cell types responsible for embryonic lethality have not been characterized. In this study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We found that Ubc was highly expressed in the embryonic liver, and the proliferation capacity of fetal liver cells was reduced in Ubc(-/-) embryos. Specifically, Ubc was most highly expressed in hematopoietic cells, and the proliferation capacity of hematopoietic cells was significantly impaired in Ubc(-/-) embryos. While hematopoietic cell and hematopoietic stem cell (HSC) frequency was maintained in Ubc(-/-) embryos, the absolute number of these cells was diminished because of reduced total liver cell number in Ubc(-/-) embryos. Transplantations of fetal liver cells into lethally irradiated recipient mice by non-competitive and competitive reconstitution methods indicated that disruption of Ubc does not significantly impair the intrinsic function of fetal liver HSCs. These findings suggest that disruption of Ubc reduces the absolute number of HSCs in embryonic livers, but has no significant effect on the autonomous function of HSCs. Thus, the lethality of Ubc(-/-) embryos is not the result of intrinsic HSC failure.

  View details for DOI 10.1371/journal.pone.0032956

  View details for Web of Science ID 000303003500114

  View details for PubMedID 22393459

• Three differentiation states risk-stratify bladder cancer into distinct subtypes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Volkmer, J., Sahoo, D., Chin, R. K., Ho, P. L., Tang, C., Kurtova, A. V., Willingham, S. B., Pazhanisamy, S. K., Contreras-Trujillo, H., Storm, T. A., Lotan, Y., Beck, A. H., Chung, B. I., Alizadeh, A. A., Godoy, G., Lerner, S. P., van de Rijng, M., Shortliffe, L. D., Weissman, I. L., Chan, K. S. 2012; 109 (6): 2078-2083

  Abstract

  Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.

  View details for DOI 10.1073/pnas.1120605109

  View details for PubMedID 22308455

• CD47 Is a Therapeutic Antibody Target in Leiomyosarcoma 101st Annual Meeting of United-States-and-Canadian-Academy-of-Pathology (USCAP) Edris, B., Weiskopf, K., Volkmer, J., Willingham, S., Volkmer, A., Fletcher, J., Beck, A., Weissman, I., van de Rijn, M. NATURE PUBLISHING GROUP. 2012: 12A–12A

  View details for Web of Science ID 000299799900039

• Programmed cell removal: a new obstacle in the road to developing cancer. Nature reviews. Cancer Chao, M. P., Majeti, R., Weissman, I. L. 2012; 12 (1): 58-67

  Abstract

  The development of cancer involves mechanisms by which aberrant cells overcome normal regulatory pathways that limit their numbers and their migration. The evasion of programmed cell death is one of several key early events that need to be overcome in the progression from normal cellular homeostasis to malignant transformation. Recently, we provided evidence in mouse and human cancers that successful cancer clones must also overcome programmed cell removal. In this Opinion article, we explore the role of programmed cell removal in both normal and neoplastic cells, and we place this pathway in the context of the initiation of programmed cell death.

  View details for DOI 10.1038/nrc3171

  View details for PubMedID 22158022

• Programmed cell removal: a new obstacle in the road to developing cancer NATURE REVIEWS CANCER Chao, M. P., Majeti, R., Weissman, I. L. 2012; 12 (1): 58-67

  Abstract

  The development of cancer involves mechanisms by which aberrant cells overcome normal regulatory pathways that limit their numbers and their migration. The evasion of programmed cell death is one of several key early events that need to be overcome in the progression from normal cellular homeostasis to malignant transformation. Recently, we provided evidence in mouse and human cancers that successful cancer clones must also overcome programmed cell removal. In this Opinion article, we explore the role of programmed cell removal in both normal and neoplastic cells, and we place this pathway in the context of the initiation of programmed cell death.

  View details for DOI 10.1038/nrc3171

  View details for Web of Science ID 000298369300014

• The Safety of Embryonic Stem Cell Therapy Relies on Teratoma Removal ONCOTARGET Tang, C., Weissman, I. L., Drukker, M. 2012; 3 (1): 7-8

  View details for Web of Science ID 000303914000004

  View details for PubMedID 22294556

• FREQUENCY OF CELLS EXPRESSING CD44, A HEAD AND NECK CANCER STEM CELL MARKER: CORRELATION WITH TUMOR AGGRESSIVENESS HEAD AND NECK-JOURNAL FOR THE SCIENCES AND SPECIALTIES OF THE HEAD AND NECK Joshua, B., Kaplan, M. J., Doweck, I., Pai, R., Weissman, I. L., Prince, M. E., Ailles, L. E. 2012; 34 (1): 42-49

  Abstract

  We previously identified by flow cytometry a Lineage-CD44+ (Lin-CD44+) subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma (HNSCC). We now correlate clinical and histologic factors with Lin-CD44+ cell frequency.The study included 31 patients with HNSCC, of whom 87% had stage IV disease. The frequency of Lin-CD44+ cells and the success of xenografting patient tumors in mice were correlated with clinical and pathologic data.The mean frequency of Lin-CD44+ cells was 25% (0.4%-81%). It was 36% in patients who had recurrence versus 15% for those without recurrence (p = .04). Successful xenograft implantation occurred in 53%. Seventy-five percent of patients with successful xenografts had recurrence versus 21% of patients with unsuccessful xenografts (p = .003).Successful xenograft implantation and a high frequency of Lin-CD44+ cells correlate with known poor prognostic factors such as advanced T classification and recurrence. These findings may support the stem cell concept in HNSCC.

  View details for DOI 10.1002/hed.21699

  View details for PubMedID 21322081

• Long-Term Outcome of Patients with Metastatic Breast Cancer Treated with High-Dose Chemotherapy and Transplantation of Purified Autologous Hematopoietic Stem Cells BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Mueller, A. M., Kohrt, H. E., Cha, S., Laport, G., Klein, J., Guardino, A. E., Johnston, L. J., Stockerl-Goldstein, K. E., Hanania, E., Juttner, C., Blume, K. G., Negrin, R. S., Weissman, I. L., Shizuru, J. A. 2012; 18 (1): 125-133

  Abstract

  Metastatic breast cancer remains a major treatment challenge. The use of high-dose chemotherapy (HDCT) with rescue by autologous mobilized peripheral blood (MPB) is controversial, in part because of contamination of MPB by circulating tumor cells. CD34(+)Thy-1(+) selected hematopoietic stem cells (HSC) represent a graft source with a greater than 250,000-fold reduction in cancer cells. Here, we present the long-term outcome of a pilot study to determine feasibility and engraftment using HDCT and purified HSC in patients with metastatic breast cancer. Twenty-two patients who had been treated with standard chemotherapy were enrolled into a phase I/II trial between December 1996 and February 1998, and underwent HDCT followed by rescue with CD34(+)Thy-1(+) HSC isolated from autologous MPB. More than 12 years after the end of the study, 23% (5 of 22) of HSC recipients are alive, and 18% (4 of 22) are free of recurrence with normal hematopoietic function. Median progression-free survival (PFS) was 16 months, and median overall survival (OS) was 60 months. Retrospective comparison with 74 patients transplanted between February 1995 and June 1999 with the identical HDCT regimen but rescue with unmanipulated MPB indicated that 9% of patients are alive, and 7% are without disease. Median PFS was 10 months, and median OS was 28 months. In conclusion, cancer-depleted HSC following HDCT resulted in better than expected 12- to 14-year PFS and OS in a cohort of metastatic breast cancer patients. These data prompt us to look once again at purified HSC transplantation in a protocol powered to test for efficacy in advanced-stage breast cancer patients.

  View details for DOI 10.1016/j.bbmt.2011.07.009

  View details for PubMedID 21767515

• Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pang, W. W., Price, E. A., Sahoo, D., Beerman, I., Maloney, W. J., Rossi, D. J., Schrier, S. L., Weissman, I. L. 2011; 108 (50): 20012-20017

  Abstract

  In the human hematopoietic system, aging is associated with decreased bone marrow cellularity, decreased adaptive immune system function, and increased incidence of anemia and other hematological disorders and malignancies. Recent studies in mice suggest that changes within the hematopoietic stem cell (HSC) population during aging contribute significantly to the manifestation of these age-associated hematopoietic pathologies. Though the mouse HSC population has been shown to change both quantitatively and functionally with age, changes in the human HSC and progenitor cell populations during aging have been incompletely characterized. To elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated immunophenotypic HSC and other hematopoietic progenitor populations from healthy, hematologically normal young and elderly human bone marrow samples. We found that aged immunophenotypic human HSC increase in frequency, are less quiescent, and exhibit myeloid-biased differentiation potential compared with young HSC. Gene expression profiling revealed that aged immunophenotypic human HSC transcriptionally up-regulate genes associated with cell cycle, myeloid lineage specification, and myeloid malignancies. These age-associated alterations in the frequency, developmental potential, and gene expression profile of human HSC are similar to those changes observed in mouse HSC, suggesting that hematopoietic aging is an evolutionarily conserved process.

  View details for DOI 10.1073/pnas.1116110108

  View details for Web of Science ID 000298034800040

  View details for PubMedID 22123971

  View details for PubMedCentralID PMC3250139

• Identification of Cardiovascular Progenitors From Human Embryonic Stem Cells Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Ardehali, R., Ali, S., Drukker, M., Abilez, O., Blauwkamp, T., Nusse, R., Weissman, I. LIPPINCOTT WILLIAMS & WILKINS. 2011

  View details for Web of Science ID 000299738707322

• Clonal Evolution of Pre-Leukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Jan, M., Snyder, T. M., Corces-Zimmerman, M. R., Weissman, I. L., Quake, S. R., Majeti, R. AMER SOC HEMATOLOGY. 2011: 4–4

  View details for Web of Science ID 000299597100005

• Quantitation of Leukemic Stem Cell Populations Predicts Clinical Outcome in Acute Myeloid Leukaemia 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Craddock, C. F., Goardon, N., Quek, L., Freeman, S., Siddique, S., Raghavan, M., Schuh, A., Grimwade, D., Hills, R. K., Brookes, C., Griffiths, M., Cavenagh, J. D., Majeti, R., Weissman, I. L., Burnett, A. K., Vyas, P. AMER SOC HEMATOLOGY. 2011: 292–92

  View details for Web of Science ID 000299597100639

• Identification of the earliest natural killer cell-committed progenitor in murine bone marrow BLOOD Fathman, J. W., Bhattacharya, D., Inlay, M. A., Seita, J., Karsunky, H., Weissman, I. L. 2011; 118 (20): 5439-5447

  Abstract

  Natural killer (NK) cells develop in the bone marrow and are known to gradually acquire the ability to eliminate infected and malignant cells, yet the cellular stages of NK lineage commitment and maturation are incompletely understood. Using 12-color flow cytometry, we identified a novel NK-committed progenitor (pre-NKP) that is a developmental intermediate between the upstream common lymphoid progenitor and the downstream NKP, previously assumed to represent the first stage of NK lineage commitment. Our analysis also refined the purity of NKPs (rNKP) by 6-fold such that 50% of both pre-NKP and rNKP cells gave rise to NKp46+ NK cells at the single-cell level. On transplantation into unconditioned Rag2-/-Il2rγc-/- recipients, both pre-NKPs and rNKPs generated mature NK cells expressing a repertoire of Ly49 family members that degranulated on stimulation ex vivo. Intrathymic injection of these progenitors, however, yielded no NK cells, suggesting a separate origin of thymic NK cells. Unlike the rNKP, the pre-NKP does not express IL-2Rβ (CD122), yet it is lineage committed toward the NK cell fate, adding support to the theory that IL-15 signaling is not required for NK commitment. Taken together, our data provide a high-resolution in vivo analysis of the earliest steps of NK cell commitment and maturation.

  View details for DOI 10.1182/blood-2011-04-348912

  View details for Web of Science ID 000297265400014

  View details for PubMedID 21931117

  View details for PubMedCentralID PMC3217348

• Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy BLOOD Chao, M. P., Tang, C., Pachynski, R. K., Chin, R., Majeti, R., Weissman, I. L. 2011; 118 (18): 4890-4901

  Abstract

  Non-Hodgkin lymphoma (NHL) presents as both localized and disseminated disease with spread to secondary sites carrying a worse prognosis. Although pathways driving NHL dissemination have been identified, there are few therapies capable of inhibiting them. Here, we report a novel role for the immunomodulatory protein CD47 in NHL dissemination, and we demonstrate that therapeutic targeting of CD47 can prevent such spread. We developed 2 in vivo lymphoma metastasis models using Raji cells, a human NHL cell line, and primary cells from a lymphoma patient. CD47 expression was required for Raji cell dissemination to the liver in mouse xenotransplants. Targeting of CD47 with a blocking antibody inhibited Raji cell dissemination to major organs, including the central nervous system, and inhibited hematogenous dissemination of primary lymphoma cells. We hypothesized that anti-CD47 antibody-mediated elimination of circulating tumor cells occurred through phagocytosis, a previously described mechanism for blocking anti-CD47 antibodies. As predicted, inhibition of dissemination by anti-CD47 antibodies was dependent on blockade of phagocyte SIRPα and required macrophage effector cells. These results demonstrate that CD47 is required for NHL dissemination, which can be therapeutically targeted with a blocking anti-CD47 antibody. Ultimately, these findings are potentially applicable to the dissemination and metastasis of other solid tumors.

  View details for DOI 10.1182/blood-2011-02-338020

  View details for Web of Science ID 000296714500018

  View details for PubMedID 21828138

  View details for PubMedCentralID PMC3208297

• In vivo Molecular MRI of Cell Survival and Teratoma Formation Following Embryonic Stem Cell Transplantation Into the Injured Murine Myocardium MAGNETIC RESONANCE IN MEDICINE Chung, J., Kee, K., Barral, J. K., Dash, R., Kosuge, H., Wang, X., Weissman, I., Robbins, R. C., Nishimura, D., Quertermous, T., Reijo-Pera, R. A., Yang, P. C. 2011; 66 (5): 1374-1381

  Abstract

  Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, nonspecific intracellular accumulation of SPIO limits long-term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO-conjugated monoclonal antibody against these antigens (SPIO-MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO-MAb. The human and mouse ESCs were transduced with the RG (ESC-RGs) and transplanted into the peri-infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO-MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC-derived teratoma formation in the injured murine myocardium.

  View details for DOI 10.1002/mrm.22929

  View details for PubMedID 21604295

• COMPARISON OF DIPG NEUROSPHERE CELL LINES FROM THREE PATIENTS 16th Annual Scientific Meeting of the Society-for-Neuro-Oncology (SNO)/AANS/CNS Section on Tumors Monje, M., Mitra, S. S., Freret, M. E., Edwards, M. S., Weissman, I. L., Beachy, P. A. OXFORD UNIV PRESS INC. 2011: 165–165

  View details for Web of Science ID 000297026600650

• Novel Hematopoietic Progenitor Populations Revealed by Direct Assessment of GATA1 Protein Expression and cMPL Signaling Events STEM CELLS Heffner, G. C., Clutter, M. R., Nolan, G. P., Weissman, I. L. 2011; 29 (11): 1774-1782

  Abstract

  Hematopoietic stem cells (HSCs) must exhibit tight regulation of both self-renewal and differentiation to maintain homeostasis of the hematopoietic system as well as to avoid aberrations in growth that may result in leukemias or other disorders. In this study, we sought to understand the molecular basis of lineage determination, with particular focus on factors that influence megakaryocyte/erythrocyte-lineage commitment, in hematopoietic stem and progenitor cells. We used intracellular flow cytometry to identify two novel hematopoietic progenitor populations within the mouse bone-marrow cKit(+) Lineage (-) Sca1(+) (KLS) Flk2 (+) compartment that differ in their protein-level expression of GATA1, a critical megakaryocyte/erythrocyte-promoting transcription factor. GATA1-high repopulating cells exhibited the cell surface phenotype KLS Flk2(+ to int), CD150(int), CD105(+), cMPL(+), and were termed "FSE cells." GATA1-low progenitors were identified as KLS Flk2(+), CD150(-), and cMPL(-), and were termed "Flk(+) CD150(-) cells." FSE cells had increased megakaryocyte/platelet potential in culture and transplant settings and exhibited a higher clonal frequency of colony-forming unit-spleen activity compared with Flk(+) CD150(-) cells, suggesting functional consequences of GATA1 upregulation in promoting megakaryocyte and erythroid lineage priming. Activation of ERK and AKT signal-transduction cascades was observed by intracellular flow cytometry in long-term HSCs and FSE cells, but not in Flk(+) CD150(-) cells in response to stimulation with thrombopoietin, an important megakaryocyte-promoting cytokine. We provide a mechanistic rationale for megakaryocyte/erythroid bias within KLS Flk2(+) cells, and demonstrate how assessment of intracellular factors and signaling events can be used to refine our understanding of lineage commitment during early definitive hematopoiesis.

  View details for DOI 10.1002/stem.719

  View details for Web of Science ID 000296565500014

  View details for PubMedID 21898686

• Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding NATURE BIOTECHNOLOGY Lu, R., Neff, N. F., Quake, S. R., Weissman, I. L. 2011; 29 (10): 928-U229

  Abstract

  Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.

  View details for DOI 10.1038/nbt.1977

  View details for Web of Science ID 000296273000020

  View details for PubMedID 21964413

  View details for PubMedCentralID PMC3196379

• Reduced ribosomal protein gene dosage and p53 activation in low-risk myelodysplastic syndrome BLOOD McGowan, K. A., Pang, W. W., Bhardwaj, R., Perez, M. G., Pluvinage, J. V., Glader, B. E., Malek, R., Mendrysa, S. M., Weissman, I. L., Park, C. Y., Barsh, G. S. 2011; 118 (13): 3622-3633

  Abstract

  Reduced gene dosage of ribosomal protein subunits has been implicated in 5q- myelodysplastic syndrome and Diamond Blackfan anemia, but the cellular and pathophysiologic defects associated with these conditions are enigmatic. Using conditional inactivation of the ribosomal protein S6 gene in laboratory mice, we found that reduced ribosomal protein gene dosage recapitulates cardinal features of the 5q- syndrome, including macrocytic anemia, erythroid hypoplasia, and megakaryocytic dysplasia with thrombocytosis, and that p53 plays a critical role in manifestation of these phenotypes. The blood cell abnormalities are accompanied by a reduction in the number of HSCs, a specific defect in late erythrocyte development, and suggest a disease-specific ontogenetic pathway for megakaryocyte development. Further studies of highly purified HSCs from healthy patients and from those with myelodysplastic syndrome link reduced expression of ribosomal protein genes to decreased RBC maturation and suggest an underlying and common pathophysiologic pathway for additional subtypes of myelodysplastic syndrome.

  View details for DOI 10.1182/blood-2010-11-318584

  View details for PubMedID 21788341

• An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells NATURE BIOTECHNOLOGY Tang, C., Lee, A. S., Volkmer, J., Sahoo, D., Nag, D., Mosley, A. R., Inlay, M. A., Ardehali, R., Chavez, S. L., Pera, R. R., Behr, B., Wu, J. C., Weissman, I. L., Drukker, M. 2011; 29 (9): 829-U86

  Abstract

  An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.

  View details for DOI 10.1038/nbt.1947

  View details for Web of Science ID 000294718400024

  View details for PubMedID 21841799

  View details for PubMedCentralID PMC3537836

• From immunological tolerance to stem cell therapy and back: an interview with Irving Weissman. Interview by Sarah Allan. Disease models & mechanisms Weissman, I. 2011; 4 (5): 559-561

  View details for DOI 10.1242/dmm.008532

  View details for PubMedID 21878457

• Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip NATURE Rinkevich, Y., Lindau, P., Ueno, H., Longaker, M. T., Weissman, I. L. 2011; 476 (7361): 409-U53

  Abstract

  The regrowth of amputated limbs and the distal tips of digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation.

  View details for DOI 10.1038/nature10346

  View details for Web of Science ID 000294209400027

  View details for PubMedID 21866153

• Enhanced survival of pluripotent stem cells under stressful conditions CELL CYCLE Ardehali, R., Ali, S. R., Inlay, M. A., Mosley, A. R., Weissman, I. L. 2011; 10 (16): 2610-2611

  View details for DOI 10.4161/cc.10.16.16527

  View details for Web of Science ID 000294155600003

  View details for PubMedID 21791974

• Isolation of Cardiovascular Progenitors from Human Embryonic Stem Cells Capable of Integration into Human Fetal Hearts 15th Annual Scientific Meeting of the Heart-Failure-Society-of-America Ardehali, R., Ali, S. R., Drukker, M., Weissman, I. L. CHURCHILL LIVINGSTONE INC MEDICAL PUBLISHERS. 2011: S32–S32

  View details for Web of Science ID 000293938700099

• CLONAL STABILITY OF MURINE HEMATOPOIETIC STEM CELLS IN VIVO ISEH 40th Annual Scientific Meeting of the Society-for-Hematology-and-Stem-Cells Lu, R., Czechowicz, A., Seita, J., Weissman, I. L. ELSEVIER SCIENCE INC. 2011: S51–S52

  View details for Web of Science ID 000293801700088

• Single-cell phospho-specific flow cytometric analysis demonstrates biochemical and functional heterogeneity in human hematopoietic stem and progenitor compartments BLOOD Gibbs, K. D., Gilbert, P. M., Sachs, K., Zhao, F., Blau, H. M., Weissman, I. L., Nolan, G. P., Majeti, R. 2011; 117 (16): 4226-4233

  Abstract

  The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations, and their functional consequences. Here, single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated, including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment, and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes, and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states.

  View details for DOI 10.1182/blood-2010-07-298232

  View details for Web of Science ID 000289807600012

  View details for PubMedID 21357764

  View details for PubMedCentralID PMC3087474

• Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jan, M., Chao, M. P., Cha, A. C., Alizadeh, A. A., Gentles, A. J., Weissman, I. L., Majeti, R. 2011; 108 (12): 5009-5014

  Abstract

  Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA. By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function.

  View details for DOI 10.1073/pnas.1100551108

  View details for Web of Science ID 000288712200061

  View details for PubMedID 21383193

  View details for PubMedCentralID PMC3064328

• Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Monje, M., Mitra, S. S., Freret, M. E., Raveh, T. B., Kim, J., Masek, M., Attema, J. L., Li, G., Haddix, T., Edwards, M. S., Fisher, P. G., Weissman, I. L., Rowitch, D. H., Vogel, H., Wong, A. J., Beachy, P. A. 2011; 108 (11): 4453-4458

  Abstract

  Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.

  View details for DOI 10.1073/pnas.1101657108

  View details for PubMedID 21368213

• In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors. Blood Richie Ehrlich, L. I., Serwold, T., Weissman, I. L. 2011; 117 (9): 2618-2624

  Abstract

  The identity of T-cell progenitors that seed the thymus has remained controversial, largely because many studies differ over whether these progenitors retain myeloid potential. Contradictory reports diverge in their use of various in vitro and in vivo assays. To consolidate these discordant findings, we compared the myeloid potential of 2 putative thymus seeding populations, common lymphoid progenitors (CLPs) and multipotent progenitors (MPPs), and the earliest intrathymic progenitor (DN1), using 2 in vitro assays and in vivo readouts. These assays gave contradictory results: CLP and DN1 displayed surprisingly robust myeloid potential on OP9-DL1 in vitro stromal cocultures but displayed little myeloid potential in vivo, as well as in methylcellulose cultures. MPP, on the other hand, displayed robust myeloid potential in all settings. We conclude that stromal cocultures reveal cryptic, but nonphysiologic, myeloid potentials of lymphoid progenitors, providing an explanation for contradictory findings in the field and underscoring the importance of using in vivo assays for the determination of physiologic lineage potentials.

  View details for DOI 10.1182/blood-2010-05-287102

  View details for PubMedID 21163922

  View details for PubMedCentralID PMC3062354

• In vitro assays misrepresent in vivo lineage potentials of murine lymphoid progenitors BLOOD Ehrlich, L. I., Serwold, T., Weissman, I. L. 2011; 117 (9): 2618-2624

  View details for DOI 10.1182/blood-2010-05-287102

  View details for Web of Science ID 000288207400014

• Irv Weissman. Nature biotechnology Weissman, I. 2011; 29 (3): 194-?

  View details for DOI 10.1038/nbt.1816

  View details for PubMedID 21390013

• Overexpression of BCL2 enhances survival of human embryonic stem cells during stress and obviates the requirement for serum factors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ardehali, R., Inlay, M. A., Ali, S. R., Tang, C., Drukker, M., Weissman, I. L. 2011; 108 (8): 3282-3287

  Abstract

  The promise of pluripotent stem cells as a research and therapeutic tool is partly undermined by the technical challenges of generating and maintaining these cells in culture. Human embryonic stem cells (hESCs) are exquisitely sensitive to culture conditions, and require constant signaling by growth factors and cell-cell and cell-matrix interactions to prevent apoptosis, senescence, and differentiation. Previous work from our laboratory demonstrated that overexpression of the prosurvival gene BCL2 in mouse embryonic stem cells overrode the requirement of serum factors and feeder cells to maintain mESCs in culture. To determine whether this prosurvival gene could similarly protect hESCs, we generated hESC lines that constitutively or inducibly express BCL2. We find that BCL2 overexpression significantly decreases dissociation-induced apoptosis, resulting in enhanced colony formation from sorted single cells, and enhanced embryoid body formation. In addition, BCL2-hESCs exhibit normal growth in the absence of serum, but require basic fibroblast growth factor to remain undifferentiated. Furthermore, they maintain their pluripotency markers, form teratomas in vivo, and differentiate into all three germ layers. Our data suggest that the BCL2 signaling pathway plays an important role in inhibiting hESC apoptosis, such that its overexpression in hESCs offers both a survival benefit in conditions of stress by resisting apoptosis and obviates the requirement for serum or a feeder layer for maintenance.

  View details for DOI 10.1073/pnas.1019047108

  View details for PubMedID 21300885

• Therapeutic Antibody Targeting of CD47 Eliminates Human Acute Lymphoblastic Leukemia CANCER RESEARCH Chao, M. P., Alizadeh, A. A., Tang, C., Jan, M., Weissman-Tsukamoto, R., Zhao, F., Park, C. Y., Weissman, I. L., Majeti, R. 2011; 71 (4): 1374-1384

  Abstract

  Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL.

  View details for DOI 10.1158/0008-5472.CAN-10-2238

  View details for Web of Science ID 000287352600020

  View details for PubMedID 21177380

  View details for PubMedCentralID PMC3041855

• Purified Hematopoietic Stem Cell Transplantation: The Next Generation of Blood and Immune Replacement HEMATOLOGY-ONCOLOGY CLINICS OF NORTH AMERICA Czechowicz, A., Weissman, I. L. 2011; 25 (1): 75-?

  Abstract

  Replacement of disease-causing stem cells with healthy ones has been achieved clinically via hematopoietic cell transplantation (HCT) for the last 40 years, as a treatment modality for a variety of cancers and immunodeficiencies with moderate, but increasing, success. This procedure has traditionally included transplantation of mixed hematopoietic populations that include hematopoietic stem cells (HSC) and other cells, such as T cells. This article explores and delineates the potential expansion of this technique to treat a variety of inherited diseases of immune function, the current barriers in HCT and pure HSC transplantation, and the up-and-coming strategies to combat these obstacles.

  View details for DOI 10.1016/j.hoc.2010.11.006

  View details for Web of Science ID 000287333600007

  View details for PubMedID 21236391

• IL-1 beta-driven neutrophilia preserves antibacterial defense in the absence of the kinase IKK beta NATURE IMMUNOLOGY Hsu, L., Enzler, T., Seita, J., Timmer, A. M., Lee, C., Lai, T., Yu, G., Lai, L., Temkin, V., Sinzig, U., Aung, T., Nizet, V., Weissman, I. L., Karin, M. 2011; 12 (2): 144-U54

  Abstract

  Transcription factor NF-κB and its activating kinase IKKβ are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKβ-NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKβ (Ikkβ(Δ) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikkβ(Δ) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikkβ(Δ)Il1r1(-/-) mice, unlike Ikkβ(Δ) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKβ-NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.

  View details for DOI 10.1038/ni.1976

  View details for Web of Science ID 000286378400008

  View details for PubMedID 21170027

  View details for PubMedCentralID PMC3677078

• LONG-TERM FOLLOW-UP OF METASTATIC BREAST CANCER PATIENTS RECEIVING HIGHLY PURIFIED AUTOLOGOUS CD34+THY-1+HEMATOPOIETIC STEM CELLS AFTER HIGH-DOSE CHEMOTHERAPY Annual Meeting of the American-Society-for-Blood-and-Marrow-Transplantation(BMT) Tandem Mueller, A. M., Kohrt, H. E., Laport, G. G., Negrin, R. S., Cha, S., Weissman, I. L., Shizuru, J. A. ELSEVIER SCIENCE INC. 2011: S198–S198

  View details for Web of Science ID 000287350500123

• VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC delta-driven migration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Razorenova, O. V., Finger, E. C., Colavitti, R., Chernikova, S. B., Boiko, A. D., Chan, C. K., Krieg, A., Bedogni, B., LaGory, E., Weissman, I. L., Broome-Powell, M., Giaccia, A. J. 2011; 108 (5): 1931-1936

  Abstract

  A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

  View details for DOI 10.1073/pnas.1011777108

  View details for PubMedID 21233420

• 50 Years Later: Remembering the Paper RADIATION RESEARCH Weissman, I. L. 2011; 175 (2): 143-144

  View details for DOI 10.1667/RRXX29.1

  View details for Web of Science ID 000287113500001

  View details for PubMedID 21268706

• Human Acute Myelogenous Leukemia Stem Cells Revisited: There's More Than Meets the Eye CANCER CELL Majeti, R., Weissman, I. L. 2011; 19 (1): 9-10

  Abstract

  In this issue of Cancer Cell, Goardon et al. revise earlier conclusions regarding acute myelogenous leukemia (AML) stem cells by demonstrating that in the majority of patients, they reside in two hierarchically related populations most similar to normal hematopoietic progenitors. These findings have implications for therapeutic targeting of these cells.

  View details for DOI 10.1016/j.ccr.2011.01.007

  View details for Web of Science ID 000287290300005

  View details for PubMedID 21251611

  View details for PubMedCentralID PMC3045274

• Activated canonical Wnt signaling in GBM is associated with increased expression of stem cell surface markers PeerEmed Cheshier, S., Ailles, L., Weissman, I., Tse, V., Skirboll, S. 2011
• Calreticulin Is the Dominant Pro-Phagocytic Signal on Multiple Human Cancers and Is Counterbalanced by CD47 SCIENCE TRANSLATIONAL MEDICINE Chao, M. P., Jaiswal, S., Weissman-Tsukamoto, R., Alizadeh, A. A., Gentles, A. J., Volkmer, J., Weiskopf, K., Willingham, S. B., Raveh, T., Park, C. Y., Majeti, R., Weissman, I. L. 2010; 2 (63)

  Abstract

  Under normal physiological conditions, cellular homeostasis is partly regulated by a balance of pro- and anti-phagocytic signals. CD47, which prevents cancer cell phagocytosis by the innate immune system, is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin's lymphoma, and bladder cancer. Blocking CD47 with a monoclonal antibody results in phagocytosis of cancer cells and leads to in vivo tumor elimination, yet normal cells remain mostly unaffected. Thus, we postulated that cancer cells must also display a potent pro-phagocytic signal. Here, we identified calreticulin as a pro-phagocytic signal that was highly expressed on the surface of several human cancers, but was minimally expressed on most normal cells. Increased CD47 expression correlated with high amounts of calreticulin on cancer cells and was necessary for protection from calreticulin-mediated phagocytosis. Blocking the interaction of target cell calreticulin with its receptor, low-density lipoprotein receptor-related protein, on phagocytic cells prevented anti-CD47 antibody-mediated phagocytosis. Furthermore, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and non-Hodgkin's lymphoma. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.

  View details for DOI 10.1126/scitranslmed.3001375

  View details for Web of Science ID 000288444900003

  View details for PubMedID 21178137

• MicroRNA-125b expands hematopoietic stem cells and enriches for the lymphoid-balanced and lymphoid-biased subsets PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ooi, A. G., Sahoo, D., Adorno, M., Wang, Y., Weissman, I. L., Park, C. Y. 2010; 107 (50): 21505-21510

  Abstract

  MicroRNAs profoundly impact hematopoietic cells by regulating progenitor cell-fate decisions, as well as mature immune effector function. However to date, microRNAs that regulate hematopoietic stem cell (HSC) function have been less well characterized. Here we show that microRNA-125b (miR-125b) is highly expressed in HSCs and its expression decreases in committed progenitors. Overexpression of miR-125b in mouse HSC enhances their function, demonstrated through serial transplantation of highly purified HSC, and enriches for the previously described Slamf1(lo)CD34(-) lymphoid-balanced and the Slamf1(neg)CD34(-) lymphoid-biased cell subsets within the multipotent HSC (CD34-KLS) fraction. Mature peripheral blood cells derived from the miR-125b-overexpressing HSC are skewed toward the lymphoid lineage. Consistent with this observation, miR-125b overexpression significantly increases the number of early B-progenitor cells within the spleen and induces the expansion and enrichment of the lymphoid-balanced and lymphoid-biased HSC subset via an antiapoptotic mechanism, reducing the mRNA expression levels of two proapoptotic targets, Bmf and KLF13. The antiapoptotic effect of miR-125b is more pronounced in the lymphoid-biased HSC subset because of their intrinsic higher baseline levels of apoptosis. These effects of miR-125b are associated with the development of lymphoproliferative disease, marked by expansion of CD8(+) T lymphocytes. Taken together, these data reveal that miR-125b regulates HSC survival and can promote lymphoid-fate decisions at the level of the HSC by preferentially expanding lymphoid-balanced and lymphoid-biased HSC.

  View details for DOI 10.1073/pnas.1016218107

  View details for Web of Science ID 000285521500053

  View details for PubMedID 21118986

  View details for PubMedCentralID PMC3003003

• Lymphocytes, Jim Gowans and in vivo veritas NATURE IMMUNOLOGY Weissman, I. 2010; 11 (12): 1073-1075

  View details for DOI 10.1038/ni1210-1073

  View details for Web of Science ID 000284262200003

  View details for PubMedID 21079628

• Haploinsufficiency of Ribosomal Protein S6 In Mice Mimics Bone Marrow Failure Syndromes In Humans 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Park, C. Y., McGowan, K. A., Glader, B., Barsh, G. S., Weissman, I. L. AMER SOC HEMATOLOGY. 2010: 89–89

  View details for Web of Science ID 000289662200195

• ABT-737 Targets Leukemic Stem Cells In Mouse Models of Mutant NRASD12/hBCL-2-Mediated Acute Myeloid Leukemia progression with Increased Survival 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Padua, R. A., Beurlet, S., Krief, P., Omidvar, N., Le Pogam, C., Auboeuf, D., de la Grange, P., Soulie, A., Janin, A., Noguera, M., Merlet, P., Sarda-Mantel, L., Fenaux, P., Konopleva, M., Andreeff, M., Tu, A., Yang, P., Fan, A. C., Kogan, S. C., Weissman, I. L., Felsher, D. W., Pla, M., West, R., Chomienne, C. AMER SOC HEMATOLOGY. 2010: 1355–56

  View details for Web of Science ID 000289662203639

• Mutant BCL2 Co-Operates with CBF beta/PEBP2 beta-MYH11 to Promote Expansion of Leukemia Initiating Cells with a Predominantly Pro-Apoptotic Mechanism Via Recruitment of Ras-GTP In a Mouse Model of Progressive Acute Myeloid Leukemia 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Omidvar, N., Le Pogam, C., Beurlet, S., Noguera, M., Janin, A., Kogan, S. C., Weissman, I. L., Pla, M., Chomienne, C., Padua, R. A. AMER SOC HEMATOLOGY. 2010: 461–61

  View details for Web of Science ID 000289662201153

• T-cell receptor-driven lymphomagenesis in mice derived from a reprogrammed T cell PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Serwold, T., Hochedlinger, K., Swindle, J., Hedgpeth, J., Jaenisch, R., Weissman, I. L. 2010; 107 (44): 18939-18943

  Abstract

  The conversion of mature somatic cells into pluripotent stem cells, both by nuclear transfer and transduction with specific "reprogramming" genes, represents a major advance in regenerative medicine. Pluripotent stem cell lines can now be generated from an individual's own cells, facilitating the generation of immunologically acceptable stem cell-based therapeutics. Many cell types can undergo nuclear reprogramming, leading to the question of whether the identity of the reprogrammed cell of origin has a biological consequence. Peripheral blood, containing a mixture of T, B, NK, and myeloid cell types, represents one potential source of reprogrammable cells. In this study, we describe the unique case of mice derived from a reprogrammed T cell. These mice have prerearranged T-cell receptor (TCR) genes in all cells. Surprisingly, ≈50% of mice with prerearranged TCR genes develop spontaneous T cell lymphomas, which originate in the thymus. The lymphomas arise from developing T cells, and contain activated Notch1, similar to most human and mouse T-cell acute lymphoblastic lymphomas. Furthermore, lymphomagenesis requires the expression of both prerearranged TCRα and TCRβ genes, indicating a critical role for TCR signaling. Furthermore, inhibitors of multiple branches of TCR signaling suppress lymphoma growth, implicating TCR signaling as an essential component in lymphoma proliferation. The lymphomagenesis in mice derived from a reprogrammed T cell demonstrates the deleterious consequences of misregulation of the TCR rearrangement and signaling pathways and illustrates one case of cellular reprogramming where the identity of the cell of origin has profound consequences.

  View details for DOI 10.1073/pnas.1013230107

  View details for Web of Science ID 000283749000039

  View details for PubMedID 20956329

• Hematopoietic stem cell: self-renewal versus differentiation WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE Seita, J., Weissman, I. L. 2010; 2 (6): 640-653

  Abstract

  The mammalian blood system, containing more than 10 distinct mature cell types, stands on one specific cell type, hematopoietic stem cell (HSC). Within the system, only HSCs possess the ability of both multipotency and self-renewal. Multipotency is the ability to differentiate into all functional blood cells. Self-renewal is the ability to give rise to HSC itself without differentiation. Since mature blood cells (MBCs) are predominantly short-lived, HSCs continuously provide more differentiated progenitors while properly maintaining the HSC pool size throughout life by precisely balancing self-renewal and differentiation. Thus, understanding the mechanisms of self-renewal and differentiation of HSC has been a central issue. In this review, we focus on the hierarchical structure of the hematopoietic system, the current understanding of microenvironment and molecular cues regulating self-renewal and differentiation of adult HSCs, and the currently emerging systems approaches to understand HSC biology.

  View details for DOI 10.1002/wsbm.86

  View details for Web of Science ID 000283713500002

  View details for PubMedID 20890962

  View details for PubMedCentralID PMC2950323

• Cholinergic activation of hematopoietic stem cells: role in tobacco-related disease? VASCULAR MEDICINE Chang, E., Forsberg, E. C., Wu, J., Wang, B., Prohaska, S. S., Allsopp, R., Weissman, I. L., Cooke, J. P. 2010; 15 (5): 375-385

  Abstract

  Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

  View details for DOI 10.1177/1358863X10378377

  View details for Web of Science ID 000282582300004

  View details for PubMedID 20926497

  View details for PubMedCentralID PMC3110740

• Comprehensive methylome map of lineage commitment from haematopoietic progenitors NATURE Ji, H., Ehrlich, L. I., Seita, J., Murakami, P., Doi, A., Lindau, P., Lee, H., Aryee, M. J., Irizarry, R. A., Kim, K., Rossi, D. J., Inlay, M. A., Serwold, T., Karsunky, H., Ho, L., Daley, G. Q., Weissman, I. L., Feinberg, A. P. 2010; 467 (7313): 338-U120

  Abstract

  Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

  View details for DOI 10.1038/nature09367

  View details for Web of Science ID 000281824900041

  View details for PubMedID 20720541

  View details for PubMedCentralID PMC2956609

• Epigenetic memory in induced pluripotent stem cells NATURE Kim, K., Doi, A., Wen, B., Ng, K., Zhao, R., Cahan, P., Kim, J., Aryee, M. J., Ji, H., Ehrlich, L. I., Yabuuchi, A., Takeuchi, A., Cunniff, K. C., Hongguang, H., McKinney-Freeman, S., Naveiras, O., Yoon, T. J., Irizarry, R. A., Jung, N., Seita, J., Hanna, J., Murakami, P., Jaenisch, R., Weissleder, R., Orkin, S. H., Weissman, I. L., Feinberg, A. P., Daley, G. Q. 2010; 467 (7313): 285-U60

  Abstract

  Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

  View details for DOI 10.1038/nature09342

  View details for Web of Science ID 000281824900030

  View details for PubMedID 20644535

  View details for PubMedCentralID PMC3150836

• Position-Dependent Silencing of Germline V beta Segments on TCR beta Alleles Containing Preassembled V beta DJ beta C beta 1 Genes JOURNAL OF IMMUNOLOGY Brady, B. L., Oropallo, M. A., Yang-Iott, K. S., Serwold, T., Hochedlinger, K., Jaenisch, R., Weissman, I. L., Bassing, C. H. 2010; 185 (6): 3564-3573

  Abstract

  The genomic organization of TCRbeta loci enables Vbeta-to-DJbeta2 rearrangements on alleles with assembled VbetaDJbetaCbeta1 genes, which could have deleterious physiologic consequences. To determine whether such Vbeta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRbeta alleles containing preassembled functional VbetaDJbetaCbeta1 genes. Vbeta10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vbeta1DJbetaCbeta1 gene, but not on alleles with a Vbeta14DJbetaCbeta1 gene. Germline Vbeta10 transcription was silenced in mature alphabeta T cells. This allele-dependent and developmental stage-specific silencing of Vbeta10 correlated with increased CpG methylation and decreased histone acetylation over the Vbeta10 promoter and coding region. Transcription, rearrangement, and expression of the Vbeta4 and Vbeta16 segments located upstream of Vbeta10 were silenced on alleles containing either VbetaDJbetaCbeta1 gene; sequences within Vbeta4, Vbeta16, and the Vbeta4/Vbeta16-Vbeta10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vbeta segments relative to assembled VbetaDJbetaCbeta1 genes influences their rearrangement and suggest that DNA sequences between Vbeta segments may form boundaries between active and inactive Vbeta chromatin domains upstream of VbetaDJbetaCbeta genes.

  View details for DOI 10.4049/jimmunol.0903098

  View details for Web of Science ID 000281559300050

  View details for PubMedID 20709953

• Anti-CD47 Antibody Synergizes with Rituximab to Promote Phagocytosis and Eradicate Non-Hodgkin Lymphoma CELL Chao, M. P., Alizadeh, A. A., Tang, C., Myklebust, J. H., Varghese, B., Gill, S., Jan, M., Cha, A. C., Chan, C. K., Tan, B. T., Park, C. Y., Zhao, F., Kohrt, H. E., Malumbres, R., Briones, J., Gascoyne, R. D., Lossos, I. S., Levy, R., Weissman, I. L., Majeti, R. 2010; 142 (5): 699-713

  Abstract

  Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

  View details for DOI 10.1016/j.cell.2010.07.044

  View details for PubMedID 20813259

• Cancer stem cells in bladder cancer: a revisited and evolving concept CURRENT OPINION IN UROLOGY Chan, K. S., Volkmer, J., Weissman, I. 2010; 20 (5): 393-397

  Abstract

  Recently, the prospective isolation and characterization of cancer stem cells (CSCs) from various human malignancies revealed that they are resistant to radiation and chemotherapies. Therefore, CSCs may be the 'roots' and ideal target for therapeutic intervention. Here, we will focus on reviewing the historical perspective, recent literatures on bladder cancer stem cells and their clinical implications.CSCs have been prospectively isolated from bladder cancer tissues from patient specimens, established cancer cell lines and xenografts, based on the expression of a combination of cell surface receptors, cytokeratin markers, drug transporters and the efficient efflux of the Hoechst 33,342 dye (side population). Further, global gene expression profiling of CSCs revealed an activated gene signature of CSCs similar to that of aggressive bladder cancer, supporting the concept that a tumor cell subpopulation is contributing to bladder cancer progression. Finally, our studies on the preclinical targeting of bladder CSCs in vitro and in xenografts using a blocking antibody for CD47 reveal promising efficacy.Functionally distinct CSCs exist in human bladder cancer and can be prospectively isolated. Continuing research will be important to identify their cell of origin, programs balancing self-renewal and differentiation and to identify additional therapeutic options to target bladder CSCs.

  View details for DOI 10.1097/MOU.0b013e32833cc9df

  View details for Web of Science ID 000280552100009

  View details for PubMedID 20657288

• Differential DNA Damage Response in Stem and Progenitor Cells CELL STEM CELL Seita, J., Rossi, D. J., Weissman, I. L. 2010; 7 (2): 145-147

  Abstract

  The long lifespan of tissue-specific stem cells suggests that they may respond differently to DNA damage than downstream cells. In this issue of Cell Stem Cell, two groups address this hypothesis by examining DNA damage responses in hematopoietic stem and progenitor cells (Milyavsky et al., 2010; Mohrin et al., 2010).

  View details for DOI 10.1016/j.stem.2010.07.006

  View details for Web of Science ID 000281107400006

  View details for PubMedID 20682442

• Patients Beware: Commercialized Stem Cell Treatments on the Web CELL STEM CELL Taylor, P. L., Barker, R. A., Blume, K. G., Cattaneo, E., Colman, A., Deng, H., Edgar, H., Fox, I. J., Gerstle, C., Goldstein, L. S., High, K. A., Lyall, A., Parkman, R., Pitossi, F. J., Prentice, E. D., Rooke, H. M., Sipp, D. A., Srivastava, A., Stayn, S., Steinberg, G. K., Wagers, A. J., Weissman, I. L. 2010; 7 (1): 43-49

  Abstract

  A report by the International Society for Stem Cell Research (ISSCR)'s Task Force on Unproven Stem Cell Treatments outlines development of resources for patients, their families, and physicians seeking information on stem cell treatments.

  View details for DOI 10.1016/j.stem.2010.06.001

  View details for Web of Science ID 000280222300012

  View details for PubMedID 20621049

• Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271 NATURE Boiko, A. D., Razorenova, O. V., van de Rijn, M., Swetter, S. M., Johnson, D. L., Ly, D. P., Butler, P. D., Yang, G. P., Joshua, B., Kaplan, M. J., Longaker, M. T., Weissman, I. L. 2010; 466 (7302): 133-U155

  Abstract

  The question of whether tumorigenic cancer stem cells exist in human melanomas has arisen in the last few years. Here we show that in melanomas, tumour stem cells (MTSCs, for melanoma tumour stem cells) can be isolated prospectively as a highly enriched CD271(+) MTSC population using a process that maximizes viable cell transplantation. The tumours sampled in this study were taken from a broad spectrum of sites and stages. High-viability cells isolated by fluorescence-activated cell sorting and re-suspended in a matrigel vehicle were implanted into T-, B- and natural-killer-deficient Rag2(-/-)gammac(-/-) mice. The CD271(+) subset of cells was the tumour-initiating population in 90% (nine out of ten) of melanomas tested. Transplantation of isolated CD271(+) melanoma cells into engrafted human skin or bone in Rag2(-/-)gammac(-/-) mice resulted in melanoma; however, melanoma did not develop after transplantation of isolated CD271(-) cells. We also show that in mice, tumours derived from transplanted human CD271(+) melanoma cells were capable of metastatsis in vivo. CD271(+) melanoma cells lacked expression of TYR, MART1 and MAGE in 86%, 69% and 68% of melanoma patients, respectively, which helps to explain why T-cell therapies directed at these antigens usually result in only temporary tumour shrinkage.

  View details for DOI 10.1038/nature09161

  View details for PubMedID 20596026

• Macrophages as mediators of tumor immunosurveillance TRENDS IN IMMUNOLOGY Jaiswal, S., Chao, M. P., Majeti, R., Weissman, I. L. 2010; 31 (6): 212-219

  Abstract

  Tumor immunosurveillance is a well-established mechanism for regulation of tumor growth. In this regard, most studies have focused on the role of T- and NK-cells as the critical immune effector cells. However, macrophages play a major role in the recognition and clearance of foreign, aged, and damaged cells. Macrophage phagocytosis is negatively regulated via the receptor SIRPalpha upon binding to CD47, a ubiquitously expressed protein. We recently showed that CD47 is up-regulated in myeloid leukemia and migrating hematopoietic progenitors, and that the level of protein expression correlates with the ability to evade phagocytosis. These results implicate macrophages in the immunosurveillance of hematopoietic cells and leukemias. The ability of macrophages to phagocytose tumor cells might be exploited therapeutically by blocking the CD47-SIRPalpha interaction.

  View details for DOI 10.1016/j.it.2010.04.001

  View details for Web of Science ID 000279427000002

  View details for PubMedID 20452821

  View details for PubMedCentralID PMC3646798

• Purified Hematopoietic Stem Cell Transplantation: The Next Generation of Blood and Immune Replacement IMMUNOLOGY AND ALLERGY CLINICS OF NORTH AMERICA Czechowicz, A., Weissman, I. L. 2010; 30 (2): 159-?

  Abstract

  Replacement of disease-causing stem cells with healthy ones has been achieved clinically via hematopoietic cell transplantation (HCT) for the last 40 years, as a treatment modality for a variety of cancers and immunodeficiencies with moderate, but increasing, success. This procedure has traditionally included transplantation of mixed hematopoietic populations that include hematopoietic stem cells (HSC) and other cells, such as T cells. This article explores and delineates the potential expansion of this technique to treat a variety of inherited diseases of immune function, the current barriers in HCT and pure HSC transplantation, and the up-and-coming strategies to combat these obstacles.

  View details for DOI 10.1016/j.iac.2010.03.003

  View details for Web of Science ID 000279115200003

  View details for PubMedID 20493393

  View details for PubMedCentralID PMC3071240

• Distinguishing Mast Cell and Granulocyte Differentiation at the Single-Cell Level CELL STEM CELL Franco, C. B., Chen, C., Drukker, M., Weissman, I. L., Galli, S. J. 2010; 6 (4): 361-368

  Abstract

  The lineage restriction of prospectively isolated hematopoietic progenitors has been traditionally assessed by bulk in vitro culture and transplantation of large number of cells in vivo. These methods, however, cannot distinguish between homogenous multipotent or heterogeneous lineage-restricted populations. Using clonal assays of 1 or 5 cells in vitro, single-cell quantitative gene expression analyses, and transplantation of mice with low numbers of cells, we show that a common myeloid progenitor (CMP) is Sca-1(lo)lin(-)c-Kit(+)CD27(+)Flk-2(-) (SL-CMP; Sca-1(lo) CMP) and a granulocyte/macrophage progenitor (GMP) is Sca-1(lo)lin(-)c-Kit(+)CD27(+)Flk-2(+)CD150(-/lo) (SL-GMP; Sca-1(lo) GMP). We found that mast cell progenitor potential is present in the SL-CMP fraction, but not in the more differentiated SL-GMP population, and is more closely related to megakaryocyte/erythrocyte specification. Our data provide criteria for the prospective isolation of SL-CMP and SL-GMP and support the conclusion that mast cells are specified during hematopoiesis earlier than and independently from granulocytes.

  View details for DOI 10.1016/j.stem.2010.02.013

  View details for Web of Science ID 000276823300014

  View details for PubMedID 20362540

  View details for PubMedCentralID PMC2852254

• MiDReG: A method of mining developmentally regulated genes using Boolean implications PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sahoo, D., Seita, J., Bhattacharya, D., Inlay, M. A., Weissman, I. L., Plevritis, S. K., Dill, D. L. 2010; 107 (13): 5732-5737

  Abstract

  We present a method termed mining developmentally regulated genes (MiDReG) to predict genes whose expression is either activated or repressed as precursor cells differentiate. MiDReG does not require gene expression data from intermediate stages of development. MiDReG is based on the gene expression patterns between the initial and terminal stages of the differentiation pathway, coupled with "if-then" rules (Boolean implications) mined from large-scale microarray databases. MiDReG uses two gene expression-based seed conditions that mark the initial and the terminal stages of a given differentiation pathway and combines the statistically inferred Boolean implications from these seed conditions to identify the relevant genes. The method was validated by applying it to B-cell development. The algorithm predicted 62 genes that are expressed after the KIT+ progenitor cell stage and remain expressed through CD19+ and AICDA+ germinal center B cells. qRT-PCR of 14 of these genes on sorted B-cell progenitors confirmed that the expression of 10 genes is indeed stably established during B-cell differentiation. Review of the published literature of knockout mice revealed that of the predicted genes, 63.4% have defects in B-cell differentiation and function and 22% have a role in the B cell according to other experiments, and the remaining 14.6% are not characterized. Therefore, our method identified novel gene candidates for future examination of their role in B-cell development. These data demonstrate the power of MiDReG in predicting functionally important intermediate genes in a given developmental pathway that is defined by a mutually exclusive gene expression pattern.

  View details for DOI 10.1073/pnas.0913635107

  View details for PubMedID 20231483

• Coronary arteries form by developmental reprogramming of venous cells NATURE Red-Horse, K., Ueno, H., Weissman, I. L., Krasnow, M. A. 2010; 464 (7288): 549-U100

  Abstract

  Coronary artery disease is the leading cause of death worldwide. Determining the coronary artery developmental program could aid understanding of the disease and lead to new treatments, but many aspects of the process, including their developmental origin, remain obscure. Here we show, using histological and clonal analysis in mice and cardiac organ culture, that coronary vessels arise from angiogenic sprouts of the sinus venosus-the vein that returns blood to the embryonic heart. Sprouting venous endothelial cells dedifferentiate as they migrate over and invade the myocardium. Invading cells differentiate into arteries and capillaries; cells on the surface redifferentiate into veins. These results show that some differentiated venous cells retain developmental plasticity, and indicate that position-specific cardiac signals trigger their dedifferentiation and conversion into coronary arteries, capillaries and veins. Understanding this new reprogramming process and identifying the endogenous signals should suggest more natural ways of engineering coronary bypass grafts and revascularizing the heart.

  View details for DOI 10.1038/nature08873

  View details for Web of Science ID 000275974200039

  View details for PubMedID 20336138

  View details for PubMedCentralID PMC2924433

• Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Beerman, I., Bhattacharya, D., Zandi, S., Sigvardsson, M., Weissman, I. L., Bryder, D., Rossi, D. J. 2010; 107 (12): 5465-5470

  Abstract

  Aging of the hematopoietic stem cell compartment is believed to contribute to the onset of a variety of age-dependent blood cell pathophysiologies. Mechanistic drivers of hematopoietic stem cell (HSC) aging include DNA damage accumulation and induction of tumor suppressor pathways that combine to reduce the regenerative capacity of aged HSCs. Such mechanisms do not however account for the change in lymphoid and myeloid lineage potential characteristic of HSC aging, which is believed to be central to the decline of immune competence and predisposition to myelogenous diseases in the elderly. Here we have prospectively isolated functionally distinct HSC clonal subtypes, based on cell surface phenotype, bearing intrinsically different capacities to differentiate toward lymphoid and myeloid effector cells mediated by quantitative differences in lineage priming. Finally, we present data supporting a model in which clonal expansion of a class of intrinsically myeloid-biased HSCs with robust self-renewal potential is a central component of hematopoietic aging.

  View details for DOI 10.1073/pnas.1000834107

  View details for Web of Science ID 000275898300037

  View details for PubMedID 20304793

  View details for PubMedCentralID PMC2851806

• microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors, biased myeloid development, and acute myeloid leukemia JOURNAL OF EXPERIMENTAL MEDICINE Han, Y., Park, C. Y., Bhagat, G., Zhang, J., Wang, Y., Fan, J., Liu, M., Zou, Y., Weissman, I. L., Gu, H. 2010; 207 (3): 475-489

  Abstract

  The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.

  View details for DOI 10.1084/jem.20090831

  View details for Web of Science ID 000275593900005

  View details for PubMedID 20212066

  View details for PubMedCentralID PMC2839143

• Molecular Signatures of Quiescent, Mobilized and Leukemia-Initiating Hematopoietic Stem Cells PLOS ONE Forsberg, E. C., Passegue, E., Prohaska, S. S., Wagers, A. J., Koeva, M., Stuart, J. M., Weissman, I. L. 2010; 5 (1)

  Abstract

  Hematopoietic stem cells (HSC) are rare, multipotent cells capable of generating all specialized cells of the blood system. Appropriate regulation of HSC quiescence is thought to be crucial to maintain their lifelong function; however, the molecular pathways controlling stem cell quiescence remain poorly characterized. Likewise, the molecular events driving leukemogenesis remain elusive. In this study, we compare the gene expression profiles of steady-state bone marrow HSC to non-self-renewing multipotent progenitors; to HSC treated with mobilizing drugs that expand the HSC pool and induce egress from the marrow; and to leukemic HSC in a mouse model of chronic myelogenous leukemia. By intersecting the resulting lists of differentially regulated genes we identify a subset of molecules that are downregulated in all three circumstances, and thus may be particularly important for the maintenance and function of normal, quiescent HSC. These results identify potential key regulators of HSC and give insights into the clinically important processes of HSC mobilization for transplantation and leukemic development from cancer stem cells.

  View details for DOI 10.1371/journal.pone.0008785

  View details for Web of Science ID 000273779000014

  View details for PubMedID 20098702

• The origin and fate of yolk sac hematopoiesis: application of chimera analyses to developmental studies INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY Ueno, H., Weissman, I. L. 2010; 54 (6-7): 1019-1031

  Abstract

  During mammalian development, as exemplified by mice, hematopoietic cells first appear in the yolk sac blood islands, then in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region and the placenta, eventually seeding into liver, spleen and then bone marrow. The formation of hematopoietic stem cells from mesodermal precursors has finished by mid-fetal life. Once established, the hematopoietic system must supply blood cells to host circulation and tissues for the entire life of the animal. Easy access to hematopoietic cells has enabled a vast number of studies over the last several decades, and much is now understood about the different hematopoietic lineages, how they differentiate, and their derivation from immature progenitors. Yet to be elucidated are the following two intriguing questions: do yolk sac and AGM hematopoietic cells arise from a common precursor or from distinct precursor cells?; and what is the lineage relationship between blood and endothelial cells. In this review, we will survey the state of our current knowledge in these areas, and discuss the potential use of multicolor chimera analyses to elucidate unresolved questions.

  View details for DOI 10.1387/ijdb.093039hu

  View details for Web of Science ID 000282481100008

  View details for PubMedID 20711980

• Identification of the Earliest Committed Natural Killer Cell Progenitor in Murine Bone Marrow 10th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Fathman, J., Bhattacharya, D., Inlay, M., Seita, J., Weissman, I. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2010: S133–S133

  View details for DOI 10.1016/j.clim.2010.03.401

  View details for Web of Science ID 000277953700385

• Functional and Transcriptional Characterization of Human Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Myocardial Infarction PLOS ONE Li, Z., Wilson, K. D., Smith, B., Kraft, D. L., Jia, F., Huang, M., Xie, X., Robbins, R. C., Gambhir, S. S., Weissman, I. L., Wu, J. C. 2009; 4 (12)

  Abstract

  Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However, the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product, both of which can limit the future clinical application of hESC-ECs. Moreover, to fully understand the beneficial effects of stem cell therapy, investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time.In this study, we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover, our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods.Taken together, we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes, form functional vessels in vivo, and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future.

  View details for DOI 10.1371/journal.pone.0008443

  View details for Web of Science ID 000273180200002

  View details for PubMedID 20046878

  View details for PubMedCentralID PMC2795856

• Differential Contribution of Chemotaxis and Substrate Restriction to Segregation of Immature and Mature Thymocytes IMMUNITY Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. 2009; 31 (6): 986-998

  Abstract

  T cell development requires sequential localization of thymocyte subsets to distinct thymic microenvironments. To address mechanisms governing this segregation, we used two-photon microscopy to visualize migration of purified thymocyte subsets in defined microenvironments within thymic slices. Double-negative (CD4(-)8(-)) and double-positive (CD4(+)8(+)) thymocytes were confined to cortex where they moved slowly without directional bias. DP cells accumulated and migrated more rapidly in a specialized inner-cortical microenvironment, but were unable to migrate on medullary substrates. In contrast, CD4 single positive (SP) thymocytes migrated directionally toward the medulla, where they accumulated and moved very rapidly. Our results revealed a requisite two-step process governing CD4 SP cell medullary localization: the chemokine receptor CCR7 mediated chemotaxis of CD4 SP cells towards medulla, whereas a distinct pertussis-toxin sensitive pathway was required for medullary entry. These findings suggest that developmentally regulated responses to both chemotactic signals and specific migratory substrates guide thymocytes to specific locations in the thymus.

  View details for DOI 10.1016/j.immuni.2009.09.020

  View details for Web of Science ID 000273616400018

  View details for PubMedID 19962328

• Self-Renewal of the Long-Term Reconstituting Subset of Hematopoietic Stem Cells is Regulated by Ikaros STEM CELLS Papathanasiou, P., Attema, J. L., Karsunky, H., Hosen, N., Sontani, Y., Hoyne, G. F., Tunningley, R., Smale, S. T., Weissman, I. L. 2009; 27 (12): 3082-3092

  Abstract

  Hematopoietic stem cells (HSCs) are rare, ancestral cells that underlie the development, homeostasis, aging, and regeneration of the blood. Here we show that the chromatin-associated protein Ikaros is a crucial self-renewal regulator of the long-term (LT) reconstituting subset of HSCs. Ikaros, and associated family member proteins, are highly expressed in self-renewing populations of stem cells. Ikaros point mutant mice initially develop LT-HSCs with the surface phenotype cKit+Thy1.1(lo)Lin(-/lo)Sca1+Flk2-CD150+ during fetal ontogeny but are unable to maintain this pool, rapidly losing it within two days of embryonic development. A synchronous loss of megakaryocyte/erythrocyte progenitors results, along with a fatal, fetal anemia. At this time, mutation of Ikaros exerts a differentiation defect upon common lymphoid progenitors that cannot be rescued with an ectopic Notch signal in vitro, with hematopoietic cells preferentially committing to the NK lineage. Althoughdispensable for the initial embryonic development of blood, Ikaros is clearly needed for maintenance of this tissue. Achieving successful clinical tissue regeneration necessitates understanding degeneration, and these data provide a striking example by a discrete genetic lesion in the cells underpinning tissue integrity during a pivotal timeframe of organogenesis.

  View details for DOI 10.1002/stem.232

  View details for Web of Science ID 000273569800021

  View details for PubMedID 19816952

  View details for PubMedCentralID PMC3401049

• Niche recycling through division-independent egress of hematopoietic stem cells JOURNAL OF EXPERIMENTAL MEDICINE Bhattacharya, D., Czechowicz, A., Ooi, A. G., Rossi, D. J., Bryder, D., Weissman, I. L. 2009; 206 (12): 2837-2850

  Abstract

  Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. To explain this apparent paradox, we calculated, through cell surface phenotyping and transplantation of unfractionated blood, that approximately 1-5% of the total pool of HSCs enters into the circulation each day. Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Consistent with this, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 d led to significantly higher levels of engraftment than did large, single-bolus transplantations of the same total number of HSCs. These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.

  View details for DOI 10.1084/jem.20090778

  View details for Web of Science ID 000272079300020

  View details for PubMedID 19887396

  View details for PubMedCentralID PMC2806613

• Single Cell Phospho-Flow Analysis of Cytokine Stimulation in Human Hematopoietic Progenitors Reveals That G-CSF Acts Directly On Human Hematopoietic Stem Cells. 51st Annual Meeting and Exposition of the American-Society-of-Hematology Gibbs, K., Gilbert, P., Weissman, I. L., Blau, H. M., Nolan, G. P., Majeti, R. AMER SOC HEMATOLOGY. 2009: 1398–98

  View details for Web of Science ID 000272725804281

• NPM1 Haploinsufficiency Results in Increased Numbers of Hematopoietic Stem Cells and Progenitor Cells 51st Annual Meeting and Exposition of the American-Society-of-Hematology Raval, A., Park, C. Y., Pang, W. W., Kusler, B., Sridhar, K. J., Gotlib, J. R., Greenberg, P. L., Weissman, I. L., Mitchell, B. S. AMER SOC HEMATOLOGY. 2009: 307–

  View details for Web of Science ID 000272725800739

• Niche Recycling through Division-Independent Egress of Hematopoietic Stem Cells. 51st Annual Meeting and Exposition of the American-Society-of-Hematology Czechowicz, A., Bhattacharya, D., Ooi, L., Rossi, D. J., Bryder, D., Weissman, I. L. AMER SOC HEMATOLOGY. 2009: 37–37

  View details for Web of Science ID 000272725800080

• In vivo Kinetics of Embryonic Stem Cell Viability Following Transplantation Into the Injured Murine Myocardium 82nd National Conference and Exhibitions and Scientific Sessions of the American-Heart-Association Chung, J., Kee, K., Barral, J. K., Dash, R., Weissman, I., Quertermous, T., Robbins, R. C., Nishimura, D. G., Reijo-Pera, R. A., Yang, P. C. LIPPINCOTT WILLIAMS & WILKINS. 2009: S310–S311

  View details for Web of Science ID 000271831500052

• Ly6d marks the earliest stage of B-cell specification and identifies the branchpoint between B-cell and T-cell development GENES & DEVELOPMENT Inlay, M. A., Bhattacharya, D., Sahoo, D., Serwold, T., Seita, J., Karsunky, H., Plevritis, S. K., Dill, D. L., Weissman, I. L. 2009; 23 (20): 2376-2381

  Abstract

  Common lymphoid progenitors (CLPs) clonally produce both B- and T-cell lineages, but have little myeloid potential in vivo. However, some studies claim that the upstream lymphoid-primed multipotent progenitor (LMPP) is the thymic seeding population, and suggest that CLPs are primarily B-cell-restricted. To identify surface proteins that distinguish functional CLPs from B-cell progenitors, we used a new computational method of Mining Developmentally Regulated Genes (MiDReG). We identified Ly6d, which divides CLPs into two distinct populations: one that retains full in vivo lymphoid potential and produces more thymocytes at early timepoints than LMPP, and another that behaves essentially as a B-cell progenitor.

  View details for DOI 10.1101/gad.1836009

  View details for PubMedID 19833765

• Evaluation of the Long-Term Reconstituting Subset of Hematopoietic Stem Cells with CD150 STEM CELLS Papathanasiou, P., Attema, J. L., Karsunky, H., Xu, J., Smale, S. T., Weissman, I. L. 2009; 27 (10): 2498-2508

  Abstract

  Blood is a tissue with a high cell turnover rate that is constantly being replenished by bone marrow hematopoietic stem cells (HSCs) seeded during fetal ontogeny from the liver. Here we show that the long-term (LT) reconstituting subset of cKit(+)Thy1.1(lo)Lin(-/lo)Sca1(+)Flk2(-) HSCs is CD150(+). HSCs sourced from the fetal liver show LT, multilineage engraftment from E14.5 onward, and the CD150 cell surface molecule can readily substitute Thy1.1 as a positive marker of LT-HSCs in this tissue. From both fetal liver and adult bone marrow, cKit(+)Thy1.1(lo)Lin(-/lo)Sca1(+)Flk2(-) CD150(+) cells exhibit robust LT competitive engraftment, self-renewal, multilineage differentiation capacity, and an accessible chromatin configuration consistent with high expression of erythroid/megakaryoid genes in purified cell subsets. Our data show that, with appropriate combinations of cell surface markers, stem cells can be accurately isolated to high purity and characterized. This is important for the clarification of lineage relationships and the identification of bona fide regulators of stem cell self-renewal and differentiation both in normal and neoplastic tissues.

  View details for DOI 10.1002/stem.170

  View details for Web of Science ID 000271830200013

  View details for PubMedID 19593793

  View details for PubMedCentralID PMC2783507

• Manganese-Guided Cellular MRI of Human Embryonic Stem Cell and Human Bone Marrow Stromal Cell Viability MAGNETIC RESONANCE IN MEDICINE Yamada, M., Gurney, P. T., Chung, J., Kundu, P., Drukker, M., Smith, A. K., Weissman, I. L., Nishimura, D., Robbins, R. C., Yang, P. C. 2009; 62 (4): 1047-1054

  Abstract

  This study investigated the ability of MnCl(2) as a cellular MRI contrast agent to determine the in vitro viability of human embryonic stem cells (hESC) and human bone marrow stromal cells (hBMSC). Basic MRI parameters including T(1) and T(2) values of MnCl(2)-labeled hESC and hBMSC were measured and viability signal of manganese (Mn(2+))-labeled cells was validated. Furthermore, the biological activity of Ca(2+)-channels was modulated utilizing both Ca(2+)-channel agonist and antagonist to evaluate concomitant signal changes. Metabolic effects of MnCl(2)-labeling were also assessed using assays for cell viability, proliferation, and apoptosis. Finally, in vivo Mn(2+)-guided MRI of the transplanted hESC was successfully achieved and validated by bioluminescence imaging.

  View details for DOI 10.1002/mrm.22071

  View details for PubMedID 19526508

• Neuroprotection of Host Cells by Human Central Nervous System Stem Cells in a Mouse Model of Infantile Neuronal Ceroid Lipofuscinosis CELL STEM CELL Tamaki, S. J., Jacobs, Y., Dohse, M., Capela, A., Cooper, J. D., Reitsma, M., He, D., Tushinski, R., Belichenko, P. V., Salehi, A., Mobley, W., Gage, F. H., Huhn, S., Tsukamoto, A. S., Weissman, I. L., Uchida, N. 2009; 5 (3): 310-319

  Abstract

  Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disease caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Ppt1 knockout mice display hallmarks of INCL and mimic the human pathology: accumulation of lipofuscin, degeneration of CNS neurons, and a shortened life span. Purified non-genetically modified human CNS stem cells, grown as neurospheres (hCNS-SCns), were transplanted into the brains of immunodeficient Ppt1(-/)(-) mice where they engrafted robustly, migrated extensively, and produced sufficient levels of PPT1 to alter host neuropathology. Grafted mice displayed reduced autofluorescent lipofuscin, significant neuroprotection of host hippocampal and cortical neurons, and delayed loss of motor coordination. Early intervention with cellular transplants of hCNS-SCns into the brains of INCL patients may supply a continuous and long-lasting source of the missing PPT1 and provide some therapeutic benefit through protection of endogenous neurons. These data provide the experimental basis for human clinical trials with these banked hCNS-SCns.

  View details for DOI 10.1016/j.stem.2009.05.022

  View details for Web of Science ID 000272545700017

  View details for PubMedID 19733542

• Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, K. S., Espinosa, I., Chao, M., Wong, D., Ailles, L., Diehn, M., Gill, H., Presti, J., Chang, H. Y., van de Rijn, M., Shortliffe, L., Weissman, I. L. 2009; 106 (33): 14016-14021

  Abstract

  Major clinical issues in bladder cancer include the identification of prediction markers and novel therapeutic targets for invasive bladder cancer. In the current study, we describe the isolation and characterization of a tumor-initiating cell (T-IC) subpopulation in primary human bladder cancer, based on the expression of markers similar to that of normal bladder basal cells (Lineage-CD44(+)CK5(+)CK20(-)). The bladder T-IC subpopulation was defined functionally by its enriched ability to induce xenograft tumors in vivo that recapitulated the heterogeneity of the original tumor. Further, molecular analysis of more than 300 bladder cancer specimens revealed heterogeneity among activated oncogenic pathways in T-IC (e.g., 80% Gli1, 45% Stat3, 10% Bmi-1, and 5% beta-catenin). Despite this molecular heterogeneity, we identified a unique bladder T-IC gene signature by gene chip analysis. This T-IC gene signature, which effectively distinguishes muscle-invasive bladder cancer with worse clinical prognosis from non-muscle-invasive (superficial) cancer, has significant clinical value. It also can predict the progression of a subset of recurring non-muscle-invasive cancers. Finally, we found that CD47, a protein that provides an inhibitory signal for macrophage phagocytosis, is highly expressed in bladder T-ICs compared with the rest of the tumor. Blockade of CD47 by a mAb resulted in macrophage engulfment of bladder cancer cells in vitro. In summary, we have identified a T-IC subpopulation with potential prognostic and therapeutic value for invasive bladder cancer.

  View details for DOI 10.1073/pnas.0906549106

  View details for PubMedID 19666525

• The ISSCR: who are we and where are we going? Cell stem cell Weissman, I. 2009; 5 (2): 151-153

  View details for DOI 10.1016/j.stem.2009.07.013

  View details for PubMedID 19664988

• A NEUROSURGEON'S GUIDE TO STEM CELLS, CANCER STEM CELLS, AND BRAIN TUMOR STEM CELLS NEUROSURGERY Cheshier, S. H., Kalani, M. Y., Lim, M., Ailles, L., Huhn, S. L., Weissman, I. L. 2009; 65 (2): 237-249

  Abstract

  Stem cells and their potential applications have become the forefront of scientific, political, and ethical discourse. Whereas stem cells were long accepted as units of development and evolution, it is now becoming increasingly clear that they are also units of oncogenesis. Although the field of stem cell biology is expanding at an astounding rate, the data attained are not readily translatable for the physicians who may eventually deliver these tools to patients. Herein, we provide a brief review of stem cell and cancer stem cell biology and highlight the scientific and clinical implications of recent findings regarding the presence of cancer-forming stem cells in brain tumors.

  View details for DOI 10.1227/01.NEU.0000349921.14519.2A

  View details for Web of Science ID 000268523200005

  View details for PubMedID 19625901

• CD47 Is an Adverse Prognostic Factor and Therapeutic Antibody Target on Human Acute Myeloid Leukemia Stem Cells CELL Majeti, R., Chao, M. P., Alizadeh, A. A., Pang, W. W., Jaiswal, S., Gibbs, K. D., van Rooijen, N., Weissman, I. L. 2009; 138 (2): 286-299

  Abstract

  Acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by a subset of self-renewing leukemia stem cells (LSC). We hypothesized that increased CD47 expression on human AML LSC contributes to pathogenesis by inhibiting their phagocytosis through the interaction of CD47 with an inhibitory receptor on phagocytes. We found that CD47 was more highly expressed on AML LSC than their normal counterparts, and that increased CD47 expression predicted worse overall survival in three independent cohorts of adult AML patients. Furthermore, blocking monoclonal antibodies directed against CD47 preferentially enabled phagocytosis of AML LSC and inhibited their engraftment in vivo. Finally, treatment of human AML LSC-engrafted mice with anti-CD47 antibody depleted AML and targeted AML LSC. In summary, increased CD47 expression is an independent, poor prognostic factor that can be targeted on human AML stem cells with blocking monoclonal antibodies capable of enabling phagocytosis of LSC.

  View details for DOI 10.1016/j.cell.2009.05.045

  View details for PubMedID 19632179

• CD47 Is Upregulated on Circulating Hematopoietic Stem Cells and Leukemia Cells to Avoid Phagocytosis CELL Jaiswal, S., Jamieson, C. H., Pang, W. W., Park, C. Y., Chao, M. P., Majeti, R., Traver, D., van Rooijen, N., Weissman, I. L. 2009; 138 (2): 271-285

  Abstract

  Macrophages clear pathogens and damaged or aged cells from the blood stream via phagocytosis. Cell-surface CD47 interacts with its receptor on macrophages, SIRPalpha, to inhibit phagocytosis of normal, healthy cells. We find that mobilizing cytokines and inflammatory stimuli cause CD47 to be transiently upregulated on mouse hematopoietic stem cells (HSCs) and progenitors just prior to and during their migratory phase, and that the level of CD47 on these cells determines the probability that they are engulfed in vivo. CD47 is also constitutively upregulated on mouse and human myeloid leukemias, and overexpression of CD47 on a myeloid leukemia line increases its pathogenicity by allowing it to evade phagocytosis. We conclude that CD47 upregulation is an important mechanism that provides protection to normal HSCs during inflammation-mediated mobilization, and that leukemic progenitors co-opt this ability in order to evade macrophage killing.

  View details for DOI 10.1016/j.cell.2009.05.046

  View details for Web of Science ID 000268277000010

  View details for PubMedID 19632178

  View details for PubMedCentralID PMC2775564

• Expression of AA4.1 marks lymphohematopoietic progenitors in early mouse development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yamane, T., Hosen, N., Yamazaki, H., Weissman, I. L. 2009; 106 (22): 8953-8958

  Abstract

  The hematopoietic system of mice is established during the early to midgestational stage of development. However, the earliest lymphohematopoietic progenitors that appear during mouse development have been less well characterized compared with the hematopoietic stem cell compartment of fetal liver and bone marrow. We isolated the earliest lymphohematopoietic progenitors by using embryonic stem (ES) cell culture in vitro. Cells with the c-Kit(+)Lin(-) cell surface phenotype were present abundantly in ES cells cocultured with stromal cell lines. We further separated the cells into two distinct cell subsets based on AA4.1 expression. Although AA4.1(+) and AA4.1(-) cells had equivalent potency to generate myeloid cell lineages, the lymphoid potential in ES-cell-derived cells was largely restricted to the cells expressing AA4.1. The same cell type was present abundantly in the early yolk sac and in fewer numbers (approximately 5% of that in the yolk sac) in the caudal half of the developing embryos. These data suggest that AA4.1 is a cell surface marker that can identify the earliest lymphohematopoietic progenitors in mouse development.

  View details for DOI 10.1073/pnas.0904090106

  View details for Web of Science ID 000266580500033

  View details for PubMedID 19458045

  View details for PubMedCentralID PMC2690003

• Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nature medicine Ootani, A., Li, X., Sangiorgi, E., Ho, Q. T., Ueno, H., Toda, S., Sugihara, H., Fujimoto, K., Weissman, I. L., Capecchi, M. R., Kuo, C. J. 2009; 15 (6): 701-706

  Abstract

  The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

  View details for DOI 10.1038/nm.1951

  View details for PubMedID 19398967

  View details for PubMedCentralID PMC2919216

• Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche NATURE MEDICINE Ootani, A., Li, X., Sangiorgi, E., Ho, Q. T., Ueno, H., Toda, S., Sugihara, H., Fujimoto, K., Weissman, I. L., Capecchi, M. R., Kuo, C. J. 2009; 15 (6): 1-U140

  Abstract

  The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

  View details for DOI 10.1038/nm.1951

  View details for Web of Science ID 000266731600031

  View details for PubMedCentralID PMC2919216

• Automated microfluidic chromatin immunoprecipitation from 2,000 cells LAB ON A CHIP Wu, A. R., Hiatt, J. B., Lu, R., Attema, J. L., Lobo, N. A., Weissman, I. L., Clarke, M. F., Quake, S. R. 2009; 9 (10): 1365-1370

  Abstract

  Chromatin immunoprecipitation (ChIP) is a powerful assay used to probe DNA-protein interactions. Traditional methods of implementing this assay are lengthy, cumbersome and require a large number of cells, making it difficult to study rare cell types such as certain cancer and stem cells. We have designed a microfluidic device to perform sensitive ChIP analysis on low cell numbers in a rapid, automated fashion while preserving the specificity of the assay. Comparing ChIP results for two modified histone protein targets, we showed our automated microfluidic ChIP (AutoChIP) from 2,000 cells to be comparable to that of conventional ChIP methods using 50,000-500,000 cells. This technology may provide a solution to the need for a high sensitivity, rapid, and automated ChIP assay, and in doing so facilitate the use of ChIP for many interesting and valuable applications.

  View details for DOI 10.1039/b819648f

  View details for Web of Science ID 000268227400008

  View details for PubMedID 19417902

• Association of reactive oxygen species levels and radioresistance in cancer stem cells NATURE Diehn, M., Cho, R. W., Lobo, N. A., Kalisky, T., Dorie, M. J., Kulp, A. N., Qian, D., Lam, J. S., Ailles, L. E., Wong, M., Joshua, B., Kaplan, M. J., Wapnir, I., Dirbas, F. M., Somlo, G., Garberoglio, C., Paz, B., Shen, J., Lau, S. K., Quake, S. R., Brown, J. M., Weissman, I. L., Clarke, M. F. 2009; 458 (7239): 780-U123

  Abstract

  The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.

  View details for DOI 10.1038/nature07733

  View details for PubMedID 19194462

• Glycogen synthase kinase 3 beta missplicing contributes to leukemia stem cell generation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Abrahamsson, A. E., Geron, I., Gotlib, J., Dao, K. T., Barroga, C. F., Newton, I. G., Giles, F. J., Durocher, J., Creusot, R. S., Karimi, M., Jones, C., Zehnder, J. L., Keating, A., Negrin, R. S., Weissman, I. L., Jamieson, C. H. 2009; 106 (10): 3925-3929

  Abstract

  Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of beta-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving beta-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus are enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/beta-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3beta, axin 1, beta-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3beta kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3beta have enhanced beta-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3beta reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3beta in GMP LSC, enabling unphosphorylated beta-catenin to participate in LSC self-renewal. Missplicing of GSK3beta represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.

  View details for DOI 10.1073/pnas.0900189106

  View details for Web of Science ID 000264036900051

  View details for PubMedID 19237556

  View details for PubMedCentralID PMC2646624

• Dysregulated gene expression networks in human acute myelogenous leukemia stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Majetl, R., Becker, M. W., Tian, Q., Lee, T. M., Yan, X., Liu, R., Chiang, J., Hood, L., Clarke, M. F., Weissman, I. L. 2009; 106 (9): 3396-3401

  Abstract

  We performed the first genome-wide expression analysis directly comparing the expression profile of highly enriched normal human hematopoietic stem cells (HSC) and leukemic stem cells (LSC) from patients with acute myeloid leukemia (AML). Comparing the expression signature of normal HSC to that of LSC, we identified 3,005 differentially expressed genes. Using 2 independent analyses, we identified multiple pathways that are aberrantly regulated in leukemic stem cells compared with normal HSC. Several pathways, including Wnt signaling, MAP Kinase signaling, and Adherens Junction, are well known for their role in cancer development and stem cell biology. Other pathways have not been previously implicated in the regulation of cancer stem cell functions, including Ribosome and T Cell Receptor Signaling pathway. This study demonstrates that combining global gene expression analysis with detailed annotated pathway resources applied to highly enriched normal and malignant stem cell populations, can yield an understanding of the critical pathways regulating cancer stem cells.

  View details for DOI 10.1073/pnas.0900089106

  View details for PubMedID 19218430

• Imaging of STAT3 Signaling Pathway During Mouse Embryonic Stem Cell Differentiation STEM CELLS AND DEVELOPMENT Xie, X., Chan, K. S., Cao, F., Huang, M., Li, Z., Lee, A., Weissman, I. L., Wu, J. C. 2009; 18 (2): 205-214

  Abstract

  Signal transducers and activators of transcription 3 (STAT3) is a pleiotropic transcription factor involved in a variety of physiological processes. STAT3 acts as a key transcriptional determinant of mouse embryonic stem (ES) cell self-renewal and plays a pivotal function in early mammalian embryogenesis because the development of many organs requires STAT3 activation. However, little is known about the role of STAT3 function during ES cell differentiation. To answer this question, we built a lentiviral construct with 7-repeat STAT3-binding sequence (enhancer) and minimal TA (promoter) driving renilla luciferase and monomeric red fluorescence protein (Rluc-mRFP), followed by a constitutive cytomegalovirus promoter driving green fluorescent protein as a selection marker. The specificity of our custom-designed 7-repeat STAT3 reporter construct was first confirmed by cotransfection with constitutively active version of STAT3 (STAT3C) into human embryonic kidney 293T cells. Next, a mouse ES cell line stably transduced with STAT3 reporter construct was isolated. This ES cell line showed a tight response in reporter gene expression with leukemia inhibitory factor (LIF) induction and was chosen as a developmental model for the STAT3 functional study. Using serial noninvasive bioluminescence imaging, we showed that the onset of embryoid body (EB) formation involved inhibition of STAT3 activity. However, during differentiation, STAT3 activity steadily increased from day 5 to 14 and was reduced by day 21. STAT3 activity was also confirmed separately by Western blots. Finally, phosphorylation of STAT3 was also found to correspond with cardiomyocyte differentiation. In summary, this is the first study to monitor real-time STAT3 activity during ES cell differentiation. This genetically modified line can be used to study the biological role of STAT3 during ES cell differentiation into different derivatives.

  View details for DOI 10.1089/scd.2008.0152

  View details for Web of Science ID 000264171300002

  View details for PubMedID 18576943

  View details for PubMedCentralID PMC3133564

• E2A proteins maintain the hematopoietic stem cell pool and promote the maturation of myelolymphoid and myeloerythroid progenitors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Semerad, C. L., Mercer, E. M., Inlay, M. A., Weissman, I. L., Murre, C. 2009; 106 (6): 1930-1935

  Abstract

  Hematopoiesis is a tightly controlled process maintained by a small pool of hematopoietic stem cells (HSCs). Here, we demonstrate that the LT-HSC, MPP, premegakaryocytic/erythroid, Pre CFU-E, Pre GM, MkP, and granulocyte-macrophage compartments were all significantly reduced in E2A-deficient bone marrow. Despite a severe depletion of erythroid progenitors, the erythrocyte and megakaryocyte compartments were equivalent in E2A-deficient bone marrow as compared with wild-type mice. E2A-deficient HSCs also failed to efficiently maintain the HSC pool on serial transplantation, and we demonstrate that the E2A proteins regulate cell cycle progression of HSCs by regulating the expression of p21(Cip1), p27(Kip1), and the thrombopoietin receptor, known regulators of HSC self-renewal activity. Based on these observations, we propose that the E2A proteins promote the developmental progression of the entire spectrum of early hematopoietic progenitors and to suppress an erythroid specific program of gene expression in alternative cell lineages. Last, the data mechanistically link E2A, cell cycle regulators, and the maintenance of the HSC pool in a common pathway.

  View details for DOI 10.1073/pnas.0808866106

  View details for Web of Science ID 000263252500048

  View details for PubMedID 19181846

• Cancer Stem Cell-Directed Therapies: Recent Data From the Laboratory and Clinic MOLECULAR THERAPY Park, C. Y., Tseng, D., Weissman, I. L. 2009; 17 (2): 219-230

  Abstract

  Cancer stem cells (CSCs) are defined by their ability to (i) fully recapitulate the tumor of origin when transplanted into immunodeficient mouse hosts, and (ii) self-renew, demonstrated by their ability to be serially transplanted. These properties suggest that CSCs are required for tumor maintenance and metastasis; thus, it has been predicted that CSC elimination is required for cure. This prediction has profoundly altered paradigms for cancer research, compelling investigators to prospectively isolate CSCs to characterize the molecular pathways regulating their behavior. Many potential strategies for CSC-directed therapy have been proposed, but few studies have rigorously demonstrated their efficacy using in vivo models. Herein, we highlight recent studies that demonstrate the utility of CSC-directed therapies and discuss the implications of the CSC hypothesis to experimental design and therapeutic strategies.

  View details for DOI 10.1038/mt.2008.254

  View details for Web of Science ID 000263287100008

  View details for PubMedID 19066601

  View details for PubMedCentralID PMC2835048

• Endochondral ossification is required for haematopoietic stem-cell niche formation NATURE Chan, C. K., Chen, C., Luppen, C. A., Kim, J., DeBoer, A. T., Wei, K., Helms, J. A., Kuo, C. J., Kraft, D. L., Weissman, I. L. 2009; 457 (7228): 490-U9

  Abstract

  Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

  View details for DOI 10.1038/nature07547

  View details for PubMedID 19078959

• CX(3)CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival BLOOD Landsman, L., Bar-On, L., Zernecke, A., Kim, K., Krauthgamer, R., Shagdarsuren, E., Lira, S. A., Weissman, I. L., Weber, C., Jung, S. 2009; 113 (4): 963-972

  Abstract

  CX(3)CR1 is a chemokine receptor with a single ligand, the membrane-tethered chemokine CX(3)CL1 (fractalkine). All blood monocytes express CX(3)CR1, but its levels differ between the main 2 subsets, with human CD16(+) and murine Gr1(low) monocytes being CX(3)CR1(hi). Here, we report that absence of either CX(3)CR1 or CX(3)CL1 results in a significant reduction of Gr1(low) blood monocyte levels under both steady-state and inflammatory conditions. Introduction of a Bcl2 transgene restored the wild-type phenotype, suggesting that the CX(3)C axis provides an essential survival signal. Supporting this notion, we show that CX(3)CL1 specifically rescues cultured human monocytes from induced cell death. Human CX(3)CR1 gene polymorphisms are risk factors for atherosclerosis and mice deficient for the CX(3)C receptor or ligand are relatively protected from atherosclerosis development. However, the mechanistic role of CX(3)CR1 in atherogenesis remains unclear. Here, we show that enforced survival of monocytes and plaque-resident phagocytes, including foam cells, restored atherogenesis in CX(3)CR1-deficent mice. The fact that CX(3)CL1-CX(3)CR1 interactions confer an essential survival signal, whose absence leads to increased death of monocytes and/or foam cells, might provide a mechanistic explanation for the role of the CX(3)C chemokine family in atherogenesis.

  View details for DOI 10.1182/blood-2008-07-170787

  View details for Web of Science ID 000262646200027

  View details for PubMedID 18971423

• Reductive isolation from bone marrow and blood implicates common lymphoid progenitors as the major source of thymopoiesis BLOOD Serwold, T., Ehrlich, L. I., Weissman, I. L. 2009; 113 (4): 807-815

  Abstract

  Ongoing thymopoiesis requires continual seeding from progenitors that reside within the bone marrow (BM), but the identity of the most proximate prethymocytes has remained controversial. Here we take a comprehensive approach to prospectively identify the major source of thymocyte progenitors that reside within the BM and blood, and find that all thymocyte progenitor activity resides within a rare Flk2(+)CD27(+) population. The BM Flk2(+)CD27(+) subset is predominantly composed of common lymphoid progenitors (CLPs) and multipotent progenitors. Of these 2 populations, only CLPs reconstitute thymopoiesis rapidly after intravenous injection. In contrast, multipotent progenitor-derived cells reconstitute the thymus with delayed kinetics only after they have reseeded the BM, self-renewed, and generated CLPs. These results identify CLPs as the major source of thymocyte progenitors within the BM.

  View details for DOI 10.1182/blood-2008-08-173682

  View details for Web of Science ID 000262646200009

  View details for PubMedID 18927436

• Two-step oligoclonal development of male germ cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ueno, H., Turnbull, B. B., Weissman, I. L. 2009; 106 (1): 175-180

  Abstract

  During mouse development, primordial germ cells (PGCs) that give rise to the entire germ line are first identified within the proximal epiblast. However, long-term tracing of the fate of the cells has not been done wherein all cells in and around the germ-cell lineage are identified. Also, quantitative estimates of the number of founder PGCs using different models have come up with various numbers. Here, we use tetrachimeric mice to show that the progenitor numbers for the entire germ line in adult testis, and for the initiating embryonic PGCs, are both 4 cells. Although they proliferate to form polyclonal germ-cell populations in fetal and neonatal testes, germ cells that actually contribute to adult spermatogenesis originate from a small number of secondary founder cells that originate in the fetal period. The rest of the "deciduous" germ cells are lost, most likely by apoptosis, before the reproductive period. The second "actual" founder germ cells generally form small numbers of large monoclonal areas in testes by the reproductive period. Our results also demonstrate that there is no contribution of somatic cells to the male germ cell pool during development or in adulthood. These results suggest a model of 2-step oligoclonal development of male germ cells in mice, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation.

  View details for DOI 10.1073/pnas.0810325105

  View details for Web of Science ID 000262263900034

  View details for PubMedID 19098099

  View details for PubMedCentralID PMC2629224

• The Adhesion Molecule Esam1 Is a Novel Hematopoietic Stem Cell Marker STEM CELLS Ooi, A. G., Karsunky, H., Majeti, R., Butz, S., Vestweber, D., Ishida, T., Quertermous, T., Weissman, I. L., Forsberg, E. C. 2009; 27 (3): 653-661

  Abstract

  Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12-14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1(-/-) mice do not exhibit severe hematopoietic defects; however, Esam1(-/-) BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1(-/-) mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.

  View details for DOI 10.1634/stemcells.2008-0824

  View details for PubMedID 19074415

• Biology of hematopoietic stem and progenitor cells Thomas' Hematopoietic Cell Transplantation Prohaska, S., Weissman, I. Wiley-Blackwell. 2009; 4: 36–63
• Identification of Conserved Gene Expression Programs in Epithelial Cancer Stem Cells 51st Annual Meeting of the American-Society-for-Radiation-Oncology (ASTRO) Diehn, M., Cho, R. W., Ailles, L., Lam, J. S., Kaplan, M. J., Somlo, G., Weissman, I. L., Clarke, M. F. ELSEVIER SCIENCE INC. 2009: S544–S545

  View details for Web of Science ID 000270573602118

• Hematopoietic Stem and Progenitor Cells and the Inflammatory Response 6th International Cancer Vaccine Symposium Jaiswal, S., Weissman, I. L. BLACKWELL PUBLISHING. 2009: 118–121

  Abstract

  Cells of the vertebrate immune system are continuously regenerated by division of hematopoietic stem cells (HSCs) into differentiated effector cells. Classically, HSCs were thought to reside primarily in the bone marrow niche where they produced mature progeny that migrated from the marrow to repopulate the peripheral immune system. However, emerging evidence has established that hematopoietic stem and progenitor cells (HSPCs) are themselves mobile and able to repopulate ectopic niches and contribute more directly to inflammatory responses in the periphery. How the HSPCs remain immune to destruction in a toxic inflammatory milieu is unknown.

  View details for Web of Science ID 000271828500015

  View details for PubMedID 19769744

• Heme oxygenase-1 deficiency leads to disrupted response to acute stress in stem cells and progenitors BLOOD Cao, Y., Wagers, A. J., Karsunky, H., Zhao, H., Reeves, R., Wong, R. J., Stevenson, D. K., Weissman, I. L., Contag, C. H. 2008; 112 (12): 4494-4502

  Abstract

  An effective response to extreme hematopoietic stress requires an extreme elevation in hematopoiesis and preservation of hematopoietic stem cells (HSCs). These diametrically opposed processes are likely to be regulated by genes that mediate cellular adaptation to physiologic stress. Herein, we show that heme oxygenase-1 (HO-1), the inducible isozyme of heme degradation, is a key regulator of these processes. Mice lacking one allele of HO-1 (HO-1(+/-)) showed accelerated hematopoietic recovery from myelotoxic injury, and HO-1(+/-) HSCs repopulated lethally irradiated recipients with more rapid kinetics. However, HO-1(+/-) HSCs were ineffective in radioprotection and serial repopulation of myeloablated recipients. Perturbations in key stem cell regulators were observed in HO-1(+/-) HSCs and hematopoietic progenitors (HPCs), which may explain the disrupted response of HO-1(+/-) HPCs and HPCs to acute stress. Control of stem cell stress response by HO-1 presents opportunities for metabolic manipulation of stem cell-based therapies.

  View details for DOI 10.1182/blood-2007-12-127621

  View details for PubMedID 18509090

• In Vivo Serial Evaluation of Superparamagnetic Iron-Oxide Labeled Stem Cells by Off-Resonance Positive Contrast MAGNETIC RESONANCE IN MEDICINE Suzuki, Y., Cunningham, C. H., Noguchi, K., Chen, I. Y., Weissman, I. L., Yeung, A. C., Robbins, R. C., Yang, P. C. 2008; 60 (6): 1269-1275

  Abstract

  MRI is emerging as a diagnostic modality to track iron-oxide-labeled stem cells. This study investigates whether an off-resonance (OR) pulse sequence designed to generate positive contrast at 1.5T can assess the location, quantity, and viability of delivered stem cells in vivo. Using mouse embryonic stem cell transfected with luciferase reporter gene (luc-mESC), multimodality validation of OR signal was conducted to determine whether engraftment parameters of superparamagnetic iron-oxide labeled luc-mESC (SPIO-luc-mESC) could be determined after cell transplantation into the mouse hindlimb. A significant increase in signal- and contrast-to-noise of the SPIO-luc-mESC was achieved with the OR technique when compared to a gradient recalled echo (GRE) sequence. A significant correlation between the quantity of SPIO-luc-mESC and OR signal was observed immediately after transplantation (R(2) = 0.74, P < 0.05). The assessment of transplanted cell viability by bioluminescence imaging (BLI) showed a significant increase of luciferase activities by day 16, while the MRI signal showed no difference. No significant correlation between BLI and MRI signals of cell viability was observed. In conclusion, using an OR sequence the precise localization and quantitation of SPIO-labeled stem cells in both space and time were possible. However, the OR sequence did not allow evaluation of cell viability.

  View details for DOI 10.1002/mrm.21816

  View details for Web of Science ID 000261225100001

  View details for PubMedID 19030159

  View details for PubMedCentralID PMC2597338

• ABT-737 Targets Intrinsic Apoptosis during Cooperation of BCL-2 and Oncogenic NRAS in An in Vivo Progression Model of Myelodysplasia/Acute Myeloid Leukaemia 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Omidvar, N., Beurlet, S., Le Pogam, C., Janin, A., Leboeuf, C., Soulie, A., Setterblad, N., Noguera, M., Sarda-Mantel, L., Merlet, P., Pla, M., Kogan, S. C., Weissman, I. L., Konopleva, M., Bormann, W., Andreeff, M., Chomienne, C., Padua, R. A. AMER SOC HEMATOLOGY. 2008: 314–14

  View details for Web of Science ID 000262104701072

• Wnt-mediated self-renewal of neural stem/progenitor cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kalani, M. Y., Cheshier, S. H., Cord, B. J., Bababeygy, S. R., Vogel, H., Weissman, I. L., Palmer, T. D., Nusse, R. 2008; 105 (44): 16970-16975

  Abstract

  In this work we have uncovered a role for Wnt signaling as an important regulator of stem cell self-renewal in the developing brain. We identified Wnt-responsive cells in the subventricular zone of the developing E14.5 mouse brain. Responding cell populations were enriched for self-renewing stem cells in primary culture, suggesting that Wnt signaling is a hallmark of self-renewing activity in vivo. We also tested whether Wnt signals directly influence neural stem cells. Using inhibitors of the Wnt pathway, we found that Wnt signaling is required for the efficient cloning and expansion of single-cell derived populations that are able to generate new stem cells as well as neurons, astrocytes, and oligodendrocytes. The addition of exogenous Wnt3a protein enhances clonal outgrowth, demonstrating not only a critical role for the Wnt pathway for the regulation of neurogenesis but also its use for the expansion of neural stem cells in cell culture and in tissue engineering.

  View details for DOI 10.1073/pnas.0808616105

  View details for PubMedID 18957545

• The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases BLOOD Weissman, I. L., Shizuru, J. A. 2008; 112 (9): 3543-3553

  Abstract

  Advances in the understanding of the cells of the hematopoietic system have provided a rich basis for improving clinical hematopoietic cell transplants; finding and using proteins and molecules to amplify or suppress particular blood cell types; understanding the stepwise progression of preleukemic stages leading first to chronic myeloid disorders, then the emergence of acute blastic leukemias; and treating malignant and nonmalignant diseases with cell subsets. As a result of intense scientific investigation, hematopoietic stem cells (HSCs) have been isolated and their key functional characteristics revealed-self-renewal and multilineage differentiation. These characteristics are now found to be present in all tissue/organ stem cell studies, and even in the analysis of pluripotent embryonic, nuclear transfer, and induced pluripotent stem cells. Studies on HSC have identified hematopoiesis as one of the best systems for studying developmental cell lineages and as the best for understanding molecular changes in cell fate decision-making and for finding preclinical and clinical platforms for tissue and organ replacement, regeneration, and oncogenesis. Here we review the steps, from our viewpoint, that led to HSC isolation and its importance in self-nonself immune recognition.

  View details for DOI 10.1182/blood-2008-08-078220

  View details for Web of Science ID 000260301800011

  View details for PubMedID 18948588

• Cancer Stem Cells: On the Verge of Clinical Translation LABMEDICINE Chao, M. P., Weissman, I. L., Park, C. Y. 2008; 39 (11): 679-686

  View details for DOI 10.1309/LMTDDRGLY374WSCQ

  View details for Web of Science ID 000260366000009

• Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes PLOS ONE Cao, F., Wagner, R. A., Wilson, K. D., Xie, X., Fu, J., Drukker, M., Lee, A., Li, R. A., Gambhir, S. S., Weissman, I. L., Robbins, R. C., Wu, J. C. 2008; 3 (10)

  Abstract

  Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

  View details for DOI 10.1371/journal.pone.0003474

  View details for Web of Science ID 000265126100005

  View details for PubMedID 18941512

  View details for PubMedCentralID PMC2565131

• Hematopoietic Stem Cell Quiescence Is Maintained by Compound Contributions of the Retinoblastoma Gene Family CELL STEM CELL Viatour, P., Somervaille, T. C., Venkatasubrahmanyam, S., Kogan, S., McLaughlin, M. E., Weissman, I. L., Butte, A. J., Passegue, E., Sage, J. 2008; 3 (4): 416-428

  Abstract

  Individual members of the retinoblastoma (Rb) tumor suppressor gene family serve critical roles in the control of cellular proliferation and differentiation, but the extent of their contributions is masked by redundant and compensatory mechanisms. Here we employed a conditional knockout strategy to simultaneously inactivate all three members, Rb, p107, and p130, in adult hematopoietic stem cells (HSCs). Rb family triple knockout (TKO) mice develop a cell-intrinsic myeloproliferation that originates from hyperproliferative early hematopoietic progenitors and is accompanied by increased apoptosis in lymphoid progenitor populations. Loss of quiescence in the TKO HSC pool is associated with an expansion of these mutant stem cells but also with an enhanced mobilization and an impaired reconstitution potential upon transplantation. The presence of a single p107 allele is sufficient to largely rescue these defects. Thus, Rb family members collectively maintain HSC quiescence and the balance between lymphoid and myeloid cell fates in the hematopoietic system.

  View details for DOI 10.1016/j.stem.2008.07.009

  View details for Web of Science ID 000260149800012

  View details for PubMedID 18940733

  View details for PubMedCentralID PMC2646421

• Identification of the Endostyle as a Stem Cell Niche in a Colonial Chordate CELL STEM CELL Voskoboynik, A., Soen, Y., Rinkevich, Y., Rosner, A., Ueno, H., Reshef, R., Ishizuka, K. J., Palmeri, K. J., Moiseeva, E., Rinkevich, B., Weissman, I. L. 2008; 3 (4): 456-464

  Abstract

  Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.

  View details for DOI 10.1016/j.stem.2008.07.023

  View details for Web of Science ID 000260149800015

  View details for PubMedID 18940736

• Multimodal evaluation of in vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Hendry, S. L., van der Bogt, K. E., Sheikh, A. Y., Arai, T., Dylla, S. J., Drukker, M., McConnell, M. V., Kutschka, I., Hoyt, G., Cao, F., Weissman, I. L., Connolly, A. J., Pelletier, M. P., Wu, J. C., Robbins, R. C., Yang, P. C. 2008; 136 (4): 1028-U14

  Abstract

  Mouse embryonic stem cells have demonstrated potential to restore infarcted myocardium after acute myocardial infarction. Although the underlying mechanism remains controversial, magnetic resonance imaging has provided reliable in vivo assessment of functional recovery after cellular transplants. Multimodal comparison of the restorative effects of mouse embryonic stem cells and mouse embryonic fibroblasts was performed to validate magnetic resonance imaging data and provide mechanistic insight.SCID-beige mice (n = 55) underwent coronary artery ligation followed by injection of 2.5 x 10(5) mouse embryonic stem cells, 2.5 x 10(5) mouse embryonic fibroblasts, or normal saline solution. In vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells was evaluated by (1) in vivo pressure-volume loops, (2) in vivo bioluminescence imaging, and (3) ex vivo TaqMan (Roche Molecular Diagnostics, Pleasanton, Calif) polymerase chain reaction and immunohistologic examination.In vivo magnetic resonance imaging demonstrated significant improvement in left ventricular ejection fraction at 1 week in the mouse embryonic stem cell group. This finding was validated with (1) pressure-volume loop analysis demonstrating significantly improved systolic and diastolic functions, (2) bioluminescence imaging and polymerase chain reaction showing superior posttransplant survival of mouse embryonic stem cells, (3) immunohistologic identification of cardiac phenotype within engrafted mouse embryonic stem cells, and (4) polymerase chain reaction measuring increased expressions of angiogenic and antiapoptotic genes and decreased expressions of antifibrotic genes.This study validates in vivo magnetic resonance imaging as an effective means of evaluating the restorative potential of mouse embryonic stem cells.

  View details for DOI 10.1016/j.jtcvs.2007.12.053

  View details for PubMedID 18954646

• ISOLATION OF BRAIN CANCER STEM CELLS FROM GLIOBLASTOMA USING AN ANTIBODY LIBRARY 13th Annual Meeting of the Society-for-Neuro-Oncology (SNO) Raveh, T., Luppen, C., Cox, D., Higgins, D., Cheshier, S., Ailles, L., Edwards, M. S., Harsh, G., Weissman, I. OXFORD UNIV PRESS INC. 2008: 904–

  View details for Web of Science ID 000259854500532

• Hematopoietic stem cells and the aging hematopoietic system SEMINARS IN HEMATOLOGY Gazit, R., Weissman, I. L., Rossi, D. J. 2008; 45 (4): 218-224

  Abstract

  The etiology of the age-associated pathophysiological changes of the hematopoietic system including the onset of anemia, diminished adaptive immune competence, and myelogenous disease development are underwritten by the loss of normal homeostatic control. As tissue and organ homeostasis in adults is primarily mediated by the activity of stem and progenitor cells, it has been suggested that the imbalances accompanying aging of the hematopoietic system may stem from alterations in the prevalence and/or functional capacity of hematopoietic stem cells (HSCs) and progenitors. In this review, we examine evidence implicating a role for stem cells in the aging of the hematopoietic system, and focus on the mechanisms suggested to contribute to stem cell aging.

  View details for DOI 10.1053/j.seminhematol.2008.07.010

  View details for Web of Science ID 000259584000003

  View details for PubMedID 18809091

• The E. Donnall Thomas lecture: Normal and neoplastic stem cells BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Weissman, I. L. 2008; 14 (8): 849-858

  Abstract

  Dr. Irving Weissman was the honored E. Donnall Thomas lecturer at the Tandem BMT Meetings, held on February 10, 2007, at Keystone, Colorado. Dr. Weissman has been a major player, and has provided us with enormous insight into many areas of biology, dating back to his high school days in Montana. He led an enormously productive career at Stanford University where he has taught us many lessons involving our understanding of lymphocyte homing, stem cell biology, both of the hematopoietic system and other types of stem cells, and also now, about cancer stem cells. Dr. Weissman has made enormous contributions to this burgeoning field that has provided us new insights and new opportunities for treatment strategies. In addition to a very productive laboratory career, he is also currently the director of both the Stem Cell Institute, as well as the Cancer Center at Stanford University. The following text is a modified transcribed version of the presentation made by Dr. Weissman.

  View details for DOI 10.1016/j.bbmt.2008.05.003

  View details for Web of Science ID 000258228200001

  View details for PubMedID 18640567

  View details for PubMedCentralID PMC2821216

• Space-time considerations for hematopoietic stem cell transplantation EUROPEAN JOURNAL OF IMMUNOLOGY Bhattacharya, D., Ehrlich, L. I., Weissman, I. L. 2008; 38 (8): 2060-2067

  Abstract

  The mammalian blood system contains a multitude of distinct mature cell lineages adapted to serving diverse functional roles. Mutations that abrogate the development or function of one or more of these lineages can lead to profound adverse consequences, such as immunodeficiency, autoimmunity, or anemia. Replacement of hematopoietic stem cells (HSC) that carry such mutations with HSC from a healthy donor can reverse such disorders, but because the risks associated with the procedure are often more serious than the blood disorders themselves, bone marrow transplantation is generally not used to treat a number of relatively common inherited blood diseases. Aside from a number of other problems, risks associated with cytoreductive treatments that create "space" for donor HSC, and the slow kinetics with which immune competence is restored following transplantation hamper progress. This review will focus on how recent studies using experimental model systems may direct future efforts to implement routine use of HSC transplantation to cure inherited blood disorders.

  View details for DOI 10.1002/eji.200838383

  View details for Web of Science ID 000258680100001

  View details for PubMedID 18651698

  View details for PubMedCentralID PMC2727747

• Multimodality Evaluation of the Viability of Stem Cells Delivered Into Different Zones of Myocardial Infarction CIRCULATION-CARDIOVASCULAR IMAGING Hung, T., Suzuki, Y., Urashima, T., Caffarelli, A., Hoyt, G., Sheikh, A. Y., Yeung, A. C., Weissman, I., Robbins, R. C., Bulte, J. W., Yang, P. C. 2008; 1 (1): 6-13

  Abstract

  We tested the hypothesis that multimodality imaging of mouse embryonic stem cells (mESCs) provides accurate assessment of cellular location, viability, and restorative potential after transplantation into different zones of myocardial infarction.Mice underwent left anterior descending artery ligation followed by transplantation of dual-labeled mESCs with superparamagnetic iron oxide and luciferase via direct injection into 3 different zones of myocardial infarction: intra-infarction, peri-infarction, and normal (remote). One day after transplantation, magnetic resonance imaging enabled assessment of the precise anatomic locations of mESCs. Bioluminescence imaging allowed longitudinal analysis of cell viability through detection of luciferase activity. Subsequent evaluation of myocardial regeneration and functional restoration was performed by echocardiography and pressure-volume loop analysis. Using 16-segment analysis, we demonstrated precise localization of dual-labeled mESCs. A strong correlation between histology and magnetic resonance imaging was established (r=0.962, P=0.002). Bioluminescent imaging data demonstrated that cell viability in the remote group was significantly higher than in other groups. Echocardiography and pressure-volume loop analysis revealed improved functional restoration in animals treated with mESCs, although myocardial regeneration was not observed.Multimodality evaluation of mESC engraftment in the heterogeneous tissue of myocardial infarction is possible. Magnetic resonance imaging demonstrated accurate anatomic localization of dual-labeled mESCs. Bioluminescent imaging enabled assessment of variable viability of mESCs transplanted into the infarcted myocardium. Echocardiography and pressure-volume loop analysis validated the restorative potential of mESCs. Although mESCs transplanted into the remote zone demonstrated the highest viability, precise delivery of mESCs into the peri-infarction region might be equally critical in restoring the injured myocardium.

  View details for DOI 10.1161/CIRCIMAGING.108.767343

  View details for PubMedID 19808509

• Flk2(+) common lymphoid progenitors possess equivalent differentiation potential for the B and T lineages BLOOD Karsunky, H., Inlay, M. A., Serwold, T., Bhattacharya, D., Weissman, I. L. 2008; 111 (12): 5562-5570

  Abstract

  Mature blood cells develop from multipotent hematopoietic stem cells through a series of sequential intermediates in which the developmental potential for particular blood lineages is progressively extinguished. We previously reported the identification of one of these developmental intermediates, the common lymphoid progenitor (CLP), which can give rise to T cells, B cells, dendritic cells (DCs), and natural killer cells (NKs), but lacks myeloid and erythroid potential. Recently, several studies have suggested that the T-cell and DC potential of CLP is limited or absent, and/or that CLP contains significant myeloid potential. Here, we show that the originally identified CLP population can be divided into functionally distinct subsets based on the expression of the tyrosine kinase receptor, Flk2. The Flk2(+) subset contains robust in vivo and in vitro T-cell, B-cell, DC, and NK potential, but lacks myeloid potential and, therefore, represents an oligopotent, lymphoid-restricted progenitor. This population of cells does not appear to be B cell-biased and robustly reconstitutes both B and T lineages in vivo, consistent with its being a physiologic progenitor of both of these subsets. Thus, Flk2 expression defines a homogeneous, readily obtainable subset of bone marrow CLP that is completely lymphoid-committed and can differentiate equivalently well into both B and T lineages.

  View details for DOI 10.1182/blood-2007-11-126219

  View details for Web of Science ID 000256786500028

  View details for PubMedID 18424665

  View details for PubMedCentralID PMC2424154

• Isolation of human fetal liver progenitors and their enhanced proliferation by three-dimensional coculture with endothelial cells TISSUE ENGINEERING PART A Xiong, A., Austin, T. W., Lagasse, E., Uchida, N., Tamaki, S., Bordier, B. B., Weissman, I. L., Glenn, J. S., Millan, M. T. 2008; 14 (6): 995-1006

  Abstract

  Liver progenitor cells, characterized by the coexpression of biliary and hepatocyte lineage markers and the ability to form colonies in culture, were isolated by flow cytometry from primary human fetal livers. These prospectively isolated liver progenitor cells supported hepatitis D virus infection, expressed, and produced albumin and alpha-fetoprotein, as tracked by albumin- and alpha-fetoprotein-driven lentiviral promoter reporter constructs and measured by ELISA, respectively. Coculture in three-dimensional (3D) fibrin gel with endothelial cells resulted in the formation of vascular structures by the endothelial cells and increased proliferation of liver progenitors. The enhanced proliferation of liver progenitors that was observed when liver progenitors and endothelial cells were cultured in direct contact was not achieved when liver progenitors and endothelial cells were cultured on adjacent but separate matrices and when they were cultured across transwell membranes. In conclusion, coculture of liver progenitors and endothelial cells in three-dimensional matrix resulted in enhanced liver progenitor proliferation and function. This coculture methodology offers a novel coculture system that could be applied for the development of engineered liver tissues.

  View details for DOI 10.1089/ten.tea.2007.0087

  View details for PubMedID 19230124

• Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Purhonen, S., Palm, J., Rossi, D., Kaskenpaa, N., Rajantie, I., Yla-Herttuala, S., Alitalo, K., Weissman, I. L., Salven, P. 2008; 105 (18): 6620-6625

  Abstract

  The mechanisms by which bone marrow (BM)-derived stem cells might contribute to angiogenesis and the origin of neovascular endothelial cells (ECs) are controversial. Neovascular ECs have been proposed to originate from VEGF receptor 2-expressing (VEGFR-2+) stem cells mobilized from the BM by VEGF or tumors, and it is thought that angiogenesis and tumor growth may depend on such endothelial precursors or progenitors. We studied the mobilization of BM cells to circulation by inoculating mice with VEGF polypeptides, adenoviral vectors expressing VEGF, or tumors. We induced angiogenesis by syngeneic melanomas, APCmin adenomas, adenoviral VEGF delivery, or matrigel plugs in four different genetically tagged universal or endothelial cell-specific chimeric mouse models, and subsequently analyzed the contribution of BM-derived cells to endothelium in a wide range of time points. To study the existence of circulating ECs in a nonmyeloablative setting, pairs of genetically marked parabiotic mice with a shared anastomosed circulatory system were created. We did not observe specific mobilization of VEGFR-2+ cells to circulation by VEGF or tumors. During angiogenesis, abundant BM-derived perivascular cells were recruited close to blood vessel wall ECs but did not form part of the endothelium. No circulation-derived vascular ECs were observed in the parabiosis experiments. Our results show that no BM-derived VEGFR-2+ or other EC precursors contribute to vascular endothelium and that cancer growth does not require BM-derived endothelial progenitors. Endothelial differentiation is not a typical in vivo function of normal BM-derived stem cells in adults, and it has to be an extremely rare event if it occurs at all.

  View details for DOI 10.1073/pnas.0710516105

  View details for Web of Science ID 000255841600021

  View details for PubMedID 18443294

• SIRT1 acts as a nutrient-sensitive growth suppressor and its loss is associated with increased AMPK and telomerase activity MOLECULAR BIOLOGY OF THE CELL Narala, S. R., Allsopp, R. C., Wells, T. B., Zhang, G., Prasad, P., Coussens, M. J., Rossi, D. J., Weissman, I. L., Vaziri, H. 2008; 19 (3): 1210-1219

  Abstract

  SIRT1, the mammalian homolog of SIR2 in Saccharomyces cerevisiae, is an NAD-dependent deacetylase implicated in regulation of lifespan. By designing effective short hairpin RNAs and a silent shRNA-resistant mutant SIRT1 in a genetically defined system, we show that efficient inhibition of SIRT1 in telomerase-immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells obtained from SIRT1-deficient mice also showed increased growth capacity and decreased dependency on growth factors. Consistent with this, SIRT1 inhibition was associated with increased telomerase activity in human cells. We also observed a significant increase in AMPK levels up on SIRT1 inhibition under glucose limiting conditions. Although SIRT1 suppression cooperated with hTERT to promote cell growth, either overexpression or suppression of SIRT1 alone had no effect on life span of human diploid fibroblasts. Our findings challenge certain models and connect nutrient sensing enzymes to the immortalization process. Furthermore, they show that in certain cell lineages, SIRT1 can act as a growth suppressor gene.

  View details for DOI 10.1091/mbc.E07-09-0965

  View details for Web of Science ID 000258951400038

  View details for PubMedID 18184747

• Stems cells and the pathways to aging and cancer CELL Rossi, D. J., Jamieson, C. H., Weissman, I. L. 2008; 132 (4): 681-696

  Abstract

  The aging of tissue-specific stem cell and progenitor cell compartments is believed to be central to the decline of tissue and organ integrity and function in the elderly. Here, we examine evidence linking stem cell dysfunction to the pathophysiological conditions accompanying aging, focusing on the mechanisms underlying stem cell decline and their contribution to disease pathogenesis.

  View details for DOI 10.1016/j.cell.2008.01.036

  View details for Web of Science ID 000253817900025

  View details for PubMedID 18295583

• Hematopoietic stem cell-derived pericytic cells in brain tumor angio-architecture STEM CELLS AND DEVELOPMENT Bababeygy, S. R., Cheshier, S. H., Hou, L. C., Higgins, D. M., Weissman, I. L., Tse, V. C. 2008; 17 (1): 11-18

  Abstract

  Bone marrow-derived cells are recruited into tumor vasculature in response to angiogenic signals, and some of the cells within the newly forming tumor vessels are hematopoietic stem cells (HSCs) in origin. Previous studies suggest that bone marrow-derived pericytes are associated with newly formed vessels in tumors. In this study, we used an orthotopic rat glioma model (RT-2/RAG) to examine the contribution of long-term hematopoietic stem cell (LT-HSC)-derived pericytic cells to brain tumor angiogenesis. Mice (RAG-2/KO5.2) were lethally irradiated, and their hematopoietic cells were repopulated by transplantation of double fluorescence-activated cell-sorted LT-HSCs that express green fluorescent protein (GFP+). RT-2/RAG cells were then injected into the striatum of the chimeric mice 6 weeks post-transplantation. The animals were sacrificed 9 days after tumor implantation, and the incorporation and lineage-specific marker expression profile of the GFP+ cells within the growing tumor and tumor periphery were analyzed. LT-HSC-derived GFP+ cells were noted to incorporate onto the surface of tumor vessels within the perivascular space. LT-HSC-derived GFP+ cells express the pericyte progenitor marker, platelet-derived growth factor receptor-beta (PDGFR beta), as well as mature perictyte markers such as nerve/glial antigen 2 proteoglycan (NG2), alpha-smooth muscle actin (alpha SMA), and desmin. These LT-HSC-derived cells may represent a population of progenitor or committed pericytes within the neovascular tree and may play a role in shaping the angio-architecture in the vascular niche of brain tumors.

  View details for DOI 10.1089/scd.2007.0117

  View details for Web of Science ID 000253628600002

  View details for PubMedID 18240955

• The PIAS-like protein Zimp10 is essential for embryonic viability and proper vascular development MOLECULAR AND CELLULAR BIOLOGY Beliakoff, J., Lee, J., Ueno, H., Aiyer, A., Weissman, I. L., Barsh, G. S., Cardiff, R. D., Sun, Z. 2008; 28 (1): 282-292

  Abstract

  Members of the PIAS (for protein inhibitor of activated STAT) family play critical roles in modulating the activity of a variety of transcriptional regulators. Zimp10, a novel PIAS-like protein, is a transcriptional coregulator and may be involved in the modification of chromatin through interactions with the SWI/SNF chromatin-remodeling complexes. Here, we investigate the biological role of Zimp10 in zimp10-deficient mice. Homozygosity for the Zimp10-targeted allele resulted in developmental arrest at approximately embryonic day 10.5. Analysis of knockout embryos revealed severe defects in the reorganization of the yolk sac vascular plexus. No significant abnormality in hematopoietic potential was observed in zimp10 null mice. Microarray and quantified reverse transcription-PCR analyses showed that the expression of the Fos family member Fra-1, which is involved in extraembryonic vascular development, was reduced in yolk sac tissues of zimp10 null embryos. Using fra-1 promoter/reporter constructs, we further demonstrate the regulatory role of Zimp10 on the transcription of Fra-1. This study provides evidence to demonstrate a crucial role for Zimp10 in vasculogenesis.

  View details for DOI 10.1128/MCB.00771-07

  View details for Web of Science ID 000251925300024

  View details for PubMedID 17967885

  View details for PubMedCentralID PMC2223308

• Investigating mechanisms of cancer stern cell radioresistance 50th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO) Diehn, M., Cho, R. W., Dorie, M., KULP, A., Weissman, I. L., Brown, M., Clarke, M. F. ELSEVIER SCIENCE INC. 2008: S29–S29

  View details for Web of Science ID 000258805300065

• In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood NATURE PROTOCOLS Park, C. Y., Majeti, R., Weissman, I. L. 2008; 3 (12): 1932-1940

  Abstract

  Establishment of robust xenograft models is critical to studying human hematopoiesis in a physiologic setting. Using a recently developed immunodeficient mouse strain, we have established long-term multilineage human grafts and demonstrated their serially transplantability using limited numbers of purified human hematopoietic stem cells (HSCs). Herein, we describe our protocol for the isolation of human HSC (Lin-CD34+CD38-CD90+) from umbilical cord blood (CB) as well as the xenotransplantation system that allows stable engraftment of human hematopoietic cells with as few as ten HSCs. Isolation of CB mononuclear cells requires 2-3 h, and cells may be cryopreserved before transplantation. Isolation of HSC requires approximately 2-3 h, and transplantation requires 1 h. Short-term and long-term engraftment is assessed 4-6 weeks and 10-12 weeks post-transplantation, respectively, with preparation and analysis time requiring 4-8 h at each time point.

  View details for DOI 10.1038/nprot.2008.194

  View details for Web of Science ID 000265781700012

  View details for PubMedID 19180077

• Establishment of a Normal Hematopoietic and Leukemia Stem Cell Hierarchy 73rd Cold Spring Harbor Symposium on Quantitative Biology Chao, M. P., Seita, J., Weissman, I. L. COLD SPRING HARBOR LABORATORY PRESS. 2008: 439–449

  Abstract

  Many types of adult tissues, especially for high turnover tissues such as the blood and intestinal system, stand on a hierarchical tissue-specific stem cell system. Tissue-specific stem cells concurrently have self-renewal capacity and potential to give rise to all types of mature cells in their tissue. The differentiation process of the tissue-specific stem cell is successive restriction of these capacities. The first progeny of tissue-specific stem cells are multipotent progenitors (MPPs) that lose long-term self-renewal capacity yet have full lineage potential. MPPs in turn give rise to oligopotent progenitors, which then commit into lineage-restricted progenitors. This hierarchical system enables a lifelong supply of matured functional cells that generally have a short life span and a relatively high turnover rate. In this chapter, we review our findings and other key experiments that have led to the establishment of the current cellular stem and progenitor hierarchy in the blood-forming systems of mice and humans for both normal and leukemic hematopoiesis. We also review select signaling pathways intrinsic to normal hematopoietic and leukemic stem cell populations as well our recent findings elucidating the possible origin of the leukemia stem cell.

  View details for Web of Science ID 000267135700050

  View details for PubMedID 19022770

• BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia CANCER RESEARCH Omidvar, N., Kogan, S., Beurlet, S., Pogam, C. I., Janin, A., West, R., Noguera, M., Reboul, M., Soulie, A., Leboeuf, C., Setterblad, N., Felsher, D., Lagasse, E., Mohamedali, A., Thomas, N. S., Fenaux, P., Fontenay, M., Pla, M., Mufti, G. J., Weissman, I., Chomienne, C., Padua, R. A. 2007; 67 (24): 11657-11667

  Abstract

  Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin(-)/Sca-1(+)/c-Kit(+)) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1(+) compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy.

  View details for DOI 10.1158/0008-5472.CAN-07-0196

  View details for Web of Science ID 000251857900025

  View details for PubMedID 18089795

• Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood CELL STEM CELL Majeti, R., Park, C. Y., Weissman, I. L. 2007; 1 (6): 635-645

  Abstract

  Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC activity have been identified, as have downstream lineage-committed progenitors, but multipotent progenitor activity has not been uniquely isolated. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. Utilizing in vivo transplantation studies and complementary in vitro assays, we demonstrate that the Lin-CD34+CD38-CD90+CD45RA- cord blood fraction contains HSC and isolate this activity to as few as 10 purified cells. Furthermore, we report the first prospective isolation of a population of candidate human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

  View details for DOI 10.1016/j.stem.2007.10.001

  View details for Web of Science ID 000251784300010

  View details for PubMedID 18371405

  View details for PubMedCentralID PMC2292126

• Cancer stem cells in head and neck squamous carcinoma Ailles, L., Prince, M., Joshua, B., Doweck, I., Kaplan, M., Clarke, M., Weissman, I. AMER ASSOC CANCER RESEARCH. 2007: 3630S–3630S

  View details for Web of Science ID 000251969000893

• Transcriptional instability is not a universal attribute of aging AGING CELL Warren, L. A., Rossi, D. J., Schiebinger, G. R., Weissman, I. L., Kim, S. K., Quake, S. R. 2007; 6 (6): 775-782

  Abstract

  It has been proposed that cumulative somatic mutations contribute to the aging process by disrupting the transcriptional networks that regulate cell structure and function. Experimental support for this model emerged from a recent study of cardiomyocytes that showed a dramatic increase in the transcriptional heterogeneity of these long-lived postmitotic cells with age. To determine if regulatory instability is a hallmark of aging in renewing tissues, we evaluated gene expression noise in four hematopoietic cell types: stem cells, granulocytes, naïve B cells and naïve T cells. We used flow cytometry to purify phenotypically equivalent cells from young and old mice, and applied multiplexed quantitative reverse transcription-polymerase chain reaction to measure the copy number of six different mRNA transcripts in 324 individual cells. There was a trend toward higher transcript levels in cells isolated from old animals, but no significant increase in transcriptional heterogeneity with age was found in the surveyed populations. Flow cytometric analysis of membrane protein expression also indicated that cell-to-cell variability was unaffected by age. We conclude that large-scale regulatory destabilization is not a universal concomitant of aging, and may be of significance as an aging mechanism primarily in nonrenewing tissues.

  View details for DOI 10.1111/j.1474-9726.2007.00337.x

  View details for Web of Science ID 000250938400007

  View details for PubMedID 17925006

• Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches SCIENCE Czechowicz, A., Kraft, D., Weissman, I. L., Bhattacharya, D. 2007; 318 (5854): 1296-1299

  Abstract

  Upon intravenous transplantation, hematopoietic stem cells (HSCs) can home to specialized niches, yet most HSCs fail to engraft unless recipients are subjected to toxic preconditioning. We provide evidence that, aside from immune barriers, donor HSC engraftment is restricted by occupancy of appropriate niches by host HSCs. Administration of ACK2, an antibody that blocks c-kit function, led to the transient removal of >98% of endogenous HSCs in immunodeficient mice. Subsequent transplantation of these mice with donor HSCs led to chimerism levels of up to 90%. Extrapolation of these methods to humans may enable mild but effective conditioning regimens for transplantation.

  View details for DOI 10.1126/science.1149726

  View details for Web of Science ID 000251086600042

  View details for PubMedID 18033883

  View details for PubMedCentralID PMC2527021

• Missplicing of glycogen synthase kinase 3 beta: A potential mechanism of blast crisis chronic myeloid leukemia stem cell generation 49th Annual Meeting of the American-Society-of-Hematology Abrahamsson, A., Geron, I., Gotlib, J., Dao, K., Giles, F., Newton, I., Kavaterchik, E., Durocher, J., Creusot, R., Karimi, M., Jones, C., Zehnder, J., Keating, A., Negrin, R., Weissman, I. L., Jamieson, C. H. AMER SOC HEMATOLOGY. 2007: 238A–239A

  View details for Web of Science ID 000251100801044

• Transcriptional profiling of antigen-dependent murine B cell differentiation and memory formation JOURNAL OF IMMUNOLOGY Bhattacharya, D., Cheah, M. T., Franco, C. B., Hosen, N., Pin, C. L., Sha, W. C., Weissman, I. L. 2007; 179 (10): 6808-6819

  Abstract

  Humoral immunity is characterized by the generation of Ab-secreting plasma cells and memory B cells that can more rapidly generate specific Abs upon Ag exposure than their naive counterparts. To determine the intrinsic differences that distinguish naive and memory B cells and to identify pathways that allow germinal center B cells to differentiate into memory B cells, we compared the transcriptional profiles of highly purified populations of these three cell types along with plasma cells isolated from mice immunized with a T-dependent Ag. The transcriptional profile of memory B cells is similar to that of naive B cells, yet displays several important differences, including increased expression of activation-induced deaminase and several antiapoptotic genes, chemotactic receptors, and costimulatory molecules. Retroviral expression of either Klf2 or Ski, two transcriptional regulators specifically enriched in memory B cells relative to their germinal center precursors, imparted a competitive advantage to Ag receptor and CD40-engaged B cells in vitro. These data suggest that humoral recall responses are more rapid than primary responses due to the expression of a unique transcriptional program by memory B cells that allows them to both be maintained at high frequencies and to detect and rapidly respond to antigenic re-exposure.

  View details for Web of Science ID 000250792700049

  View details for PubMedID 17982071

• Hematopoietic stem cell quiescence attenuates DNA damage response and permits DNA damage accumulation during aging CELL CYCLE Rossi, D. J., Seita, J., Czechowicz, A., Bhattacharya, D., Bryder, D., Weissman, I. L. 2007; 6 (19): 2371-2376

  Abstract

  The aging of tissue-specific stem and progenitor cells is believed to be central to the pathophysiological conditions arising in aged individuals. While the mechanisms driving stem cell aging are poorly understood, mounting evidence points to age-dependent DNA damage accrual as an important contributing factor. While it has been postulated that DNA damage may deplete stem cell numbers with age, recent studies indicate that murine hematopoietic stem cell (HSC) reserves are in fact maintained despite the accrual of genomic damage with age. Evidence suggests this to be a result of the quiescent (G0) cell cycle status of HSC, which results in an attenuation of checkpoint control and DNA damage responses for repair or apoptosis. When aged stem cells that have acquired damage are called into cycle under conditions of stress or tissue regeneration however, their functional capacity was shown to be severely impaired. These data suggest that age-dependent DNA damage accumulation may underlie the diminished capacity of aged stem cells to mediate a return to homeostasis after acute stress or injury. Moreover, the cytoprotection afforded by stem cell quiescence in stress-free, steady-state conditions suggests a mechanism through which potentially dangerous lesions can accumulate in the stem cell pool with age.

  View details for Web of Science ID 000251085700012

  View details for PubMedID 17700071

• Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy CELL STEM CELL Pronk, C. J., Rossi, D. J., Mansson, R., Attema, J. L., Norddahl, G. L., Chan, C. K., Sigvardsson, M., Weissman, I. L., Bryder, D. 2007; 1 (4): 428-442

  Abstract

  The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.

  View details for DOI 10.1016/j.stem.2007.07.005

  View details for Web of Science ID 000251055300012

  View details for PubMedID 18371379

• Isolation of brain cancer stem cells using signaling pathway reporters 12th Annual Meeting of the Society-for-Neuro-Oncology Ailles, L., Cheshier, S., Raveh, T., Higgins, D., Harsh, G., Edwards, M., Weissman, I. OXFORD UNIV PRESS INC. 2007: 593–93

  View details for Web of Science ID 000249999100474

• Cancer stem cells in solid tumors CURRENT OPINION IN BIOTECHNOLOGY Ailles, L. E., Weissman, I. L. 2007; 18 (5): 460-466

  Abstract

  Cancer stem cells (CSCs) are cells that drive tumorigenesis, as well as giving rise to a large population of differentiated progeny that make up the bulk of the tumor, but that lack tumorigenic potential. CSCs have been identified in a variety of human tumors, as assayed by their ability to initiate tumor growth in immunocompromised mice. Further characterization studies have demonstrated that gene expression profiles in breast cancer correlate with patient prognosis, and brain CSCs are specifically resistant to radiation through DNA damage repair. In addition, specific signaling pathways play a functional role in CSC self renewal and/or differentiation, and early studies indicate that CSCs are associated with a microenvironmental niche. Thus the biological properties of CSCs are just beginning to be revealed, and the continuation of these studies should lead to the development of CSC-targeted therapies for cancer treatment.

  View details for DOI 10.1016/j.copbio.2007.10.007

  View details for Web of Science ID 000251512700013

  View details for PubMedID 18023337

• The effect of bleeding on hematopoietic stem cell cycling and self-renewal STEM CELLS AND DEVELOPMENT Cheshier, S. H., Prohaska, S. S., Weissman, I. L. 2007; 16 (5): 707-717

  Abstract

  Hematopoietic stem cells (HSCs) divide and give rise to more committed progenitors, which ultimately produce all lineages of blood cells. HSCs can be induced to enter the cell cycle in vitro and in vivo by stimulatory cytokines and in vivo by ablation of bone marrow (BM) cells with irradiation or chemotherapeutic agents. Although it has been postulated that rates of HSC proliferation increase with normal hematopoietic stresses, such as infection or hemorrhage, this hypothesis has never been directly tested. The ability to analyze HSCs prospectively by cell-surface phenotype c-kit(+), Thy1.1(lo), Sca-1(+), Linage(neg/lo) has allowed us to perform a detailed examination of the effects of bleeding on the cell cycle kinetics of HSCs. Our results demonstrate for the first time that HSCs in both the BM and the spleen proliferate and self-renew in response to tail-vein bleeding in mice. This response was suppressed when red blood cells, but not when white blood cells, were transferred after bleeding. Thus, regulators of HSC proliferation can sense and respond to red blood cell levels.

  View details for DOI 10.1089/scd.2007.0017

  View details for Web of Science ID 000251266900003

  View details for PubMedID 17999593

• Toward understanding the molecular mechanisms of lineage determination in hematopoietic stem cells 36th Annual Meeting of the International-Society-for-Experimental-Hematology Heffner, G. C., Clutter, M. R., Nolan, G. P., Weissman, I. L. ELSEVIER SCIENCE INC. 2007: 19–19

  View details for Web of Science ID 000249535200043

• Reversal of autoimmune disease in lupus-prone New Zealand black/New Zealand white mice by nom-nyeloablative transplantation of purified allogeneic hematopoietic stem cells Tandem BMT Meeting 2007 Smith-Berdan, S., Gille, D., Weissman, I. L., Christensen, J. L. AMER SOC HEMATOLOGY. 2007: 1370–78

  Abstract

  Patients with severe systemic lupus erythematosus (SLE) refractory to conventional treatment are candidates for autologous hematopoietic stem cell (HSC) transplantation if the intent is to reset the immunologic clock. These patients might be candidates for allotransplantation with (SLE)-resistant major histocompatibility complex (MHC) haplotype-matched HSC if partial or complete replacement of an autoimmune-prone system is the intent. Using lupus-prone New Zealand black x New Zealand white (NZBW) mice, we investigated the use of highly enriched, haplomismatched, allogeneic HSC to prevent development of or to treat established autoimmune pathology. Young NZBW mice receiving purified allogeneic HSC transplants had improved survival, decreased proteinuria, circulating immune complexes, and autoantibodies to nuclear antigens than did untreated mice or mice given NZBW HSCs. NZBW mice with established lupus-like disease that received nonmyeloablative conditioning and transplants of (MHC) haplomismatched allogeneic HSCs also had greatly increased overall survival. Mice that received transplants exhibited stabilization or reversal of their lupus symptoms; stabilized or decreased proteinuria, and a lower frequency of elevated circulating immune complexes or autoantibodies than did control mice. Induction of durable mixed chimerism by transplantation of purified allogeneic HSCs after nonmyeloablative conditioning has the potential to reverse symptoms of established NZBW lupus.

  View details for DOI 10.1182/blood-2007-03-081497

  View details for Web of Science ID 000248655300046

  View details for PubMedID 17435112

• The Wilms' tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis LEUKEMIA Hosen, N., Shirakata, T., Nishida, S., Yanagihara, M., Tsuboi, A., Kawakami, M., Oji, Y., Oka, Y., Okabe, M., Tan, B., Sugiyama, H., Weissman, I. L. 2007; 21 (8): 1783-1791

  Abstract

  The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.

  View details for DOI 10.1038/sj.leu.2404752

  View details for Web of Science ID 000248170100021

  View details for PubMedID 17525726

• Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Attema, J. L., Papathanasiou, P., Forsberg, E. C., Xu, J., Smale, S. T., Weissman, I. L. 2007; 104 (30): 12371-12376

  Abstract

  Hematopoietic stem cells (HSC) produce all blood cell lineages by virtue of their capacity to self-renew and differentiate into progenitors with decreasing cellular potential. Recent studies suggest that epigenetic mechanisms play an important role in controlling stem cell potency and cell fate decisions. To investigate this hypothesis in HSC, we have modified the conventional chromatin immunoprecipitation assay allowing for the analysis of 50,000 prospectively purified stem and progenitor cells. Together with bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides colocalize across defined regulatory regions of lineage-affiliated genes in HSC. These active epigenetic histone modifications either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with gene expression. We also observed bivalent histone modifications at a lymphoid-affiliated gene in HSC and downstream transit-amplifying progenitors. Together, these data support a model in which epigenetic modifications serve as an important mechanism to control HSC multipotency.

  View details for DOI 10.1073/pnas.0704468104

  View details for Web of Science ID 000248472100026

  View details for PubMedID 17640913

  View details for PubMedCentralID PMC1924790

• Pioneer factor interactions and unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xu, J., Pope, S. D., Jazirehi, A. R., Attema, J. L., Papathanasiou, P., Watts, J. A., Zaret, K. S., Weissman, I. L., Smale, S. T. 2007; 104 (30): 12377-12382

  Abstract

  Recent studies have suggested that, in ES cells, inactive genes encoding early developmental regulators possess bivalent histone modification domains and are therefore poised for activation. However, bivalent domains were not observed at typical tissue-specific genes. Here, we show that windows of unmethylated CpG dinucleotides and putative pioneer factor interactions mark enhancers for at least some tissue-specific genes in ES cells. The unmethylated windows expand in cells that express the gene and contract, disappear, or remain unchanged in nonexpressing tissues. However, in ES cells, they do not always coincide with common histone modifications. Genomic footprinting and chromatin immunoprecipitation demonstrated that transcription factor binding underlies the unmethylated windows at enhancers for the Ptcra and Alb1 genes. After stable integration of premethylated Ptcra enhancer constructs into the ES cell genome, the unmethylated windows readily appeared. In contrast, the premethylated constructs remained fully methylated and silent after introduction into Ptcra-expressing thymocytes. These findings provide initial functional support for a model in which pioneer factor interactions in ES cells promote the assembly of a chromatin structure that is permissive for subsequent activation, and in which differentiated tissues lack the machinery required for gene activation when these ES cell marks are absent. The enhancer marks may therefore represent important features of the pluripotent state.

  View details for DOI 10.1073/pnas.0704579104

  View details for Web of Science ID 000248472100027

  View details for PubMedID 17640912

  View details for PubMedCentralID PMC1941477

• Early TCR expression and aberrant T cell development in mice with endogenous prerearranged T cell receptor genes JOURNAL OF IMMUNOLOGY Serwold, T., Hochedlinger, K., Inlay, M. A., Jaenisch, R., Weissman, I. L. 2007; 179 (2): 928-938

  Abstract

  The factors that regulate the rate of production of T cells by the thymus remain incompletely defined. To test whether generation of functional T cell receptors limits the rate of thymic T cell export, we made use of a line of mice, LN3alphabeta, that have endogenously prerearranged TCR genes. The prerearranged TCR genes were expressed abnormally early in hemopoietic development, indicating that RAG-mediated recombination, rather than transcription factor expression, is the key determinant of the initiation of robust TCR transcription. Thymic T cell export rates were similar between wild-type (wt) and LN3alphabeta mice, indicating that T cell maturation rates in these mice are determined by factors other than TCR gene rearrangement. In competitive bone marrow chimeras, however, LN3alphabeta thymocytes were out-competed by wt cells and failed to develop beyond the double-negative 4 stage. Furthermore, wt progenitors transplanted intrathymically into LN3alphabeta mice proliferated excessively, suggesting that increased proliferative signals in the LN3alphabeta thymus compensate for faulty T cell development driven by early TCR expression.

  View details for Web of Science ID 000247752100029

  View details for PubMedID 17617584

• Bmi-1-green fluorescent protein-knock-in mice reveal the dynamic regulation of Bmi-1 expression in normal and leukemic hematopoietic cells STEM CELLS Hosen, N., Yamane, T., Muijtjens, M., Pham, K., Clarke, M. F., Weissman, I. L. 2007; 25 (7): 1635-1644

  Abstract

  The ability to self-renew is essential for all kinds of stem cells regardless of tissue type. One of the best candidate genes involved in conferring self-renewal capacity is Bmi-1, which has been proven to be essential for the maintenance of both normal adult hematopoietic and leukemia stem cells, as well as adult neural stem cells. To investigate the possible role of Bmi-1 in other cell types that also self-renew, we generated Bmi-1-green fluorescent protein (GFP)-knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of the Bmi-1 gene. Using these targeted reporter mice, we demonstrated that Bmi-1 is expressed in hematopoietic stem cells (HSCs) at its highest levels and downregulated upon commitment to differentiation. An in vivo reconstitution assay revealed that the frequency of HSCs was 1/16 in Bmi-1high c-kit+ lin -Sca-1+ bone marrow (BM) cells and 1/49 in Bmi-1 high lin- BM cells, suggesting that Bmi-1 may serve as a marker for normal HSCs. In murine leukemia models induced by P210BCR/ABL or TEL/PDGFbetaR + AML1/ETO, Bmi-1 was not overexpressed in leukemic HSCs, despite the increase in the HSC numbers. Bmi-1 was expressed at its highest levels in undifferentiated leukemia cells. Furthermore, in several other nonhematopoietic tissues, cells could be separated into distinct subpopulations with differential Bmi-1 expression. Thus, these mice allow for the isolation of viable Bmi-1-expressing cells and have the potential to become a useful tool for understanding the role of Bmi-1 in normal and cancer stem cells in multiple tissue types. Disclosure of potential conflicts of interest is found at the end of this article.

  View details for DOI 10.1634/stemcells.2006-0229

  View details for Web of Science ID 000247722100006

  View details for PubMedID 17395774

• CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hosen, N., Park, C. Y., Tatsumi, N., Oji, Y., Sugiyama, H., Gramatzki, M., Krensky, A. M., Weissman, I. L. 2007; 104 (26): 11008-11013

  Abstract

  Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34(+)CD38(-) AML cells in many cases (74.0 +/- 25.3% in 19 of 29 cases), whereas only a few (4.9 +/- 1.6%) cells in the normal HSC-enriched population (Lin(-)CD34(+)CD38(-)CD90(+)) expressed CD96 weakly. To examine whether CD96(+) AML cells are enriched for LSC activity, we separated AML cells into CD96(+) and CD96(-) fractions and transplanted them into irradiated newborn Rag2(-/-) gamma(c)(-/-) mice. In four of five samples, only CD96(+) cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.

  View details for DOI 10.1073/pnas.0704271104

  View details for Web of Science ID 000247641900048

  View details for PubMedID 17576927

  View details for PubMedCentralID PMC1904175

• Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age NATURE Rossi, D. J., Bryder, D., Seita, J., Nussenzweig, A., Hoeijmakers, J., Weissman, I. L. 2007; 447 (7145): 725-U15

  Abstract

  A diminished capacity to maintain tissue homeostasis is a central physiological characteristic of ageing. As stem cells regulate tissue homeostasis, depletion of stem cell reserves and/or diminished stem cell function have been postulated to contribute to ageing. It has further been suggested that accumulated DNA damage could be a principal mechanism underlying age-dependent stem cell decline. We have tested these hypotheses by examining haematopoietic stem cell reserves and function with age in mice deficient in several genomic maintenance pathways including nucleotide excision repair, telomere maintenance and non-homologous end-joining. Here we show that although deficiencies in these pathways did not deplete stem cell reserves with age, stem cell functional capacity was severely affected under conditions of stress, leading to loss of reconstitution and proliferative potential, diminished self-renewal, increased apoptosis and, ultimately, functional exhaustion. Moreover, we provide evidence that endogenous DNA damage accumulates with age in wild-type stem cells. These data are consistent with DNA damage accrual being a physiological mechanism of stem cell ageing that may contribute to the diminished capacity of aged tissues to return to homeostasis after exposure to acute stress or injury.

  View details for DOI 10.1038/nature05862

  View details for Web of Science ID 000247030700046

  View details for PubMedID 17554309

• B-cell development fails in the absence of the Pbx1 proto-oncogene BLOOD Sanyal, M., Tung, J. W., Karsunky, H., Zeng, H., Selleri, L., Weissman, I. L., Herzenberg, L. A., Cleary, M. L. 2007; 109 (10): 4191-4199

  Abstract

  Pbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B-cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B-cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T-cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B-cell development from CLPs following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B-cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19(+)) cells and thereafter revealed that Pbx1 is not necessary for B-cell development to proceed from the pro-B-cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B-cell commitment and, therefore, is one of the earliest-acting transcription factors that regulate de novo B-lineage lymphopoiesis.

  View details for DOI 10.1182/blood-2006-10-054213

  View details for Web of Science ID 000246609100023

  View details for PubMedID 17244677

  View details for PubMedCentralID PMC1885499

• Striving for normality: whole body regeneration through a series of abnormal generations FASEB JOURNAL Voskoboynik, A., Simon-Blecher, N., Soen, Y., Rinkevich, B., De Tomaso, A. W., Ishizuka, K. J., Weissman, I. L. 2007; 21 (7): 1335-1344

  Abstract

  Embryogenesis and asexual reproduction are commonly considered to be coordinated developmental processes, which depend on accurate progression through a defined sequence of developmental stages. Here we report a peculiar developmental scenario in a simple chordate, Botryllus schlosseri, wherein a normal colony of individuals (zooids and buds) is regenerated from the vasculature (vascular budding) through a sequence of morphologically abnormal developmental stages. Vascular budding was induced by surgically removing buds and zooids from B. schlosseri colonies, leaving only the vasculature and the tunic that connects them. In vivo imaging and histological sections showed that the timing and morphology of developing structures during vascular budding deviated significantly from other asexual reproduction modes (the regular asexual reproduction mode in this organism and vascular budding in other botryllid species). Subsequent asexual reproduction cycles exhibited gradual regaining of normal developmental patterns, eventually leading to regeneration of a normal colony. The conversion into a normal body form suggests the activation of an alternative pathway of asexual reproduction, which involves gradual regaining of normal positional information. It presents a powerful model for studying the specification of the same body plan by different developmental programs.

  View details for DOI 10.1096/fj.06-7337com

  View details for Web of Science ID 000246117000009

  View details for PubMedID 17289924

• Hematopoietic stem cell aging: Mechanism and consequence 1st European Congress of Aging Research in Immunology - Impact of Genomics Rossi, D. J., Bryder, D., Weissman, I. L. PERGAMON-ELSEVIER SCIENCE LTD. 2007: 385–90

  Abstract

  Advancing age is frequented by the onset of a variety of hematological conditions characterized by diminished homeostatic control of blood cell production. The fact that upstream hematopoietic stem and progenitor cells are obligate mediators of homeostatic control of all blood lineages, has implicated the involvement of these cells in the pathophysiology of these conditions. Indeed, evidence from our group and others has suggested that two of the most clinically significant age-associated hematological conditions, namely, the diminution of the adaptive immune system and the elevated incidence of myeloproliferative diseases, have their origin in cell autonomous changes in the functional capacity of hematopoietic stem cells.

  View details for DOI 10.1016/j.exger.2006.11.019

  View details for Web of Science ID 000246532900002

  View details for PubMedID 17275237

  View details for PubMedCentralID PMC1892213

• Stem cells - Blood lines from embryo to adult NATURE Ueno, H., Weissman, I. L. 2007; 446 (7139): 996-997

  View details for DOI 10.1038/446996a

  View details for Web of Science ID 000245950400033

  View details for PubMedID 17460657

• Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation CLONING AND STEM CELLS Cao, F., Drukker, M., Lin, S., Sheikh, A. Y., Xie, X., Li, Z., Connolly, A. J., Weissman, I. L., Wu, J. C. 2007; 9 (1): 107-117

  Abstract

  Numerous studies have demonstrated the potential use of stem cells for the repair and regeneration of injured tissues. However, tracking transplanted stem cell fate and function in vivo remains problematic. To address these issues, murine embryonic stem (ES) cells were stably transduced with self-inactivating lentiviral vectors carrying either a triple fusion (TF) or double fusion (DF) reporter gene construct. The TF consisted of monomeric red fluorescence protein (mrfp), firefly luciferase (Fluc), and herpes simplex virus truncated thymidine kinase (HSV-ttk) reporter genes. The DF consisted of enhanced green fluorescence protein (egfp) and Fluc reporter genes but lacked HSV-ttk. Stably transduced ES-TF or ES-DF cells were selected by fluorescence activated cell sorting based on either mrfp (TF) or egfp (DF) expression. Afterwards, cells were injected subcutaneously into the right (ES-TF cells) and left (ES-DF cells) shoulders of adult female nude mice. Cell survival was tracked noninvasively by bioluminescence and positron emission tomography imaging of Fluc and HSV-ttk reporter genes, respectively. Imaging signals progressively increased from day 2 to day 14, consistent with ES cell survival and proliferation in vivo. However, teratoma formation occurred in all nude mice after 5 weeks. Administration of ganciclovir (GCV), targeting the HSV-ttk gene, resulted in selective ablation of teratomas arising from the ES-TF cells but not ES-DF cells. These data demonstrate the novel use of multimodality imaging techniques to (1) monitor transplanted ES cell survival and proliferation in vivo and (2) assess the efficacy of suicide gene therapy as a backup safety measure against teratoma formation.

  View details for DOI 10.1089/clo.2006.0016

  View details for Web of Science ID 000245390300015

  View details for PubMedID 17386018

• The ISSCR guidelines for human embryonic stem cell research SCIENCE Daley, G. Q., Ahrlund-Richter, L., Auerbach, J. M., Benvenisty, N., Charo, R. A., Chen, G., Deng, H., Goldstein, L. S., Hudson, K. L., Hyun, I., Junn, S. C., Love, J., Lee, E. H., McLaren, A., Mummery, C. L., Nakatsuji, N., Racowsky, C., Rooke, H., Rossant, J., Schoeler, H. R., Solbakk, J. H., Taylor, P., Trounson, A. O., Weissman, I. L., Wilmut, I., Yu, J., Zoloth, L. 2007; 315 (5812): 603-604

  View details for DOI 10.1126/science.1139337

  View details for Web of Science ID 000243909400027

  View details for PubMedID 17272706

• Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells JOURNAL OF EXPERIMENTAL MEDICINE Serafini, M., Dylla, S. J., Oki, M., Heremans, Y., Tolar, J., Jiang, Y., Buckley, S. M., Pelacho, B., Burns, T. C., Frommer, S., Rossi, D. J., Bryder, D., Panoskaltsis-Mortari, A., O'Shaughnessy, M. J., Nelson-Holte, M., Fine, G. C., Weissman, I. L., Blazar, B. R., Verfaillie, C. M. 2007; 204 (1): 129-139

  Abstract

  For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

  View details for DOI 10.1084/jem.20061115

  View details for Web of Science ID 000243753600017

  View details for PubMedID 17227908

  View details for PubMedCentralID PMC2118428

• Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Prince, M. E., Sivanandan, R., Kaczorowski, A., Wolf, G. T., Kaplan, M. J., Dalerba, P., Weissman, I. L., Clarke, M. F., Ailles, L. E. 2007; 104 (3): 973-978

  Abstract

  Like many epithelial tumors, head and neck squamous cell carcinoma (HNSCC) contains a heterogeneous population of cancer cells. We developed an immunodeficient mouse model to test the tumorigenic potential of different populations of cancer cells derived from primary, unmanipulated human HNSCC samples. We show that a minority population of CD44(+) cancer cells, which typically comprise <10% of the cells in a HNSCC tumor, but not the CD44(-) cancer cells, gave rise to new tumors in vivo. Immunohistochemistry revealed that the CD44(+) cancer cells have a primitive cellular morphology and costain with the basal cell marker Cytokeratin 5/14, whereas the CD44(-) cancer cells resemble differentiated squamous epithelium and express the differentiation marker Involucrin. The tumors that arose from purified CD44(+) cells reproduced the original tumor heterogeneity and could be serially passaged, thus demonstrating the two defining properties of stem cells: ability to self-renew and to differentiate. Furthermore, the tumorigenic CD44(+) cells differentially express the BMI1 gene, at both the RNA and protein levels. By immunohistochemical analysis, the CD44(+) cells in the tumor express high levels of nuclear BMI1, and are arrayed in characteristic tumor microdomains. BMI1 has been demonstrated to play a role in self-renewal in other stem cell types and to be involved in tumorigenesis. Taken together, these data demonstrate that cells within the CD44(+) population of human HNSCC possess the unique properties of cancer stem cells in functional assays for cancer stem cell self-renewal and differentiation and form unique histological microdomains that may aid in cancer diagnosis.

  View details for DOI 10.1073/pnas.0610117104

  View details for Web of Science ID 000243761100053

  View details for PubMedID 17210912

• Generation of a monoclonal antibody library against human embryonic stem cells. Methods in molecular biology (Clifton, N.J.) Drukker, M., Muscat, C., Weissman, I. L. 2007; 407: 63-81

  Abstract

  Differentiated cell types derived from human embryonic stem cells (hESCs) may serve in the future to treat various human diseases and to model early human embryonic development in vitro. Fulfilling this potential, however, requires extensive development of methods and reagents for studying hESCs self-renewal and differentiation. One of the most widely used experimental approaches in the field of stem cell research is the identification of cell surface markers that can be used to prospectively define and isolate specific populations of stem cells and their progenitors. Here, we review an efficient method for generating monoclonal antibodies against cell surface antigens expressed by hESCs and stem cells at different stages of differentiation. This method may have profound implications for many aspects of hESC research and therapeutics.

  View details for DOI 10.1007/978-1-59745-536-7_6

  View details for PubMedID 18453249

• Stem cells; lessons from the past, lessons for the future Stem Cell Technology and Other Innovative Therapies Weissman, I. Pontificia Academia Scientiarum. 2007
• The cancer stem cell hypothesis: a work in progress LABORATORY INVESTIGATION Tan, B. T., Park, C. Y., Ailles, L. E., Weissman, I. L. 2006; 86 (12): 1203-1207

  Abstract

  There is a growing body of evidence that supports the idea that malignant tumors are initiated and maintained by a population of tumor cells that share similar biologic properties to normal adult stem cells. This model, the cancer stem cell (CSC) hypothesis, is based on the observation that tumors, like adult tissues, arise from cells that exhibit the ability to self-renew as well as give rise to differentiated tissue cells. Although the concept of the CSC is not entirely new, advances made over the past two decades in our understanding of normal stem cell biology in conjunction with the recent application of these concepts to experimentally define CSCs have resulted in the identification of CSCs in several human malignancies.

  View details for DOI 10.1038/labinvest.3700488

  View details for Web of Science ID 000242442400001

  View details for PubMedID 17075578

• Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Warren, L., Bryder, D., Weissman, I. L., Quake, S. R. 2006; 103 (47): 17807-17812

  Abstract

  We report here a systematic, quantitative population analysis of transcription factor expression within developmental progenitors, made possible by a microfluidic chip-based "digital RT-PCR" assay that can count template molecules in cDNA samples prepared from single cells. In a survey encompassing five classes of early hematopoietic precursor, we found markedly heterogeneous expression of the transcription factor PU.1 in hematopoietic stem cells and divergent patterns of PU.1 expression within flk2- and flk2+ common myeloid progenitors. The survey also revealed significant differences in the level of the housekeeping transcript GAPDH across the surveyed populations, which demonstrates caveats of normalizing expression data to endogenous controls and underscores the need to put gene measurement on an absolute, copy-per-cell basis.

  View details for DOI 10.1073/pnas.0608512103

  View details for Web of Science ID 000242464900042

  View details for PubMedID 17098862

  View details for PubMedCentralID PMC1693828

• Heme oxygenase 1 deficiency compromises stress responses of hematopoietic stem cells. 48th Annual Meeting of the American-Society-of-Hematology Cao, Y., Wagers, A. J., Karsunky, H., Zhao, H., Reeves, R., Wang, R. J., Stevenson, D. K., Weissman, I. L., Contag, C. H. AMER SOC HEMATOLOGY. 2006: 395A–395A

  View details for Web of Science ID 000242440001612

• Aberrant regulation of Wnt/beta-catenin pathway mediators in chronic myelogenous leukemia stem cells 48th Annual Meeting of the American-Society-of-Hematology Abrahamsson, A., Geron, I., Gotlib, J., Durocher, J., Creusot, R., Kavalerchik, E., Goff, D., Fathman, C. G., Lilleberg, S. L., Giles, F., Weissman, I., Jamieson, C. AMER SOC HEMATOLOGY. 2006: 605A–605A

  View details for Web of Science ID 000242440002656

• The Wilms' tumor gene WT1 is over-expressed in immature leukemia cells but not necessary for leukemia development in mouse leukemia models. 48th Annual Meeting of the American-Society-of-Hematology Hosen, N., Sugiyama, H., Weissman, I. L. AMER SOC HEMATOLOGY. 2006: 415A–415A

  View details for Web of Science ID 000242440001688

• AML1/ETO and PML/RAR alpha can immortalize committed myeloid progenitor cells in-vitro but not expand them in-vivo. 48th Annual Meeting of the American-Society-of-Hematology Hosen, N., Passegue, E., Weissman, I. L. AMER SOC HEMATOLOGY. 2006: 719A–719A

  View details for Web of Science ID 000242440003334

• Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer research Clarke, M. F., Dick, J. E., Dirks, P. B., Eaves, C. J., Jamieson, C. H., Jones, D. L., Visvader, J., Weissman, I. L., Wahl, G. M. 2006; 66 (19): 9339-9344

  View details for PubMedID 16990346

• Flow cytometric analysis of neural stem cell markers on pediatric brain tumors 7th Congress of the European-Association-for-Neuro-Oncology (EANO) Cheshier, S. H., Ailles, L. E., Lim, M., Laddis, P., Tse, V., Weissman, I. L., Huhn, S. OXFORD UNIV PRESS INC. 2006: 466–66

  View details for Web of Science ID 000240877301290

• CD90 expression segregates tumor-sphere forming cells in human glioblastoma multiforme 7th Congress of the European-Association-for-Neuro-Oncology (EANO) Cheshier, S. H., Ailles, L., Higgins, D. M., Lim, M., Kalani, M. Y., Bababeygy, S., Weissman, I. L. OXFORD UNIV PRESS INC. 2006: 471–71

  View details for Web of Science ID 000240877301309

• Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands DEVELOPMENTAL CELL Ueno, H., Weissman, I. L. 2006; 11 (4): 519-533

  Abstract

  Direct clonal analysis of tissue and organ maturation in vivo is a critical step in the interpretation of in vitro cell precursor-progeny relationships. We have developed a method to analyze clonal progenitor contributions in vivo using ES cells stably expressing separate fluorescent proteins and placed into normal blastocysts to form tetrachimeras. Here we applied this method to the analysis of embryonic yolk sac blood islands. In most vertebrates, yolk sac blood islands are the initial sites of appearance of hematopoietic and endothelial cells. It has been proposed that these lineages arise from a common clonal progenitor, the hemangioblast, but this hypothesis has not been tested directly in physiological development in vivo. Our analysis shows that each island has contributions from multiple progenitors. Moreover, contribution by individual hemangioblast progenitors to both endothelial and hematopoietic lineages within an island, if it happens at all, is an infrequent event.

  View details for DOI 10.1016/j.devcel.2006.08.001

  View details for Web of Science ID 000241123300013

  View details for PubMedID 17011491

• Proliferation and differentiation of brain cancer stem cells in organotypic slices 16th International Congress of Neuropathology D'Apuzzo, M. M., Cheshier, S., Ailles, L., Vogel, H., Weissman, I. WILEY-BLACKWELL. 2006: S117–S117

  View details for Web of Science ID 000239938600262

• Incorporation of bone marrow-derived Flk-1-expressing CD34+ cells in the endothelium of tumor vessels in the mouse brain NEUROSURGERY Santarelli, J. G., Udani, V., Yung, Y. C., Cheshier, S., Wagers, A., Brekken, R. A., Weissman, I., Tse, V. 2006; 59 (2): 374-381

  Abstract

  Neoangiogenesis is a prerequisite for the full phenotypic expression and growth of a malignant tumor mass. It is believed to be triggered by tissue hypoxia and involves proliferation and sprouting of the preexisting vessels and the recruitment of endothelial progenitor cells from bone marrow.A chimeric mouse model was used to examine the contribution of these progenitor cells to the neovasculature of brain tumor. T-cell knockout (RAG/KO5.2) mice were irradiated lethally, and their bone marrow was repopulated with T-cell depleted green fluorescent protein (GFP)-expressing bone marrow cells. RAG/RT-2 glioma cells were implanted into the striatum of the animals. Neovascular formation at various times of tumor growth was monitored together with the extent of incorporation of GFP+ bone marrow-derived cells within the vascular tree, in particular, cells carrying the endothelial progenitor markers CD34 and Flk-1.The recruitment of GFP+ cells to the growing tumor and their incorporation into the vascular network occurred during the period of increasing vascular density and preceded the expansion of the tumor. The number of marrow-derived cells with endothelial morphology and phenotype was small but significant (4% of all endothelial cells at Day 12); 54% of all tumor vessels contained at least one GFP+ cell.Our results suggest that bone marrow cells are recruited to newly formed and remodeled tumor vessels. Their recruitment may occur in response to signals from a highly proliferating milieu, and their role is to support the neovascular complex and to promote tumor growth.

  View details for DOI 10.1227/01.NEU.0000222658.66878.CC

  View details for Web of Science ID 000239763800047

  View details for PubMedID 16883178

• Hematopoietic stem cells - The paradigmatic tissue-specific stem cell AMERICAN JOURNAL OF PATHOLOGY Bryder, D., Rossi, D. J., Weissman, I. L. 2006; 169 (2): 338-346

  Abstract

  The recent prospective isolation of a wide variety of somatically derived stem cells has affirmed the notion that homeostatic maintenance of most tissues and organs is mediated by tissue-specific stem and progenitor cells and fueled enthusiasm for the use of such cells in strategies aimed at repairing or replacing damaged, diseased, or genetically deficient tissues and organs. Hematopoietic stem cells (HSCs) are arguably the most well-characterized tissue-specific stem cell, with decades of basic research and clinical application providing not only a profound understanding of the principles of stem cell biology, but also of its potential pitfalls. It is our belief that emerging stem cell fields can benefit greatly from an understanding of the lessons learned from the study of HSCs. In this review we discuss some general concepts regarding stem cell biology learned from the study of HSCs with a highlight on recent work pertaining to emerging topics of interest for stem cell biology.

  View details for DOI 10.2353/jmoldx.2006.050079

  View details for Web of Science ID 000239471100002

  View details for PubMedID 16877336

  View details for PubMedCentralID PMC1698791

• New evidence supporting megakaryocyte-erythrocyte potential of Flk2/Flt3(+) multipotent hematopoietic progenitors CELL Forsberg, E. C., Serwold, T., Kogan, S., Weissman, I. L., Passegue, E. 2006; 126 (2): 415-426

  Abstract

  A model of hematopoietic development wherein multipotentiality is conserved until segregation of myeloid and lymphoid potential has recently been challenged, proposing that megakaryocyte/erythrocyte (MegE) potential is lost in Flk2/Flt3-expressing early progenitors. Here, we used sensitive in vivo approaches to quantitatively and kinetically assess the MegE potential of hematopoietic stem cells and various Flk2(+) early progenitors and compared it with the MegE potential of downstream committed myeloid and lymphoid progenitors and with their ability to give rise to mature myelomonocytic and lymphoid cells. We demonstrate that Flk2(+) early progenitors retain MegE potential in vivo both at the population and clonal levels. These results indicate that Flk2 expression by early progenitors is not at the expense of full multipotency and support the current model of hematopoietic development with segregation of myeloid and lymphoid lineages from multipotent progenitors.

  View details for DOI 10.1016/j.cell.2006.06.037

  View details for Web of Science ID 000239552600025

  View details for PubMedID 16873070

• fester, a Candidate allorecognition receptor from a primitive chordate IMMUNITY Nyholm, S. V., Passegue, E., Ludington, W. B., Voskoboynik, A., Mitchel, K., Weissman, I. L., De Tomaso, A. W. 2006; 25 (1): 163-173

  Abstract

  Histocompatibility in the primitive chordate, Botryllus schlosseri, is controlled by a single, highly polymorphic locus, the FuHC. By taking a forward genetic approach, we have identified a locus encoded near the FuHC, called fester, which is polymorphic, polygenic, and inherited in distinct haplotypes. Somatic diversification occurs through extensive alternative splicing, with each individual expressing a unique repertoire of splice forms, both membrane bound and potentially secreted, all expressed in tissues intimately associated with histocompatibility. Functional studies, via both siRNA-mediated knockdown and direct blocking by monoclonal antibodies raised against fester, were able to disrupt predicted histocompatibility outcomes. The genetic and somatic diversity, coupled to the expression and functional data, suggests that fester is a receptor involved in histocompatibility.

  View details for DOI 10.1016/j.immuni.2006.04.011

  View details for Web of Science ID 000239713000019

  View details for PubMedID 16860765

• Rapid lymphocyte reconstitution of unconditioned immunodeficient mice with non-self-renewing multipotent hematopoietic progenitors CELL CYCLE Bhattacharya, D., Bryder, D., Rossi, D. J., Weissman, I. L. 2006; 5 (11): 1135-1139

  Abstract

  The replacement of abnormal hematopoietic stem cells (HSCs) with normal transplanted HSCs can correct a wide range of hematologic disorders. Here, we provide evidence that transplantation of more differentiated progenitor cells can be used to more rapidly correct lymphoid deficiencies in unconditioned immunocompromised mice. Transplantation of flk2+ multipotent progenitors led to robust B and T cell reconstitution that was maintained for at least 16 weeks. Antigenic challenge at 16 weeks post-transplantation revealed that reconstituted lymphocytes maintained a functional repertoire. In contrast to the persistent lymphocytic engraftment, myeloid chimerism was lost by 12 weeks post-transplantation consistent with the fact that flk2+ progenitors are non-self-renewing. Thus, while more differentiated progenitors are capable of rescuing lymphoid deficiencies, transplantation of HSCs must be used for the correction of non-lymphoid disorders, and, we propose, very long-term immune reconstitution. Based on recent evidence, we discuss novel strategies to achieve the replacement of abnormal HSCs without the use of cytotoxic conditioning regimens.

  View details for Web of Science ID 000238581100003

  View details for PubMedID 16760650

• Pten, tumorigenesis, and stem cell self-renewal CELL Rossi, D. J., Weissman, I. L. 2006; 125 (2): 229-231

  Abstract

  Self-renewal pathways crucial for maintaining stem cells are deregulated in cancer, raising the spectre that cancer therapies targeting such pathways might also ablate normal stem cells. As Yilmaz et al. (2006) report in a recent Nature paper, this may not be the case for the tumor suppressor protein Pten, which drives the self-renewal of normal hematopoietic stem cells and the formation of leukemia cells through different mechanisms.

  View details for DOI 10.1016/j.cell.2006.04.006

  View details for Web of Science ID 000237241500013

  View details for PubMedID 16630811

• The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jamieson, C. H., Gotlib, J., Durocher, J. A., Chao, M. P., Mariappan, M. R., Lay, M., Jones, C., Zehnder, J. L., Lilleberg, S. L., Weissman, I. L. 2006; 103 (16): 6224-6229

  Abstract

  Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

  View details for DOI 10.1073/pnas.0601462103

  View details for Web of Science ID 000236999000031

  View details for PubMedID 16603627

  View details for PubMedCentralID PMC1434515

• Differential expression of alpha 2 integrin separates long-term and short-term reconstituting Lin(-/lo)Thy1.1(lo)c-kit(+)Sca-1(+) hematopoietic stem cells STEM CELLS Wagers, A. J., Weissman, I. L. 2006; 24 (4): 1087-1094

  Abstract

  Self-renewing, multipotent hematopoietic stem cells are highly enriched within the Lin- Thy1.1(lo)c-kit+ Sca-1+ subset of mouse bone marrow. However, heterogeneous expression within this population of certain cell surface markers raises the possibility that it may be further fractionated phenotypically and perhaps functionally. We previously identified alpha2-integrin (CD49b) as a surface marker with heterogeneous expression on Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ stem cells. To determine whether differences in alpha2 expression were indicative of differences in stem cell function, we purified alpha2- and alpha2hi stem cells by fluorescence-activated cell sorting and analyzed their function in long- and short-term hematopoietic reconstitution assays. Both alpha2- and alpha2hi cells could give rise to mature lymphoid and myeloid cells after transplantation into lethally irradiated congenic recipients. However, alpha2hi cells supported hematopoiesis for only a short time (<4 weeks), whereas alpha2- cells reproducibly yielded robust, long-term (>20 weeks) reconstitution, suggesting that alpha2- cells represent a more primitive population than do alpha2hi cells. Consistent with this idea, alpha2- Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ cells exhibited an approximately sixfold decreased frequency of spleen colony-forming units (day 12) versus alpha2hi cells. Furthermore, bone marrow cells isolated from animals transplanted >20 weeks previously with 20 alpha2- Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ cells included both alpha2- and alpha2hi stem cells of donor origin, indicating that alpha2hi cells are likely lineal descendents of alpha2- cells. Interestingly, alpha2 integrin expression is significantly reduced on lineage-restricted oligopotent progenitors in the marrow, suggesting that high level expression of alpha2 selectively marks a subset of primitive hematopoietic cells which retains multilineage reconstitution potential but exhibits reduced self-renewal capacity.

  View details for DOI 10.1634/stemcells.2005-0396

  View details for Web of Science ID 000240636300031

  View details for PubMedID 16373693

• Memory T and memory B cells share a transcriptional program of self-renewal with long-term hematopoietic stem cells Annual Meeting of the American-Association-of-Immunologists Luckey, C. J., Bhattacharya, D., Goldrath, A. W., Weissman, I. L., Benoist, C., Mathis, D. AMER ASSOC IMMUNOLOGISTS. 2006: S298–S299

  View details for Web of Science ID 000238837102385

• Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning. Annual Meeting of the American-Association-of-Immunologists Bhattacharya, D., Rossi, D. J., Bryder, D., Weissman, I. L. AMER ASSOC IMMUNOLOGISTS. 2006: S308–S308

  View details for Web of Science ID 000238837102430

• Early TCR expression and aberrant T cell development in mice with clone-derived T cell receptor genes Annual Meeting of the American-Association-of-Immunologists Serwold, T., Hochedlinger, K., Inlay, M. A., Jaenisch, R., Weissman, I. L. AMER ASSOC IMMUNOLOGISTS. 2006: S314–S315

  View details for Web of Science ID 000238837102462

• Stem cells: Biology, transplantation, and political ethics PROCEEDINGS OF THE AMERICAN PHILOSOPHICAL SOCIETY Weissman, I. L. 2006; 150 (1): 121-147

  View details for Web of Science ID 000240669300006

• Memory T and memory B cells share a transcriptional program of self-renewal with long-term hematopoietic stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Luckey, C. J., Bhattacharya, D., Goldrath, A. W., Weissman, I. L., Benoist, C., MATHIS, D. 2006; 103 (9): 3304-3309

  Abstract

  The only cells of the hematopoietic system that undergo self-renewal for the lifetime of the organism are long-term hematopoietic stem cells and memory T and B cells. To determine whether there is a shared transcriptional program among these self-renewing populations, we first compared the gene-expression profiles of naïve, effector and memory CD8(+) T cells with those of long-term hematopoietic stem cells, short-term hematopoietic stem cells, and lineage-committed progenitors. Transcripts augmented in memory CD8(+) T cells relative to naïve and effector T cells were selectively enriched in long-term hematopoietic stem cells and were progressively lost in their short-term and lineage-committed counterparts. Furthermore, transcripts selectively decreased in memory CD8(+) T cells were selectively down-regulated in long-term hematopoietic stem cells and progressively increased with differentiation. To confirm that this pattern was a general property of immunologic memory, we turned to independently generated gene expression profiles of memory, naïve, germinal center, and plasma B cells. Once again, memory-enriched and -depleted transcripts were also appropriately augmented and diminished in long-term hematopoietic stem cells, and their expression correlated with progressive loss of self-renewal function. Thus, there appears to be a common signature of both up- and down-regulated transcripts shared between memory T cells, memory B cells, and long-term hematopoietic stem cells. This signature was not consistently enriched in neural or embryonic stem cell populations and, therefore, appears to be restricted to the hematopoeitic system. These observations provide evidence that the shared phenotype of self-renewal in the hematopoietic system is linked at the molecular level.

  View details for DOI 10.1073/pnas.0511137103

  View details for Web of Science ID 000235780700055

  View details for PubMedID 16492737

  View details for PubMedCentralID PMC1413911

• In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery CIRCULATION Cao, F., Lin, S., Xie, X. Y., Ray, P., Patel, M., Zhang, X. Z., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X. Y., Weissman, I. L., Gambhir, S. S., Wu, J. C. 2006; 113 (7): 1005-1014

  Abstract

  Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities.Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, 1x10(7) of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7x10(7)+/-5.8x10(6) photons.s(-1).cm(-2) per steradian (sr) and 0.08+/-0.03% injected dose/g, respectively (P<0.05 versus control). Both signals increased progressively from week 1 to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (n=4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (n=4) treated with saline (1 mL/kg).This is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. This versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.

  View details for DOI 10.1161/CIRCULATIONHA.105.588954

  View details for PubMedID 16476845

• Flk2(+) myeloid progenitors are the main source of Langerhans cells BLOOD Mende, I., Karsunky, H., Weissman, I. L., Engleman, E. G., Merad, M. 2006; 107 (4): 1383-1390

  Abstract

  Langerhans cells (LCs) are antigen-presenting cells (APCs) residing in the epidermis that play a major role in skin immunity. Our earlier studies showed that when skin is inflamed LCs are replaced by bone marrow-derived progenitor cells, while during steady-state conditions LCs are able to self-renew in the skin. Identification of the LC progenitors in bone marrow would represent a critical step toward identifying the factors that regulate LC generation as well as their trafficking to the skin. To determine LC lineage origin, we reconstituted lethally irradiated CD45.2 mice with rigorously purified lymphoid and myeloid progenitors from CD45.1 congenic mice. Twenty-four hours later, we exposed the mice to UV light to deplete resident LCs and induce their replacement by progenitors. Reconstitution with common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-macrophage progenitors (GMPs), or early thymic progenitors led to LC generation within 2 to 3 weeks. CMPs were at least 20 times more efficient at generating LCs than CLPs. LCs from both lineages were derived almost entirely from fetal liver kinase-2+ (Flk-2+) progenitors, displayed typical dendritic-cell (DC) morphology, and showed long-term persistence in the skin. These results indicate that LCs are derived mainly from myeloid progenitors and are dependent on Flt3-ligand for their development.

  View details for DOI 10.1182/blood-2005-05-1878

  View details for Web of Science ID 000235296100026

  View details for PubMedID 16263793

• Adult human hematopoietic cells differentiate into mature T cells via a CD3-4+8-intermediate within the mouse thymic microenvironment; A new model system for the study of human thymocyte development further enhanced by antimurine c-Kit mAB 32nd Annual Meeting of the American-Society-for-Blood-and-Marrow-Transplantation Kraft, D. L., Czechowicz, A., Weissman, I. J. ELSEVIER SCIENCE INC. 2006: 131–31

  View details for Web of Science ID 000235344100379

• Differential amplification of murine bipotent megakaryocytic/erythroid progenitor and precursor cells during recovery from acute and chronic erythroid stress STEM CELLS Sanchez, M., Weissman, I. L., Pallavicini, M., Valeri, M., Guglielmelli, P., Vannucchi, A. M., Migliaccio, G., Migliaccio, A. R. 2006; 24 (2): 337-348

  Abstract

  Two murine bipotent erythroid/megakaryocytic cells, the progenitor (MEP) and precursor (PEM) cells, recently have been identified on the basis of the phenotypes of linnegc-kitposSca-1neg CD16/CD32lowCD34low and TER119pos4A5pos or 2D5pos, respectively. However, the functional relationship between these two subpopulations and their placement in the hemopoietic hierarchy is incompletely understood. We compared the biological properties of these subpopulations in marrow and spleen of mice with and without acute or chronic erythroid stress. MEP cells, but not PEM cells, express c-kit, respond to stem cell factor in vitro, and form spleen colonies in vivo. PEM cells comprise up to 50%-70% of the cells in BFU-E-derived colonies but are not present among the progeny of purified MEP cells cultured under erythroid and megakaryocytic permissive conditions. PEM cells increase 10- to 20-fold under acute and chronic stress, whereas MEP cell increases (21%-84%) are observed only in acutely stressed animals. These data suggest that MEP and PEM cells represent distinct cell populations that may exist in an upstream-downstream differentiation relationship under conditions of stress. Whereas the dynamics of both populations are altered by stress induction, the differential response to acute and chronic stress suggests different regulatory mechanisms. A model describing the relationship between MEP, PEM, and common myeloid progenitor cells is presented.

  View details for DOI 10.1634/stemcells.2005-0023

  View details for Web of Science ID 000240635900016

  View details for PubMedID 16144876

• Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning JOURNAL OF EXPERIMENTAL MEDICINE Bhattacharya, D., Rossi, D. J., Bryder, D., Weissman, I. L. 2006; 203 (1): 73-85

  Abstract

  In the absence of irradiation or other cytoreductive conditioning, endogenous hematopoietic stem cells (HSCs) are thought to fill the unique niches within the bone marrow that allow maintenance of full hematopoietic potential and thus prevent productive engraftment of transplanted donor HSCs. By transplantation of purified exogenous HSCs into unconditioned congenic histocompatible strains of mice, we show that approximately 0.1-1.0% of these HSC niches are available for engraftment at any given point and find no evidence that endogenous HSCs can be displaced from the niches they occupy. We demonstrate that productive engraftment of HSCs within these empty niches is inhibited by host CD4+ T cells that recognize very subtle minor histocompatibility differences. Strikingly, transplantation of purified HSCs into a panel of severe combined immunodeficient (SCID) mice leads to a rapid and complete rescue of lymphoid deficiencies through engraftment of these very rare niches and expansion of donor lymphoid progenitors. We further demonstrate that transient antibody-mediated depletion of CD4+ T cells allows short-term HSC engraftment and regeneration of B cells in a mouse model of B(-) non-SCID. These experiments provide a general mechanism by which transplanted HSCs can correct hematopoietic deficiencies without any host conditioning or with only highly specific and transient lymphoablation.

  View details for DOI 10.1084/jem.20051714

  View details for Web of Science ID 000235003600011

  View details for PubMedID 16380511

  View details for PubMedCentralID PMC2118067

• Medicine: Politic stem cells NATURE Weissman, I. L. 2006; 439 (7073): 145-?

  View details for DOI 10.1038/439145a

  View details for Web of Science ID 000234538400025

  View details for PubMedID 16407938

• Hematopoietic stem cells - Expression profiling and beyond STEM CELL REVIEWS Forsberg, E. C., Bhattacharya, D., Weissman, I. L. 2006; 2 (1): 23-30

  Abstract

  This review focuses on the genomics of mouse hematopoiesis, but also draws parallels to other systems and discusses issues common to the analysis of rare populations such as stem cells. As examples from the mouse blood forming system are used to illustrate several points, the authors first give a brief introduction to mouse hematopoiesis as a model system. We review the multiple microarray analyses that have been performed on various mouse hematopoietic subpopulations and comment on both technical and biological aspects of such experiments. The concept of stemness is discussed, and the importance of biological function of gene products, protein-protein interactions and molecular pathways highlighted. Finally, the authors discuss some major unresolved issues in hematopoiesis and discuss the potential uses of future microarray analysis as well as other genomic and functional approaches that might prove useful to further our understanding of hematopoiesis and other stem cell systems.

  View details for Web of Science ID 000240469100005

  View details for PubMedID 17142883

• Stem cells are units of natural selection in a colonial ascidian CELL Laird, D. J., De Tomaso, A. W., Weissman, I. L. 2005; 123 (7): 1351-1360

  Abstract

  Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent, self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates, but not to both, is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele, these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.

  View details for DOI 10.1016/j.cell.2005.10.026

  View details for Web of Science ID 000234584500021

  View details for PubMedID 16377573

• Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates JOURNAL OF EXPERIMENTAL MEDICINE Passegue, E., Wagers, A. J., Giuriato, S., Anderson, W. C., Weissman, I. L. 2005; 202 (11): 1599-1611

  Abstract

  Knowledge of the molecular networks controlling the proliferation and fate of hematopoietic stem cells (HSC) is essential to understand their function in maintaining blood cell production during normal hematopoiesis and upon clinical transplantation. Using highly purified stem and progenitor cell populations, we define the proliferation index and status of the cell cycle machinery at discrete stages of hematopoietic differentiation and during cytokine-mediated HSC mobilization. We identify distinct sets of cell cycle proteins that specifically associate with differentiation, self-renewal, and maintenance of quiescence in HSC and progenitor cells. Moreover, we describe a striking inequality of function among in vivo cycling and quiescent HSC by demonstrating that their long-term engraftment potential resides predominantly in the G(0) fraction. These data provide a direct link between HSC proliferation and function and identify discrete molecular targets in regulating HSC cell fate decisions that could have implications for both the therapeutic use of HSC and the understanding of leukemic transformation.

  View details for DOI 10.1084/jem.20050967

  View details for Web of Science ID 000233753900015

  View details for PubMedID 16330818

  View details for PubMedCentralID PMC2213324

• Identification of a novel gene involved in asexual organogenesis in the budding ascidian Botryllus schlosseri DEVELOPMENTAL DYNAMICS Laird, D. J., Chang, W. T., Weissman, I. L., Lauzon, R. J. 2005; 234 (4): 997-1005

  Abstract

  Development via regeneration or budding shares some known genetic pathways with embryogenesis, but no concerted effort has been made to identify genes unique to asexual development. We have identified a novel gene that plays a role in cyclical bud formation and asexual organogenesis in the colonial ascidian Botryllus schlosseri. Athena mRNA is transcribed at high levels during the 24- to 36-hr interval of programmed cell death and new bud initiation at the conclusion of the budding cycle (takeover). Knockdown of Athena by RNAi and antisense morpholinos induced defects in the development of new buds ranging from retardation in growth and abnormal organogenesis to hollow buds lacking organs. As genetic intervention in this organism has not been possible, this study establishes the use of RNAi and morpholinos in Botryllus as well as describing the knockdown phenotype of a new gene.

  View details for DOI 10.1002/dvdy.20583

  View details for Web of Science ID 000233715500018

  View details for PubMedID 16193502

  View details for PubMedCentralID PMC2821222

• Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells BLOOD Lawrence, H. J., Christensen, J., Fong, S., Hu, Y. L., Weissman, I., Sauvageau, G., Humphries, R. K., Largman, C. 2005; 106 (12): 3988-3994

  Abstract

  The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells, and overexpression of Hoxa-9 markedly expands hematopoietic stem cells, suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However, sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia, indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays, Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability, a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures, Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation, with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo.

  View details for DOI 10.1182/blood-2005-05-2003

  View details for Web of Science ID 000233662400055

  View details for PubMedID 16091451

• Isolation and characterization of a protochordate histocompatibility locus NATURE De Tomaso, A. W., Nyholm, S. V., Palmeri, K. J., Ishizuka, K. J., Ludington, W. B., Mitchel, K., Weissman, I. L. 2005; 438 (7067): 454-459

  Abstract

  Histocompatibility--the ability of an organism to distinguish its own cells and tissue from those of another--is a universal phenomenon in the Metazoa. In vertebrates, histocompatibility is a function of the immune system controlled by a highly polymorphic major histocompatibility complex (MHC), which encodes proteins that target foreign molecules for immune cell recognition. The association of the MHC and immune function suggests an evolutionary relationship between metazoan histocompatibility and the origins of vertebrate immunity. However, the MHC of vertebrates is the only functionally characterized histocompatibility system; the mechanisms underlying this process in non-vertebrates are unknown. A primitive chordate, the ascidian Botryllus schlosseri, also undergoes a histocompatibility reaction controlled by a highly polymorphic locus. Here we describe the isolation of a candidate gene encoding an immunoglobulin superfamily member that, by itself, predicts the outcome of histocompatibility reactions. This is the first non-vertebrate histocompatibility gene described, and may provide insights into the evolution of vertebrate adaptive immunity.

  View details for DOI 10.1038/nature04150

  View details for Web of Science ID 000233458200040

  View details for PubMedID 16306984

  View details for PubMedCentralID PMC1401502

• Bioluminescent imaging of human leukemic stem cell engraftment. 47th Annual Meeting of the American-Society-of-Hematology Jamieson, C., Karimi, M., Creusot, R., Negrin, R., Gotlib, J., Chao, M., Jones, C., Keating, A., Fathman, C. G., Zehnder, J., Weissman, I. L. AMER SOC HEMATOLOGY. 2005: 205A–205A

  View details for Web of Science ID 000233426001165

• Molecular progenitor profiling in human myeloproliferative disorders. 47th Annual Meeting of the American-Society-of-Hematology Jamieson, C. H., Gotlib, J., Chao, M., Mariappan, M. R., LayRaj, M., Jones, C., Zehnder, J., Durocher, J., Lilleberg, S., Coutre, S., Weissman, I. L. AMER SOC HEMATOLOGY. 2005: 38A–39A

  View details for Web of Science ID 000233426000119

• Identification of phenotypic neural stem cells in a pediatric astroblastoma JOURNAL OF NEUROSURGERY Huhn, S. L., Yung, Y., Cheshier, S., Harsh, G., Ailles, L., Weissman, I., Vogel, H., Tse, V. 2005; 103 (5): 446-450

  Abstract

  The goal of this study was to illustrate the findings of a significant subpopulation of cells within a pediatric astroblastoma that have the specific cell surface phenotype found on known human neural stem cells.Cells with a cell surface marker profile characteristic of human neural stem cells were isolated using fluorescence-activated cell sorting from a mostly nonmitotic astroblastoma removed from the brain of an 11-year-old girl. An unusually high proportion (24%) of the cells were CD133 positive and CD24, CD34, and CD45 negative (CD133(+)CD24(-)CD34(-)CD45(-) cells), the phenotypic antigenic pattern associated with neural stem cells; very few CD133-positive cells were not also CD24, CD34, and CD45 negative. Some cells (12%) were CD34 positive, indicating the presence within the tumor of hematopoietic stem cells. Cells formed cytospheres that resembled neurospheres when seeded into stem cell media and coexpressed beta-tubulin and glial fibrillary acidic protein (GFAP) but did not express the oligodendrocyte marker O4. Cell proliferation was demonstrated by incorporation of bromodeoxyuridine. The cells lost their capacity for self-renewal in vitro after four to six passages, although they continued to coexpress beta-tubulin and GFAP. The cells did not differentiate into neurons or astrocytes when placed in differentiation medium.Although this astroblastoma contained a high proportion of phenotypic neural stemlike cells, the cells had limited proliferative capacity and multipotency. Their role in astroblastoma formation and growth is unknown.

  View details for PubMedID 16302618

• Simple and efficient isolation of hematopoietic stem cells from H2K-zFP transgenic mice STEM CELLS Surdez, D., Kunz, B., Wagers, A. J., Weissman, I. L., Terskikh, A. V. 2005; 23 (10): 1617-1625

  Abstract

  We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP(bright) cells are also c-kit(high), Sca-1(high), Lin(neg), Flk-2(neg), which is a bona fide phenotype for long-term HSCs. Double-sorted zFP(bright) HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.

  View details for Web of Science ID 000233708700021

  View details for PubMedID 16091556

• Hematopoietic stem cells give rise to perivascular endothelial-like cells during brain tumor angiogenesis 54th Annual Meeting of the Congress-of-Neurological-Surgeons Udani, M., Santarelli, G., Yung, Y. C., Wagers, A. J., Cheshier, S. H., Weissman, I. L., Tse, V. MARY ANN LIEBERT INC. 2005: 478–86

  Abstract

  Bone marrow (BM) cells have recently been shown to give rise to skeletal, hepatic, cardiac, neural, and vascular endothelial tissues. However, it has been shown that this is the result of cell fusion rather than transdifferentiation of hematopoietic stem cells (HSC). For this study, we established a mouse model of brain tumor growth to investigate the differentiation potential of HSC into endothelial cells during brain tumor-induced angiogenesis. Nontransgenic (GFP(neg)) recipient mice were lethally irradiated, and their hematopoietic cells were subsequently repopulated by transplantation of a single green fluorescent protein (GFP)-expressing HSC. Rat glioma (RT-2/RAG) cells were then injected into the striatum of the chimeric mice 6-8 weeks post-transplantation. The animals were sacrificed 3-9 days after tumor implantation, and the mobilization, temporal-spatial distribution, and lineage-specific marker expression profile of the GFP(+) cells within the growing tumor were analyzed. We saw that GFP(+) cells gave rise to elongated, CD34(+)/Flk-1(+) cells that incorporated into the endothelium of tumor blood vessels. However, all GFP(+) cells were also CD45(+), and the presence of CD45 on the HSC-derived endothelial-like cells supports the hypothesis that the hematopoietic cells were recruited into the tumor milieu. The fact that we failed to demonstrate the expression of von Willebrand factor in these cells argues against a true endothelial identity. Nevertheless, the recruitment of HSC-derived endothelial-like cells was an extremely rare event in normal brain parenchyma, and, thus, the permissive influence afforded by the growing tumor appeared to enhance the perivascular tropism and acquisition of an endothelial phenotypes by a population of HSC-derived cells.

  View details for Web of Science ID 000233904500003

  View details for PubMedID 16305333

• Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus JOURNAL OF IMMUNOLOGY Garcia-Ojeda, M. E., Dejbakhsh-Jones, S., Chatterjea-Matthes, D., Mukhopadhyay, A., Bitmansour, A., Weissman, I. L., Brown, J. M., Strober, S. 2005; 175 (7): 4363-4373

  Abstract

  We identified committed T cell progenitors (CTPs) in the mouse bone marrow that have not rearranged the TCRbeta gene; express a variety of genes associated with commitment to the T cell lineage, including GATA-3, T cell-specific factor-1, Cbeta, and Id2; and show a surface marker pattern (CD44+ CD25- CD24+ CD5-) that is similar to the earliest T cell progenitors in the thymus. More mature committed intermediate progenitors in the marrow have rearranged the TCR gene loci, express Valpha and Vbeta genes as well as CD3epsilon, but do not express surface TCR or CD3 receptors. CTPs, but not progenitors from the thymus, reconstituted the alphabeta T cells in the lymphoid tissues of athymic nu/nu mice. These reconstituted T cells vigorously secreted IFN-gamma after stimulation in vitro, and protected the mice against lethal infection with murine CMV. In conclusion, CTPs in wild-type bone marrow can generate functional T cells via an extrathymic pathway in athymic nu/nu mice.

  View details for Web of Science ID 000232092600027

  View details for PubMedID 16177077

• Stem cell research - Paths to cancer therapies and regenerative medicine JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Weissman, I. 2005; 294 (11): 1359-1366

  Abstract

  Most tissues in complex metazoans contain a rare subset of cells that, at the single-cell level, can self-renew and also give rise to mature daughter cells. Such stem cells likely in development build tissues and are retained in adult life to regenerate them. Cancers and leukemias are apparently not an exception: rare leukemia stem cells and cancer stem cells have been isolated that contain all of the tumorigenicity of the whole tumor, and it is their properties that will guide future therapies. None of this was apparent just 20 years ago, yet this kind of stem cell thinking already provides new perspectives in medical science and could usher in new therapies. Today, political, religious, and ethical issues surround embryonic stem cell and patient-specific pluripotent stem cell research and are center stage in the attempts by governments to ban these fields for discovery and potential therapies. These interventions require physicians and physician-scientists to determine for themselves whether patient welfare or personal ethics will dominate in their practices, and whether all aspects of stem cell research can be pursued in a safe and regulated fashion.

  View details for Web of Science ID 000231987800009

  View details for PubMedID 16174694

• They are not stealthy in the heart: embryonic stem cells trigger cell infiltration, humoral and T- lymphocyte-based host immune response 18th Annual Meeting of the European-Association-for-Cardiothoracic-Surgery/12th Annual Meeting of the European-Society-of-Thoracic-Surgeons Kofidis, T., deBruin, J. L., Tanaka, M., Zwierzchoniewska, M., Weissman, I., Fedoseyeva, E., Haverich, A., Robbins, R. C. OXFORD UNIV PRESS INC. 2005: 461–66

  Abstract

  The in vivo immunogenicity of Embryonic Stem Cells is controversial. At present, there is only in vitro evidence of MHC I expression by this cell population but vivid speculation about their immune-privileged state. The immunology aspect of ESC transplantation deserves thorough investigation.We injected mouse ESC (expressing Green Fluorescent Protein, GFP) into injured myocardium of syngeneic, allogeneic and SCID recipients. Furthermore, we monitored host response for up to 4 weeks post cell transfer. We determined local response (CD 3, CD 11c expression by host cells), MHC I expression by donor cells, MHC-II expression within and around the graft, humoral response of allogeneic hosts using Flow Cytometry and evaluated the hosts' cytokine response using stimulated spleenocytes by means of ELISPOT. Cell survival was estimated by morphometry, by calculating the area of the GFP+ graft over the area of infarction at multiple sections of the harvested heart.There was significant cellular infiltration into and around the graft consisting of T-lymphocytes (CD3+) and dendritic cells (CD 11c). Infiltration was detectable at 1 week and progressed through 4 weeks following cell transplantation. The humoral Ab response was moderate at 2 weeks but frank at 4 weeks. ELISPOT demonstrated a Th1 pathway of donor specific T-lymphocyte response with strong IFN-gamma and Il-2 production (figure A). MHC I expression was significant within the graft and maximal in the allogeneic groups.An immune response against transplanted ESC was demonstrated and the future use of ESC will likely require the use of systemic immunosuppression.

  View details for DOI 10.1016/j.ejcts.2005.03.049

  View details for Web of Science ID 000232069300018

  View details for PubMedID 15990327

• Differential expression of novel potential regulators in hematopoietic stem cells PLOS GENETICS Forsberg, E. C., Prohaska, S. S., Katzman, S., Heffner, G. C., Stuart, J. M., Weissman, I. L. 2005; 1 (3): 281-294

  Abstract

  The hematopoietic system is an invaluable model both for understanding basic developmental biology and for developing clinically relevant cell therapies. Using highly purified cells and rigorous microarray analysis we have compared the expression pattern of three of the most primitive hematopoietic subpopulations in adult mouse bone marrow: long-term hematopoietic stem cells (HSC), short-term HSC, and multipotent progenitors. All three populations are capable of differentiating into a spectrum of mature blood cells, but differ in their self-renewal and proliferative capacity. We identified numerous novel potential regulators of HSC self-renewal and proliferation that were differentially expressed between these closely related cell populations. Many of the differentially expressed transcripts fit into pathways and protein complexes not previously identified in HSC, providing evidence for new HSC regulatory units. Extending these observations to the protein level, we demonstrate expression of several of the corresponding proteins, which provide novel surface markers for HSC. We discuss the implications of our findings for HSC biology. In particular, our data suggest that cell-cell and cell-matrix interactions are major regulators of long-term HSC, and that HSC themselves play important roles in regulating their immediate microenvironment.

  View details for DOI 10.1371/journal.pgen.0010028

  View details for Web of Science ID 000234714300002

  View details for PubMedID 16151515

  View details for PubMedCentralID PMC1200425

• Identification of mast cell progenitors in adult mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, C. C., Grimbaldeston, M. A., Tsai, M., Weissman, I. L., Galli, S. J. 2005; 102 (32): 11408-11413

  Abstract

  It is well known that mast cells are derived from hematopoietic stem cells. However, in adult hematopoiesis, a committed mast cell progenitor has not yet been identified in any species, nor is it clear at what point during adult hematopoiesis commitment to the mast cell lineage occurs. We identified a cell population in adult mouse bone marrow, characterized as Lin(-)c-Kit(+)Sca-1(-)-Ly6c(-)FcepsilonRIalpha(-)CD27(-)beta7(+)T1/ST2+, that gives rise only to mast cells in culture and that can reconstitute the mast cell compartment when transferred into c-kit mutant mast cell-deficient mice. In addition, our experiments strongly suggest that these adult mast cell progenitors are derived directly from multipotential progenitors instead of, as previously proposed, common myeloid progenitors or granulocyte/macrophage progenitors.

  View details for DOI 10.1073/pnas.0504197102

  View details for Web of Science ID 000231253400051

  View details for PubMedID 16006518

  View details for PubMedCentralID PMC1183570

• Surface phenotype of Peyer's patch germinal center cells: Implications for the role of germinal centers in B cell differentiation (Reprinted) JOURNAL OF IMMUNOLOGY BUTCHER, E. C., Rouse, R. V., Coffman, R. L., Nottenburg, C. N., Hardy, R. R., Weissman, I. L. 2005; 175 (3): 1363-1372

  View details for Web of Science ID 000233648000002

  View details for PubMedID 16034071

• Cell intrinsic alterations underlie hematopoietic stem cell aging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rossi, D. J., Bryder, D., Zahn, J. M., Ahlenius, H., Sonu, R., Wagers, A. J., Weissman, I. L. 2005; 102 (26): 9194-9199

  Abstract

  Loss of immune function and an increased incidence of myeloid leukemia are two of the most clinically significant consequences of aging of the hematopoietic system. To better understand the mechanisms underlying hematopoietic aging, we evaluated the cell intrinsic functional and molecular properties of highly purified long-term hematopoietic stem cells (LT-HSCs) from young and old mice. We found that LT-HSC aging was accompanied by cell autonomous changes, including increased stem cell self-renewal, differential capacity to generate committed myeloid and lymphoid progenitors, and diminished lymphoid potential. Expression profiling revealed that LT-HSC aging was accompanied by the systemic down-regulation of genes mediating lymphoid specification and function and up-regulation of genes involved in specifying myeloid fate and function. Moreover, LT-HSCs from old mice expressed elevated levels of many genes involved in leukemic transformation. These data support a model in which age-dependent alterations in gene expression at the stem cell level presage downstream developmental potential and thereby contribute to age-dependent immune decline, and perhaps also to the increased incidence of leukemia in the elderly.

  View details for DOI 10.1073/pnas.0503280102

  View details for Web of Science ID 000230191400021

  View details for PubMedID 15967997

  View details for PubMedCentralID PMC1153718

• Stimulation of paracrine pathways with growth factors enhances embryonic stem cell engraftment and host-specific differentiation in the heart after ischemic myocardial injury CIRCULATION Kofidis, T., de Bruin, J. L., Yamane, T., Tanaka, M., Lebl, D. R., Swijnenburg, R. J., Weissman, I. L., Robbins, R. C. 2005; 111 (19): 2486-2493

  Abstract

  Growth factors play an essential role in organogenesis. We examine the potential of growth factors to enhance cell engraftment and differentiation and to promote functional improvement after transfer of undifferentiated embryonic stem cells into the injured heart.Green fluorescent protein (GFP)-positive embryonic stem cells derived from 129sv mice were injected into the ischemic area after left anterior descending artery ligation in allogenic (BALB/c) mice. Fifty nanograms of recombinant mouse vascular endothelial growth factor, fibroblast growth factor (FGF), and transforming growth factor (TGF) was added to the cell suspension. Separate control groups were formed in which only the growth factors were given. Echocardiography was performed 2 weeks later to evaluate heart function (fractional shortening [FS]), end-diastolic diameter, and left ventricular wall thickness). Hearts were harvested for histology (connexin 43, alpha-sarcomeric actin, CD3, CD11c, major histocompatability complex class I, hematoxylin-eosin). Degree of restoration (GFP-positive graft/infarct area ratio), expression of cardiac markers, host response, and tumorigenicity were evaluated. Cell transfer resulted in improved cardiac function. TGF-beta led to better restorative effect and a stronger expression of connexin 43, alpha-sarcomeric actin, and major histocompatability complex class I. TGF-beta and FGF retained left ventricular diameter. FS was better in the TGF-beta, FGF, and embryonic stem cells-only group compared with left anterior descending artery-ligated controls. Growth factors with cells (TGF-beta, FGF) resulted in higher FS and smaller end-diastolic diameter than growth factors alone.Growth factors can promote in vivo organ-specific differentiation of early embryonic stem cells and improve myocardial function after cell transfer into an area of ischemic lesion. TGF-beta should be considered as an adjuvant for myocardial restoration with the use of embryonic stem cells.

  View details for DOI 10.1161/01.CIR.0000165063.09283.A8

  View details for Web of Science ID 000229126900012

  View details for PubMedID 15883216

• Hematopoietic cells maintain hematopoietic fates upon entering the brain JOURNAL OF EXPERIMENTAL MEDICINE Massengale, M., Wagers, A. J., Vogel, H., Weissman, I. L. 2005; 201 (10): 1579-1589

  Abstract

  Several studies have reported that bone marrow (BM) cells may give rise to neurons and astrocytes in vitro and in vivo. To further test this hypothesis, we analyzed for incorporation of neural cell types expressing donor markers in normal or injured brains of irradiated mice reconstituted with whole BM or single, purified c-kit(+)Thy1.1(lo)Lin(-)Sca-1(+) (KTLS) hematopoietic stem cells (HSCs), and of unirradiated parabionts with surgically anastomosed vasculature. Each model showed low-level parenchymal engraftment of donor-marker(+) cells with 96-100% immunoreactivity for panhematopoietic (CD45) or microglial (Iba1 or Mac1) lineage markers in all cases studied. Other than one arborizing structure in the olfactory bulb of one BM-transplanted animal, possibly representing a neuronal or glial cell process, we found no donor-marker-expressing astrocytes or non-Purkinje neurons among >10,000 donor-marker(+) cells from 21 animals. These data strongly suggest that HSCs and their progeny maintain lineage fidelity in the brain and do not adopt neural cell fates with any measurable frequency.

  View details for DOI 10.1084/jem.20050030

  View details for PubMedID 15897275

• Frizzled 9 knock-out mice have abnormal B-cell development BLOOD Ranheim, E. A., Kwan, H. C., Reya, T., Wang, Y. K., Weissman, I. L., FRANCKE, U. 2005; 105 (6): 2487-2494

  Abstract

  The binding of frizzled (Fzd) receptors by their Wnt ligands results in the inhibition of beta-catenin degradation and subsequent transcription of beta-catenin/LEF-inducible genes. The beta-catenin pathway is known to be involved in development, tumorigenesis, and stem cell self-renewal. In humans, the FZD9 gene lies in the region of chromosome 7q11.23 deleted in the neurodevelopmental disorder, Williams-Beuren syndrome (WBS). Fzd9-/- mice show no obvious features of WBS, but reveal a role for Fzd9 in lymphoid development and maturation. Fzd9-/- mice show pronounced splenomegaly, thymic atrophy, and lymphadenopathy with age, with accumulation of plasma cells in lymph nodes. There is a depletion of developing B cells in the bone marrow (BM), particularly in the pre-B stage where immunoglobulin heavy chains are expressed and the cells are undergoing clonal expansion prior to light chain rearrangement. The pre-B defect is partially intrinsic to the hematopoietic system; as in competitive BM reconstitution studies, Fzd9-/- -derived BM exhibits defective B-cell development when implanted into a wild-type host. Mature B cells are present in normal numbers in lymph node and spleen. These findings suggest a role for Fzd9 signaling in lymphoid development, particularly at points where B cells undergo self-renewal prior to further differentiation.

  View details for DOI 10.1182/blood-2004-06-2334

  View details for Web of Science ID 000227630500047

  View details for PubMedID 15572594

• Enforced Bcl-2 expression overrides serum and feeder cell requirements for mouse embryonic stem cell self-renewal PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yamane, T., Dylla, S. J., Muijtjens, M., Weissman, I. L. 2005; 102 (9): 3312-3317

  Abstract

  Leukemia inhibitory factor (LIF) is required, but not sufficient, for pluripotent mouse embryonic stem (ES) cell expansion in vitro in the absence of serum or a feeder cell layer, suggesting that additional signals are provided by serum or feeders that are necessary to support self-renewal. Here we show that transgenic ES cell lines expressing Bcl-2, an antiapoptotic protein, continue to self-renew in serum- and feeder-free conditions when supplemented with LIF; even in the absence of bone morphogenic proteins. Bcl-2-expressing clones sustain the characteristics of undifferentiated, pluripotent ES cells during long-term culture, and maintain their potential to differentiate into mature cell types. These results suggest that LIF and Bcl-2 overexpression are sufficient to expand these mouse pluripotent stem cells in vitro.

  View details for DOI 10.1073/pnas.0500167102

  View details for Web of Science ID 000227423700028

  View details for PubMedID 15728354

  View details for PubMedCentralID PMC552922

• Rejuvenation of aged progenitor cells by exposure to a young systemic environment NATURE Conboy, I. M., Conboy, M. J., Wagers, A. J., Girma, E. R., Weissman, I. L., Rando, T. A. 2005; 433 (7027): 760-764

  Abstract

  The decline of tissue regenerative potential is a hallmark of ageing and may be due to age-related changes in tissue-specific stem cells. A decline in skeletal muscle stem cell (satellite cell) activity due to a loss of Notch signalling results in impaired regeneration of aged muscle. The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-alpha and the chromatin remodelling factor brahma (Brm) inhibits the regenerative capacity of aged liver. To examine the influence of systemic factors on aged progenitor cells from these tissues, we established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells. The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis increased aged hepatocyte proliferation and restored the cEBP-alpha complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.

  View details for DOI 10.1038/nature03260

  View details for Web of Science ID 000227039200043

  View details for PubMedID 15716955

• Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors EXPERIMENTAL HEMATOLOGY Karsunky, H., Merad, M., Mende, I., Manz, M. G., Engleman, E. G., Weissman, I. L. 2005; 33 (2): 173-181

  Abstract

  The aim of this study was to determine the lineage origin of interferon-alpha-producing cells (IPCs), also called plasmacytoid dendritic cells, in mice by evaluating the ability of common lymphoid (CLP) and myeloid (CMP) progenitors to give rise to IPCs.Sublethally irradiated C57Bl/6 mice were intravenously transplanted with rigorously purified lymphoid and myeloid progenitors from a congenic mouse strain. At various time points posttransplantation mice were analyzed for donor-derived cells by flow cytometry. The developmental potential of all progenitor populations was also tested in in vitro cultures. In addition, in vitro and in vivo derived IPCs were functionally assessed for their interferon-alpha production after virus challenge.Transplantation of 1 x 10(4) common myeloid progenitors, 1 x 10(4) common lymphoid progenitors or 2.5 x 10(4) granulocyte/macrophage progenitors all led to the generation of IPCs within 2 to 3 weeks. In general, IPC reconstitution in spleen and liver by CMPs was more efficient than by CLP. Adding Flt3L alone to in vitro cultures was sufficient to support the development of IPCs from myeloid progenitors whereas CLPs required additional survival factors provided either by stroma cells or by introduction of transgenic Bcl-2. Both myeloid- and lymphoid-derived IPC were indistinguishable by function, gene expression, and morphology.Surprisingly, our results clearly show that murine IPCs differentiate from both lineages but are mainly of myeloid origin. These results extend to IPCs the observation made originally in classical dendritic cells that cellular expression of so called lineage markers does not correlate with lineal origin.

  View details for DOI 10.1016/j.exphem.2004.10.010

  View details for Web of Science ID 000227147000007

  View details for PubMedID 15676211

• Prognostic progenitor profiling in chronic myelomonocytic leukemia Joint Meeting of the American-Society-for-Blood-and-Marrow-Transplantation/Center-for-International-Blood-and-Marrow-Transplant-Research Jamieson, C. H., Li, K., Gotlib, J., Coutre, S. E., Lagasse, E., Weissman, I. L. ELSEVIER SCIENCE INC. 2005: 59–59

  View details for Web of Science ID 000227329000175

• Bioluminescent tracking of candidate leukemic stem cell engraftment in immunocompromised mice Joint Meeting of the American-Society-for-Blood-and-Marrow-Transplantation/Center-for-International-Blood-and-Marrow-Transplant-Research Jamieson, C. H., Karimi, M., Creusot, R., Fathman, C. G., Negrin, R., Weissman, I. L. ELSEVIER SCIENCE INC. 2005: 86–86

  View details for Web of Science ID 000227329000253

• Hematopoietic stem and progenitor cells: Clinical and preclinical regeneration of the hematolymphoid system ANNUAL REVIEW OF MEDICINE Shizuru, J. A., Negrin, R. S., Weissman, I. L. 2005; 56: 509-538

  Abstract

  A vast literature exists on the biology of blood formation and regeneration under experimental and clinical conditions. The field of hematopoiesis was recently advanced by the capacity to purify to homogeneity primitive hematopoietic stem and progenitor cells. Isolation of cells at defined maturational stages has enhanced the understanding of the fundamental nature of stem cells, including how cell fate decisions are made, and this understanding is relevant to the development of other normal as well as malignant tissues. This review updates the basic biology of hematopoietic stem cells (HSC) and progenitors, the evolving use of purified HSC as grafts for clinical hematopoietic cell transplantation (HCT) including immune tolerance induction, and the application of HSC biology to other stem cell fields.

  View details for DOI 10.1146/annurev.med.54.101601.152334

  View details for PubMedID 15660525

• Preuss Resident Research Award: bone marrow-derived Flk-1-expressing CD34+ cells contribute to the endothelium of tumor vessels in mouse brain. Clinical neurosurgery Santarelli, J. G., Udani, V., Yung, C. Y., Cheshier, S., Wagers, A., Brekken, R. A., Weissman, I., Tse, V. 2005; 52: 384-388

  View details for PubMedID 16626098

• Normal and neoplastic stem cells. Novartis Foundation symposium Weissman, I. L. 2005; 265: 35-50

  Abstract

  Stem cells are cells that at the single cell level both self-renew and give rise to differentiated progeny. Self renewal is the property that distinguishes stem cells and progenitors, and in the blood-forming system explains why haematopoietic stem cells (HSCs), not progenitors, are the only cells capable of providing rapid and sustained regeneration of the blood-forming system after ablation by cancer chemo- and radiotherapies. Cancer-free prospectively purified HSCs regenerate the haematopoietic system of patients as rapidly as a marrow or mobilized blood transplant, but without the risk of re-seeding the body with cancer cells. Further, purified allogeneic HSCs can establish donor-specific tolerance to subsequent tissue grafts. However, in contrast to widely-publicized reports of HSC plasticity, we have not been able to show transdifferentiation of HSC to muscle, heart, brain or gut, and conclude that rare cell fusions and incomplete purifications are likely explanations for the other published results. The ability to self-renew is also potentially dangerous, as poorly regulated self renewal is, we believe, a central lesion in all cancers. We have recently shown that myeloid leukaemias in mouse and human are often driven by rare leukaemia (cancer) stem cells which are at the progenitor stage of differentiation, but have activated the self-renewing cell division pathway normally used only by HSCs. Similar cancer stem cells have been isolated in other tumours.

  View details for PubMedID 16050249

• Chronic versus acute myelogenous leukemia: A question of self-renewal CANCER CELL Jamieson, C. H., Weissman, I. L., Passegue, E. 2004; 6 (6): 531-533

  Abstract

  Leukemia stem cells are defined as transformed hematopoietic stem cells or committed progenitor cells that have amplified or acquired the stem cell capacity for self-renewal, albeit in a poorly regulated fashion. In this issue of Cancer Cell, Huntly and colleagues report a striking difference in the ability of two leukemia-associated fusion proteins, MOZ-TIF2 and BCR-ABL, to transform myeloid progenitor populations. This rigorous study supports the idea of a hierarchy among leukemia-associated protooncogenes for their ability to endow committed myeloid progenitors with the self-renewal capacity driving leukemic stem cell propagation, and sheds new light on the pathogenesis of chronic and acute myelogenous leukemias.

  View details for Web of Science ID 000226076600002

  View details for PubMedID 15607956

• Selection of aberrant class II restricted CD8(+) T cells in NOD mice expressing a glutamic acid decarboxylase (GAD) 65-specific T cell receptor transgene AUTOIMMUNITY Ranheim, E. A., Tarbell, K. V., Krogsgaard, M., Mallet-Designe, V., Teyton, L., McDevitt, H. O., Weissman, I. L. 2004; 37 (8): 555-567

  Abstract

  We previously described the generation of non-obese diabetic (NOD) mice expressing a transgenic T cell receptor (TCR) specific for peptide epitope 286-300 of the diabetes related self antigen, glutamic acid decarboxylase (GAD)65 in the context of I-A(g7) class II MHC, that are paradoxically protected from diabetes. In this report, we examine the atypical CD8+ cells in these mice. Unlike typical class II restricted TCR transgenic mice, GAD286 mice have normal numbers of CD8+ cells, half of which express high levels of the transgenic TCR. These MHC mismatched CD8+ cells persist in the periphery and proliferate to GAD286-300 peptide in vitro and in vivo in a class II restricted fashion. Interestingly, the CD8+ tetramer(-) T cells that are expressing endogenous TCR can delay diabetes induction in a transfer model, as we previously showed for CD4+ tetramer+ T cells in these mice. The MHC mismatched CD8+ cells appear to be positively selected in an atypical fashion, in that they do not upregulate CD69 or reexpress CD44, and they escape negative selection. We find that production of these CD8+ cells is not dependent on NOD thymus or high affinity of the TCR, but is dependent on the atypical TCR transgenic thymic environment.

  View details for DOI 10.1080/08916930400020545

  View details for Web of Science ID 000228026000003

  View details for PubMedID 15763918

• Incorporation of naive bone marrow derived cells into the vascular architecture of brain tumor MICROCIRCULATION Yung, Y. C., Cheshier, S., Santarelli, J. G., Huang, Z., Wagers, A., Weissman, I., Tse, V. 2004; 11 (8): 699-708

  Abstract

  Neovascularization is essential for tumor growth and invasion. Mounting evidence suggests that tumor cells recruit circulating endothelial progenitor cells to promote vasculogenesis to compliment tumor angiogenesis. This study examines the constitutive role of bone marrow-derived cells in this process.Rat glioma cells were implanted into brains of T-cell-depleted knockout mice. At various timepoints after tumor implantation, naïve bone marrow cells from ubiquitous transgenic mice expressing green fluorescent protein (GFP) were infused into these animals. The incorporation of GFP-positive cells into the vascular architecture was visualized by fluorescence confocal microscopy in conjunction with the transcription profiles of vascular endothelial growth factor (VEGF) and angiopoietin-1 and -2 (Ang-1 and Ang-2).Of the cells infused, 8 days after tumor implantation, 0.49% were found exclusively sequestered in the vicinity of tumor vessels. This coincided with a decline in the expression of Ang-1 and a rise in the expression of VEGF and Ang-2. A few of these cells (0.66 of the 0.49%) localized onto the vascular wall. They resembled endothelial cells and expressed vWF.The incorporation of bone marrow-derived unpurified endothelial cells into the tumor vascular bed is both time-limited and infrequent. These cells may play a supportive rather than a constitutive role in tumor neovascularization.

  View details for DOI 10.1080/10739680490521005

  View details for Web of Science ID 000225641500007

  View details for PubMedID 15726837

• Predictive progenitor profiling in chronic myelomonocytic leukemia. 46th Annual Meeting of the American-Society-of-Hematology Jamieson, C., Gotlib, J., Coutre, S., Li, K., Weissman, I. AMER SOC HEMATOLOGY. 2004: 268B–268B

  View details for Web of Science ID 000225127701065

• Isolation of adult mouse myogenic progenitors: Functional heterogeneity of cells within and engrafting skeletal muscle CELL Sherwood, R. I., Christensen, J. L., Conboy, I. M., Conboy, M. J., Rando, T. A., Weissman, I. L., Wagers, A. J. 2004; 119 (4): 543-554

  Abstract

  Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.

  View details for Web of Science ID 000225183200012

  View details for PubMedID 15537543

• JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells CELL Passegue, E., Wagner, E. F., Weissman, I. L. 2004; 119 (3): 431-443

  Abstract

  The AP-1 transcription factor JunB is a transcriptional regulator of myelopoiesis. Inactivation of JunB in postnatal mice results in a myeloproliferative disorder (MPD) resembling early human chronic myelogenous leukemia (CML). Here, we show that JunB regulates the numbers of hematopoietic stem cells (HSC). JunB overexpression decreases the frequency of long-term HSC (LT-HSC), while JunB inactivation specifically expands the numbers of LT-HSC and granulocyte/macrophage progenitors (GMP) resulting in chronic MPD. Further, we demonstrate that junB inactivation must take place in LT-HSC, and not at later stages of myelopoiesis, to induce MPD and that only junB-deficient LT-HSC are capable of transplanting the MPD to recipient mice. These results demonstrate a stem cell-specific role for JunB in normal and leukemic hematopoiesis and provide experimental evidence that leukemic stem cells (LSC) can reside at the LT-HSC stage of development in a mouse model of MPD.

  View details for Web of Science ID 000224908300013

  View details for PubMedID 15507213

• Telomerase maintained in self-renewing tissues during serial regeneration of the urochordate Botryllus schlosseri DEVELOPMENTAL BIOLOGY Laird, D. J., Weissman, I. L. 2004; 273 (2): 185-194

  Abstract

  Telomerase is critical for the protection of germ line and stem cell chromosomes from fatal shortening during replication. In most organisms, telomerase activity is suppressed in progressively committed cells and falls to basal rates in terminally differentiated lineages. The colonial ascidian Botryllus schlosseri propagates asexually and sexually, presumably from pools of stem cells that self-renew throughout the 2- to 5-year colony life span. Asexual budding takes place continuously from the parental body wall. When the colony reaches a critical size, sexual reproduction commences with the generation of gonads. Here, we establish the existence of 6-15 kb telomeres on the ends of Botryllus chromosomes. We develop a real-time quantitative PCR telomeric repeat amplification protocol (TRAP) assay that reliably detects 0.2-100 TPG units in cells and tissues. We find highest levels of enzymatic activity in the gonads, developing embryos, and tissues containing the earliest asexual buds. Telomerase activity appears to be suppressed in later buds during organogenesis and falls to basal rates in mature zooids. We postulate that this pattern reflects maximum telomere restoration in somatic stem cells of early buds and suppression of telomerase activity in progenitors and terminally differentiated cells, indicative of an alternate role for stem cells as repeated body regenerators in colonial life histories.

  View details for DOI 10.1016/j.ydbio.2004.05.029

  View details for Web of Science ID 000223681000002

  View details for PubMedID 15328006

• Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors Workshop on Malignant Stem Cells in Leukemia and Solid Tumors Ayton, P. M., Cozzio, A., Passegue, E., Karsunky, H., Cleary, M. L., Weissman, I. L. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2004: 105–

  View details for Web of Science ID 000223709500013

• Identification of hematopoietic cell populations with activated Wnt signaling using a lentiviral vector containing a reporter cassette Workshop on Malignant Stem Cells in Leukemia and Solid Tumors Ailles, L. E., Serwold, T., Weissman, I. L. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2004: 104–5

  View details for Web of Science ID 000223709500010

• Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML NEW ENGLAND JOURNAL OF MEDICINE Jamieson, C. H., Ailles, L. E., Dylla, S. J., Muijtjens, M., Jones, C., Zehnder, J. L., Gotlib, J., Li, K., Manz, M. G., Keating, A., Sawyers, C. L., Weissman, I. L. 2004; 351 (7): 657-667

  Abstract

  The progression of chronic myelogenous leukemia (CML) to blast crisis is supported by self-renewing leukemic stem cells. In normal mouse hematopoietic stem cells, the process of self-renewal involves the beta-catenin-signaling pathway. We investigated whether leukemic stem cells in CML also use the beta-catenin pathway for self-renewal.We used fluorescence-activated cell sorting to isolate hematopoietic stem cells, common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors from marrow during several phases of CML and from normal marrow. BCR-ABL, beta-catenin, and LEF-1 transcripts were compared by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay in normal and CML hematopoietic stem cells and granulocyte-macrophage progenitors. Confocal fluorescence microscopy and a lymphoid enhancer factor/T-cell factor reporter assay were used to detect nuclear beta-catenin in these cells. In vitro replating assays were used to identify self-renewing cells as candidate leukemic stem cells, and the dependence of self-renewal on beta-catenin activation was tested by lentiviral transduction of hematopoietic progenitors with axin, an inhibitor of the beta-catenin pathway.The granulocyte-macrophage progenitor pool from patients with CML in blast crisis and imatinib-resistant CML was expanded, expressed BCR-ABL, and had elevated levels of nuclear beta-catenin as compared with the levels in progenitors from normal marrow. Unlike normal granulocyte-macrophage progenitors, CML granulocyte-macrophage progenitors formed self-renewing, replatable myeloid colonies, and in vitro self-renewal capacity was reduced by enforced expression of axin.Activation of beta-catenin in CML granulocyte-macrophage progenitors appears to enhance the self-renewal activity and leukemic potential of these cells.

  View details for PubMedID 15306667

• Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kelly, S., Bliss, T. M., Shah, A. K., Sun, G. H., Ma, M., Foo, W. C., Masel, J., Yenari, M. A., Weissman, I. L., Uchida, N., Palmer, T., Steinberg, G. K. 2004; 101 (32): 11839-11844

  Abstract

  We characterize the survival, migration, and differentiation of human neurospheres derived from CNS stem cells transplanted into the ischemic cortex of rats 7 days after distal middle cerebral artery occlusion. Transplanted neurospheres survived robustly in naive and ischemic brains 4 wk posttransplant. Survival was influenced by proximity of the graft to the stroke lesion and was negatively correlated with the number of IB4-positive inflammatory cells. Targeted migration of the human cells was seen in ischemic animals, with many human cells migrating long distances ( approximately 1.2 mm) predominantly toward the lesion; in naive rats, cells migrated radially from the injection site in smaller number and over shorter distances (0.2 mm). The majority of migrating cells in ischemic rats had a neuronal phenotype. Migrating cells between the graft and the lesion expressed the neuroblast marker doublecortin, whereas human cells at the lesion border expressed the immature neuronal marker beta-tubulin, although a small percentage of cells at the lesion border also expressed glial fibrillary acid protein (GFAP). Thus, transplanted human CNS (hCNS)-derived neurospheres survived robustly in naive and ischemic brains, and the microenvironment influenced their migration and fate.

  View details for DOI 10.1073/pnas.0404474101

  View details for Web of Science ID 000223276700056

  View details for PubMedID 15280535

  View details for PubMedCentralID PMC511061

• Progress and prospects in hematopoietic stem cell expansion and transplantation EXPERIMENTAL HEMATOLOGY Brown, J. M., Weissman, I. L. 2004; 32 (8): 693-695

  View details for DOI 10.1016/j.exphem.2004.07.001

  View details for Web of Science ID 000223584200003

  View details for PubMedID 15308317

• Varicella-zoster virus infection of human neural cells in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Baiker, A., Fabel, K., Cozzio, A., Zerboni, L., Fabel, K., Sommer, M., Uchida, N., He, D. P., Weissman, I., Arvin, A. M. 2004; 101 (29): 10792-10797

  Abstract

  Varicella-zoster virus (VZV) establishes latency in sensory ganglia and causes herpes zoster upon reactivation. These investigations in a nonobese diabetic severe combined immunodeficient mouse-human neural cell model showed that VZV infected both neurons and glial cells and spread efficiently from cell to cell in vivo. Neural cell morphology and protein synthesis were preserved, in contrast to destruction of epithelial cells by VZV. Expression of VZV genes in neural cells was characterized by nuclear retention of the major viral transactivating protein and a block in synthesis of the predominant envelope glycoprotein. The attenuated VZV vaccine strain retained infectivity for neurons and glial cells in vivo. VZV gene expression in differentiated human neural cells in vivo differs from neural infection by herpes simplex virus, which is characterized by latency-associated transcripts, and from lytic VZV replication in skin. The chimeric nonobese diabetic severe combined immunodeficient mouse model may be useful for investigating other neurotropic human viruses.

  View details for DOI 10.1073/pnas.0404016101

  View details for Web of Science ID 000222842700055

  View details for PubMedID 15247414

  View details for PubMedCentralID PMC490013

• Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host disease NATURE MEDICINE Merad, M., Hoffmann, P., Ranheim, E., Slaymaker, S., Manz, M. G., Lira, S. A., Charo, I., Cook, D. N., Weissman, I. L., Strober, S., Engleman, E. G. 2004; 10 (5): 510-517

  Abstract

  Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.

  View details for DOI 10.1038/nm1038

  View details for Web of Science ID 000221242400029

  View details for PubMedID 15098028

• Leukemic transformation of hematopoietic progenitors by MLL-GAS7 in the absence of Hoxa7 or Hoxa9 BLOOD So, C. W., Karsunky, H., Wong, P., Weissman, I. L., Cleary, M. L. 2004; 103 (8): 3192-3199

  Abstract

  Differential expression of Hox genes is associated with normal hematopoiesis, whereas inappropriate maintenance of Hox gene expression, particularly Hoxa7 and Hoxa9, is a feature of leukemias harboring mixed-lineage leukemia (MLL) mutations. To understand the pathogenic roles of Hox genes in MLL leukemias, we assessed the impact of Hoxa7 or Hoxa9 nullizygosity on hematopoietic progenitor compartments and their susceptibility to MLL-induced leukemias. Selective reductions in the absolute numbers of committed progenitors, but not of hematopoietic stem cells, distinguished Hoxa7- and Hoxa9-deficient mice. Megakaryocytic/erythroid progenitor (MEP) reductions in Hoxa7(-/-) mice correlated with reticulocytosis and thrombocytopenia without anemia. Conversely, Hoxa9(-/-) mice displayed marked lymphopenia and substantial reductions of common lymphoid progenitors (CLPs) and lymphoid precursors, in addition to significant reductions of common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs). In retroviral transduction/transplantation assays, Hoxa7- and Hoxa9-deficient progenitors remained susceptible to transformation by MLL-GAS7, which activates MLL through a dimerization-dependent mechanism. However, Hoxa7(-/-) or Hoxa9(-/-) progenitors were less efficient in generating transformed blast colony-forming units (CFUs) in vitro and induced leukemias with longer disease latencies, reduced penetrance, and less mature phenotypes. Thus, Hoxa7 and Hoxa9 contribute to hematopoietic progenitor homeostasis but are not necessary for MLL-GAS7-mediated leukemogenesis, yet they appear to affect disease latency, penetrance, and phenotypes consistent with their critical roles as downstream targets of MLL fusion proteins.

  View details for Web of Science ID 000222163500056

  View details for PubMedID 15070702

• Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium NATURE Balsam, L. B., Wagers, A. J., Christensen, J. L., Kofidis, T., Weissman, I. L., Robbins, R. C. 2004; 428 (6983): 668-673

  Abstract

  Under conditions of tissue injury, myocardial replication and regeneration have been reported. A growing number of investigators have implicated adult bone marrow (BM) in this process, suggesting that marrow serves as a reservoir for cardiac precursor cells. It remains unclear which BM cell(s) can contribute to myocardium, and whether they do so by transdifferentiation or cell fusion. Here, we studied the ability of c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1(lo) Lin- Sca-1+ long-term reconstituting haematopoietic stem cells to regenerate myocardium in an infarct model. Cells were isolated from transgenic mice expressing green fluorescent protein (GFP) and injected directly into ischaemic myocardium of wild-type mice. Abundant GFP+ cells were detected in the myocardium after 10 days, but by 30 days, few cells were detectable. These GFP+ cells did not express cardiac tissue-specific markers, but rather, most of them expressed the haematopoietic marker CD45 and myeloid marker Gr-1. We also studied the role of circulating cells in the repair of ischaemic myocardium using GFP+-GFP- parabiotic mice. Again, we found no evidence of myocardial regeneration from blood-borne partner-derived cells. Our data suggest that even in the microenvironment of the injured heart, c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1(lo) Lin- Sca-1+ long-term reconstituting haematopoietic stem cells adopt only traditional haematopoietic fates.

  View details for DOI 10.1038/nature02460

  View details for Web of Science ID 000220697200048

  View details for PubMedID 15034594

• Plasticity of adult stem cells CELL Wagers, A. J., Weissman, I. L. 2004; 116 (5): 639-648

  Abstract

  Recent years have seen much excitement over the possibility that adult mammalian stem cells may be capable of differentiating across tissue lineage boundaries, and as such may represent novel, accessible, and very versatile effectors of therapeutic tissue regeneration. Yet studies proposing such "plasticity" of adult somatic stem cells remain controversial, and in general, existing evidence suggests that in vivo such unexpected transformations are exceedingly rare and in some cases can be accounted for by equally unexpected alternative explanations.

  View details for Web of Science ID 000221499700005

  View details for PubMedID 15006347

• Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS biology Christensen, J. L., Wright, D. E., Wagers, A. J., Weissman, I. L. 2004; 2 (3): E75-?

  Abstract

  The major site of hematopoiesis transitions from the fetal liver to the spleen and bone marrow late in fetal development. To date, experiments have not been performed to evaluate functionally the migration and seeding of hematopoietic stem cells (HSCs) during this period in ontogeny. It has been proposed that developmentally timed waves of HSCs enter the bloodstream only during distinct windows to seed the newly forming hematopoietic organs. Using competitive reconstitution assays to measure HSC activity, we determined the localization of HSCs in the mid-to-late gestation fetus. We found that multilineage reconstituting HSCs are present at low numbers in the blood at all timepoints measured. Seeding of fetal bone marrow and spleen occurred over several days, possibly while stem cell niches formed. In addition, using dual-chamber migration assays, we determined that like bone marrow HSCs, fetal liver HSCs migrate in response to stromal cell-derived factor-1alpha (SDF-1alpha); however, unlike bone marrow HSCs, the migratory response of fetal liver HSCs to SDF-1alpha is greatly increased in the presence of Steel factor (SLF), suggesting an important role for SLF in HSC homing to and seeding of the fetal hematopoietic tissues. Together, these data demonstrate that seeding of fetal organs by fetal liver HSCs does not require large fluxes of HSCs entering the fetal bloodstream, and that HSCs constitutively circulate at low levels during the gestational period from 12 to 17 days postconception. Newly forming hematopoietic tissues are seeded gradually by HSCs, suggesting initial seeding is occurring as hematopoietic niches in the spleen and bone marrow form and become capable of supporting HSC self-renewal. We demonstrate that fetal and adult HSCs exhibit specific differences in chemotactic behavior. While both migrate in response to SDF-1alpha, fetal HSCs also respond significantly to the cytokine SLF. In addition, the combination of SDF-1alpha and SLF results in substantially enhanced migration of fetal HSCs, leading to migration of nearly all fetal HSCs in this assay. This finding indicates the importance of the combined effects of SLF and SDF-1alpha in the migration of fetal HSCs, and is, to our knowledge, the first demonstration of a synergistic effect of two chemoattractive agents on HSCs.

  View details for PubMedID 15024423

• Continuous development precludes radioprotection in a colonial ascidian DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Laird, D. J., Weissman, I. L. 2004; 28 (3): 201-209

  Abstract

  Colonial organisms provide a unique experimental system for stem cell biology. The colonial Urochordate Botryllus schlosseri reproduces sexually as well as by continuous asexual budding. Adjacent colonies with a shared histocompatibility allele undergo vascular fusion and establish a common blood circulation, performing natural transplantation. Fused colonies become chimeras, often with complete somatic replacement of the host cell genotype by the fused parabiont. We attempted to establish a radioprotection assay for the somatic stem cells that induce long-term chimerism in Botryllus. We demonstrate over a range of radiation doses that neither autologous nor allogeneic cell transplantation enhances survival of host colonies. This suggests that high mitotic index associated with continuous asexual development leads to radiosensitivity of organs and structures essential to survival during engraftment. We observe that radiation induces uncontrolled epithelial cell proliferation in abnormally terminated buds, suggesting that stem cells are not required for the initial stages of bud development.

  View details for DOI 10.1016/j.dci.2003.08.007

  View details for Web of Science ID 000188375700003

  View details for PubMedID 14642887

• Evolution of a protochordate allorecognition locus SCIENCE De Tomaso, A. W., Weissman, I. L. 2004; 303 (5660): 977-977

  View details for Web of Science ID 000188918000035

  View details for PubMedID 14963321

• Therapeutic implications of cancer stem cells CURRENT OPINION IN GENETICS & DEVELOPMENT Al-Hajj, M., Becker, M. W., Wichal, M., Weissman, I., Clarke, M. F. 2004; 14 (1): 43-47

  Abstract

  Most cancers comprise a heterogenous population of cells with marked differences in their proliferative potential as well as the ability to reconstitute the tumor upon transplantation. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. In addition, dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. This new model for cancer will have significant ramifications for the way we study and treat cancer. In addition, through targeting the cancer stem cell and its dysregulated self-renewal, our therapies for treating cancer are likely to improve.

  View details for DOI 10.1016/j.gde.2003.11.007

  View details for Web of Science ID 000188978200008

  View details for PubMedID 15108804

• Shifting foci of hematopoiesis during reconstitution from single stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cao, Y. A., Wagers, A. J., Beilhack, A., Dusich, J., Bachmann, M. H., Negrin, R. S., Weissman, I. L., Contag, C. H. 2004; 101 (1): 221-226

  Abstract

  To reveal the early events and dynamics of hematopoietic reconstitution in living animals in real-time, we used bioluminescence imaging to monitor engraftment from single luciferase-labeled hematopoietic stem cells (HSC) in irradiated recipients. Transplanted HSC generated discrete foci in the spleen and bone marrow (BM), at a frequency that correlated with BM compartment size. Initially detected foci could expand locally, seed other sites in BM or spleen, and/or recede with different kinetics. These studies reveal dynamic and variable patterns of engraftment from highly purified HSC and indicate that the final overall contribution of individual HSC to hematopoietic chimerism does not depend on the specific site of initial engraftment and expansion.

  View details for PubMedID 14688412

• Insulin-like growth factor promotes engraftment, differentiation, and functional improvement after transfer of embryonic stem cells for myocardial restoration STEM CELLS Kofidis, T., de Bruin, J. L., Yamane, T., Balsam, L. B., Lebl, D. R., Swijnenburg, R. J., Tanaka, M., Weissman, I. L., Robbins, R. C. 2004; 22 (7): 1239-1245

  Abstract

  Insulin-like growth factor-1 (IGF-1) promotes myocyte proliferation and can reverse cardiac abnormalities when it is administered in the early fetal stage. Supplementation of a mouse embryonic stem cell (ESC) suspension with IGF-1 might enhance cellular engraftment and host organ-specific differentiation after injection in the area of acute myocardial injury. In the study reported here, we sought to enhance the restorative effect of ESCs in the injured heart by adding IGF-1 to the injected cell population. Green fluorescent protein (GFP)-labeled sv129 ESCs (2.5 x 10(5)) were injected into the ischemic area after left anterior descending (LAD) artery ligation in BalbC mice. Recombinant mouse IGF-1 (25 ng) was added to the cell suspension prior to the injection (n = 5). Echocardiography was performed before organ harvest 2 weeks later. The degree of restoration (ratio of GFP+ to infarct area), expression of cardiac markers by GFP+ cells, inflammatory response, and tumorigenicity were evaluated. Mice with LAD ligation only (n = 5) and ESC transfer without IGF-1 (n = 5) served as controls. ESCs formed viable grafts and improved cardiac function. Left ventricular wall thickness was higher in the IGF-1 group (p = .025). There was a trend toward higher fractional shortening in the IGF-treated group. Histological analysis demonstrated that IGF-1 promoted expression of alpha-sarcomeric actin (p = .015) and major histocompatibility complex class I (p = .01). IGF did not affect the cellular response to the donor cells or tumorigenicity. IGF-1 promotes expression of cardiomyocyte phenotype in ESCs in vivo. It should be considered as an adjuvant to cell transfer for myocardial restoration.

  View details for DOI 10.1634/stem-cells.2004-0127

  View details for Web of Science ID 000225759300011

  View details for PubMedID 15579642

• Leukemia and leukemic stem cells Stem Cells in the Nervous System: Functional and clinical implications Jamieson, C., Passegue, E., Weissman, I. Springer-Verlag. 2004
• Stem cells, cell differentiation and cancer Clinical Oncology Clarke, M., Weissman, I. Elsevier, Inc.. 2004
• Biology of hematopoietic stem and progenitor cells Thomas' Hematopoietic Cell Transplantation Manz, M., Akashi, K., Weissman, I. Blackwell Publishing. 2004: 69–95
• Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells STEM CELLS Sherwood, R. I., Christensen, J. L., Weissman, I. L., Wagers, A. J. 2004; 22 (7): 1292-1304

  Abstract

  To investigate the factors that regulate incorporation into uninjured or damaged skeletal muscle of donor markers derived from unfractionated bone marrow (BM) cells or from highly purified c-kit+ Thy1.1lo Lin- Sca-1+ hematopoietic stem cells (HSCs), we evaluated myofiber chimerism of multiple muscle groups in irradiated and transplanted recipient mice and in unirradiated parabiotic animals. Uninjured panniculus carnosus, diaphragm, and abdominal muscles infrequently incorporated donor markers into myofibers in a subset of animals after either BM or HSC transplantation; however, acute muscle injury was essential to elicit contributions to triceps surae (TS) and tibialis anterior muscles. The low level of incorporation of donor marker-expressing myofibers could not be enhanced either by transplantation into newborn recipients or by induced migration of HSCs into the periphery. Analysis of muscle chimerism in unirradiated animals joined surgically by parabiosis revealed that contributions of circulating cells to myofibers in the TS were injury dependent and that at least some circulating cells with the potential to contribute to regenerating muscle derive from BM, suggesting that hematoablative preconditioning is not required for such contributions. In all cases tested, donor-derived myofibers expressed both donor-specific and host-specific markers, suggesting that they arise by low-level fusion into skeletal muscle of cells that can include the progeny of HSCs. It is not yet clear whether such events represent a normal myogenic pathway or a pathological response to muscle damage.

  View details for DOI 10.1634/stemcells.2004-0090

  View details for Web of Science ID 000225759300016

  View details for PubMedID 15579647

• Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors GENES & DEVELOPMENT Cozzio, A., Passegue, E., Ayton, P. M., Karsunky, H., Cleary, M. L., Weissman, I. L. 2003; 17 (24): 3029-3035

  Abstract

  We have used the hematopoietic system as a model to investigate whether acute myeloid leukemia arises exclusively from self-renewing stem cells or also from short-lived myeloid progenitors. When transduced with a leukemogenic MLL fusion gene, prospectively isolated stem cells and myeloid progenitor populations with granulocyte/macrophage differentiation potential are efficiently immortalized in vitro and result in the rapid onset of acute myeloid leukemia with similar latencies following transplantation in vivo. Regardless of initiating cell, leukemias displayed immunophenotypes and gene expression profiles characteristic of maturation arrest at an identical late stage of myelomonocytic differentiation, putatively a monopotent monocytic progenitor stage. Our findings unequivocally establish the ability of transient repopulating progenitors to initiate myeloid leukemias in response to an MLL oncogene, and support the existence of cancer stem cells that do not necessarily overlap with multipotent stem cells of the tissue of origin.

  View details for DOI 10.1101/gad.1143403

  View details for Web of Science ID 000187803000006

  View details for PubMedID 14701873

  View details for PubMedCentralID PMC305255

• Neural progenitor genes - Germinal zone expression and analysis of genetic overlap in stem cell populations DEVELOPMENTAL BIOLOGY Easterday, M. C., Dougherty, J. D., JACKSON, R. L., Ou, J., Nakano, I., Paucar, A. A., Roobini, B., Dianati, M., Irvin, D. K., Weissman, I. L., Terskikh, A. V., Geschwind, D. H., Kornblum, H. I. 2003; 264 (2): 309-322

  Abstract

  The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.

  View details for DOI 10.1016/j.ydbio.2003.09.003

  View details for Web of Science ID 000187216900001

  View details for PubMedID 14651920

• Differential amplification of bipotent megakaryocytic/erythroid "progenitor" and "precursor" cells during the recovery from acute and chronic erythroid stress in mice. 45th Annual Meeting and Exhibition of the American-Society-of-Hematology Migliaccio, A. R., Sanchez, M., Vannucchi, A. M., Migliaccio, G., Nakorn, T. N., Weissman, I. L. AMER SOC HEMATOLOGY. 2003: 565A–565A

  View details for Web of Science ID 000186536702075

• Initial characterization of a protochordate histocompatibility locus IMMUNOGENETICS De Tomaso, A. W., Weissman, I. L. 2003; 55 (7): 480-490

  Abstract

  The colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction which is controlled by a single, highly polymorphic locus called the Fu/HC. We are using map-based cloning to identify Fu/HC gene(s), and have currently delineated their location to an approximately 1-cM region of the B. schlosseri genome. The Fu/HC physical map currently consists of 85 sequence-tagged sites mapped on a minimum tiling path of 800 kb which consists of five contigs, with four gaps remaining to be crossed, and is estimated to be 75% completed. Approximately half this region has been sequenced throughout the locus, allowing the first analysis of a metazoan histocompatibility locus outside of vertebrates. This has resulted in the identification of 18 predicted genes, a number of which have been found to be expressed. Several of these genes are well conserved among the chordates; however, none of the predicted or expressed genes are linked within the genome of any organism in the databases. In addition, the Fu/HC is one of the most polymorphic loci ever described, and physical mapping has revealed that the locus is quite dynamic, and includes features such as hotspots of recombination.

  View details for DOI 10.1007/s00251-003-0612-7

  View details for Web of Science ID 000186201200006

  View details for PubMedID 14520503

• Effects of allogeneic contact on life-history traits of the colonial ascidian Botryllus schlosseri in Monterey Bay BIOLOGICAL BULLETIN Chadwick-Furman, N. E., Weissman, I. L. 2003; 205 (2): 133-143

  Abstract

  The formation of chimeric colonies following allogeneic contact between benthic invertebrates may strongly affect colony fitness. Here we show that, in a field population of the colonial ascidian Botryllus schlosseri in Monterey Bay, California, more than 20% of all colonies occur in allogeneic contact with conspecifics. We experimentally assessed the effects of allogeneic contact on the following life-history traits under natural field conditions: growth, age and size at first reproduction, and egg production (fecundity). When compared with isolated colonies, and in some cohorts also with colonies that rejected allogeneic neighbors, colonies that fused with neighbors incurred reduced fitness in terms of most life-history traits measured. We propose that one of the benefits of precise allorecognition is that, in fused colonies, it limits the unit of selection to chimeric individuals composed of closely related kin.

  View details for Web of Science ID 000186338800006

  View details for PubMedID 14583511

• Normal and leukemic hematopoiesis: Are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Arthur M Sackler Colloquium of the National-Academy-of-Sciences on Regenerative Medicine Passegue, E., Jamieson, C. H., Ailles, L. E., Weissman, I. L. NATL ACAD SCIENCES. 2003: 11842–11849

  Abstract

  Leukemia can be viewed as a newly formed, abnormal hematopoietic tissue initiated by a few leukemic stem cells (LSCs) that undergo an aberrant and poorly regulated process of organogenesis analogous to that of normal hematopoietic stem cells. A hallmark of all cancers is the capacity for unlimited self-renewal, which is also a defining characteristic of normal stem cells. Given this shared attribute, it has been proposed that leukemias may be initiated by transforming events that take place in hematopoietic stem cells. Alternatively, leukemias may also arise from more committed progenitors caused by mutations and/or selective expression of genes that enhance their otherwise limited self-renewal capabilities. Identifying the LSCs for each type of leukemia is a current challenge and a critical step in understanding their respective biologies and may provide key insights into more effective treatments. Moreover, LSC identification and purification will provide a powerful diagnostic, prognostic, and therapeutic tool in the clinic.

  View details for Web of Science ID 000185805000006

  View details for PubMedID 14504387

  View details for PubMedCentralID PMC304096

• Over-expression of Bcl-2 provides protection in septic mice by a trans effect JOURNAL OF IMMUNOLOGY Iwata, A., Stevenson, V. M., Minard, A., Tasch, M., Tupper, J., Lagasse, E., Weissman, I., Harlan, J. M., Winn, R. K. 2003; 171 (6): 3136-3141

  Abstract

  Transgenic mice that over-express B cell leukemia/lymphomas (Bcl)-2 in myeloid cells under control of the human MRP8 promoter (hMRP8-Bcl-2) or in T lymphocytes under the E micro promoter (E micro -Bcl-2) were compared with C57BL/6 control mice following cecal ligation and puncture (CLP). There was a significant difference in outcome between the hMRP8-Bcl-2 and control mice with 100% survival in the hMRP8-Bcl-2 mice vs 25% survival in the control mice. In separate experiments there was a significant difference between E micro -Bcl-2 and control mice with 87.5 and 22.2% survival, respectively. Adoptive transfer of CD11b-positive bone marrow cells from hMRP8-Bcl-2 or C57BL/6 mice to C57BL/6 mice subjected to CLP resulted in 100 and 0% survival, respectively. Adoptive transfer of CD11b-positive cells from either hMRP8-Bcl-2 or C57BL/6 mice to Rag-1(-/-) mice (no mature T or B cells) subjected to CLP resulted in survival of 87.5 and 12.5%, respectively. The hMRP8-Bcl-2 mice had significantly more neutrophils and fewer bacteria in the peritoneum compared with C57BL/6 mice 24 h after CLP. These experiments show that Bcl-2 over-expression is protective in CLP and that protection is independent of lymphocytes. We propose that over-expression of Bcl-2 in T cells or myeloid cells induce release of a molecule(s) that protects against death following CLP.

  View details for Web of Science ID 000185247300050

  View details for PubMedID 12960340

• Stem cells in clinical practice JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS Evers, B. M., Weissman, I. L., Flake, A. W., Tabar, V., Weisel, R. D. 2003; 197 (3): 458-478

  View details for DOI 10.1016/S1072-7515(03)00655-0

  View details for Web of Science ID 000185124800019

  View details for PubMedID 12946802

• Expression of BCR/ABL and BCL-2 in myeloid progenitors leads to myeloid leukemias PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jaiswal, S., Traver, D., Miyamoto, T., Akashi, K., Lagasse, E., Weissman, I. L. 2003; 100 (17): 10002-10007

  Abstract

  Chronic myelogenous leukemia is a myeloproliferative disorder (MPD) that, over time, progresses to acute leukemia. Both processes are closely associated with the t(9;22) chromosomal translocation that creates the BCR/ABL fusion gene in hematopoietic stem cells (HSCs) and their progeny. Chronic myelogenous leukemia is therefore classified as an HSC disorder in which a clone of multipotent HSCs is likely to be malignantly transformed, although direct evidence for malignant t(9;22)+ HSCs is lacking. To test whether HSC malignancy is required, we generated hMRP8p210BCR/ABL transgenic mice in which expression of BCR/ABL is absent in HSCs and targeted exclusively to myeloid progenitors and their myelomonocytic progeny. Four of 13 BCR/ABL transgenic founders developed a chronic MPD, but only one progressed to blast crisis. To address whether additional oncogenic events are required for progression to acute disease, we crossed hMRP8p210BCR/ABL mice to apoptosis-resistant hMRP8BCL-2 mice. Of 18 double-transgenic animals, 9 developed acute myeloid leukemias that were transplantable to wild-type recipients. Taken together, these data indicate that a MPD can arise in mice without expression of BCR/ABL in HSCs and that additional mutations inhibiting programmed cell death may be critical in the transition of this disease to blast-crisis leukemia.

  View details for DOI 10.1073/pnas.1633833100

  View details for Web of Science ID 000184926000069

  View details for PubMedID 12890867

  View details for PubMedCentralID PMC187741

• Early defect prethymic in bone marrow T cell progenitors in athymic nu/nu mice JOURNAL OF IMMUNOLOGY Chatterjea-Matthes, D., Garcia-Ojeda, M. E., Dejbakhsh-Jones, S., Jerabek, L., Manz, M. G., Weissman, I. L., Strober, S. 2003; 171 (3): 1207-1215

  Abstract

  nu/nu mice fail to develop a thymus and mature T cells due to a defect in the whn gene encoding a transcription factor necessary for terminal epithelial cell differentiation. We investigated whether early T cell progenitor development in the nu/nu bone marrow is also defective. We demonstrated a maturation arrest of nu/nu marrow T cell progenitors associated with a lack of pTalpha gene expression and a failure to give rise to mature T cells in adoptive euthymic hosts. Wild-type hemopoietic stem cells rapidly matured into functional T cell progenitors in the marrow of euthymic or thymectomized but not nu/nu hosts. We show that defects in bone marrow prethymic T cell development can also contribute to T cell deficiency in nu/nu mice.

  View details for Web of Science ID 000184327600012

  View details for PubMedID 12874207

• Flt3 ligand regulates dendritic cell development from Flt3(+) lymphoid and myeloid-committed progenitors to Flt3(+) dendritic cells in vivo JOURNAL OF EXPERIMENTAL MEDICINE Karsunky, H., Merad, M., Cozzio, A., Weissman, I. L., Manz, M. G. 2003; 198 (2): 305-313

  Abstract

  Stimulation of Flt3 receptor tyrosine kinase through its cognate ligand expands early hematopoietic progenitor and dendritic cells (DCs) in humans and mice. The exact developmental stages at which hematopoietic progenitors express Flt3, are responsive to its ligand, and subsequently develop to DCs, are not known. Here we show that common lymphoid and common myeloid progenitors, as well as steady state DCs in thymus, spleen, and epidermis, express Flt3. The receptor is down-regulated once definitive B cell, T cell, and megakaryocyte/erythrocyte commitment occurs, and Flt3 is not detectable on other steady state hematopoietic cell populations. Upon in vivo Flt3 ligand (Flt3L) administration, Flt3+ progenitor cells and their progeny DCs are expanded, whereas Flt3- downstream progenitors are not, or are only slightly increased. Transplantation of common lymphoid and common myeloid progenitors and subsequent Flt3L injection increases progeny DCs of both precursor populations. These findings provide a definitive map of Flt3 expression in the hematopoietic hierarchy and directly demonstrate that Flt3L can drive DC development along both the lymphoid and myeloid developmental pathways from Flt3+ progenitors to Flt3+ DCs.

  View details for DOI 10.1084/jem.20030323

  View details for Web of Science ID 000184368200012

  View details for PubMedID 12874263

• Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation BLOOD Arber, C., Bitmansour, A., Sparer, T. E., Higgins, J. P., Mocarski, E. S., Weissman, I. L., Shizuru, J. A., Brown, J. M. 2003; 102 (2): 421-428

  Abstract

  Lymphoid deficiency after allogeneic hematopoietic cell transplantation (HCT) results in increased susceptibility to infection; however, transplantation of mature lymphocytes frequently results in a serious complication known as graft-versus-host disease (GVHD). Here we demonstrate in mice that both congenic as well as allogeneic transplantation of low numbers of highly purified common lymphoid progenitors (CLPs)-a rare population of lymphoid-lineage-committed bone marrow cells-accelerates immune reconstitution after lethal irradiation and rescue with hematopoietic stem cells (HSCs). After congenic transplantation, 3 x 10(3) CLPs protected against murine cytomegalovirus (MCMV) infection at a level roughly equivalent to 107 unfractionated lymph node cells. In the allogeneic model of matched unrelated donor HSC transplantation, cotransplantation of 3 x 10(3) CLPs protected thymus-bearing as well as thymectomized hosts from MCMV infection and attenuated disease severity. Immunohistochemistry in combination with antibody depletion of T and natural killer (NK) cells confirmed that CLP-derived as well as residual host lymphocytes contribute to antiviral protection. Importantly, transplantation of allogeneic CLPs provided a durable antiviral immunity without inducing GVHD. These data support the potential for composing grafts with committed progenitors to reduce susceptibility to viral infection following HCT.

  View details for DOI 10.1182/blood-2002-12-3834

  View details for Web of Science ID 000184083500010

  View details for PubMedID 12663447

• Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation BLOOD Allsopp, R. C., Morin, G. B., DePinho, R., Harley, C. B., Weissman, I. L. 2003; 102 (2): 517-520

  Abstract

  Telomere shortening ultimately limits the replicative life span of cultured human somatic cells. Telomeres also shorten during replicative aging in vivo in hematopoietic cells, including early hematopoietic progenitors and hematopoietic stem cells (HSCs), from humans and mice, despite readily detectable levels of telomerase in these cells. To assess the relevance of telomerase to the long-term replicative capacity of HSCs in vivo, we serially transplanted HSCs from wild-type and telomerase-deficient mice until exhaustion and monitored telomere length in HSCs during this process. Telomerase-deficient HSCs could be serially transplanted for only 2 rounds, whereas wild-type HSCs could be serially transplanted for at least 4 rounds. Furthermore, the rate of telomere shortening was increased approximately 2-fold during serial transplantation of telomerase-deficient HSCs. These findings suggest that one role for telomerase in the HSC is to partially counter the rate of telomere shortening during division of HSCs, thereby preventing premature loss of telomere function and providing added replicative capacity.

  View details for DOI 10.1182/blood-2002-07-2334

  View details for Web of Science ID 000184083500024

  View details for PubMedID 12663456

• Gene expression analysis of purified hematopoietic stem cells and committed progenitors BLOOD Terskikh, A. V., Miyamoto, T., Chang, C., Diatchenko, L., Weissman, I. L. 2003; 102 (1): 94-101

  Abstract

  Lifelong self-renewal is a unique property of somatic stem cells. Recently, several primitive multipotent yet committed (non-self-renewing) hematopoietic progenitor populations were identified in mouse bone marrow. We have characterized the expression of 1200 selected mouse genes using the Atlas cDNA array in highly purified hematopoietic stem cells (HSCs) and 6 closely related progenitor populations: common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythrocyte progenitors (MEPs), common lymphoid progenitors (CLPs), and pro-T and pro-B cells. Cluster analysis revealed that nearly half of all differentially expressed transcripts are associated with HSCs, supporting the notion of an active transcriptional status of HSCs. Genes found enriched in the HSC cluster encompass many developmentally regulated genes, some previously associated with HSC self-renewal. In contrast, genes that are enriched in committed progenitors are mostly associated with hematopoietic differentiation, immune regulation, and metabolism. Thus, the transition from HSCs toward committed progenitors correlates with the down-regulation of a large number of HSC-associated genes and progressive up-regulation of a limited number of lineage-specific genes. These genetic analyses revealed both quantitative and qualitative differences between the transcripts associated with HSCs versus downstream progenitors and produced a list of the candidate genes, potentially involved in HSC self-renewal.

  View details for DOI 10.1182/blood-2002-08-2509

  View details for Web of Science ID 000183820300023

  View details for PubMedID 12623852

• Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2 EXPERIMENTAL HEMATOLOGY Domen, J., Weissman, I. L. 2003; 31 (7): 631-639

  Abstract

  Objective. Chemotherapeutic agents function by inducing apoptosis and their effectiveness depends on the balance of pro- and anti-apoptotic proteins in cells. Due to the complicated interactions of the many proteins involved, it has been difficult to determine in tumors whether overexpression of single genes is prognostic for increased resistance. Therefore, we studied the influence of bcl-2 overexpression on resistance to chemotherapeutics in a transgenic mouse system. This allowed us to study a wide variety of cells, including important but rare populations such as hematopoietic stem cells (HSC).Methods. H2K-bcl-2 transgenic and wild-type (WT) mice were treated with several agents(5-fluoruracil, cyclophosphamide, and busulfan) to determine the contribution of increased amounts of bcl-2 to the response to these chemotherapeutics in vivo. Populations were enumerated using flow cytometry. HSC were studied by FACS purification and long-term reconstitution assays in vivo and resistance was confirmed by short-term proliferation assays with different amounts of chemotherapeutics in vitro.Results. bcl-2 overexpression alone protects many cell types, though protection levels differ between populations and agents. However, even sensitive populations return to pretreatment levels faster in transgenic mice. bcl-2 overexpression also prevents the dramatic changes in HSC following 5-FU treatment (downregulation of c-kit, upregulation of Lin, less efficient long-term reconstitution). In vitro studies directly demonstrate increased resistance of bcl-2 overexpressing HSC to chemotherapeutic agents.Conclusions. Increased expression of bcl-2 in HSC and their progeny endows these cells with broad resistance to chemotherapeutic agents. The ability to (differentially) regulate sensitivity to apoptosis of bystander and tumor cells is clinically important.

  View details for DOI 10.1016/S0301-472X(03)00084-5

  View details for Web of Science ID 000184052300008

  View details for PubMedID 12842708

• Wnt proteins are lipid-modified and can act as stem cell growth factors NATURE Willert, K., BROWN, J. D., Danenberg, E., Duncan, A. W., Weissman, I. L., Reya, T., Yates, J. R., Nusse, R. 2003; 423 (6938): 448-452

  Abstract

  Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.

  View details for DOI 10.1038/nature01611

  View details for Web of Science ID 000183012000044

  View details for PubMedID 12717451

• A role for Wnt signalling in self-renewal of haematopoietic stem cells NATURE Reya, T., Duncan, A. W., Ailles, L., Domen, J., Scherer, D. C., Willert, K., Hintz, L., Nusse, R., Weissman, I. L. 2003; 423 (6938): 409-414

  Abstract

  Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

  View details for DOI 10.1038/nature01593

  View details for Web of Science ID 000183012000034

  View details for PubMedID 12717450

• Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells NATURE Park, I. K., Qian, D. L., Kiel, M., Becker, M. W., Pihalja, M., Weissman, I. L., Morrison, S. J., Clarke, M. F. 2003; 423 (6937): 302-305

  Abstract

  A central issue in stem cell biology is to understand the mechanisms that regulate the self-renewal of haematopoietic stem cells (HSCs), which are required for haematopoiesis to persist for the lifetime of the animal. We found that adult and fetal mouse and adult human HSCs express the proto-oncogene Bmi-1. The number of HSCs in the fetal liver of Bmi-1-/- mice was normal. In postnatal Bmi-1-/- mice, the number of HSCs was markedly reduced. Transplanted fetal liver and bone marrow cells obtained from Bmi-1-/- mice were able to contribute only transiently to haematopoiesis. There was no detectable self-renewal of adult HSCs, indicating a cell autonomous defect in Bmi-1-/- mice. A gene expression analysis revealed that the expression of stem cell associated genes, cell survival genes, transcription factors, and genes modulating proliferation including p16Ink4a and p19Arf was altered in bone marrow cells of the Bmi-1-/- mice. Expression of p16Ink4a and p19Arf in normal HSCs resulted in proliferative arrest and p53-dependent cell death, respectively. Our results indicate that Bmi-1 is essential for the generation of self-renewing adult HSCs.

  View details for DOI 10.1038/nature01587

  View details for Web of Science ID 000182853100046

  View details for PubMedID 12714971

• A transgenic TCR recognizing a GAD peptide plus I-ag7 produces regulatory CD4+and aberrant, class II reactive CD8+T cells in NOD mice 90th Annual Meeting of the American-Association-for-Immunologists Ranheim, E. A., Tarbell, K., McDevitt, H., Weissman, I. L. FEDERATION AMER SOC EXP BIOL. 2003: C259–C259

  View details for Web of Science ID 000182367001206

• Did the molecules of adaptive immunity evolve from the innate immune system? Annual Meeting of the Society-for-Integrative-and-Comparative-Biology Bartl, S., Baish, M., Weissman, I. L., Diaz, M. OXFORD UNIV PRESS INC. 2003: 338–46

  Abstract

  The antigen receptors on cells of innate immune systems recognize broadly expressed markers on non-host cells while the receptors on lymphocytes of the adaptive immune system display a higher level of specificity. Adaptive immunity, with its exquisite specificity and immunological memory, has only been found in the jawed vertebrates, which also display innate immunity. Jawless fishes and invertebrates only have innate immunity. In the adaptive immune response, T and B-lymphocytes detect foreign agents or antigens using T cell receptors (TCR) or immunoglobulins (Ig), respectively. While Ig can bind free intact antigens, TCR only binds processed antigenic fragments that are presented on molecules encoded in the major histocompatibility complex (MHC). MHC molecules display variation through allelic polymorphism. A diverse repertoire of Ig and TCR molecules is generated by gene rearrangement and junctional diversity, processes carried out by the recombinase activating gene (RAG) products and terminal deoxynucleotidyl transferase (TdT). Thus, the molecules that define adaptive immunity are TCR, Ig, MHC molecules, RAG products and TdT. No direct predecessors of these molecules have been found in the jawless fishes or invertebrates. In contrast, the complement cascade can be activated by either adaptive or innate immune systems and contains examples of molecules that gradually evolved from non-immune functions to being part of the innate and then adaptive immune system. In this paper we examine the molecules of the adaptive immune system and speculate on the existence of direct predecessors that were part of innate immunity.

  View details for Web of Science ID 000184416000016

  View details for PubMedID 21680442

• Effect of TERT over-expression on the long-term transplantation capacity of hematopoietic stem cells NATURE MEDICINE Allsopp, R. C., Morin, G. B., Horner, J. W., DePinho, R., Harley, C. B., Weissman, I. L. 2003; 9 (4): 369-U6

  View details for DOI 10.1038/nm0403-369

  View details for Web of Science ID 000181987400003

  View details for PubMedID 12669037

• Construction and characterization of large-insert genomic libraries (BAC and fosmid) from the ascidian Botryllus schlosseri and initial physical mapping of a histocompatibility locus MARINE BIOTECHNOLOGY De Tomaso, A. W., Weissman, I. L. 2003; 5 (2): 103-115

  Abstract

  The colonial protochordate Botryllus schlosseri is genetically manipulable and represents a potential model organism for a variety of biological disciplines, including immunology, stem cell biology and development. This article presents the construction and characterization of both BAC and fosmid genomic libraries of the 725-Mbp B. schlosseri genome. The BAC library currently consists of 2x genome coverage with an average insert size of 80 kb. The fosmid library is at 11x genome coverage with an average insert of 40 kb. B. schlosseri is a small organism containing a large number of compounds that hinder DNA purification. Thus a number of protocols had to be modified in order to make purified, high molecular weight inserts for cloning, including both gel purification and insert concentration techniques. Both libraries were characterized by using them in initial physical mapping of a single histocompatibility locus, and were found to be representative and functional. These libraries are important tools for physical mapping and positional cloning in the B. schlosseri genome, and the techniques adapted to make them are suitable for use on other organisms in which high molecular weight DNA is difficult to purify.

  View details for DOI 10.1007/s10126-002-0071-1

  View details for Web of Science ID 000182588700001

  View details for PubMedID 12876644

• MLL-GAS7 transforms multipotent hematopoietic progenitors and induces mixed lineage leukemias in mice CANCER CELL So, C. W., Karsunky, H., Passegue, E., Cozzio, A., Weissman, I. L., Cleary, M. L. 2003; 3 (2): 161-171

  Abstract

  A specific association with mixed lineage leukemias suggests that MLL oncoproteins may selectively target early multipotent hematopoietic progenitors or stem cells. We demonstrate here that a representative MLL fusion protein, MLL-GAS7, impairs the differentiation and enhances the in vitro growth of murine hematopoietic cells with multipotent features. The multilineage differentiation potential of these cells was suggested by their immuno-phenotypes and transcriptional programs and confirmed by their ability to induce three pathologically distinct leukemias in mice, including an acute biphenotypic leukemia (ABL) that recapitulates the distinctive hallmark features of many MLL-associated leukemias in humans. This experimental modeling of ABL in mice highlights its origin from multipotential progenitors that arrest at a bipotential stage specifically targeted or induced by MLL oncogenes.

  View details for Web of Science ID 000181240100009

  View details for PubMedID 12620410

• Characterization of mouse clonogenic megakaryocyte progenitors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nakorn, T. N., Miyamoto, T., Weissman, I. L. 2003; 100 (1): 205-210

  Abstract

  Although it has been shown that unfractionated bone marrow, hematopoietic stem cells, common myeloid progenitors, and bipotent megakaryocyteerythrocyte progenitors can give rise to megakaryocyte colonies in culture, monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here, we use a monoclonal antibody to the megakaryocyte-associated surface protein, CD9, to purify MKPs from the c-kit(+)Sca-1(-)IL7Ralpha(-)Thy1.1(-)Lin(-) fraction of adult C57BLKa-Thy1.1 bone marrow. The CD9(+) fraction contained a subset of CD41(+)FcgammaR(lo)CD34(+)CD38(+) cells that represent approximately 0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit-megakaryocytes and occasionally burst-forming unit-megakaryocytes, with a plating efficiency >60% at the single-cell level. In vivo, MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for approximately 3 weeks. Common myeloid progenitors and megakaryocyteerythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures, indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns.

  View details for DOI 10.1073/pnas.262655099

  View details for Web of Science ID 000180307100038

  View details for PubMedID 12490656

  View details for PubMedCentralID PMC140928

• Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice DIABETES Beilhack, G. F., Scheffold, Y. C., Weissman, I. L., TAYLOR, C., Jerabek, L., Burge, M. J., Masek, M. A., Shizuru, J. A. 2003; 52 (1): 59-68

  Abstract

  Purified hematopoietic stem cells (HSCs) were transplanted into NOD mice to test whether development of hyperglycemia could be prevented. Engraftment of major histocompatibility complex-mismatched HSCs was compared with bone marrow (BM) grafts. HSCs differed from BM because HSCs were more strongly resisted and HSC recipients retained significant levels of NOD T-cells, whereas BM recipients were full donor chimeras. Despite persistent NOD T-cells, all HSC chimeras were protected from hyperglycemia, and attenuation of islet lesions was observed. T-cell selection was altered in allogeneic HSC recipients as demonstrated by deletion of both donor and host superantigen-specific T-cells. Syngeneic and congenic hematopoietic cell transplants were also performed to differentiate the influence of the preparative regimen(s) versus the allografts. Unlike the allogeneic HSC transplantations, syngeneic or congenic grafts did not retard diabetes development. In a pilot study, overtly diabetic NOD mice were cured by co-transplantation of allogeneic HSCs and donor-matched islets. We conclude that allogeneic HSC transplants block allo- and autoimmunity, despite residual host T-cell presence. These data demonstrate for the first time that purified HSC grafts block development of autoimmune diabetes and illuminate how HSC grafts alter thymic and peripheral T-cell responses against auto- and alloantigens.

  View details for Web of Science ID 000180157300009

  View details for PubMedID 12502494

• Biology of hematopoietic stem cells and progenitors: Implications for clinical application ANNUAL REVIEW OF IMMUNOLOGY Kondo, M., Wagers, A. J., Manz, M. G., Prohaska, S. S., Scherer, D. C., Beilhack, G. E., Shizuru, J. A., Weissman, I. L. 2003; 21: 759-806

  Abstract

  Stem cell biology is scientifically, clinically, and politically a current topic. The hematopoietic stem cell, the common ancestor of all types of blood cells, is one of the best-characterized stem cells in the body and the only stem cell that is clinically applied in the treatment of diseases such as breast cancer, leukemias, and congenital immunodeficiencies. Multicolor cell sorting enables the purification not only of hematopoietic stem cells, but also of their downstream progenitors such as common lymphoid progenitors and common myeloid progenitors. Recent genetic approaches including gene chip technology have been used to elucidate the gene expression profile of hematopoietic stem cells and other progenitors. Although the mechanisms that control self-renewal and lineage commitment of hematopoietic stem cells are still ambiguous, recent rapid advances in understanding the biological nature of hematopoietic stem and progenitor cells have broadened the potential application of these cells in the treatment of diseases.

  View details for DOI 10.1146/annurev.immunol.21.120601.141007

  View details for Web of Science ID 000182523500023

  View details for PubMedID 12615892

• Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation BLOOD Bitmansour, A., Burns, S. M., Traver, D., Akashi, K., Contag, C. H., Weissman, I. L., Brown, J. M. 2002; 100 (13): 4660-4667

  Abstract

  Myelotoxic treatments for oncologic diseases are often complicated by neutropenia, which renders patients susceptible to potentially lethal infections. In these studies of murine hematopoietic stem cell transplantation (HSCT), cotransplantation of lineage-restricted progenitors known as common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP) protects against death following otherwise lethal challenge with either of 2 pathogens associated with neutropenia: Aspergillus fumigatus and Pseudomonas aeruginosa. Cotransplantation of CMP/GMP resulted in a significant and rapid increase in the absolute number of myeloid cells in the spleen, most of which were derived from the donor CMP/GMP. Despite persistent peripheral neutropenia, improved survival correlated with the measurable appearance of progenitor-derived myeloid cells in the spleen. A marked reduction or elimination of tissue pathogen load was confirmed by culture and correlated with survival. Localization of infection by P aeruginosa and extent of disease was also assessed by in vivo bioluminescent imaging using a strain of P aeruginosa engineered to constitutively express a bacterial luciferase. Imaging confirmed that transplantation with a graft containing hematopoietic stem cells and CMP/GMP reduced the bacterial load as early as 18 hours after infection. These results demonstrate that enhanced reconstitution of a tissue myeloid pool offers protection against lethal challenge with serious fungal and bacterial pathogens.

  View details for DOI 10.1182/blood-2002-05-1552

  View details for Web of Science ID 000179759800058

  View details for PubMedID 12393415

• Telomerase activation and rejuvenation of telomere length in stimulated T cells derived from serially transplanted hematopoietic stem cells JOURNAL OF EXPERIMENTAL MEDICINE Allsopp, R. C., Cheshier, S., Weissman, I. L. 2002; 196 (11): 1427-1433

  Abstract

  Telomeres shorten in hematopoietic cells, including hematopoietic stem cells (HSCs), during aging and after transplantation, despite the presence of readily detectable levels of telomerase in these cells. In T cells, antigenic stimulation has been shown to result in a marked increase in the level of telomerase activity. We now show that stimulation of T cells derived from serially transplanted HSC results in a telomerase-dependent elongation of telomere length to a size similar to that observed in T cells isolated directly from young mice. Southern analysis of telomere length in resting and anti-CD3/CD28 stimulated donor-derived splenic T cells revealed an increase in telomere size by approximately 7 kb for the population as a whole. Stimulation of donor-derived T cells from recipients of HSCs from telomerase-deficient mice did not result in regeneration of telomere length, demonstrating a dependence on telomerase. Furthermore, clonal anti-CD3/CD28 stimulation of donor-derived T cells followed by fluorescent in situ hybridization (FISH) analysis of telomeric signal intensity showed that telomeres had increased in size by approximately 50% for all clonal expansions. Together, these results imply that one role for telomerase in T cells may be to renew or extend replicative potential via the rejuvenation of telomere length.

  View details for DOI 10.1084/jem.20021003

  View details for Web of Science ID 000179682000004

  View details for PubMedID 12461078

  View details for PubMedCentralID PMC2194261

• Developmental plasticity of lymphoid progenitors SEMINARS IN IMMUNOLOGY Prohaska, S. S., Scherer, D. C., Weissman, I. L., Kondo, M. 2002; 14 (6): 377-384

  Abstract

  The identification of the common lymphoid progenitors in mouse bone marrow allows us to directly assess the regulatory mechanisms of lymphoid lineage commitment. The unexpected finding of a latent myeloid differentiation potential in lymphoid progenitors sheds light on the importance of cytokine receptor expression at this stage. We will discuss the biological nature of common lymphoid progenitors as a model of differentiation from multipotent to lineage committed progenitors. Elucidation of this hidden differentiation potential in progenitors will help further our understanding of the molecular mechanisms that control the cell fate determination of not only common lymphoid progenitors, but also their ancestors, hematopoietic stem cells, and their descendents such as committed T and B cell progenitors.

  View details for DOI 10.1016/S1044-5323(02)00072-6

  View details for Web of Science ID 000179881000004

  View details for PubMedID 12457610

• Langerhans cells renew in the skin throughout life under steady-state conditions NATURE IMMUNOLOGY Merad, M., Manz, M. G., Karsunky, H., Wagers, A., Peters, W., Charo, I., Weissman, I. L., Cyster, J. G., Engleman, E. G. 2002; 3 (12): 1135-1141

  Abstract

  Langerhans cells (LCs) are bone marrow (BM)-derived epidermal dendritic cells (DCs) that represent a critical immunologic barrier to the external environment, but little is known about their life cycle. Here, we show that in lethally irradiated mice that had received BM transplants, LCs of host origin remained for at least 18 months, whereas DCs in other organs were almost completely replaced by donor cells within 2 months. In parabiotic mice with separate organs, but a shared blood circulation, there was no mixing of LCs. However, in skin exposed to ultraviolet light, LCs rapidly disappeared and were replaced by circulating LC precursors within 2 weeks. The recruitment of new LCs was dependent on their expression of the CCR2 chemokine receptor and on the secretion of CCR2-binding chemokines by inflamed skin. These data indicate that under steady-state conditions, LCs are maintained locally, but inflammatory changes in the skin result in their replacement by blood-borne LC progenitors.

  View details for DOI 10.1038/ni852

  View details for Web of Science ID 000179467800010

  View details for PubMedID 12415265

• Flk2/Flt3 receptor expression at defined stages of early hematopoietic development and expansion of progenitors and downstream dendritic cells through cognate ligand stimulation. 44th Annual Meeting of the American-Society-of-Hematology Manz, M. M., Karsunky, H., Merad, M., Cozzio, A., Weissman, I. L. AMER SOC HEMATOLOGY. 2002: 290A–290A

  View details for Web of Science ID 000179184701112

• The role of Wnt signaling in myeloid leukemogenesis. 44th Annual Meeting of the American-Society-of-Hematology Jamieson, C. H., Ailles, L. E., Reya, T., Muijtjens, M., Weissman, I. L. AMER SOC HEMATOLOGY. 2002: 26A–26A

  View details for Web of Science ID 000179184700087

• Enforced expression of Bcl-2 restores the number of NK cells, but does not rescue the impaired development of NKT cells or intraepithelial lymphocytes, in IL-2/IL-15 receptor beta-chain-deficient mice JOURNAL OF IMMUNOLOGY Minagawa, M., Watanabe, H., Miyaji, C., Tomiyama, K., Shimura, H., Ito, A., Ito, M., Domen, J., Weissman, I. L., Kawai, K. 2002; 169 (8): 4153-4160

  Abstract

  IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.

  View details for Web of Science ID 000178512000014

  View details for PubMedID 12370344

• Little evidence for developmental plasticity of adult hematopoietic stem cells SCIENCE Wagers, A. J., Sherwood, R. I., Christensen, J. L., Weissman, I. L. 2002; 297 (5590): 2256-2259

  Abstract

  To rigorously test the in vivo cell fate specificity of bone marrow (BM) hematopoietic stem cells (HSCs), we generated chimeric animals by transplantation of a single green fluorescent protein (GFP)-marked HSC into lethally irradiated nontransgenic recipients. Single HSCs robustly reconstituted peripheral blood leukocytes in these animals, but did not contribute appreciably to nonhematopoietic tissues, including brain, kidney, gut, liver, and muscle. Similarly, in GFP+:GFP- parabiotic mice, we found substantial chimerism of hematopoietic but not nonhematopoietic cells. These data indicate that "transdifferentiation" of circulating HSCs and/or their progeny is an extremely rare event, if it occurs at all.

  View details for DOI 10.1126/science.1074807

  View details for Web of Science ID 000178222000046

  View details for PubMedID 12215650

• Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: A model for development and regeneration DEVELOPMENTAL BIOLOGY Lauzon, R. J., Ishizuka, K. J., Weissman, I. L. 2002; 249 (2): 333-348

  Abstract

  Botryllus schlosseri is a colonial marine urochordate in which all adult organisms (called zooids) in a colony die synchronously by apoptosis (programmed cell death) in cyclical fashion. During this death phase called takeover, cell corpses within the dying organism are engulfed by circulating phagocytic cells. The "old" zooids and their organs are resorbed within 24-36 h (programmed cell removal). This process coincides temporally with the growth of asexually derived primary buds, that harbor a small number of undifferentiated cells, into mature zooids containing functional organs and tissues with the same body plan as adult zooids from which they budded. Within these colonies, all zooids share a ramifying network of extracorporeal blood vessels embedded in a gelatinous tunic. The underlying mechanisms regulating programmed cell death and programmed cell removal in this organism are unknown. In this study, we extirpated buds or zooids from B. schlosseri colonies in order to investigate the interplay that exists between buds, zooids, and the vascular system during takeover. Our findings indicate that, in the complete absence of buds (budectomy), organs from adult zooids underwent programmed cell death but were markedly impaired in their ability to be resorbed despite engulfment of zooid-derived cell corpses by phagocytes. However, when buds were removed from only half of the flower-shaped systems of zooids in a colony (hemibudectomy), the budectomized zooids were completely resorbed within 36-48 h following onset of programmed cell death. Furthermore, if hemibudectomies were carried out by using small colonies, leaving only a single functional bud, zooids from the old generation were also resorbed, albeit delayed to 48-60 h following onset of programmed cell death. This bud eventually reached functional maturity, but grew significantly larger in size than any control zooid, and exhibited hyperplasia. This finding strongly suggested that components of the dying zooid viscera could be reutilized by the developing buds, possibly as part of a colony-wide recycling mechanism. In order to test this hypothesis, zooids were surgically removed (zooidectomy) at the onset of takeover, and bud growth was quantitatively determined. In these zooidectomized colonies, bud growth was severely curtailed. In most solitary, long-lived animals, organs and tissues are maintained by processes of continual death and removal of aging cells counterbalanced by regeneration with stem and progenitor cells. In the colonial tunicate B. schlosseri, the same kinds of processes ensure the longevity of the colony (an animal) by cycles of death and regeneration of its constituent zooids (also animals).

  View details for DOI 10.1006/dbio.2002.0772

  View details for Web of Science ID 000178133300010

  View details for PubMedID 12221010

• Engraftment of sorted/expanded human central nervous system stem cells from fetal brain JOURNAL OF NEUROSCIENCE RESEARCH Tamaki, S., Eckert, K., He, D. P., Sutton, R., Doshe, M., Jain, G., Tushinski, R., Reitsma, M., Harris, B., Tsukamoto, A., Gage, F., Weissman, I., Uchida, N. 2002; 69 (6): 976-986

  Abstract

  Direct isolation of human central nervous system stem cells (CNS-SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS-SC were isolated from fetal brain tissue using the cell surface markers CD133(+), CD34(-), CD45(-), and CD24(-/lo) (CD133(+) cells). Fluorescence-activated cell sorted (FACS) CD133(+) cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for >10 passages. CD133(-), CD34(-), and CD45(-) sorted cells (approximately 95% of total fetal brain tissue) fail to initiate neurospheres. Neurosphere cells transplanted into neonatal immunodeficient NOD-SCID mice proliferated, migrated, and differentiated in a site-specific manner. However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (beta-tubulin III and glial fibrillary acidic protein) are not species specific. To trace the progeny of human cells after transplantation, CD133(+)-derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter. After transduction, GFP(+) cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD-SCID brain. The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP. GFP(+) cells were present in the subventricular zone-rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum. Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells. Thus, marking human CNS-SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model.

  View details for DOI 10.1002/jnr.10412

  View details for Web of Science ID 000177792600031

  View details for PubMedID 12205691

• Prospective isolation of human clonogenic common myeloid progenitors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Manz, M. G., Miyamoto, T., Akashi, K., Weissman, I. L. 2002; 99 (18): 11872-11877

  Abstract

  The hierarchical development from hematopoietic stem cells to mature cells of the hematolymphoid system involves progressive loss of self-renewal capacity, proliferation ability, and lineage potentials. Here we show the prospective isolation of early developmental intermediates, the human clonogenic common myeloid progenitors and their downstream progeny, the granulocyte/macrophage and megakaryocyte/erythrocyte progenitors. All three populations reside in the lineage-negative (lin(-)) CD34(+)CD38(+) fraction of adult bone marrow as well as in cord blood. They are distinguishable by the expression of the IL-3R alpha chain, the receptor of an early-acting hematopoietic cytokine, and CD45RA, an isoform of a phosphotyrosine phosphatase involved in negative regulation of cytokine signaling. Multipotent progenitors, early lymphoid progenitors, and the here-defined myeloid progenitors express distinct profiles of hematopoiesis-affiliated genes. The isolation of highly purified hematopoietic intermediates provides tools to better understand developmental programs underlying normal and leukemic hematopoiesis.

  View details for DOI 10.1073/pnas.172384399

  View details for Web of Science ID 000177843100061

  View details for PubMedID 12193648

  View details for PubMedCentralID PMC129361

• The road ended up at stem cells IMMUNOLOGICAL REVIEWS Weissman, I. L. 2002; 185: 159-174

  Abstract

  For me the search for hematopoietic stem cells (HSC) actually started with the discovery by Till, McCulloch, and colleagues (1-3) that bone marrow contained single cells that could give rise to myeloerythroid colonies in the spleen, and sometimes these colonies contained cells that made more spleen colonies as well as radioprotected and reconstituted lethally irradiated mice (3). But in retrospect, it should have started with the remarkable observation of Ray Owen in 1945 that bovine fraternal twins sharing a single placenta and blood circulation retained production of blood cells genetically defined to be from both throughout their life (4). It could be argued that this was the experiment that began both modern experimental hematology as well as modern cellular immunology. The Till, McCulloch, Wu, Becker, and Simonovitch experiments were elegant demonstrations that single, genetically marked cells existed (random DNA breaks and translocations induced by sublethal irradiation of the donor bone marrow) that could both self-renew and differentiate (2, 5). But these experiments did not put the pure cells in the hands of scientists, and so most of their functions for the next 25 years were implied rather than directly analyzed. Just as genetics is the complement to biochemistry (when one considers genes and gene products), cell marking is the complement to cell purification in the fields of developmental and cellular biology. The first attempts at such cellular purification came from the 'school' of Till & McCulloch (6, 7), and independently the school of Van Bekkum in the Netherlands (8). But what was lacking in those experiments and at that time were both a comprehensive approach that would take into account the clonal activity of stem cells in both self-renewal and differentiation to all blood cell outcomes, and the tools with which one could separate what turned out to be an extremely rare population in the bone marrow. And, it wasn't known until much later that most day 8-10 spleen colonies were the progeny of progenitors, not stem cells (9). Two inventions facilitated the technology of purification of HSC: the advent of monoclonal antibody technology by Kohler & Milstein (10), and the development of the multiparameter fluorescence activated cell sorter by the Herzenberg group (11). My laboratory had established assays for the clonal precursors of T cells and B cells, and we had been using the Till-McCulloch spleen colony clonal assays since the mid-1960s. In the late 1970s and early 1980s we began in earnest the search for mouse early hematopoietic progenitors, including HSCs (12-16). The purification of HSCs proved to be much like the purification of an enzyme, or a cell surface receptor, or a gene. Successive enrichments finally led to the isolation of a population, which could no longer be subdivided and which contained precursors that read out in all clonal assays as well as in radioprotection of lethally irradiated hosts (17). Our first experiments transplanting single HSC in 1991 and 1992, led to the definitive demonstration that these were indeed HSCs (18-20). But these experiments and the ideas that led to them were developed in the context of immunology and experimental hematology as they were emerging in the 1950s and 60 s. This volume of Immunological Reviews is a rich testimony to the kinds of ideas and experiments that, at least in retrospect, turned out to be critical. Many roads were taken, but only one ended up at stem cells.

  View details for Web of Science ID 000177588900014

  View details for PubMedID 12190929

• Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment DEVELOPMENTAL CELL Miyamoto, T., Iwasaki, H., Reizis, B., Ye, M., Graf, T., Weissman, I. L., Akashi, K. 2002; 3 (1): 137-147

  Abstract

  We demonstrate here that "promiscuous" expression of myeloid or lymphoid genes precedes lineage commitment in hematopoiesis. Prospectively purified single common myeloid progenitors (CMPs) coexpress myelo-erythroid but not lymphoid genes, whereas single common lymphoid progenitors (CLPs) coexpress T and B lymphoid but not myeloid genes. Genes unrelated to the adopted lineage are downregulated in bipotent and monopotent descendants of CMPs and CLPs. Promiscuous gene expression does not alter the biological potential of multipotent progenitors: CMPs with an activated endogenous M lysozyme locus yield normal proportions of myelo-erythroid colonies, and CLPs expressing the pre-T cell receptor alpha gene differentiate into normal numbers of B cells. Thus, the accessibility for multiple myeloid or lymphoid programs promiscuously may allow flexibility in fate commitments at these multipotent stages.

  View details for Web of Science ID 000176769500016

  View details for PubMedID 12110174

• Myeloerythroid-restricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S JOURNAL OF CLINICAL INVESTIGATION Nakorn, T. N., Traver, D., Weissman, I. L., Akashi, K. 2002; 109 (12): 1579-1585

  Abstract

  Whole-body irradiation at the minimal lethal dose causes bone marrow failure and death within 12-18 days. To identify the principal components of the hematopoietic system that are radioprotective, we transplanted lethally irradiated mice with purified progenitors: common myeloid progenitors (CMPs), megakaryocyte/erythrocyte-restricted progenitors (MEPs), or granulocyte/monocyte-restricted progenitors (GMPs). Transplanted CMPs gave rise to cells both of the granulocyte/monocyte (GM) series and the megakaryocyte/erythrocyte series, whereas GMPs or MEPs showed reconstitution of only GM or ME cells, respectively. CMPs and MEPs but not GMPs protected mice in a dose-dependent manner, suggesting that erythrocytes, platelets, or both are the critical effectors of radioprotection. Accordingly, CMPs and MEPs formed robust colonies in recipient bone marrow and spleen, whereas GMPs formed small colonies that rapidly disappeared. Direct comparisons of spleen CFU (CFU-S) potentials among each progenitor subset showed that MEPs contain the vast majority of day 8 CFU-S activity, suggesting that day 8 CFU-S are the precursors of radioprotective cell subsets. All animals radioprotected for 30 days subsequently survived for at least 6 months post-transplant, and showed only host-derived hematopoiesis after 30 days. These findings suggest that rare hematopoietic stem cells survive myeloablation that can eventually repopulate irradiated hosts if myeloerythroid-restricted progenitors transiently rescue ablated animals through the critical window of bone marrow failure.

  View details for DOI 10.1172/JCI200215272

  View details for Web of Science ID 000176318600011

  View details for PubMedID 12070305

  View details for PubMedCentralID PMC151014

• Characterization of murine leukemic myeloid progenitors. Jamieson, C. H., Nanakorn, T., Jaiswal, S., Muijtjens, M., Weissman, I. L. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2002: S71–S71

  View details for Web of Science ID 000176439600217

• Stem cells - Scientific, medical, and political issues NEW ENGLAND JOURNAL OF MEDICINE Weissman, I. L. 2002; 346 (20): 1576-1579

  View details for Web of Science ID 000175563900012

  View details for PubMedID 11994551

• Replicative senescence of hematopoietic stem cells during serial transplantation: does telomere shortening play a role? ONCOGENE Allsopp, R. C., Weissman, I. L. 2002; 21 (21): 3270-3273

  Abstract

  Hematopoietic stem cells (HSC) have a finite proliferative lifespan, based upon the limited number of times they can be serially transplanted in mice. Telomeres have been shown to shorten during the division of many normal somatic cells in humans, and the attrition of telomeres has been shown to ultimately cause replicative senescence in vitro for a number of different human cell strains. Whereas most human cell types have little to no detectable levels of telomerase activity, hematopoietic cells, including HSC, express low to moderate levels of telomerase, and yet telomeres shorten considerably during replicative aging of these cells. Here we consider the role telomerase may play in the hematopoietic system as well as the effect that over-expression of telomerase reverse transcriptase may have on the replicative capacity of hematopoietic stem cells during transplantation.

  View details for DOI 10.1038/sj/onc/1205314

  View details for Web of Science ID 000175633300003

  View details for PubMedID 12032768

• Hematopoietic stem cells are uniquely selective in their migratory response to chemokines JOURNAL OF EXPERIMENTAL MEDICINE Wright, D. E., Bowman, E. P., Wagers, A. J., BUTCHER, E. C., Weissman, I. L. 2002; 195 (9): 1145-1154

  Abstract

  Although hematopoietic stem cell (HSC) migration into and out of sites of active hematopoiesis is poorly understood, it is a critical process that underlies modern clinical stem cell transplantation and may be important for normal hematopoietic homeostasis. Given the established roles of chemotactic cytokine (chemokine)-directed migration of other leukocyte subsets, the migration of murine HSC to a large panel of CC and CXC chemokines was investigated. HSC migrated only in response to stromal derived factor-1alpha, the ligand for the CXC chemokine receptor 4 (CXCR4). CXCR4 expression by HSC was confirmed by reverse transcription polymerase chain reaction analysis. Surprisingly, HSC also expressed mRNA for CCR3 and CCR9, although they failed to migrate to the ligands for these receptors. The sharply restricted chemotactic responsiveness of HSC is unique among leukocytes and may be necessary for the specific homing of circulating HSC to bone marrow, as well as for the maintenance of HSC in hematopoietic microenvironments.

  View details for DOI 10.1084/jem.20011284

  View details for Web of Science ID 000176110700006

  View details for PubMedID 11994419

  View details for PubMedCentralID PMC2193709

• Cell fate determination from stem cells GENE THERAPY Wagers, A. J., Christensen, J. L., Weissman, I. L. 2002; 9 (10): 606-612

  Abstract

  In the adult, tissue-specific stem cells are thought to be responsible for the replacement of differentiated cells within continuously regenerating tissues, such as the liver, skin, and blood system. In this review, we will consider the factors that influence stem cell fate, taking as a primary example the cell fate determination of hematopoietic stem cells.

  View details for DOI 10.1038/sj/gt/3301717

  View details for Web of Science ID 000175525800002

  View details for PubMedID 12032706

• Lineage infidelity in myeloid cells with TCR gene rearrangement: A latent developmental potential of proT cells revealed by ectopic cytokine receptor signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA King, A. G., Kondo, M., Scherer, D. C., Weissman, I. L. 2002; 99 (7): 4508-4513

  Abstract

  The most immature lymphoid-committed progenitors in both the bone marrow (common lymphoid progenitor) and thymus (proT1) maintain a latent granulocyte/macrophage (G/M) differentiation potential that can be initiated by signals emanating from exogenously expressed IL-2 receptors. In this study, we investigate at which developmental stage thymocytes lose this G/M differentiation potential. We demonstrate that the next maturational stage after proT1 cells (proT2), but not preT (TN3) cells, can convert cell fate from lymphoid to myeloid in response to ectopic IL-2 receptor signaling in human IL-2Rbeta transgenic mice. It is significant that approximately 10% of clonogenic G/M colonies derived from proT cells of IL-2Rbeta transgenic mice have DJ rearrangement specifically at the Dbeta1 but not Dbeta2 segment in the TCRbeta locus. No TCR gene rearrangement is observed in G/M cells from nontransgenic mice, suggesting that the G/M cells we observe in this system were truly lymphoid-committed before stimulation with IL-2. In addition, Dbeta1 and Dbeta2 DJ rearrangement of the TCRbeta gene may be differentially regulated and thus serve as markers for distinct proT cell maturational stages.

  View details for DOI 10.1073/pnas.072087899

  View details for Web of Science ID 000174856000068

  View details for PubMedID 11917122

  View details for PubMedCentralID PMC123678

• Changes in integrin expression are associated with altered homing properties of Lin(-/lo)Thy(1.1lo)Sca-1(+) (+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor EXPERIMENTAL HEMATOLOGY Wagers, A. J., Allsopp, R. C., Weissman, I. L. 2002; 30 (2): 176-185

  Abstract

  Although migration of hematopoietic stem cells (HSC) is essential for normal hematopoiesis and successful hematopoietic cell transplantation, little is known about the mechanisms that underlie this movement. We have sought to characterize the factors that regulate HSC migration by analyzing changes in expression of particular adhesion receptors associated with cyclophosphamide/granulocyte colony-stimulating factor (Cy/G-CSF)-induced HSC mobilization.Expression by Lineage(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) HSC of members of the beta1 integrin family of adhesion molecules was assessed in untreated or Cy/G-CSF-treated mice by multiparameter flow cytometry. In parallel, the in vivo homing properties of normal and mobilized HSC were compared following intravenous transfer of fluorescently marked HSC.Normal adult HSC express high levels of several beta1 integrin family members. Following Cy/G treatment, bone marrow HSC selectively downregulate alpha 2 integrin expression and upregulate alpha 5 expression. HSC found in the blood following Cy/G-CSF treatment express significantly lower levels of multiple integrins than their bone marrow and/or splenic counterparts. Changes in integrin expression by blood-borne HSC correlate with a 50% decrease in their ability to home to the bone marrow in short-term assays, and with previously observed defects in competitive engraftment by these HSC. Similar reductions in bone marrow (BM) homing are observed for BM HSC treated with alpha 4 integrin function blocking mAb prior to injection. Modulation of integrin expression induced by mobilization was not associated with cell-cycle progression.Changes in integrin expression and function are associated with HSC mobilization and likely significantly affect the engraftment potential of hematopoietic stem cells.

  View details for Web of Science ID 000174129400010

  View details for PubMedID 11823053

• A genetic determinant that specifically regulates the frequency of hematopoietic stem cells JOURNAL OF IMMUNOLOGY Morrison, S. J., Qian, D., Jerabek, L., Thiel, B. A., Park, I. K., Ford, P. S., Kiel, M. J., Schork, N. J., Weissman, I. L., Clarke, M. F. 2002; 168 (2): 635-642

  Abstract

  The regulation of hematopoietic stem cell (HSC) homeostasis is not well understood. We screened for genetic polymorphisms that were linked to differences between mouse strains in the numbers of long-term reconstituting HSCs or restricted progenitors in the bone marrow. AKR/J mice had significantly higher frequencies and numbers of both HSCs and restricted progenitors in their bone marrow than C57BL/Ka-Thy-1.1 mice. The C57BL/Ka-Thy-1.1 alleles were partially dominant. A locus on chromosome 17, including the H-2 complex, was significantly linked to the frequency of long-term self-renewing HSCs but showed no evidence of linkage to the frequency of restricted progenitors. Conversely, a chromosome 1 locus exhibited suggestive linkage to restricted progenitor frequencies but was not linked to HSC frequency. This demonstrates that there are distinct genetic determinants of the frequencies of HSCs and restricted progenitors in vivo. The AKR/J chromosome 17 locus was not sufficient to increase HSC frequencies when bred onto a C57BL background. This suggests that to affect HSC frequencies, the product(s) of this locus likely depend on interactions with unlinked modifying loci.

  View details for Web of Science ID 000173193700014

  View details for PubMedID 11777956

• Differential gene expression profiling of adult murine hematopoietic stem cells BLOOD Park, I. K., He, Y. Q., Lin, F. M., Laerum, O. D., Tian, Q., Bumgarner, R., Klug, C. A., Li, K. J., Kuhr, C., Doyle, M. J., Xie, T., Schummer, M., Sun, Y., GOLDSMITH, A., Clarke, M. F., Weissman, I. L., Hood, L., Li, L. H. 2002; 99 (2): 488-498

  Abstract

  Hematopoietic stem cells (HSCs) have self-renewal capacity and multilineage developmental potentials. The molecular mechanisms that control the self-renewal of HSCs are still largely unknown. Here, a systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs and a mixed population of stem and early multipotent progenitor (MPP) cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA clones with very low hybridization signals were selected for sequencing and further analysis using microarrays on glass slides. Two populations of cells, HSCs and MPP cells, were compared for differential gene expression using microarray analysis. HSCs have the ability to self-renew, while MPP cells have lost the capacity for self-renewal. A large number of genes that were differentially expressed by enriched populations of HSCs and MPP cells were identified. These included transcription factors, signaling molecules, and previously unknown genes.

  View details for Web of Science ID 000173215900013

  View details for PubMedID 11781229

• Intrathymic injection for analysis of T-cell progenitor activity. Methods in molecular medicine Jerabek, L., Weissman, I. L. 2002; 63: 161-165

  Abstract

  Within our field, improvement in fluorescence-activated cell sorting (FACS) and molecular technologies has led to various types of correlative studies that imply the developmental sequence and subsequent emigration of thymic-lymphocyte subsets. Unfortunately, the implied conclusions are often accepted unequivocally by most of the immunology community. In fact, direct demonstration of precursor progeny relationships by specific cell marking within the thymus, or specific delivery of purified cells at a particular stage of isolation back into the thymus, are the only methods that reproducibly identify cell stages and intermediates (1-11).

  View details for DOI 10.1385/1-59259-140-X:161

  View details for PubMedID 21437807

• Correspondence - Dr. Weissman Replies New England Journal of Medicine Weissman, I. 2002; 347 (20): 1621
• Something in the eye of the beholder: Response Science Wagers, A., Sherwood, R., Christensen, J., Weissman, I. 2002; 298 (5592): 362-363
• Genetic variability of Botryllus schlosseri invasions to the east and west coasts of the USA Marine Ecology Progress Series Stoner, D., Ben-Shlomo, R., Rinkevich, B., Weissman, I. 2002; 243: 93-100
• Scientific and medical aspects of human reproductive cloning Weissman, I., et al National Academies Press. 2002
• Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Christensen, J. L., Weissman, I. L. 2001; 98 (25): 14541-14546

  Abstract

  Clonogenic multipotent mouse hematopoietic stem cells (HSCs) and progenitor cells are contained within the c-kit(+) (K) lineage(-/lo) (L) Sca-1(+) (S) population of hematopoietic cells; long-term (LT) and short-term (ST) HSCs are Thy-1.1(lo). c-kit is a member of the receptor tyrosine kinase family, a class of receptors that are important in the proliferation and differentiation of hematopoietic cells. To establish whether the Flk-2/Flt3 receptor tyrosine kinase was expressed on the most primitive LT-HSCs, we sorted highly purified multipotent stem and progenitor cells on the basis of Flk-2 surface expression and used them in competitive reconstitution assays. Low numbers of Flk-2(-) HSCs gave rise to long-term multilineage reconstitution in the majority of recipients, whereas the transfer of Flk-2(+) multipotent cells resulted in mostly short-term multilineage reconstitution. The KLS subset of adult mouse bone marrow was analyzed for Flk-2 and Thy-1.1 expression. Three phenotypically and functionally distinct populations were isolated: Thy(lo) Flk-2(-) (LT-HSCs), Thy(lo) Flk-2(+) (ST-HSCs), and Thy(-) Flk-2(+) multipotent progenitors. The loss of Thy-1.1 and gain of Flk-2 expression marks the loss of self-renewal in HSC maturation. The addition of Flk-2 antibody to the lineage mix allows direct isolation of LT-HSC from adult bone marrow as c-kit(+) lin(-) Sca-1(+) Flk-2(-) from many strains of mice. Fetal liver HSCs are contained within Flk-2(-) and Flk-2(+) KTLS cells.

  View details for Web of Science ID 000172576900065

  View details for PubMedID 11724967

• Physiological migration of hematopoietic stem and progenitor Celts SCIENCE Wright, D. E., Wagers, A. J., Gulati, A. P., Johnson, F. L., Weissman, I. L. 2001; 294 (5548): 1933-1936

  Abstract

  Hematopoietic stem cells (HSCs) reside predominantly in bone marrow, but low numbers of HSCs are also found in peripheral blood. We examined the fate of blood-borne HSCs using genetically marked parabiotic mice, which are surgically conjoined and share a common circulation. Parabionts rapidly established stable, functional cross engraftment of partner-derived HSCs and maintained partner-derived hematopoiesis after surgical separation. Determination of the residence time of injected blood-borne progenitor cells suggests that circulating HSCs/progenitors are cleared quickly from the blood. These data demonstrate that HSCs rapidly and constitutively migrate through the blood and play a physiological role in, at least, the functional reengraftment of unconditioned bone marrow.

  View details for Web of Science ID 000172465000061

  View details for PubMedID 11729320

• A transgenic TCR recognizing a potentially diabetogenic self antigen in the context of MHC class II (I-A(g7)) produces a regulatory CD4+and an aberrant, class II reactive CD8+T cell population in NOD mice. Ranheim, E. A., Tarbell, K., Lee, M., Teyton, L., Davis, M., McDevitt, H., Weissman, I. L. AMER SOC HEMATOLOGY. 2001: 701A–701A

  View details for Web of Science ID 000172134102945

• Stem cells, cancer, and cancer stem cells NATURE Reya, T., Morrison, S. J., Clarke, M. F., Weissman, I. L. 2001; 414 (6859): 105-111

  Abstract

  Stem cell biology has come of age. Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine. Perhaps the most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells, and cancer cells may include 'cancer stem cells' - rare cells with indefinite potential for self-renewal that drive tumorigenesis.

  View details for Web of Science ID 000171898900054

  View details for PubMedID 11689955

• Lymphocyte development from hematopoietic stem cells CURRENT OPINION IN GENETICS & DEVELOPMENT Kondo, M., Scherer, D. C., King, A. G., Manz, M. G., Weissman, I. L. 2001; 11 (5): 520-526

  Abstract

  The recent application of new techniques, such as multi-color cell sorting and the production of transgenic and gene-knockout mice, has contributed to a better understanding of lymphocyte development from hematopoietic stem cells. Now that we can purify progenitors at different maturational stages during lymphocyte development, the challenge is to understand the processes that govern each developmental stage transition.

  View details for Web of Science ID 000171477500005

  View details for PubMedID 11532393

• AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yuan, Y. Z., Zhou, L. M., Miyamoto, T., Iwasaki, H., Harakawa, N., Hetherington, C. J., Burel, S. A., Lagasse, E., Weissman, I. L., Akashi, K., Zhang, D. E. 2001; 98 (18): 10398-10403

  Abstract

  The t(8;21) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). The translocation, which involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generates an AML1-ETO fusion transcription factor. To examine the effect of the AML1-ETO fusion protein on leukemogenesis, we made transgenic mice in which expression of AML1-ETO is under the control of the human MRP8 promoter (hMRP8-AML1-ETO). AML1-ETO is specifically expressed in myeloid cells, including common myeloid progenitors of hMRP8-AML1-ETO transgenic mice. The transgenic mice were healthy during their life spans, suggesting that AML1-ETO alone is not sufficient for leukemogenesis. However, after treatment of newborn hMRP8-AML1-ETO transgenic mice and their wild-type littermates with a strong DNA-alkylating mutagen, N-ethyl-N-nitrosourea, 55% of transgenic mice developed AML and the other 45% of transgenic mice and all of the wild-type littermates developed acute T lymphoblastic leukemia. Our results provide direct evidence that AML1-ETO is critical for causing myeloid leukemia, but one or more additional mutations are required for leukemogenesis. The hMRP8-AML1-ETO-transgenic mice provide an excellent model that can be used to isolate additional genetic events and to further understand the molecular pathogenesis of AML1-ETO-related leukemia.

  View details for Web of Science ID 000170738000068

  View details for PubMedID 11526243

• Immunity to infections following hematopoietic cell transplantation CURRENT OPINION IN IMMUNOLOGY Brown, J. M., Weissman, I. L., Shizuru, J. A. 2001; 13 (4): 451-457

  Abstract

  Hematopoietic cell transplantation has progressed from the use of unpurified bone marrow cells or mobilized peripheral blood cells to the use of purified stem cells and progenitor cells. These kinds of transplants can be designed to provide not only hematopoietic rescue but also augmented innate and acquired immunity.

  View details for Web of Science ID 000169648600010

  View details for PubMedID 11498301

• Fetal liver myelopoiesis occurs through distinct, prospectively isolatable progenitor subsets BLOOD Traver, D., Miyamoto, T., Christensen, J., Iwasaki-Arai, J., Akashi, K., Weissman, I. L. 2001; 98 (3): 627-635

  Abstract

  Hematopoietic fate maps in the developing mouse embryo remain imprecise. Definitive, adult-type hematopoiesis first appears in the fetal liver, then progresses to the spleen and bone marrow. Clonogenic common lymphoid progenitors and clonogenic common myeloid progenitors (CMPs) in adult mouse bone marrow that give rise to all lymphoid and myeloid lineages, respectively, have recently been identified. Here it is shown that myelopoiesis in the fetal liver similarly proceeds through a CMP equivalent. Fetal liver CMPs give rise to megakaryocyte-erythrocyte-restricted progenitors (MEPs) and granulocyte-monocyte-restricted progenitors (GMPs) that can also be prospectively isolated by cell surface phenotype. MEPs and GMPs generate mutually exclusive cell types in clonogenic colony assays and in transplantation experiments, suggesting that the lineage restriction observed within each progenitor subset is absolute under normal conditions. Purified progenitor populations were used to analyze expression profiles of various hematopoiesis-related genes. Expression patterns closely matched those of the adult counterpart populations. These results suggest that adult hematopoietic hierarchies are determined early in the development of the definitive immune system and suggest that the molecular mechanisms underlying cell fate decisions within the myeloerythroid lineages are conserved from embryo to adult. (Blood. 2001;98:627-635)

  View details for Web of Science ID 000170094800022

  View details for PubMedID 11468160

• The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver BLOOD DiMartino, J. F., SELLERI, L., Traver, D., Firpo, M. T., Rhee, J., Warnke, R., O'Gorman, S., Weissman, I. L., Cleary, M. L. 2001; 98 (3): 618-626

  Abstract

  Pbx1 is the product of a proto-oncogene originally discovered at the site of chromosomal translocations in acute leukemias. It binds DNA as a complex with a broad subset of homeodomain proteins, but its contributions to hematopoiesis have not been established. This paper reports that Pbx1 is expressed in hematopoietic progenitors during murine embryonic development and that its absence results in severe anemia and embryonic lethality at embryonic day 15 (E15) or E16. Definitive myeloerythroid lineages are present in Pbx1(-/-) fetal livers, but the total numbers of colony-forming cells are substantially reduced. Fetal liver hypoplasia reflects quantitative as well as qualitative defects in the most primitive multilineage progenitors and their lineage-restricted progeny. Hematopoietic stem cells from Pbx1(-/-) embryos have reduced colony-forming activity and are unable to establish multilineage hematopoiesis in competitive reconstitution experiments. Common myeloid progenitors (CMPs), the earliest known myeloerythroid-restricted progenitors, are markedly depleted in Pbx1(-/-) embryos at E14 and display clonogenic defects in erythroid colony formation. Comparative cell-cycle indexes suggest that these defects result largely from insufficient proliferation. Megakaryocyte- and erythrocyte-committed progenitors are also reduced in number and show decreased erythroid colony-forming potential. Taken together, these data indicate that Pbx1 is essential for the function of hematopoietic progenitors with erythropoietic potential and that its loss creates a proliferative constriction at the level of the CMP. Thus, Pbx1 is required for the maintenance, but not the initiation, of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets through which mature erythrocytes are generated. (Blood. 2001;98:618-626)

  View details for Web of Science ID 000170094800021

  View details for PubMedID 11468159

• From hematopoiesis to neuropoiesis: Evidence of overlapping genetic programs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Terskikh, A. V., Easterday, M. C., Li, L. H., Hood, L., Kornblum, H. I., Geschwind, D. H., Weissman, I. L. 2001; 98 (14): 7934-7939

  Abstract

  It is reasonable to propose that gene expression profiles of purified stem cells could give clues for the molecular mechanisms of stem cell behavior. We took advantage of cDNA subtraction to identify a set of genes selectively expressed in mouse adult hematopoietic stem cells (HSC) as opposed to bone marrow (BM). Analysis of HSC-enriched genes revealed several key regulatory gene candidates, including two novel seven transmembrane (7TM) receptors. Furthermore, by using cDNA microarray techniques we found a large set of HSC-enriched genes that are expressed in mouse neurospheres (a population greatly enriched for neural progenitor cells), but not present in terminally differentiated neural cells. In situ hybridization demonstrated that many of them, including one HSC-enriched 7TM receptor, were selectively expressed in the germinal zones of fetal and adult brain, the regions harboring mouse neural stem cells. We propose that at least some of the transcripts that are selectively and commonly expressed in two or more types of stem cells define a functionally conserved group of genes evolved to participate in basic stem cell functions, including stem cell self-renewal.

  View details for Web of Science ID 000169744200054

  View details for PubMedID 11438738

• Dendritic cell potentials of early lymphoid and myeloid progenitors BLOOD Manz, M. G., Traver, D., Miyamoto, T., Weissman, I. L., Akashi, K. 2001; 97 (11): 3333-3341

  Abstract

  It has been proposed that there are at least 2 classes of dendritic cells (DCs), CD8alpha(+) DCs derived from the lymphoid lineage and CD8alpha(-) DCs derived from the myeloid lineage. Here, the abilities of lymphoid- and myeloid-restricted progenitors to generate DCs are compared, and their overall contributions to the DC compartment are evaluated. It has previously been shown that primitive myeloid-committed progenitors (common myeloid progenitors [CMPs]) are efficient precursors of both CD8alpha(+) and CD8alpha(-) DCs in vivo. Here it is shown that the earliest lymphoid-committed progenitors (common lymphoid progenitors [CLPs]) and CMPs and their progeny granulocyte-macrophage progenitors (GMPs) can give rise to functional DCs in vitro and in vivo. CLPs are more efficient in generating DCs than their T-lineage descendants, the early thymocyte progenitors and pro-T cells, and CMPs are more efficient DC precursors than the descendant GMPs, whereas pro-B cells and megakaryocyte-erythrocyte progenitors are incapable of generating DCs. Thus, DC developmental potential is preserved during T- but not B-lymphoid differentiation from CLP and during granulocyte-macrophage but not megakaryocyte-erythrocyte development from CMP. In vivo reconstitution experiments show that CLPs and CMPs can reconstitute CD8alpha(+) and CD8alpha(-) DCs with similar efficiency on a per cell basis. However, CMPs are 10-fold more numerous than CLPs, suggesting that at steady state, CLPs provide only a minority of splenic DCs and approximately half the DCs in thymus, whereas most DCs, including CD8alpha(+) and CD8alpha(-) subtypes, are of myeloid origin. (Blood. 2001;97:3333-3341)

  View details for Web of Science ID 000168927900004

  View details for PubMedID 11369621

• The fetal liver counterpart of adult common lymphoid progenitors gives rise to all lymphoid lineages, CD45(+)CD4(+)CD3(-) cells, as well as macrophages JOURNAL OF IMMUNOLOGY Mebius, R. E., Miyamoto, T., Christensen, J., Domen, J., Cupedo, T., Weissman, I. L., Akashi, K. 2001; 166 (11): 6593-6601

  Abstract

  We identified an IL-7Ralpha(+)Sca-1(low)c-Kit(low) population in E14 fetal liver, which is the phenotypical analog of common lymphoid progenitors (CLP) in adult bone marrow. After transfer into newborn mice, the IL-7Ralpha(+)Sca-1(low)c-Kit(low) population rapidly differentiated into CD45(+)CD4(+)CD3(-) cells, which are candidate cells for initiating lymph node and Peyer's patch formation. In addition, this population also gave rise to B, T, NK, and CD8alpha(+) and CD8alpha(-) dendritic cells. The fetal liver precursors expressed a significantly lower level of the myeloid-suppressing transcription factor Pax-5, than adult CLP, and retained differentiation activity for macrophages in vitro. We propose that the transition from fetal liver IL-7Ralpha(+)Sca-1(low)c-Kit(low) cells to adult CLP involves a regulated restriction of their developmental potential, controlled, at least in part, by Pax-5 expression.

  View details for Web of Science ID 000170948900017

  View details for PubMedID 11359812

• Disappearing stem cells, disappearing science SCIENCE Weissman, I. L., Baltimore, D. 2001; 292 (5517): 601-601

  View details for Web of Science ID 000168478300001

  View details for PubMedID 11330301

• Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells JOURNAL OF EXPERIMENTAL MEDICINE Allsopp, R. C., Cheshier, S., Weissman, I. L. 2001; 193 (8): 917-924

  Abstract

  Reactivation of telomerase and maintenance of telomere length can lead to the prevention of replicative senescence in some human somatic cells grown in vitro. To investigate whether telomere shortening might also play a role in the limitation of hematopoietic stem cell (HSC) division capacity in vivo, we analyzed telomere length during serial transplantation of murine HSCs. Southern blot analysis of telomere length in donor bone marrow cells revealed extensive shortening ( approximately 7 kb) after just two rounds of HSC transplantation. The number of cycling HSCs increased after transplantation and remained elevated for at least 4 mo, while the frequency of HSCs in the bone marrow was completely regenerated by 2 mo after transplantation. Direct analysis of telomeres in HSCs by fluorescent in situ hybridization during serial transplantation also revealed a reduction in telomere size. Together, these data show that telomeres shorten during division of HSCs in vivo, and are consistent with the hypothesis that telomere shortening may limit the replicative capacity of HSCs.

  View details for Web of Science ID 000168199900004

  View details for PubMedID 11304552

• Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle BLOOD Wright, D. E., Cheshier, S. H., Wagers, A. J., Randall, T. D., Christensen, J. L., Weissman, I. L. 2001; 97 (8): 2278-2285

  Abstract

  Cytokine-mobilized peripheral blood hematopoietic stem cells (MPB HSC) are widely used for transplantation in the treatment of malignancies, but the mechanism of HSC mobilization is unclear. Although many HSC in bone marrow (BM) cycle rapidly and expand their numbers in response to cytoreductive agents, such as cyclophosphamide (CY), and cytokines, such as granulocyte colony-stimulating factor (G-CSF), MPB HSC are almost all in the G(0) or G(1) phase of the cell cycle. This has raised the question of whether a subset of noncycling BM HSC is selectively released, or whether cycling BM HSC are mobilized after M phase, but before the next S phase of the cell cycle. To distinguish between these possibilities, mice were treated with one dose of CY followed by daily doses of G-CSF, and dividing cells were marked by administration of bromodeoxyuridine (BrdU) during the interval that BM HSC are expanding. After CY and 4 days of G-CSF, 98.5% of the 2n DNA content long-term repopulating MPB (LT)-HSC stained positively for BrdU, and therefore derived from cells that divided during the treatment interval. Next, LT-HSC from mice previously treated with a single dose of CY, which kills cycling cells, and 3 daily doses of G-CSF, were nearly all killed by a second dose of CY, suggesting that CY/G-CSF causes virtually all LT-HSC to cycle. Analysis of cyclin D2 messenger RNA (mRNA) expression and total RNA content of MPB HSC suggests that these cells are mostly in G(1) phase. After CY/G-CSF treatment, virtually all BM LT-HSC enter the cell cycle; some of these HSC then migrate into the blood, specifically after M phase, and are rapidly recruited to particular hematopoietic organs.

  View details for Web of Science ID 000168516100011

  View details for PubMedID 11290588

• Toward regenerative medicine IMMUNITY Lagasse, E., Shizuru, J. A., Uchida, N., Tsukamoto, A., Weissman, I. L. 2001; 14 (4): 425-436

  View details for Web of Science ID 000168246700010

  View details for PubMedID 11336688

• Can stem cells cross lineage boundaries? NATURE MEDICINE ANDERSON, D. J., Gage, F. H., Weissman, I. L. 2001; 7 (4): 393-395

  View details for Web of Science ID 000167960500017

  View details for PubMedID 11283651

• L-selectin can facilitate metastasis to lymph nodes in a transgenic mouse model of carcinogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Qian, F., Hanahan, D., Weissman, I. L. 2001; 98 (7): 3976-3981

  Abstract

  L-selectin mediates homing of lymphocytes to lymph nodes (LN). Transgenic mice that express rat insulin promoter regulated simian virus 40 Tag (RIP-Tag) develop large, local cancers that metastasize to liver but not LN. To test whether this lack of LN metastases reflects their absence from the circulation, transgenic mice were produced that express Tag (T), L-selectin (L), and Escherichia coli LacZ (Z), in pancreatic beta cells. LTZ mice developed insulinomas that specifically had LN metastases; metastasis was blocked by an anti L-selectin mAb. LacZ(+) tumor cells from these LN homed to secondary LN upon transfer. These results suggest that the highly vascularized islet carcinomas are shedding tumor cells into the bloodstream, which is a necessary but insufficient condition for metastasis to occur; L-selectin can facilitate homing of such tumor cells to LN, resulting in metastasis.

  View details for Web of Science ID 000167833700067

  View details for PubMedID 11274419

  View details for PubMedCentralID PMC31164

• BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor alpha chimeric protein (PMLRAR alpha) to block neutrophil differentiation and initiate acute leukemia JOURNAL OF EXPERIMENTAL MEDICINE Kogan, S. C., Brown, D. E., Shultz, D. B., Truong, B. T., Lallemand-Breitenbach, V., Guillemin, M. C., Lagasse, E., Weissman, I. L., BISHOP, J. M. 2001; 193 (4): 531-543

  Abstract

  The promyelocytic leukemia retinoic acid receptor alpha (PMLRARalpha) chimeric protein is associated with acute promyelocytic leukemia (APL). PMLRARalpha transgenic mice develop leukemia only after several months, suggesting that PMLRARalpha does not by itself confer a fully malignant phenotype. Suppression of apoptosis can have a central role in tumorigenesis; therefore, we assessed whether BCL-2 influenced the ability of PMLRARalpha to initiate leukemia. Evaluation of preleukemic animals showed that whereas PMLRARalpha alone modestly altered neutrophil maturation, the combination of PMLRARalpha and BCL-2 caused a marked accumulation of immature myeloid cells in bone marrow. Leukemias developed more rapidly in mice coexpressing PMLRARalpha and BCL-2 than in mice expressing PMLRARalpha alone, and all mice expressing both transgenes succumbed to leukemia by 7 mo. Although both preleukemic, doubly transgenic mice and leukemic animals had abundant promyelocytes in the bone marrow, only leukemic mice exhibited thrombocytopenia and dissemination of immature cells. Recurrent gain of chromosomes 7, 8, 10, and 15 and recurrent loss of chromosome 2 were identified in the leukemias. These chromosomal changes may be responsible for the suppression of normal hematopoiesis and dissemination characteristic of the acute leukemias. Our results indicate that genetic changes that inhibit apoptosis can cooperate with PMLRARalpha to initiate APL.

  View details for Web of Science ID 000167114900013

  View details for PubMedID 11181704

  View details for PubMedCentralID PMC2195904

• A genetic analysis of neural progenitor differentiation NEURON Geschwind, D. H., Ou, J., Easterday, M. C., Dougherty, J. D., JACKSON, R. L., Chen, Z. G., Antoine, H., Terskikh, A., Weissman, I. L., Nelson, S. F., Kornblum, H. I. 2001; 29 (2): 325-339

  Abstract

  Genetic mechanisms regulating CNS progenitor function and differentiation are not well understood. We have used microarrays derived from a representational difference analysis (RDA) subtraction in a heterogeneous stem cell culture system to systematically study the gene expression patterns of CNS progenitors. This analysis identified both known and novel genes enriched in progenitor cultures. In situ hybridization in a subset of clones demonstrated that many of these genes were expressed preferentially in germinal zones, some showing distinct ventricular or subventricular zone labeling. Several genes were also enriched in hematopoietic stem cells, suggesting an overlap of gene expression in neural and hematopoietic progenitors. This combination of methods demonstrates the power of using custom microarrays derived from RDA-subtracted libraries for both gene discovery and gene expression analysis in the central nervous system.

  View details for Web of Science ID 000167141700009

  View details for PubMedID 11239426

• Molecular cloning and characterization of a novel regulator of G-protein signaling from mouse hematopoietic stem cells JOURNAL OF BIOLOGICAL CHEMISTRY Parks, I. K., Klug, C. A., Li, K. J., Jerabek, L., Li, L. H., Nanamori, M., Neubig, R. R., Hood, L., Weissman, I. L., Clarke, M. F. 2001; 276 (2): 915-923

  Abstract

  A novel regulator of G-protein signaling (RGS) has been isolated from a highly purified population of mouse long-term hematopoietic stem cells, and designated RGS18. It has 234 amino acids consisting of a central RGS box and short divergent NH(2) and COOH termini. The calculated molecular weight of RGS18 is 27,610 and the isoelectric point is 8.63. Mouse RGS18 is expressed from a single gene and shows tissue specific distribution. It is most highly expressed in bone marrow followed by fetal liver, spleen, and then lung. In bone marrow, RGS18 level is highest in long-term and short-term hematopoietic stem cells, and is decreased as they differentiate into more committed multiple progenitors. The human RGS18 ortholog has a tissue-specific expression pattern similar to that of mouse RGS18. Purified RGS18 interacts with the alpha subunit of both G(i) and G(q) subfamilies. The results of in vitro GTPase single-turnover assays using Galpha(i) indicated that RGS18 accelerates the intrinsic GTPase activity of Galpha(i). Transient overexpression of RGS18 attenuated inositol phosphates production via angiotensin receptor and transcriptional activation through cAMP-responsive element via M1 muscarinic receptor. This suggests RGS18 can act on G(q)-mediated signaling pathways in vivo.

  View details for Web of Science ID 000166430900009

  View details for PubMedID 11042171

• Stem and progenitor cells: Origins, phenotypes, lineage commitments, and transdifferentiations ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Weissman, I. L., Anderson, D. J., Gage, F. 2001; 17: 387-403

  Abstract

  Multipotent stem cells are clonal cells that self-renew as well as differentiate to regenerate adult tissues. Whereas stem cells and their fates are known by unique genetic marker studies, the fate and function of these cells are best studied by their prospective isolation. This review is about the properties of various highly purified tissue-specific multipotent stem cells and purified oligolineage progenitors. We contend that unless the stem or progenitor cells in question have been purified to near homogeneity, one cannot know whether their generation of expected (or unexpected) progeny is a property of a known cell type. It is interesting that in the hematopoietic system the only long-term self-renewing cells in the stem and progenitors pool are the hematopoietic stem cells. This fact is discussed in the context of normal and leukemic hematopoiesis.

  View details for Web of Science ID 000172448800013

  View details for PubMedID 11687494

• Stem cells and hematolymphoid development Hematopoiesis, A Developmental Approach Akashi, K., Weissman, I. 2001: 15–34
• Formation and differentiation of leukocytes Physiology of Inflammation Wright, D., Weissman, I. Oxford Press. 2001: 11–51
• Dendritic cell development from common myeloid progenitors 3rd International Conference on Hematopoietic Stem Cells: Genetics and Medicine Manz, M. G., Traver, D., Akashi, K., Merad, M., Miyamoto, T., Engleman, E. G., Weissman, I. L. NEW YORK ACAD SCIENCES. 2001: 167–174

  Abstract

  Dendritic cells (DCs) are professional antigen-presenting cells which both initiate adaptive immune responses and control tolerance to self-antigens. It has been suggested that these different effects on responder cells depend on subsets of DCs arising from either myeloid or lymphoid hematopoietic origins. In this model, CD8 alpha+ Mac-1- DCs are supposed to be of lymphoid while CD8 alpha- Mac-1+ DCs are supposed to be of myeloid origin. Here we summarize our findings that both CD8 alpha+ and CD8 alpha- DCs can arise from clonogenic common myeloid progenitors (CMPs) in both thymus and spleen. Therefore CD8 alpha expression DCs does not indicate a lymphoid origin and differences among CD8 alpha+ and CD8 alpha- DCs might rather reflect maturation status than ontogeny. On the basis of transplantation studies, it seems likely that most of the DCs in secondary lymphoid organs and a substantial fraction of thymic DCs are myeloid-derived.

  View details for Web of Science ID 000172028500019

  View details for PubMedID 11458504

• Direct isolation of human central nervous system stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Uchida, N., Buck, D. W., He, D. P., Reitsma, M. J., Masek, M., Phan, T. V., Tsukamoto, A. S., Gage, F. H., Weissman, I. L. 2000; 97 (26): 14720-14725

  Abstract

  Stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, have the potential to replace or repair damaged tissue. We have directly isolated clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue, using antibodies to cell surface markers and fluorescence-activated cell sorting. These hCNS-SC are phenotypically 5F3 (CD133)(+), 5E12(+), CD34(-), CD45(-), and CD24(-/lo). Single CD133(+) CD34(-) CD45(-) sorted cells initiated neurosphere cultures, and the progeny of clonogenic cells could differentiate into both neurons and glial cells. Single cells from neurosphere cultures initiated from CD133(+) CD34(-) CD45(-) cells were again replated as single cells and were able to reestablish neurosphere cultures, demonstrating the self-renewal potential of this highly enriched population. Upon transplantation into brains of immunodeficient neonatal mice, the sorted/expanded hCNS-SC showed potent engraftment, proliferation, migration, and neural differentiation.

  View details for Web of Science ID 000165993700130

  View details for PubMedID 11121071

• Hematopoietic stem cells need two signals to prevent apoptosis; BCL-2 can provide one of these, Kitl/c-Kit signaling the other JOURNAL OF EXPERIMENTAL MEDICINE Domen, J., Weissman, I. L. 2000; 192 (12): 1707-1718

  Abstract

  Growth factors can cause cells to proliferate, differentiate, survive, or die. Distinguishing between these responses is difficult in multicellular, multiparameter systems. Yet this is essential to understand the impact on cells like hematopoietic stem cells (HSCs), which have strict and still poorly understood growth factor requirements. Single cell plating in serum-free medium allows direct assessment of growth factor responses. The range of tested factors can be expanded if the cells are protected from growth factor deprivation-induced apoptosis. BCL-2 is overexpressed in HSCs of H2K-BCL-2 transgenic mice, protecting them from many apoptotic stimuli. The response of single wild-type and transgenic HSCs to stimulations with individual factors was tested. Surprisingly, we find that high level BCL-2 expression does not prevent rapid death under serum-free conditions, even though it does in the presence of serum. We also find that transgenic, but not wild-type cells, survive and proliferate rapidly in response to steel factor (Kit ligand). These studies show that two separate signals are necessary to prevent apoptosis in HSCs, and that Kit ligand by itself provides a strong proliferative stimulus to HSCs. However, the proliferative response does not result in self-renewal, but in differentiation to all known hematopoietic oligolineage progenitors.

  View details for Web of Science ID 000166013100004

  View details for PubMedID 11120768

• Development of CD8 alpha-positive dendritic cells from a common myeloid progenitor SCIENCE Traver, D., Akashi, K., Manz, M., Merad, M., Miyamoto, T., Engleman, E. G., Weissman, I. L. 2000; 290 (5499): 2152-2154

  Abstract

  Dendritic cells (DCs) are critical in both initiating adaptive immune responses and maintaining tolerance to self antigens. These apparently contradictory roles have been suggested to depend on different subsets of DCs that arise from either myeloid or lymphoid hematopoietic origins, respectively. Although DC expression of CD8alpha is attributed to a lymphoid origin, here we show that both CD8alpha+ and CD8alpha- DCs can arise from clonogenic common myeloid progenitors in both thymus and spleen. Thus, expression of CD8alpha is not indicative of a lymphoid origin, and phenotypic and functional differences among DC subsets are likely to reflect maturation status rather than ontogeny.

  View details for Web of Science ID 000165870600058

  View details for PubMedID 11118150

• "Fluorescent timer": Protein that changes color with time SCIENCE Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X. N., Lukyanov, S., Matz, M., Kim, S., Weissman, I., Siebert, P. 2000; 290 (5496): 1585-1588

  Abstract

  We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

  View details for Web of Science ID 000165446200053

  View details for PubMedID 11090358

• Purified hematopoietic stem cells can differentiate into hepatocytes in vivo NATURE MEDICINE Lagasse, E., Connors, H., Al-Dhalimy, M., Reitsma, M., Dohse, M., Osborne, L., Wang, X., Finegold, M., Weissman, I. L., Grompe, M. 2000; 6 (11): 1229-1234

  Abstract

  The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that intravenous injection of adult bone marrow cells in the FAH(-/-) mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.

  View details for Web of Science ID 000165114800029

  View details for PubMedID 11062533

• Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines NATURE Kondo, M., Scherer, D. C., Miyamoto, T., King, A. G., Akashi, K., Sugamura, K., Weissman, I. L. 2000; 407 (6802): 383-386

  Abstract

  The primary role of cytokines in haemato-lymphopoiesis is thought to be the regulation of cell growth and survival. But the instructive action of cytokines in haematopoiesis has not been well addressed. Here we show that a clonogenic common lymphoid progenitor, a bone marrow-resident cell that gives rise exclusively to lymphocytes (T, B and natural killer cells), can be redirected to the myeloid lineage by stimulation through exogenously expressed interleukin (IL)-2 and GM-CSF (granulocyte/macrophage colony-stimulating factor) receptors. Analysis of mutants of the beta-chain of the IL-2 receptor revealed that the granulocyte- and monocyte-differentiation signals are triggered by different cytoplasmic domains, showing that the signalling pathway(s) responsible for these unique developmental outcomes are separable. Finally, we show that the endogenous myelomonocytic cytokine receptors for GM-CSF and macrophage colony-stimulating factor (M-CSF) are expressed at low to moderate levels on the more primitive haematopoietic stem cells, are absent on common lymphoid progenitors, and are upregulated after myeloid lineage induction by IL-2. We conclude that cytokine signalling can regulate cell-fate decisions and propose that a critical step in lymphoid commitment is downregulation of cytokine receptors that drive myeloid cell development.

  View details for Web of Science ID 000089390700048

  View details for PubMedID 11014194

• Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shizuru, J. A., Weissman, I. L., Kernoff, R., Masek, M., Scheffold, Y. C. 2000; 97 (17): 9555-9560

  Abstract

  Engraftment of allogeneic bone marrow (BM) has been shown to induce tolerance to organs genotypically matched with the BM donor. Immune reconstitution after BM transplantation therefore involves re-establishment of a T cell pool tolerant to antigens present on both donor and host tissues. However, how hematopoietic grafts exert their influence over the regenerating immune system is not completely understood. Prior studies suggest that education of the newly arising T cell pool involves distinct contributions from donor and host stromal elements. Specifically, negative selection is thought to be mediated primarily by donor BM-derived antigen-presenting cells, whereas positive selection is dictated by radio-resistant host-derived thymic stromal cells. In this report we studied the effect of highly purified allogeneic hematopoietic stem cells (HSCs) on organ transplantation tolerance induction and immune reconstitution. In contrast to engraftment of BM that results in near-complete donor T cell chimerism, HSC engraftment results in mixed T cell chimerism. Nonetheless we observed that HSC grafts induce tolerance to donor-matched neonatal heart grafts, and one way the HSC grafts alter host immune responses is via deletion of newly arising donor as well as radiation-resistant host T cells. Furthermore, using an in vivo assay of graft rejection to study positive selection we made the unexpected observation that T cells in chimeric mice rejected grafts only in the context of the donor MHC type. These latter findings conflict with the conventionally held view that radio-resistant host elements primarily dictate positive selection.

  View details for Web of Science ID 000088840500041

  View details for PubMedID 10920206

• Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny BLOOD Klug, C. A., Cheshier, S., Weissman, I. L. 2000; 96 (3): 894-901

  Abstract

  Hematopoietic stem cell gene therapy holds promise for the treatment of many hematologic disorders. One major variable that has limited the overall success of gene therapy to date is the lack of sustained gene expression from viral vectors in transduced stem cell populations. To understand the basis for reduced gene expression at a single-cell level, we have used a murine retroviral vector, MFG, that expresses the green fluorescent protein (GFP) to transduce purified populations of long-term self-renewing hematopoietic stem cells (LT-HSC) isolated using the fluorescence-activated cell sorter. Limiting dilution reconstitution of lethally irradiated recipient mice with 100% transduced, GFP(+) LT-HSC showed that silencing of gene expression occurred rapidly in most integration events at the LT-HSC level, irrespective of the initial levels of GFP expression. When inactivation occurred at the LT-HSC level, there was no GFP expression in any hematopoietic lineage clonally derived from silenced LT-HSC. Inactivation downstream of LT-HSC that stably expressed GFP( )in long-term reconstituted animals was restricted primarily to lymphoid cells. These observations suggest at least 2 distinct mechanisms of silencing retrovirally expressed genes in hematopoietic cells.

  View details for Web of Science ID 000088394000017

  View details for PubMedID 10910902

• AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Miyamoto, T., Weissman, I. L., Akashi, K. 2000; 97 (13): 7521-7526

  Abstract

  Leukemia-specific AML1/ETO transcripts are detectable in most patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. To understand the inconsistency between the clinical cure and the presence of "residual disease" at a molecular level, we separated and identified the cells expressing AML1/ETO by phenotype and function. Here we demonstrate that AML1/ETO transcripts are present in a fraction of stem cells, monocytes, and B cells in remission marrow, and in a fraction of B cells in leukemic marrow, but not in T cells. AML1/ETO transcripts also were demonstrated in a fraction of colony-forming cells of erythroid, granulocyte-macrophage, and/or megakaryocyte lineages in both leukemic and remission marrow. These data strongly suggest that the acquisition of the t(8;21) occurs at the level of stem cells capable of differentiating into B cells as well as all myeloid lineages, and that a fraction of the AML1/ETO-expressing stem cells undergo additional oncogenic event(s) that ultimately leads to transformation into AML.

  View details for Web of Science ID 000087811600106

  View details for PubMedID 10861016

• 50 million years of chordate evolution: Seeking the origins of adaptive immunity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Laird, D. J., De Tomaso, A. W., COOPER, M. D., Weissman, I. L. 2000; 97 (13): 6924-6926

  View details for Web of Science ID 000087811600002

  View details for PubMedID 10860947

• A morphological study of nonrandom senescence in a colonial urochordate BIOLOGICAL BULLETIN Lauzon, R. J., Rinkevich, B., Patton, C. W., Weissman, I. L. 2000; 198 (3): 367-378

  Abstract

  Botryllus schlosseri is a clonally modular ascidian, in which individuals (zooids) have a finite life span that is intimately associated with a weekly budding process called blastogenesis. Every blastogenic cycle concludes with a synchronized phase of regression called takeover, during which all zooids in a colony die, primarily by apoptosis, and are replaced by a new generation of asexually derived zooids. We have previously documented that, in addition to this cyclical death phase, entire colonies undergo senescence during which all asexually derived individuals in a colony, buds and zooids, die in concert. In addition, when a specific parent colony (genet) is experimentally separated into a number of clonal replicates (ramets), ramets frequently undergo senescence simultaneously, indicating that mortality can manifest itself in nonrandom fashion. Here, we document a morphological portrait of senescence in laboratory-maintained colonies from Monterey Bay, California, that exhibit nonrandom mortality. Nonrandom senescence proceeded according to a series of characteristic changes within the colony over a period of about one week. These changes included systemic constriction and congestion of the vasculature accompanied by massive accumulation of pigment cells in the zooid body wall (mantle), blood vessels, and ampullae; gradual shrinkage of individual zooids; loss of colonial architecture, and ultimately death. At the ultrastructural level, individual cells exhibited changes typical of ischemic cell death, culminating in necrotic cell lysis rather than apoptosis. Collectively, these observations indicate that senescence is accompanied by unique morphological changes that occur systemically, and which are distinct from those occurring during takeover. We discuss our findings in relation to current experimental models of aging and the possible role of a humoral factor in bringing about the onset of senescence.

  View details for Web of Science ID 000087933000006

  View details for PubMedID 10897450

• B lymphopoiesis in the thymus JOURNAL OF IMMUNOLOGY Akashi, K., Richie, L. I., Miyamoto, T., Carr, W. H., Weissman, I. L. 2000; 164 (10): 5221-5226

  Abstract

  The thymus has been regarded as the major site of T cell differentiation. We find that in addition to alphabeta and gammadelta T cells, a significant number (approximately 3 x 104 per day) of B220+IgM+ mature B cells are exported from the thymus of C57BL/6 mice. Of these emigrating B cells, we estimate that at least approximately 2 x 104 per day are cells which developed intrathymically, whereas a maximum of approximately 0.8 x 104 per day are cells which circulated through the thymus from the periphery. The thymus possesses a significant number of pro-B and pre-B cells that express CD19, VpreB, lambda5, and pax-5. These B cell progenitors were found in the thymic cortex, whereas increasingly mature B cells were found in the corticomedullar and medullary regions. Other lymphoid cells, including NK cells and lymphoid dendritic cells, are not exported from the thymus at detectable levels. Thus, the thymus contributes to the formation of peripheral pools of B cells as well as of alphabeta and gammadelta T cells.

  View details for Web of Science ID 000086947900034

  View details for PubMedID 10799882

• The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage BRITISH JOURNAL OF HAEMATOLOGY Kina, T., Ikuta, K., Takayama, E., Wada, K., Majumdar, A. S., Weissman, I. L., Katsura, Y. 2000; 109 (2): 280-287

  Abstract

  The antigen specificity of a rat monoclonal antibody TER-119 was investigated. In adult mice, TER-119 reacted with mature erythrocytes, 20-25% of bone marrow cells and 2-3% of spleen cells but not with thymocytes nor lymph node cells. In fetal haematopoietic tissues, 30-40% of d 10 yolk sac cells, 80-90% of d 14 fetal liver cells and 40-50% of newborn liver cells were reactive with TER-119. TER-119+ cells in adult bone marrow expressed significant levels of CD45 but not myeloid (Mac-1, Gr-1) or B-cell (B220) markers. Morphological examination and haematopoietic colony-forming assays for isolated TER-119+ cells revealed that TER-119 reacts with erythroid cells at differentiation stages from early proerythroblast to mature erythrocyte, but not with cells showing typical erythroid blast-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) activities. Erythroleukaemia cell lines do not express the TER-119 antigen even after stimulation with dimethylsulphoxide. TER-119 immunoprecipitated protein bands with molecular masses of 110 kDa, 60 kDa, 52 kDa and 32 kDa from erythrocyte membrane, whereas only a 52-kDa band was detected by TER-119 in Western blot analysis. Further molecular and cellular analyses indicated that the TER-119 antigen is a molecule associated with cell-surface glycophorin A but not with glycophorin A itself.

  View details for Web of Science ID 000087491900005

  View details for PubMedID 10848813

• Reconstitution of T cells in vivo by committed T cell progenitors from the bone marrow Garcia-Ojeda, M. E., Dejbakhsh-Jones, S., Chatterjea-Matthes, D., Weissman, I. L., Strober, S. FEDERATION AMER SOC EXP BIOL. 2000: A921–A921

  View details for Web of Science ID 000086643100051

• Lymphoid precursors CURRENT OPINION IN IMMUNOLOGY Akashi, K., Reya, T., Dalma-Weiszhausz, D., Weissman, I. L. 2000; 12 (2): 144-150

  Abstract

  Lymphopoiesis of mature and diverse populations of T, B and NK (natural killer) cells from multipotent hematopoietic stem cells is an ideal model of tissue generation and regeneration. Identification and isolation of hematolymphoid stem and progenitor cells in several laboratories over the past several years have provided populations that can be studied biologically for lineage commitment and biochemically for receptor function, signal transduction and selective gene expression. These studies may ultimately provide candidate genes involved in lineage commitment, cell death or survival, self-renewal and migratory capacities of progenitors.

  View details for Web of Science ID 000085786300002

  View details for PubMedID 10712944

• In vivo natural killer cell activities revealed by natural killer cell-deficient mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, S., Iizuka, K., Aguila, H. L., Weissman, I. L., Yokoyama, W. M. 2000; 97 (6): 2731-2736

  Abstract

  Studies of natural killer (NK) cell function in vivo have been challenging primarily due to the lack of animal models in which NK cells are genetically and selectively deficient. Here, we describe a transgenic mouse with defective natural killing and selective deficiency in NK1.1(+) CD3(-) cells. Despite functionally normal B, T, and NK/T cells, transgenic mice displayed impaired acute in vivo rejection of tumor cells. Adoptive transfer experiments confirmed that NK1.1(+) CD3(-) cells were responsible for acute tumor rejection, establishing the relationship of NK1.1(+) CD3(-) cells to NK cells. Additional studies provided evidence that (i) NK cells play an important role in suppressing tumor metastasis and outgrowth; (ii) NK cells are major producers of IFNgamma in response to bacterial endotoxin but not to interleukin-12, and; (iii) NK cells are not essential for humoral responses to T cell-independent type 2 antigen or the generalized Shwartzman reaction, both of which were previously proposed to involve NK cells.

  View details for Web of Science ID 000085941400060

  View details for PubMedID 10694580

• Posttranscriptional regulation of Bruton's tyrosine kinase expression in antigen receptor-stimulated splenic B cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nisitani, S., Satterthwaite, A. B., Akashi, K., Weissman, I. L., Witte, O. N., Wahl, M. I. 2000; 97 (6): 2737-2742

  Abstract

  Mutation of Bruton's tyrosine kinase (Btk) causes human X-linked agammaglobulinemia and murine X-linked immunodeficiency syndrome (xid). Quantitative aspects of B lymphocyte development and function have been demonstrated to depend on Btk level in vivo by using a murine transgenic model system. A sensitive intracellular immunofluorescent assay was developed to measure Btk protein on a per cell basis to test the hypothesis that its dosage is dynamically regulated during B cell development or functional responses. Marrow-derived hematopoietic stem cells, common lymphoid progenitor cells, and developing B and myeloid lineages expressed Btk protein at comparable levels. Resting peripheral B lineage cells had a significantly lower amount of Btk than marrow-derived cells in both wild-type and xid mice. Activation of the B cell antigen receptor up-regulated Btk protein level 10-fold within several hours by a phosphatidylinositol 3-kinase-dependent, posttranscriptional mechanism. In contrast, the protein level of Btk R28C in activated B lymphocytes from xid mice remained low. Bypass of the antigen receptor signaling pathways by treatment of cells with phorbol myristic acid and ionomycin rescued up-regulation of Btk protein in xid splenic B cells. These combined results suggest that certain receptor signals mediated by Btk regulate the level of expression of Btk protein in responding B lymphocytes to potentiate signal transduction. Dynamic regulation of Btk protein dosage is an additional mechanism to modulate B lymphocyte immune functions.

  View details for Web of Science ID 000085941400061

  View details for PubMedID 10688914

• A clonogenic common myeloid progenitor that gives rise to all myeloid lineages NATURE Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. 2000; 404 (6774): 193-197

  Abstract

  Haematopoietic stem cells give rise to progeny that progressively lose self-renewal capacity and become restricted to one lineage. The points at which haematopoietic stem cell-derived progenitors commit to each of the various lineages remain mostly unknown. We have identified a clonogenic common lymphoid progenitor that can differentiate into T, B and natural killer cells but not myeloid cells. Here we report the prospective identification, purification and characterization, using cell-surface markers and flow cytometry, of a complementary clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Common myeloid progenitors give rise to either megakaryocyte/erythrocyte or granulocyte/macrophage progenitors. Purified progenitors were used to provide a first-pass expression profile of various haematopoiesis-related genes. We propose that the common lymphoid progenitor and common myeloid progenitor populations reflect the earliest branch points between the lymphoid and myeloid lineages, and that the commitment of common myeloid progenitors to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events.

  View details for Web of Science ID 000085870900054

  View details for PubMedID 10724173

• Translating stem and progenitor cell biology to the clinic: Barriers and opportunities SCIENCE Weissman, I. L. 2000; 287 (5457): 1442-1446

  Abstract

  Stem cells are the natural units of embryonic generation, and also adult regeneration, of a variety of tissues. Recently, the list of tissues that use the model of differentiation from stem to progenitor to mature cell has increased from blood to include a variety of tissues, including both central and peripheral nervous systems and skeletal muscle; it is also possible that all organs and tissues are derived from, and still contain, stem cells. Because the number and activities of stem cells and their progeny are homeostatically regulated, clinical stem cell transplantation could greatly add to the physician's armamentarium against degenerative diseases.

  View details for Web of Science ID 000085531600039

  View details for PubMedID 10688785

• The role of apoptosis in the regulation of hematopoietic stem cells: Overexpression of BCL-2 increases both their number and repopulation potential JOURNAL OF EXPERIMENTAL MEDICINE Domen, J., Cheshier, S. H., Weissman, I. L. 2000; 191 (2): 253-263

  Abstract

  Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2-transgenic mice have increased numbers of HSC in bone marrow (2.4x wild type), but fewer of these cells are in the S/G(2)/M phases of the cell cycle (0.6x wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.

  View details for Web of Science ID 000084908000006

  View details for PubMedID 10637270

• Stem cells: Units of development, units of regeneration, and units in evolution CELL Weissman, I. L. 2000; 100 (1): 157-168

  View details for Web of Science ID 000084722600014

  View details for PubMedID 10647940

• Function of cytokines in lymphocyte development Workshop on Lymphoid Organogenesis Kondo, M., Weissman, I. L. SPRINGER-VERLAG BERLIN. 2000: 59–65

  View details for Web of Science ID 000167097700008

  View details for PubMedID 11036759

• Hematopoietic stem cells: biological targets and therapeutic tools Clinical Bone Marrow and Blood Stem Cell Transplantation Weissman, I., Uchida, N. Cambridge Press. 2000
• Transplantation of highly purified CD34(+)Thy-I+ hematopoietic stem cells in patients with metastatic breast cancer BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Negrin, R. S., Atkinson, K., Leemhuis, T., Hanania, E., Juttner, C., Tierney, K., Hu, W. W., JOHNSTON, L. J., Shizuru, J. A., Stockerl-Goldstein, K. E., Blume, K. G., Weissman, I. L., Bower, S., Baynes, R., Dansey, R., Karanes, C., Peters, W., Klein, J. 2000; 6 (3): 262-271

  Abstract

  We report here the transplantation of extensively purified "mobilized" peripheral blood CD34Thy-1 hematopoietic stem cells from 22 patients with recurrent or metastatic breast cancer. Patients were mobilized with either high-dose granulocyte colony-stimulating factor (G-CSF) alone or cyclophosphamide plus G-CSE Median purity of the stem cell product at cryopreservation was 95.3% (range, 91.1%-98.3%), and viability was 98.6% (range, 96.5%-100%). After high-dose chemotherapy with carmustine, cisplatin, and cyclophosphamide, CD34+Thy-1 cells at a median dose of 11.3 x 10(5) per kilogram (range, 4.7-163 x 10(5) per kilogram) were infused. No infusion-related toxicity was observed. Neutrophil recovery was prompt, with median absolute neutrophil count >500/microL by day 10 (range, 8-15 days) and >1000/microL by day 11 (range, 8-17 days). Median platelet recovery (>20,000/microL) was observed by day 14 (range, 9-42 days) and >50,000/microL by day 17 (range, 11-49 days). Tumor cell depletion below the limits of detection of a sensitive immunofluorescence-based assay was accomplished in all patients who had detectable tumor cells in apheresis products before processing. Although CD4+ T-cell reconstitution was slow, no unusual infections were observed. Neither early nor late graft failure was observed, and no patient required infusion of unmanipulated backup cells. At a median follow-up of approximately 1.4 years and a maximum follow-up of 2.5 years, 16 of the 22 patients remain alive, with 9 free of disease progression, and have stable blood counts. In summary, highly purified CD34+Thy-1+ cells used as the sole source of the hematopoietic graft result in rapid and sustained hematopoietic engraftment.

  View details for Web of Science ID 000090022300006

  View details for PubMedID 10871151

• CD8(+)TCR(+) and CD8(+)TCR(-) cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers IMMUNITY Gandy, K. L., Domen, J., Aguila, H., Weissman, I. L. 1999; 11 (5): 579-590

  Abstract

  Although purified hematopoietic stem cells (HSC) are sufficient to engraft irradiated allogeneic recipients, bone marrow (BM) contains other cells that facilitate engraftment. Here, several candidate facilitators were tested by cotransplantation with HSC. Both TCR+ and TCR- CD8alpha+ BM subpopulations have facilitative potential. CD8+TCR+ cells are typical T lymphocytes. CD8+TCR- facilitators are CD3 , not CD3+, have a granular morphology, and are CD8beta- and CD11c+; they share phenotypic characteristics with CD8(alpha)alpha lymphoid dendritic cells and veto cells. We also demonstrate that lytic function is nqt necessary for facilitation and that the CD8alpha molecule is either important for facilitation or in the development of facilitators.

  View details for Web of Science ID 000083952200010

  View details for PubMedID 10591183

• Immune reconstitution NEW ENGLAND JOURNAL OF MEDICINE Weissman, I. L., Shizuru, J. A. 1999; 341 (16): 1227-1229

  View details for Web of Science ID 000083087400010

  View details for PubMedID 10519902

• Heritable germ and somatic cell lineage competitions in chimeric colonial protochordates PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Stoner, D. S., Rinkevich, B., Weissman, I. L. 1999; 96 (16): 9148-9153

  Abstract

  Theories of evolution that state natural selection acts on individuals have been modified to include multiple levels of selection. Here we demonstrate in chimeric protochordates that primitive germ cell (pgc) and somatic cell (psc) lineages have traits that also make them likely units of natural selection. Specifically, by using microsatellites to determine the genetic identity of various somatic and gametic tissues within vascularly fused Botryllus schlosseri chimeras, we show that genetically distinct pgc and psc can compete for access to developing gonads and somatic organs, and that this competition is hierarchical, reproducible, and heritable. Given that a single, highly polymorphic locus (Fu/HC) controls whether two contacting colonies fuse or reject, our findings also support a leading hypothesis for why the highly polymorphic histocompatibility loci common to many metazoa may have arisen or been maintained: to limit supercompetitor lineages to histocompatible kin.

  View details for Web of Science ID 000081835500068

  View details for PubMedID 10430910

• Induction of germline transcription in the TCR gamma locus by Stat5: Implications for accessibility control by the IL-7 receptor IMMUNITY Ye, S. K., Maki, K., Kitamura, T., Sunaga, S., Akashi, K., Domen, J., Weissman, I. L., Honjo, T., Ikuta, K. 1999; 11 (2): 213-223

  Abstract

  IL-7 receptor (IL-7R) plays critical roles in lymphocyte development by promoting survival and proliferation and by inducing V(D)J recombination in TCR and Ig loci. Here, we demonstrate that IL-7R-activated Stat5 binds to consensus motifs in the 5' regions of Jgamma segments and induces germline transcripts. We also show that a constitutively active form of Stat5 restores V-J recombination of TCRgamma genes and partially rescues T cell development from IL-7R(-/-) T cell precursors, especially in favor of gammadelta T cells. Therefore, this study reveals a potential role of Stat5 in T cell development and also implies that IL-7R may control the accessibility of the TCRgamma locus through Stat5-induced germline transcription.

  View details for Web of Science ID 000082383400010

  View details for PubMedID 10485656

• Towards animal models of myeloid leukemia. Padua, R. A., Kogan, S., Le Pogam, C., Lagasse, E., Traver, D., Weissman, I., Chomienne, C., BISHOP, J. M. ELSEVIER SCIENCE INC. 1999: 70–70

  View details for Web of Science ID 000081030100131

• Antibodies to CD44 and integrin alpha(4), but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brocke, S., Piercy, C., Steinman, L., Weissman, I. L., VEROMAA, T. 1999; 96 (12): 6896-6901

  Abstract

  The role of various adhesion molecules in lymphocyte homing to the brain and in inflammatory autoimmune disease of the central nervous system (CNS) was examined in mice. Activated T cell lines and clones expressed CD44 and integrin alpha4, but not L-selectin, and entered the CNS independent of their antigen specificity. mAbs directed against CD44 and integrin alpha4 prevented the transfer of experimental autoimmune encephalomyelitis (EAE) by myelin basic protein-specific T cells. T cells preincubated with anti-CD44 or antiintegrin alpha4 were blocked only partially from entering the brain parenchyma. However, both antibodies efficiently prevented CNS inflammation and clinical expression of EAE when injected in vivo. This effect lasted as long as antibodies were administered. Antibodies specific for L-selectin had no effect on homing of encephalitogenic T cells to the brain or development of EAE. Antiintegrin alpha4 and anti-CD44 did not impair the activation and function of encephalitogenic T cells in vitro and did not deplete integrin alpha4- or CD44-positive cells in vivo. These data suggest that, in the absence of leukocyte recruitment, the entry of a reduced number of activated myelin basic protein-reactive T cells in the CNS is not sufficient for the development and expression of EAE. We propose that antibodies to integrin alpha4 and CD44 prevent clinical disease by partially targeting the primary influx of encephalitogenic T cells and by preventing the secondary influx of leukocytes to lesions initiated by the transferred T cells.

  View details for Web of Science ID 000080842200059

  View details for PubMedID 10359810

• Lymphoid development from hematopoietic stem cells INTERNATIONAL JOURNAL OF HEMATOLOGY Akashi, K., Traver, D., Kondo, M., Weissman, I. L. 1999; 69 (4): 217-226

  Abstract

  Mechanisms and pathways for commitment to the lymphoid lineage from hematopoietic stem cells (HSC) remain controversial. The interleukin-7 receptor (IL-7R) transduces nonredundant signals for both T- and B-cell development. Recently, we identified a clonogenic common lymphoid progenitor population in mouse bone marrow that can give rise to T, B, and natural killer (NK) cells, but lacks myeloid differentiation capacity. These cells are not self-renewing stem cells, but progenitors that have a limited life span. HSC do not express IL-7R, and the upregulation of the IL-7R occurs at the stage of common lymphoid progenitors. The IL-7R mediates nonredundant signals to reinforce the survival of developing T cells, and to promote rearrangement of immunoglobulin heavy chain genes in B-cell progenitors. Thus, common lymphoid progenitors exist in early hematopoiesis, and expression of the IL-7R is a critical step in the initiation of lymphoid development from HSC.

  View details for Web of Science ID 000085346900001

  View details for PubMedID 10407577

• Self-renewal, differentiation or death: regulation and manipulation of hematopoietic stem cell fate MOLECULAR MEDICINE TODAY Domen, J., Weissman, I. L. 1999; 5 (5): 201-208

  Abstract

  Hematopoietic stem cells (HSCs) are the rare cells from which all hematopoietic cells are derived. The absence of HSCs is not compatible with life because many essential cells, such as myeloid and erythroid cells, are short lived. The hematopoietic system is the first essential organ system that fails following cytotoxic treatments. It is the vulnerability of HSCs that prevents regeneration following treatment and thus long-term survival. Because HSCs have the capacity to regenerate a functional hematopoietic system, the manipulation of these cells in vitro holds many promises for gene-therapeutic and other applications; however, these are severely curtailed by current difficulties in maintaining and expanding HSCs in culture. This review focuses on recent approaches towards understanding how the HSC compartment is regulated in vivo and discusses how this knowledge might be applied to manipulating HSC numbers.

  View details for Web of Science ID 000081646700007

  View details for PubMedID 10322312

• In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cheshier, S. P., Morrison, S. J., Liao, X. S., Weissman, I. L. 1999; 96 (6): 3120-3125

  Abstract

  A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, approximately 5% of LT-HSC were in S/G2/M phases of the cell cycle and another 20% were in G1 phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription-PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although approximately 75% of LT-HSC are quiescent in G0 at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.

  View details for Web of Science ID 000079224500101

  View details for PubMedID 10077647

• Cyclophilin C-associated protein: A normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Trahey, M., Weissman, I. L. 1999; 96 (6): 3006-3011

  Abstract

  Mouse cyclophilin C-associated protein (CyCAP) is a member of the scavenger-receptor cysteine-rich domain superfamily and is 69% identical to the human Mac-2 binding protein. Here, we show that CyCAP is a widely expressed secreted glycoprotein that modulates the host response to endotoxin. Gene-targeted CyCAP-deficient mice are more sensitive to the lethal effects of endotoxin. In response to endotoxin, CyCAP-deficient mice overproduced interleukin 12 and interferon-gamma systemically and tumor necrosis factor alpha locally; these are proinflammatory molecules that also promote T helper 1 responses. Furthermore, macrophages stimulated in vitro with endotoxin in serum deficient in CyCAP secreted more tumor necrosis factor alpha, supporting the proposal that CyCAP specifically down-modulates endotoxin signaling.

  View details for Web of Science ID 000079224500081

  View details for PubMedID 10077627

• Allorecognition in colonial tunicates: protection against predatory cell lineages? IMMUNOLOGICAL REVIEWS Magor, B. G., De Tomaso, A., Rinkevich, B., Weissman, I. L. 1999; 167: 69-79

  Abstract

  The MHC molecules have been historically perceived as transplantation antigens, though it is now recognized that their primary, if not sole, role is in eliminating parasites and in surveillance and clearance of aberrant self. Indeed, pregnancy in mammals would represent the closest to a natural transplantation process that occurs in vertebrates. However, among the immediate ancestors to the vertebrates, natural intraspecific allorecognition processes are common. Among members of the colonial tunicate Botryllus schlosseri, two individuals that share a single allele of the highly polymorphic fusibility/histocompatibility (Fu/HC) locus are able to fuse with one another. Could this Fu/HC be related to the MHC such that the MHC really did have its origins as a transplantation antigen? Presently we review the genetics and biology of natural transplantation processes in colonial tunicates, comparing it with allorecognition as mediated through the vertebrate T-cell receptor, killer cell inhibitory receptor/Ly49, and MHC. Experimental approaches to determining if the molecules regulating allorecognition in tunicates have any ancestral relationship to the vertebrate MHC are discussed, as is a genomic approach to isolating novel mediators of allorecognition. We also explore the biological basis for allorecognition in colonial tunicates and recent work that highlights the costs of not maintaining a system for allorecognition.

  View details for Web of Science ID 000079851100006

  View details for PubMedID 10319252

• Lymphoid development from stem cells and the common lymphocyte progenitors 64th Symposia: Signaling and Gene Expression in the Immune System Akashi, K., Kondo, M., Cheshier, S., Shizuru, J., Gandy, K., Domen, J., Mebius, R., Traver, D., Weissman, I. L. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1999: 1–12

  View details for Web of Science ID 000087225400002

  View details for PubMedID 11232274

• International Benchmarking of US Immunology Research Weissman, I., et al The National Academies Press. 1999
• Isolation and characterization of hematopoietic progenitor and stem cells Hematopoietic Cell Transplantation Shizuru, J., Weissman, I. Blackwell Sciences. 1999: 63–78
• Transfer of primitive stem/progenitor bone marrow cells from LT alpha(-/-) donors to wild-type hosts: Implications for the generation of architectural events in lymphoid B cell domains JOURNAL OF IMMUNOLOGY Mebius, R. E., van Tuijl, S., Weissman, I. L., Randall, T. D. 1998; 161 (8): 3836-3843

  Abstract

  To analyze whether the phenotypic abnormalities observed in lymphotoxin-alpha(-/-) (LT alpha-/-) mice are intrinsic to the hemolymphoid system itself or dependent on stromal elements, wild-type (WT) mice were reconstituted with bone marrow (BM) cells enriched for hemopoietic stem cells from LT alpha-/- animals. WT mice reconstituted with LT alpha-/- c-kit+ Lin- Sca-1+ BM cells do not maintain follicular dendritic cell (FDC) networks and do not form primary follicles, while clear segregation of B and T cells could be observed. Furthermore, IgM+ IgD- B cells, MOMA-1 (anti-metallophilic macrophages), ERTR-9 (anti-marginal zone macrophages), and MECA-367 (anti-MAdCAM-1) were all absent from the splenic marginal zone. Surprisingly, however, the expression of MOMA-1, ERTR-9, and MAdCAM-1 was normal in the lymph nodes of mice reconstituted with LT alpha-/- cells. In addition, peanut agglutinin-positive germinal centers were observed in both the spleen and mesenteric lymph nodes, although in the absence of detectable FDC. Furthermore, in animals reconstituted with a mixture of LT alpha-/- and WT c-kit+ Lin- Sca-1+, GC contained either predominantly LT alpha-/- B cells or WT B cells. These results suggest that although the formation of primary follicles, FDC networks, and the splenic marginal zone are all dependent on hemopoietically derived LT alpha, germinal center formation and the expression of MAdCAM-1, MOMA-1, and ERTR-9 in lymph nodes are not. Our results also suggest that the disturbed B-T cell separation in LT alpha-/- mice is unrelated to defects in the marginal zone.

  View details for Web of Science ID 000076343300008

  View details for PubMedID 9780148

• Role of interleukin-7 in T-cell development from hematopoietic stem cells IMMUNOLOGICAL REVIEWS Akashi, K., Kondo, M., Weissman, I. L. 1998; 165: 13-28

  Abstract

  All lymphocytes are derived from hematopoietic stem cells (HSC). The interleukin-7 receptor (IL-7R) transduces non-redundant signals for both T and B-cell development from HSC. The upregulation of the IL-7R occurs at the stage of the clonogenic common lymphoid progenitor, a recently identified population that can give rise to all lymphoid lineages (T, B and natural killer cells) at a single cell level. The IL-7R plays a critical role in the rearrangement of immunoglobulin heavy chain genes required for B-cell development. IL-7R expression is critically regulated in developing thymocytes; thymocytes that fail the positive selection process downregulate the IL-7R, but those undergoing positive selection upregulate or maintain IL-7R expression. Recent data indicate that IL-7 signaling enhances the survival of developing thymocytes and mature T cells, presumably by its upregulating Bcl-2. Detailed analysis of the signaling cascades activated by the IL-7R may help to reveal the differential roles of IL-7 signaling in T and B-cell development.

  View details for Web of Science ID 000077188400002

  View details for PubMedID 9850848

• Transplantation of Fu/HC-incompatible zooids in Botryllus schlosseri results in chimerism BIOLOGICAL BULLETIN Rinkevich, B., Weissman, I. L., De Tomaso, A. W. 1998; 195 (2): 98-106

  Abstract

  The colonial urochordate Botryllus schlosseri undergoes a genetically defined, natural transplantation reaction that is controlled by a single Mendelian locus (called the Fu/HC). This Fu/HC-based allorecognition system is initiated when peripheral elements of the vasculature interact on the edges of two asexually expanding colonies. To better understand the spatial organization of the cellular elements responsible for Fu/HC-based allorecognition, we bypassed the normal site of interaction (the ampullae) and experimentally transplanted zooids between Fu/HC-noncompatible Botryllus schlosseri pairs. The results show that (1) instead of the expected rejections (tissue necroses) that develop after natural contacts between peripheral blood vessels, the transplanted organs are morphologically eliminated within a few days in conjunction with the normal blastogenic cycle; and (2) donor-recipient chimerism is established after complete morphological elimination of transplanted tissues. These results suggest that Fu/HC-based allorecognition responses in Botryllus schlosseri occur exclusively at the ampullae and that once cells have crossed this barrier, they are able to survive and proliferate in the new host colony.

  View details for Web of Science ID 000076917600002

  View details for PubMedID 9818360

• The PEBP2 beta MYH11 fusion created by Inv(16)(p13;q22) in myeloid leukemia impairs neutrophil maturation and contributes to granulocytic dysplasia PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kogan, S. C., Lagasse, E., Atwater, S., Bae, S. C., Weissman, I., Ito, Y., BISHOP, J. M. 1998; 95 (20): 11863-11868

  Abstract

  Chromosomal translocations involving the genes encoding the alpha and beta subunits of the Pebp2/Cbf transcription factor have been associated with human acute myeloid leukemia and the preleukemic condition, myelodysplasia. Inv(16)(p13;q22) fuses the gene encoding the beta subunit of Pebp2 to the MYH11 gene encoding a smooth muscle myosin heavy chain (Smmhc). To examine the effect of the inv(16)(p13;q22) on myelopoiesis, we used the hMRP8 promoter element to generate transgenic mice expressing the Pebp2betaSmmhc chimeric fusion protein in myeloid cells. Neutrophil maturation was impaired in PEBP2betaMYH11 transgenic mice. Although the transgenic mice had normal numbers of circulating neutrophils, their bone marrow contained increased numbers of immature neutrophilic cells, which exhibited abnormal characteristics. In addition, PEBP2betaMYH11 inhibited neutrophilic differentiation in colonies derived from hematopoietic progenitors. Coexpression of both PEBP2betaMYH11 and activated NRAS induced a more severe phenotype characterized by abnormal nuclear morphology indicative of granulocytic dysplasia. These results show that PEBP2betaMYH11 can impair neutrophil development and provide evidence that alterations of Pebp2 can contribute to the genesis of myelodysplasia.

  View details for Web of Science ID 000076222200065

  View details for PubMedID 9751756

  View details for PubMedCentralID PMC21731

• HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G(0)/G(1) human hematopoietic stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Uchida, N., Sutton, R. E., Friera, A. M., He, D. P., Reitsma, M. J., Chang, W. C., Veres, G., Scollay, R., Weissman, I. L. 1998; 95 (20): 11939-11944

  Abstract

  Recent studies have opened the possibility that quiescent, G0/G1 hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34(+) cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G0/G1. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G0/G1 HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP+ HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.

  View details for Web of Science ID 000076222200078

  View details for PubMedID 9751769

• Characterization of a polymorphic protein localized to vascular epithelium in Botryllus schlosseri: role in tunic synthesis? MOLECULAR MARINE BIOLOGY AND BIOTECHNOLOGY Fagan, M. B., Weissman, I. L. 1998; 7 (3): 204-213

  Abstract

  To develop an antibody-based screen for epitopes involved in botryllid historecognition, BALB/c mice were immunized with whole Botryllus schlosseri colonies. Resulting monoclonal antibodies were screened for alpha or beta fusibility types using enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. One monoclonal antibody (109) that recognized a polymorphic epitope was further analyzed by Western blotting. It binds a species-specific epitope localized to the atrial siphon and blood vessels. The epitope does not cosegregate with fusibility type. A complementary DNA clone encoding this antigen contains an endoplasmic reticulum retention motif. Polymorphism observed on Western blots was confirmed by Northern blot analysis. This antigen provides a new polymorphic marker that may be useful in studies of tunic formation.

  View details for Web of Science ID 000075452000006

  View details for PubMedID 9701615

• Chronic myelomonocytic leukemia: Demonstration of failed monocyte apoptosis as a pathogenic event in mouse and man Lagasse, E., Coutre, S. E., Weissman, I. L. ELSEVIER SCIENCE INC. 1998: 787–87

  View details for Web of Science ID 000075830600387

• Mice defective in two apoptosis pathways in the myeloid lineage develop acute myeloblastic leukemia IMMUNITY Traver, D., Akashi, K., Weissman, I. L., Lagasse, E. 1998; 9 (1): 47-57

  Abstract

  Fas-deficient (Fas(lpr/lpr)) mice constitutively expressing Bcl-2 in myeloid cells by the hMRP8 promoter often develop a fatal disease analogous to human acute myeloblastic leukemia (AML-M2). Hematopoietic cells from leukemic Fas(lpr/lpr)hMRP8bcl-2 animals form clonogenic blast colonies in vitro and can transfer disease to wild-type mice. In vitro ligation of Fas on Fas+/+ hMRP8bcl-2 marrow cells depletes approximately 50% of myeloid progenitor activity, demonstrating that Bcl-2 can only partially block Fas-mediated death signals in myelomonocytic progenitors. In addition, Fas(lpr/lpr) marrow contains greatly increased numbers of myeloid colony-forming cells as compared to Fas+/+ controls. Taken together, these data suggest that Fas has a novel role in the regulation of myelopoiesis and that Fas may act as a tumor suppressor to control leukemogenic transformation in myeloid progenitor cells.

  View details for Web of Science ID 000075066600005

  View details for PubMedID 9697835

• An alternate pathway for T cell development supported by the bone marrow microenvironment: Recapitulation of thymic maturation JOURNAL OF EXPERIMENTAL MEDICINE Garcia-Ojeda, M. E., Dejbakhsh-Jones, S., Weissman, I. L., Strober, S. 1998; 187 (11): 1813-1823

  Abstract

  In the principal pathway of alpha/beta T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.

  View details for Web of Science ID 000074120200009

  View details for PubMedID 9607922

• EVOLUTION OF ALLORECOGNITION IN BOTRYLLID ASCIDIANS INFERRED FROM A MOLECULAR PHYLOGENY. Evolution; international journal of organic evolution Cohen, C. S., Saito, Y., Weissman, I. L. 1998; 52 (3): 746-756

  Abstract

  Despite the functional and phyletic ubiquity of highly polymorphic genetic recognition systems, the evolution and maintenance of these remarkable loci remain an empirical and theoretical puzzle. Many clonal invertebrates use polymorphic genetic recognition systems to discriminate kin from unrelated individuals during behavioral interactions that mediate competition for space. Space competition may have been a selective force promoting the evolution of highly polymorphic recognition systems, or preexisting polymorphic loci may have been coopted for the purpose of mediating space competition. Ascidian species in the family Botryllidae have an allorecognition system in which fusion or rejection between neighboring colonies is controlled by allele-sharing at a single, highly polymorphic locus. The behavioral sequence involved in allorecognition varies in a species-specific fashion with some species requiring extensive intercolony tissue integration prior to the allorecognition response, while other species contact opposing colonies at only a few points on the outer surface before resolving space conflicts. Due to an apparent species-specific continuum of behavioral variation in the degree of intercolony tissue integration required for allorecognition, this system lends itself to a phylogenetic analysis of the evolution of an allorecognition system. We constructed a molecular phylogeny of the botryllids based on 18S rDNA sequence and mapped allorecognition behavioral variation onto the phylogeny. Our phylogeny shows the basal allorecognition condition for the group is the most internal form of the recognition reaction. More derived species show progressively more external allorecognition responses, and in some cases loss of some features of internal function. We suggest that external allorecognition appears to be a secondary function of a polymorphic discriminatory system that was already in place due to other selective pressures such as gamete, pathogen, or developmental cell lineage recognition.

  View details for DOI 10.1111/j.1558-5646.1998.tb03699.x

  View details for PubMedID 28565254

• Evolution of allorecognition in botryllid ascidians inferred from a molecular phylogeny EVOLUTION Cohen, C. S., Saito, Y., Weissman, I. L. 1998; 52 (3): 746-756

  View details for Web of Science ID 000074621000010

• Arrest of B lymphocyte terminal differentiation by CD40 signaling: Mechanism for lack of antibody-secreting cells in germinal centers IMMUNITY Randall, T. D., Heath, A. W., Santos-Argumedo, L., Howard, M. C., Weissman, I. L., Lund, F. E. 1998; 8 (6): 733-742

  Abstract

  Despite extensive research, the role of CD40 signaling in B cell terminal differentiation remains controversial. Here we show that CD40 engagement arrests B cell differentiation prior to plasma cell formation. This arrest is manifested at a molecular level as a reduction in mRNA levels of secretory immunoglobulin gene products such as mu(s) and J chain as well as the loss of the transcriptional regulator BLIMP-1. Furthermore, the inhibition of B cell differentiation by CD40 engagement could not be overcome by either mitogens or cytokines, but could be reversed by antibodies that interfere with the CD40/gp39 interaction. These data suggest that secretory immunoglobulin is not produced by B cells that are actively engaged by gp39-expressing T cells.

  View details for Web of Science ID 000074396300009

  View details for PubMedID 9655487

• Linkage analysis of HSP70 genes and historecognition locus in Botryllus schlosseri IMMUNOGENETICS Fagan, M. B., Weissman, I. L. 1998; 47 (6): 468-476

  Abstract

  The protochordate allorecognition system has long invited comparison with the vertebrate major histocompatibility complex (MHC). In the colonial species Botryllus schlosseri, a rapid fusion or rejection response resembling graft acceptance or rejection in vertebrates is controlled by a single highly polymorphic genetic region. Because linkage between heat shock protein 70 (HSP70) genes and the MHC appears to be conserved within the vertebrate lineage, linkage relationships between two HSP70 genes (HSP70.1 and HSP70.2) and the historecognition locus (FuHC) have been analyzed in B. schlosseri. Segregation patterns of restriction fragment length polymorphisms located in the 3' flanking regions of HSP70.1 and HSP70.2 were determined for progeny of defined crosses. These progeny were also analyzed for fusibility type by an in vivo cut colony assay. No close linkage was detected between any of the three loci. These results do not support the hypothesis that the allorecognition response in B. schlosseri is determined by an MHC homologue. However, it remains a possibility that orthologues of other MHC-linked genes will be linked to the B. schlosseri FuHC.

  View details for Web of Science ID 000073621900006

  View details for PubMedID 9553153

• Mapping the genome of a model protochordate. I. A low resolution genetic map encompassing the fusion/histocompatibility (Fu/HC) locus of Botryllus schlosseri GENETICS De Tomaso, A. W., Saito, Y., Ishizuka, K. J., Palmeri, K. J., Weissman, I. L. 1998; 149 (1): 277-287

  Abstract

  The colonial protochordate, Botryllus schlosseri, undergoes a genetically defined, natural transplantation reaction when the edges of two growing colonies interact. Peripheral blood vessels of each colony touch and will either fuse together to form a common vasculature between the colonies, or reject each other in an active blood-based inflammatory process in which the interacting vessels are cut off and the two colonies no longer interact. Previous studies have demonstrated that allorecognition in Botryllus is principally controlled by a single Mendelian locus named the fusion/histocompatibility (Fu/HC) locus, with multiple codominantly expressed alleles. However, identification and cloning of this locus has been difficult. We are taking a genomic approach in isolating this locus by creating a detailed genetic linkage map of the 725 Mbp Botryllus genome using DNA polymorphisms (primarily identified as AFLPs) as molecular genetic markers. DNA polymorphisms are identified in inbred laboratory strains of Fu/HC defined Botryllus, and their segregation and linkage is analyzed in a series of defined crosses. Using bulk segregant analysis, we have focused our mapping efforts on the Fu/HC region of the genome, and have generated an initial map which delineates the Fu/HC locus to a 5.5 cM region.

  View details for Web of Science ID 000073672500022

  View details for PubMedID 9584102

• Systemic overexpression of BCL-2 in the hematopoietic system protects transgenic mice from the consequences of lethal irradiation BLOOD Domen, J., Gandy, K. L., Weissman, I. L. 1998; 91 (7): 2272-2282

  Abstract

  A new transgenic mouse has been generated in which the proto-oncogene BCL-2 is ubiquitously overexpressed. H2K-BCL-2 transgenic mice overexpress BCL-2 in all cells of the hematolymphoid system and have been used to assess the role of BCL-2 in protecting cells of the hematolymphoid system from the consequences of ionizing radiation. We have expanded on previous studies that have demonstrated protection for specific (lymphoid) cell populations and show that systemic overexpression of BCL-2 can protect the hematopoietic system as a whole, including hematopoietic stem cells (HSC), thus increasing the radioresistance of the animal. The increase in radioresistance in H2K-BCL-2 transgenic mice has two components: an increase in the radioresistance of individual cells and, to a lesser extent, an increase in the size of certain critically important cell populations, such as HSC. Bone marrow transplantation experiments show that the increased radioresistance of the transgenic animals is provided by cells of the hematopoietic system. Protection against the consequences of irradiation is not limited to the increased expression levels of BCL-2 in transgenic mice; levels of endogenous BCL-2 are higher in lymphocyte populations that survive irradiation in wild-type mice. We show that ubiquitous overexpression of BCL-2 in the hematopoietic system can be used to increase the resistance of animals to lethal challenges such as irradiation.

  View details for Web of Science ID 000072671900008

  View details for PubMedID 9516125

• Helios, a T cell-restricted Ikaros family member that quantitatively associates with Ikaros at centromeric heterochromatin GENES & DEVELOPMENT Hahm, K., Cobb, B. S., McCarty, A. S., Brown, K. E., Klug, C. A., LEE, R., Akashi, K., Weissman, I. L., Fisher, A. G., Smale, S. T. 1998; 12 (6): 782-796

  Abstract

  The Ikaros gene encodes multiple protein isoforms that contribute critical functions during the development of lymphocytes and other hematopoietic cell types. The intracellular functions of Ikaros are not known, although recent studies have shown that Ikaros proteins colocalize with inactive genes and centromeric heterochromatin. In this study, Ikaros proteins were found to be components of highly stable complexes. The complexes from an immature T cell line were purified, revealing associated proteins of 70 and 30 kD. The p70 gene, named Helios, encodes two protein isoforms with zinc finger domains exhibiting considerable homology to those within Ikaros proteins. Helios and Ikaros recognize similar DNA sequences and, when overexpressed, Helios associates indiscriminately with the various Ikaros isoforms. Although Ikaros is present in most hematopoietic cells, Helios was found primarily in T cells. The relevance of the Ikaros-Helios interaction in T cells is supported by the quantitative association of Helios with a fraction of the Ikaros. Interestingly, the Ikaros-Helios complexes localize to the centromeric regions of T cell nuclei, similar to the Ikaros localization previously observed in B cells. Unlike the B cell results, however, only a fraction of the Ikaros, presumably the fraction associated with Helios, exhibited centromeric localization in T cells. These results establish immunoaffinity chromatography as a useful method for identifying Ikaros partners and suggest that Helios is a limiting regulatory subunit for Ikaros within centromeric heterochromatin.

  View details for Web of Science ID 000072770300004

  View details for PubMedID 9512513

  View details for PubMedCentralID PMC316626

• Two distinct pathways of positive selection for thymocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Akashi, K., Kondo, M., Weissman, I. L. 1998; 95 (5): 2486-2491

  Abstract

  Most mouse thymocytes undergoing positive selection are found on one of two pathways; the c-Kit+ and the c-Kit- pathways. Here, we show that c-Kit and interleukin-7 receptor (IL-7R)-mediated signals support positive selection during the transition from the subpopulation that first expresses cell surface T cell receptor (TCR)-the TCRalpha/betaloCD4(int)/CD8(int) (DPint) c-Kit+ cells to TCRalpha/betamedc-Kit+ transitional intermediate cells (the c-Kit+ pathway). Cells that fail positive selection on the c-Kit+ pathway become TCRalpha/betaloc-Kit- (DPhi) blasts that appear to undergo alternative TCRalpha rearrangements. The rare DPhic-Kit- blast cells that thus are salvaged for positive selection by expressing a self-major histocompatibility complex selectable TCRalpha/beta up-regulate IL-7R, but not c-Kit, and are the principal progenitors on the c-Kit- pathway; this c-Kit-IL-7R+ pathway is mainly CD4 lineage committed. Cell division is a feature of the TCRlo-medc-Kit+ transition, but is not essential for CD4 lineage maturation from DPhic-Kit- blasts. In this view, positive selection on the c-Kit- path results from a salvage of cells that failed positive selection on the c-Kit+ path.

  View details for Web of Science ID 000072366600098

  View details for PubMedID 9482912

• High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts JOURNAL OF CLINICAL INVESTIGATION Uchida, N., Tsukamoto, A., He, D. P., Friera, A. M., Scollay, R., Weissman, I. L. 1998; 101 (5): 961-966

  Abstract

  In humans, autologous transplants derived from bone marrow (BM) usually engraft more slowly than transplants derived from mobilized peripheral blood. Allogeneic BM transplants show a further delay in engraftment and have an apparent requirement for donor T cells to facilitate engraftment. In mice, Thy-1.1(lo)Lin-/loSca-1+ hematopoietic stem cells (HSCs) are the principal population in BM which is responsible for engraftment in syngeneic hosts at radioprotective doses, and higher doses of HSCs can radioprotect an allogeneic host in the absence of donor T cells. Using the mouse as a preclinical model, we wished to test to what extent engraftment kinetics was a function of HSC content, and whether at high doses of c-Kit+Thy-1.1(lo)Lin-/loSca-1+ (KTLS) cells rapid allogeneic engraftment could also be achieved. Here we demonstrate that engraftment kinetics varied greatly over the range of KTLS doses tested (100-10,000 cells), with the most rapid engraftment being obtained with a dose of 5,000 or more syngeneic cells. Mobilized splenic KTLS cells and the rhodamine 123(lo) subset of KTLS cells were also able to engraft rapidly. Higher doses of allogeneic cells were needed to produce equivalent engraftment kinetics. This suggests that in mice even fully allogeneic barriers can be traversed with high doses of HSCs, and that in humans it may be possible to obtain rapid engraftment in an allogeneic context with clinically achievable doses of purified HSCs.

  View details for Web of Science ID 000072445800007

  View details for PubMedID 9486965

• Tolerance of allogeneic heart grafts in mice simultaneously reconstituted with purified allogeneic hematopoietic stem cells TRANSPLANTATION Gandy, K. L., Weissman, I. L. 1998; 65 (3): 295-304

  Abstract

  Animals reconstituted with allogeneic whole bone marrow (WBM) are often tolerant of donor-specific solid organ grafts. Clinical application of bone marrow transplantation in solid organ transplantation has been limited, however, principally by graft-versus-host disease. We previously demonstrated that hematopoietic stem cells (HSCs) reconstitute lethally irradiated allogeneic mice without producing graft-versus-host disease. The purpose of this study was to determine whether tolerance to solid organ grafts could be induced in mice reconstituted with HSCs.BALB/c mice were lethally irradiated and reconstituted with allogeneic C57BL/Ka, Thy-1.1 WBM or HSCs. An isolated group was given a limited number of HSCs (250 cells) and a subpopulation of allogeneic cells known to facilitate HSC engraftment (facilitators). C57BL/Ka, Thy-1.1 neonatal heart grafts were placed in reconstituted animals either at the time of hematopoietic transplant or 35 days later. Third-party C3H grafts were placed over 2 months after hematopoietic reconstitution. Tolerance was defined as the persistence of cardiac contraction for the duration of evaluation (125-270 days).All surviving mice that were reconstituted with C57BL/Ka, Thy-1.1 HSCs, WBM, or HSCs and facilitators were tolerant of C57BL/Ka grafts long-term. Third-party C3H grafts placed in reconstituted animals were rejected by day 12, whereas those placed in unmanipulated mice were rejected by day 9.These data indicate that tolerance to concurrently or subsequently placed solid organ grafts can be reliably achieved with limited numbers of purified HSCs in a model where immunocompetence to third-party major histocompatibility complex antigens is delayed but intact.

  View details for Web of Science ID 000072089100001

  View details for PubMedID 9484743

• Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Klug, C. A., Morrison, S. J., Masek, M., Hahm, K., Smale, S. T., Weissman, I. L. 1998; 95 (2): 657-662

  Abstract

  The generation of lymphoid cells in mice depends on the function of the Ikaros protein. Ikaros has been characterized as a lymphoid-restricted, zinc-finger transcription factor that is derived from an alternatively spliced message. Ikaros knockout mice have defects in multiple cell lineages, raising the question of whether the protein regulates multiple committed progenitors and/or multipotent stem cells. To address this issue, we examined Ikaros expression in purified populations of multipotent cells and more committed progenitors. We found that the DNA-binding isoforms of Ikaros were localized in the nucleus of the most primitive hematopoietic stem cell subset. Changes in the RNA splicing pattern of Ikaros occurred at two stages: (i) as long-term self-renewing stem cells differentiated into short-term self-renewing stem cells and (ii) as non-self-renewing multipotent progenitors differentiated into lymphoid-committed progenitors. Unexpectedly, we found Ikaros localized to heterochromatin in Abelson-transformed pre-B lymphocytes by using immunogold electron microscopy. These observations suggest a complex role for Ikaros in lymphoid development.

  View details for Web of Science ID 000071606000041

  View details for PubMedID 9435248

• Characterization of a population of cells in the bone marrow that phenotypically mimics hematopoietic stem cells: Resting stem cells or mystery population? STEM CELLS Randall, T. D., Weissman, I. L. 1998; 16 (1): 38-48

  Abstract

  We have identified a population of cells in murine bone marrow that has many of the phenotypic characteristics attributed to resting hematopoietic stem cells but does not reconstitute irradiated mice. These cells express high levels of Sca-1, H-2K and CD38 and low levels of Thy-1.1, but do not express CD34 nor any of the lineage markers including CD3, CD4, CD5, CD8 NK1.1, I-A, B220, Ig(MGA), CD40, kappa, Mac-1, Gr-1 or Ter119. In addition, this population can be found at normal frequency in nu/nu as well as rag-1-/- mice. These cells incorporate only low levels of Rh123, are resistant to the cytotoxic effects of 5-fluorouracil and, consistent with their resting phenotype, less than 2% of these cells are in the S/G2/M phases of the cell cycle. The only phenotypic characteristic that distinguishes these cells from the lineage- Sca-1+, Thy-1.1low long-term reconstituting hematopoietic stem cell population is their lack of c-kit expression. Here we have explored the possibility that these cells represent a truly resting population of hematopoietic stem cells. We found that the lineage-, Sca-1+, c-kit- cells do not respond to hematopoietic growth factors in vitro, either alone or in combination with stromal layers. Furthermore, these cells do not form in vivo spleen colonies nor do they have the ability to reconstitute irradiated mice. Thus, this population may represent either a population of resting stem cells for which we lack the appropriate activating stimulus, or simply represent a "mystery population" that phenotypically mimics most of the physical properties of resting stem cells. Given the close phenotypic similarity of the c-kit- mystery population cells to the c-kit+ long-term reconstituting stem cells, investigators must be rigorous to exclude their effects from other stem cell assays.

  View details for Web of Science ID 000071663000006

  View details for PubMedID 9474746

• Evolution of allorecognition in botryllid ascidians inferred from a molecular phylogeny Evolution Cohen, C., Saito, Y., Weissman, I. 1998; 52: 746-756
• MAdCAM-1 dependent colonization of developing lymph nodes involves a unique subset of CD4+CD3- hematolymphoid cells CELL ADHESION AND COMMUNICATION Mebius, R. E., Schadee-Eestermans, I. L., Weissman, I. L. 1998; 6 (2-3): 97-103

  Abstract

  During fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules briefly express the Peyer's patch addressin MAdCAM-1. This allows initial seeding by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta 7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. It was found that the CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1. They can differentiate into natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. In addition to LN, CD4+CD3- cells can also be found in fetal spleen starting at 13.5 dpc, while absent from fetal liver. In view of the necessity of lymphotoxin in lymphoid organ development, it is thought that the novel subset of CD4+CD3- LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.

  View details for Web of Science ID 000076971300003

  View details for PubMedID 9823459

• T–cell development from hematopoietic stem cells Molecular Biology of B–Cell and T–Cell Development Akashi, K., Kondo, M., Schlageter, A., Weissman, I. Humana Press. 1998: 305–336
• Hydroxyurea can be used to increase mouse c-kit(+)Thy-1.1(lo)Lin(-/lo)Sca-1(+) hematopoietic cell number and frequency in cell cycle in vivo BLOOD Uchida, N., Friera, A. M., He, D. P., Reitsma, M. J., Tsukamoto, A. S., Weissman, I. L. 1997; 90 (11): 4354-4362

  Abstract

  The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1(lo)Lin-/loSca-1(+) (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

  View details for Web of Science ID A1997YH76200013

  View details for PubMedID 9373246

• Identification of clonogenic common lymphoid progenitors in mouse bone marrow CELL Kondo, M., Weissman, I. L., Akashi, K. 1997; 91 (5): 661-672

  Abstract

  The existence of a common lymphoid progenitor that can only give rise to T cells, B cells, and natural killer (NK) cells remains controversial and constitutes an important gap in the hematopoietic lineage maps. Here, we report that the Lin(-)IL-7R(+)Thy-1(-)Sca-1loc-Kit(lo) population from adult mouse bone marrow possessed a rapid lymphoid-restricted (T, B, and NK) reconstitution capacity in vivo but completely lacked myeloid differentiation potential either in vivo or in vitro. A single Lin(-)IL-7R(+)Thy-1(-)Sca-1loc-Kit(lo) cell could generate at least both T and B cells. These data provide direct evidence for the existence of common lymphoid progenitors in sites of early hematopoiesis.

  View details for Web of Science ID A1997YH96000013

  View details for PubMedID 9393859

• Tolerance induction to solid organs and the immune response after transplantation of purified allogeneic hematopoietic stem cells. Shizuru, J. A., Kernoff, R., Masek, M., Weissman, I. L. AMER SOC HEMATOLOGY. 1997: 2506–

  View details for Web of Science ID A1997YG42402502

• Developing lymph nodes collect CD4(+)CD3(-) LT beta(+) cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells IMMUNITY Mebius, R. E., Rennert, P., Weissman, I. L. 1997; 7 (4): 493-504

  Abstract

  For a brief period during fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules surprisingly express the Peyer's patch addressin MAdCAM-1. This expression allows initial seeding of this incipient structure by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. We show here that CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1 and that can become natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. Since the necessity of lymphotoxin in lymphoid organ development has been shown, we propose that the novel subset of CD4+CD3-LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.

  View details for Web of Science ID A1997YD59300006

  View details for PubMedID 9354470

• Highly polymorphic microsatellite loci in the colonial ascidian Botryllus schlosseri MOLECULAR MARINE BIOLOGY AND BIOTECHNOLOGY Stoner, D. S., Quattro, O. M., Weissman, I. L. 1997; 6 (3): 163-171

  Abstract

  Five mircosatellite loci are characterized for the colonial ascidian Botryllus schlosseri. Within one population from Monterey, California, these loci have 3 to 17 alleles, observed heterozygosities from 0.40 to 0.63, expected heterozygosities from 0.52 to 0.84, and an overall paternity exclusion rate (QT) of 0.78. Three of the five loci demonstrated Mendelian patterns of inheritance in laboratory crosses. The size distribution of alleles suggests that most allelic diversity within these loci is generated by single-step and less frequently multistep mutations. However, several alleles may also have been generated by single based insertions or deletions. Mutation rates for the five microsatellite loci are less than 1 x 10(-2) per generation. Because or their highly polymorphic nature, these loci should prove useful for exploring issues of identity, kinship, population structure, and phylogenetics.

  View details for Web of Science ID A1997XT50600002

  View details for PubMedID 9284556

• Purified hematopoietic stem cell grafts induce tolerance to donor matched solid organs. Shizuru, J. A., Kernoff, R., Masek, M. A., Weissman, I. L. ELSEVIER SCIENCE INC. 1997: 642–42

  View details for Web of Science ID A1997XR33000628

• Hematopoietic stem cells: Biology and transplantation. Weissman, I. L., Morrison, S., Cheshier, S., Uchida, N. ELSEVIER SCIENCE INC. 1997: 2–2

  View details for Web of Science ID A1997XR33000002

• Bcl-2 rescues T lymphopoiesis, but not B or NK cell development, in common gamma chain-deficient mice IMMUNITY Kondo, M., Akashi, K., Domen, J., Sugamura, K., Weissman, I. L. 1997; 7 (1): 155-162

  Abstract

  The common cytokine receptor gamma chain (gamma(c)) is an indispensable subunit for the formation of lymphoid-related cytokine receptors, including IL-7 and IL-15 receptors, that mediate nonredundant or critical signals for the differentiation of T and B cells and natural killer (NK) cells, respectively. We introduced the bcl-2 transgene driven by E mu or H-2K promoters into gamma(c)-deficient mice that lack all three lymphoid subclasses. The forced expression of Bcl-2 restored all stages of T lymphopoiesis, but not B or NK cell development, indicating that a primary function of gamma(c)-mediated signals in the T lineage might be to maintain cell survival. Therefore, the development of T, B, and NK cells may be influenced by distinct intracytoplasmic signaling cascades that are activated by coupling of gamma(c)-related receptors.

  View details for Web of Science ID A1997XN69300014

  View details for PubMedID 9252128

• Enforced expression of Bcl-2 in monocytes rescues macrophages and partially reverses osteopetrosis in op/op mice CELL Lagasse, E., Weissman, I. L. 1997; 89 (7): 1021-1031

  Abstract

  Osteopetrotic (op/op) mice lack functional M-CSF and have depressed levels of macrophages and osteoclasts. We prepared transgenic mice (hMRP8bcl-2) that express human Bcl-2 in monocytes. In vitro hMRP8bcl-2 monocytes do not undergo apoptosis in the absence of serum and M-CSF, while op/op and wild-type monocytes die. These Bcl-2-expressing monocytes spontaneously undergo macrophage differentiation. In vivo, the op/op hMRP8bcl-2 mice show significant replenishment of tissue macrophages. Their long bone osteopetrosis is largely reversed, and extensive medullary hematopoiesis appears in the bone marrow. We propose that M-CSF augments monocyte survival, permitting them to respond to internal and external cues for their differentiation.

  View details for Web of Science ID A1997XG83000007

  View details for PubMedID 9215625

• Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice CELL Akashi, K., Kondo, M., VONFREEDENJEFFRY, U., Murray, R., Weissman, I. L. 1997; 89 (7): 1033-1041

  Abstract

  Mice lacking functional IL-7 or IL-7R alpha genes are severely deficient in developing thymocytes, T cells, and B cells. IL-7 and IL-7 receptor functions are believed to result in lymphoid cell proliferation and cell maturation, implying signal transduction pathways directly involved in mitogenesis and elaboration of developmentally specific new gene programs. Here, we show that enforced expression of the bcl-2 gene in T-lymphoid cells (by crossing in the Emu-bcl-2 transgene) in IL-7R alpha-deficient mice results in a significant restoration of thymic positive selection and T cell numbers and function. We propose cell survival signals to be the principal function of IL-7R engagement in thymic and T cell development.

  View details for Web of Science ID A1997XG83000008

  View details for PubMedID 9215626

• From stem cells to lymphocytes: Biology and transplantation IMMUNOLOGICAL REVIEWS Aguila, H. L., Akashi, K., Domen, J., Gandy, K. L., Lagasse, E., Mebius, R. E., Morrison, S. J., Shizuru, J., Strober, S., Uchida, N., Wright, D. E., Weissman, I. L. 1997; 157: 13-40

  Abstract

  We review the development of the hematopoietic system, focusing on the transition from hematopoietic stem cells (HSCs) to T cells. This includes the isolation of HSCs, and recent progress in understanding their ontogeny, homing properties, and differentiation. HSC transplantation is reviewed, including the kinetics of reconstitution, engraftment across histocompatibility barriers, the facilitation of allogeneic engraftment, and the mechanisms of graft rejection. We describe progress in understanding T-cell development in the bone marrow and thymus as well as the establishment of lymph nodes. Finally, the role of bcl-2 in regulating homeostasis in the hematopoietic system is discussed.

  View details for Web of Science ID A1997XL05000002

  View details for PubMedID 9255619

• Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment BLOOD Randall, T. D., Weissman, I. L. 1997; 89 (10): 3596-3606

  Abstract

  A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU-enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU-treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

  View details for Web of Science ID A1997XD97600012

  View details for PubMedID 9160664

• Identification of a lineage of multipotent hematopoietic progenitors DEVELOPMENT Morrison, S. J., WANDYCZ, A. M., Hemmati, H. D., Wright, D. E., Weissman, I. L. 1997; 124 (10): 1929-1939

  Abstract

  All multipotent hematopoietic progenitors in C57BL-Thy-1.1 bone marrow are divided among three subpopulations of Thy-1.1(lo) Sca-1+ Lin(-/lo) c-kit+ cells: long-term reconstituting Mac-1- CD4- c-kit+ cells and transiently reconstituting Mac-1(lo) CD4- or Mac-1(lo) CD4(lo) cells. This study shows that the same populations, with similar functional activities, exist in mice whose hematopoietic systems were reconstituted by hematopoietic stem cells after lethal irradiation. We demonstrate that these populations form a lineage of multipotent progenitors from long-term self-renewing stem cells to the most mature multipotent progenitor population. In reconstituted mice, Mac-1- CD4- c-kit+ cells gave rise to Mac-1(lo) CD4- cells, which gave rise to Mac-1(lo) CD4(lo) cells. Mac-1- CD4- c-kit+ cells had long-term self-renewal potential, with each cell being capable of giving rise to more than 10(4) functionally similar Mac-1- CD4- c-kit+ cells. At least half of Mac-1(lo) CD4- cells had transient self-renewal potential, detected in the spleen 7 days after reconstitution. Mac-1(lo) CD4(lo) cells did not have detectable self-renewal potential. The identification of a lineage of multipotent progenitors provides an important tool for identifying genes that regulate self-renewal and lineage commitment.

  View details for Web of Science ID A1997XD82500009

  View details for PubMedID 9169840

• Hematopoietic stem cells: Challenges to expectations CURRENT OPINION IN IMMUNOLOGY Morrison, S. J., Wright, D. E., Cheshier, S. H., Weissman, I. L. 1997; 9 (2): 216-221

  Abstract

  The past year provided a number of challenges to our expectations regarding hematopoietic stem cell (HSC) biology. Evidence has emerged that HSCs arise intraembryonically before they can be detected in the yolk sac. A number of genes that may regulate the formation, self-renewal, or differentiation of HSC have been identified. New markers for purifying HSCs have also been described. Although different groups have attributed different properties to HSCs, it now appears that the differences may be explained by variations in assay conditions rather than by differences in the HSCs themselves. Finally, insights have emerged into the complexity of the regulation of HSC proliferation and adhesion properties.

  View details for Web of Science ID A1997WV04000008

  View details for PubMedID 9099790

• A PMLRAR alpha transgene initiates murine acute promyelocytic leukemia PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brown, D., Kogan, S., Lagasse, E., Weissman, I., Alcalay, M., Pelicci, P. G., Atwater, S., BISHOP, J. M. 1997; 94 (6): 2551-2556

  Abstract

  The malignant cells of acute promyelocytic leukemia (APL) contain a reciprocal chromosomal translocation that fuses the promyelocytic leukemia gene (PML) with the retinoic acid receptor alpha gene (RAR alpha). To test the hypothesis that the chimera PMLRAR alpha plays a role in leukemogenesis, we expressed a PMLRAR alpha cDNA in myeloid cells of transgenic mice. PMLRAR alpha transgenic mice exhibited impaired neutrophil maturation early in life, which progressed at a low frequency over the course of several months to overt APL. Both the preleukemic state and the leukemia could be transplanted to nontransgenic mice, and the transplanted preleukemia could progress to APL. The APL recapitulated features of the human disease, including a response to retinoic acid. Retinoic acid caused the leukemic cells to differentiate in vitro and in vivo, eliciting remissions of both the preleukemic state and APL in mice. Our results demonstrate that PMLRAR alpha impairs neutrophil differentiation and initiates the development of APL. The transgenic mice described here provide an apparently accurate model for human APL that includes clear evidence of tumor progression. The model should be useful for exploring the molecular pathogenesis of APL and the mechanisms of the therapeutic response to retinoic acid, as well as for preclinical studies of therapeutic regimens.

  View details for Web of Science ID A1997WP33400084

  View details for PubMedID 9122233

  View details for PubMedCentralID PMC20126

• Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Morrison, S. J., Wright, D. E., Weissman, I. L. 1997; 94 (5): 1908-1913

  Abstract

  We isolated hematopoietic stem cells (HSC) from mice treated with cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). All mobilized multipotent progenitor activity was contained in two populations: Thy-1(lo) Sca-1+ Lin- Mac-1- CD4- c-kit+ long-term reconstituting progenitors and Thy-1(lo) Sca-1+ Lin- Mac-1(lo) CD4- transiently reconstituting progenitors. CY/G-CSF treatment drove both long-term and transient multipotent progenitors into cycle, leading to a more than 12-fold expansion in the number of long-term self-renewing HSC prior to mobilization. After CY and 2 days of G-CSF treatment the number of bone marrow HSC began to decline and the number of blood and splenic HSC increased. HSC continued to proliferate in the bone marrow and spleen through 8 days of G-CSF treatment, but HSC released into the blood tended to be in G0/G1 phase. Mobilized multipotent progenitors isolated from the spleen were less efficient than normal bone marrow multipotent progenitors in engrafting irradiated mice but did not differ in colony forming unit-spleen (CFU-S) activity or single cell in vitro assays of primitive progenitor activity. The data suggest that mobilized HSC isolated from the spleen are less efficient at homing to and engrafting the bone marrow of irradiated recipient mice.

  View details for Web of Science ID A1997WM05900058

  View details for PubMedID 9050878

• HSP70 genes and historecognition in Botryllus schlosseri: Implications for MHC evolution 5th International Workshop on MHC Evolution Fagan, M. B., Weissman, I. L. WILEY-BLACKWELL PUBLISHING, INC. 1997: 25–35

  Abstract

  The colonial protochordate Botryllus schlosseri possesses a historecognition system which has long invited comparison to the vertebrate MHC. Upon contact, colonies either fuse or reject one another in a manner resembling graft acceptance or rejection in vertebrates. This response is controlled by a single highly polymorphic genetic region, the FuHC locus. Colonial protochordates such as B. schlosseri are among the closest relatives of the vertebrate lineage, and therefore may possess a recognizable MHC homologue. Since linkage between heat shock protein 70 (HSP70) genes and MHC appears to be conserved within the vertebrate lineage, we have analyzed HSP70 genes from B. schlosseri as a first step toward isolating the historecognition locus. Two HSP70 genes (HSP70.1 and HSP70.2) have been cloned and sequenced, and exhibit 93.6% sequence identity within the predicted coding regions. The B. schlosseri genes share a number of characteristics with vertebrate MHC-linked HSP70 genes: Northern blotting and sequence analysis suggest that the protochordate genes are cytoplasmically-expressed heat-inducible members of the HSP70 gene family (FAGAN and WEISSMAN 1996). However, unlike vertebrate MHC-linked HSP70 genes, HSP70.1 and HSP70.2 are not closely linked (FAGAN and WEISSMAN 1997). Furthermore, neither is closely linked to the locus determining historecognition (FAGAN and WEISSMAN 1997). These results do not support the hypothesis that the B. schlosseri FuHC locus is an MHC homolog. A discussion of the implications of these results for evolution of the vertebrate MHC is included.

  View details for Web of Science ID 000071313300006

  View details for PubMedID 9420467

• Stem cells: the lessons from hematopoises Isolation, Characterization and Utilization of CNS Stem Cells Weissman, I. Springer-Verlag. 1997: 1–8
• Somatic and germ cell parasitism in a colonial ascidian: Possible role for a highly polymorphic allorecognition system PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Stoner, D. S., Weissman, I. L. 1996; 93 (26): 15254-15259

  Abstract

  A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell "parasitism" (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337-5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss's hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.

  View details for Web of Science ID A1996WC20400049

  View details for PubMedID 8986797

• More or less hematopoietic stem cells - Reply NATURE MEDICINE Morrison, S. J., Weissman, I. L. 1996; 2 (12): 1282-1283

  View details for Web of Science ID A1996VW51500007

• Flow cytometric identification of murine neutrophils and monocytes JOURNAL OF IMMUNOLOGICAL METHODS Lagasse, E., Weissman, I. L. 1996; 197 (1-2): 139-150

  Abstract

  Flow cytometry and cell sorting have been employed in order to identify and purify murine neutrophils and monocytes. Using a combination of antibodies, we were able to distinguish between these two subsets of myeloid cells. The method described in this paper is simple to perform and can be applied to characterize myeloid cells from blood, spleen, bone marrow and after an induced inflammation.

  View details for Web of Science ID A1996VM82600013

  View details for PubMedID 8890901

• A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+CD3- cells to colonize lymph nodes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mebius, R. E., STREETER, P. R., Michie, S., BUTCHER, E. C., Weissman, I. L. 1996; 93 (20): 11019-11024

  Abstract

  IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

  View details for Web of Science ID A1996VL33300091

  View details for PubMedID 8855301

• Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and thy-1 and is highly enriched for Abelson leukemia virus targets (vol 9, pg 2665, 1989) MOLECULAR AND CELLULAR BIOLOGY Tidmarsh, G. F., Heimfeld, S., WHITLOCK, C. A., Weissman, I. L., MULLERSIEBURG, C. E. 1996; 16 (10): 5946-5946

  View details for Web of Science ID A1996VH85200075

  View details for PubMedID 8927055

• The aging of hematopoietic stem cells NATURE MEDICINE Morrison, S. J., WANDYCZ, A. M., Akashi, K., Globerson, A., Weissman, I. L. 1996; 2 (9): 1011-1016

  Abstract

  We have purified hematopoietic stem cells (HSCs) from the bone marrow of old mice and compared their properties to HSCs in young and middle-aged mice. Single, reconstituting HSCs (by limit dilution) from old and young mice exhibited indistinguishable progenitor activities in vivo. HSCs were five times as frequent in the bone marrow of old mice; however, HSCs from old mice were only one-quarter as efficient at homing to and engrafting the bone marrow of irradiated recipients. HSCs in young and middle-aged mice rarely were in the S/G2/M phases of the cell cycle, but HSCs in old mice were frequently in cycle. We speculate that the unexpected proliferation of HSCs in old mice might be related to the increased incidence of leukemia in old mice. HSCs change with age, but it is unknown whether these changes are determined intrinsically or caused by the aging of their environment.

  View details for Web of Science ID A1996VF49700036

  View details for PubMedID 8782459

• Telomerase activity in hematopoietic cells is associated with self-renewal potential IMMUNITY Morrison, S. J., Prowse, K. R., Ho, P., Weissman, I. L. 1996; 5 (3): 207-216

  Abstract

  It has been proposed that the biological clock underlying the limited division potential of eukaryotic cells is telomere length. We assayed telomerase activity in single cells of the hematopoietic and immune systems. We examined hematopoietic stem cells at four stages of differentiation, lineage-committed progenitors, and mature myeloid and lymphoid cells. The frequency of telomerase-expressing cells within each population was proportional to the frequency of cells thought to have self-renewal potential. Among bone marrow hematopoietic stem cells, 70% exhibited detectable telomerase activity. The telomerase-expressing somatic cells observed in this study are not thought to be immortal, and expression was not correlated with cell cycle distribution or differentiation state. This study demonstrates that the developmental characteristic most consistently associated with telomerase expression is self-renewal potential.

  View details for Web of Science ID A1996VJ79700003

  View details for PubMedID 8808676

• The c-kit(+) maturation pathway in mouse thymic T cell development: Lineages and selection IMMUNITY Akashi, K., Weissman, I. L. 1996; 5 (2): 147-161

  Abstract

  Positive selection of T cells begins with TCR alpha beta lo thymic progenitors. Here, we show that the most efficient TCRlo progenitors are c-kit+ with intermediate levels of CD4 and CD8 (DPint). Positive selection of DPint TCRlo c-kit+ cells results in TCRmed CD69+ c-kit+ transitional intermediates that show increased TCRV beta frequencies to selecting superantigen (SAg) that are committed to the CD4 or CD8 pathway. The cells on the c-kit+ maturation pathway maintain Bcl-2 expression. Most DPint c-kit+ progenitors fail positive selection, and become DPhi c-kit- cells that lose Bcl-2 expression. Some DPhi c-kit blast cells can be salvaged to produce mature single-positive (SP) cells. DPint c-kit+ maturation to SP cells can occur in <12 hr in vitro on thymic stromal monolayers.

  View details for Web of Science ID A1996VE58900005

  View details for PubMedID 8769478

• CD3(-)4(-)8(-) thymocyte precursors with interleukin-2 receptors differentiate phenotypically in coculture with thymic stromal cells SCANDINAVIAN JOURNAL OF IMMUNOLOGY Small, M., Weissman, I. L. 1996; 44 (2): 115-121

  Abstract

  CD3-4-8- interleukin-2 receptor positive (IL-2R+) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R+CD3-4-8- thymocytes were obtained by positive panning of IL-2R+ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2 1/2 days significant numbers of CD4+8+ and CD3+4+8- cell populations developed, the latter expressing the alpha beta T-cell receptor (alpha beta-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R+ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.

  View details for Web of Science ID A1996UZ88300004

  View details for PubMedID 8711423

• Activation of physiological cell death mechanisms by a necrosis-causing agent MICROSCOPY RESEARCH AND TECHNIQUE Vaux, D. L., Whitney, D., Weissman, I. L. 1996; 34 (3): 259-266

  Abstract

  Cell death is an important physiological process, but it can be triggered by both physiological and nonphysiological stimuli. The product of the bcl-2 gene has the ability to inhibit a physiological cell death process that can be activated by a variety of physiological signals, such as growth factor deprivation. This report describes the use of electron microscopy to examine the effects of two cytotoxic drugs on factor-dependent cells that constitutively express the human bcl-2 gene. Although all cells treated with sodium azide showed changes typical of necrosis, in the absence of Bcl-2 the cells died more rapidly and also displayed features of apoptosis. The fact that Bcl-2 could delay cell death argues that cells can activate internal cell death mechanisms to commit suicide before they are killed by a cytotoxin. Northern analysis showed that growth factor did not preserve viability of the cells through induction of bcl-2. However, growth factor may prevent activation of the physiological cell death mechanisms that bcl-2 can control. This process may constitute a primitive defense response, and blocking it may provide a means of limiting damage caused by otherwise sublethal injuries.

  View details for Web of Science ID A1996UL95900008

  View details for PubMedID 8743413

• The role of cell adhesion molecules in the development of IDDM - Implications for pathogenesis and therapy DIABETES Yang, X. D., Michie, S. A., Mebius, R. E., Tisch, R., Weissman, I., McDevitt, H. O. 1996; 45 (6): 705-710

  Abstract

  IDDM is a chronic inflammatory disease in which there is autoimmune-mediated organ-specific destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The migration of autoreactive lymphocytes and other leukocytes from the bloodstream into the target organ is of clear importance in the etiology of many organ-specific autoimmune/inflammatory disorders, including IDDM. In IDDM, this migration results in lymphocytic invasion of the islets (formation of insulitis) and subsequent destruction of beta-cells. Migration of lymphocytes from the bloodstream into tissues is a complex process involving sequential adhesion and activation events. This migration is controlled in part by selective expression and functional regulation of cell adhesion molecules (CAMs) on the surface of lymphocytes and vascular endothelial cells or in the extracellular matrix. Understanding the mechanisms that regulate lymphocyte migration to the pancreatic islets will lead to further understanding of the pathogenesis of IDDM. In this article, we summarize the recent advances regarding the function of CAMs in the development of IDDM in animal models and in humans and discuss the potential for developing CAM-based therapies for IDDM.

  View details for Web of Science ID A1996UN72400001

  View details for PubMedID 8635641

• Heterogeneity of N insertion capacity in fetal hematopoietic stem cells INTERNATIONAL IMMUNOLOGY Komagata, Y., Weissman, I. L., Ikuta, K. 1996; 8 (6): 837-845

  Abstract

  TCR gene rearrangement is strictly regulated during mouse ontogeny. The V-(D)-J junctions of alphabeta and gammadelta TCR transcripts expressed in the adult thymus are more highly diverse than those in the fetal thymus. We previously showed that adult hematopoietic stem cells (HSC) have a higher capacity to insert N nucleotides into Vgamma4 TCR transcripts than fetal HSC and that the level of N nucleotide insertion is determined, at least in part, at the level of HSC. To analyze this developmental change of HSC at the single cell level, we investigated N nucleotide insertions in three TCR transcripts (Vgamma4, Vgamma2 and Vbeta8) derived from limiting numbers of fetal liver HSC by fetal thymic organ culture. Eight day-14 fetal liver HSC clones showed various levels of N nucleotide insertions in Vgamma transcripts (0-78%). On the other hand, the level of N insertions was similarly regulated in Vgamma4, Vgamma2, and Vbeta8 TCR transcripts in a clone-specific way. These results suggested that the level of N insertion is programmed at the level of single HSC and that fetal liver contains a heterogeneous population of HSC in terms of N insertion capacity. After 3 weeks culture with a stromal cell line, fetal HSC showed higher levels of N insertion capacity than before culture. This result and the presence of HSC with intermediate N insertion capacity support the hypothesis that the developmental potential of individual HSC gradually changes from fetal to adult type in one stem cell lineage.

  View details for Web of Science ID A1996UU91500004

  View details for PubMedID 8671673

• Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells BLOOD Randall, T. D., Lund, F. E., Howard, M. C., Weissman, I. L. 1996; 87 (10): 4057-4067

  Abstract

  Using a monoclonal antibody to murine CD38, we showed that a population of adult bone marrow cells that expressed the markers Sca-1 and c-kit but lacked the lineage markers Mac-1, GR-1, B220, IgM, CD3, CD4, CD8 and CD5 could be subdivided by the expression of CD38. We showed that CD38high c-kit+ Sca-1+, linlow/-cells sorted from adult bone marrow cultured with interleukin-3 (IL-3), IL-6, and kit-L produced much larger colonies in liquid culture at a greater frequency than their CD38low/- counterparts. In addition, we found that CD36low/ - cells contained most of the day-12 colony-forming units-spleen (CFU-S) but were not long-term reconstituting cells, whereas the population that expressed higher levels of CD38 contained few, but significant, day-12 CFU-S and virtually all the long-term reconstituting stem cells. Interestingly, the CD38high Sca-1+ c-kit+ linlow/- cells isolated from day-E14.5 fetal liver were also found to be long-term reconstituting stem cells. This is in striking contrast to human hematopoietic progenitors in which the most primitive hematopoietic cells from fetal tissues lack the expression of CD38. Furthermore, because antibodies to CD38 could functionally replace antibodies to Thy-1.1 in a stem cell purification procedure, the use of anti-CD38 may be more generally applicable to the purification of hematopoietic stem cells from mouse strains that do not express the Thy-1.1 allele.

  View details for Web of Science ID A1996UK87900004

  View details for PubMedID 8639761

• Searching for hematopoietic stem cells .2. The heterogeneity of Thy-1.1(lo)Lin(-/lo)Sca-1(+) mouse hematopoietic stem cells separated by counterflow centrifugal elutriation EXPERIMENTAL HEMATOLOGY Uchida, N., Jerabek, L., Weissman, I. L. 1996; 24 (5): 649-659

  Abstract

  Mouse hematopoietic stem cells (HSCs) are highly enriched in the rare (approximately 0.05%) Thy-1.1(lo)Lin(-/lo)Sca-1+ fraction of hematopoietic tissues. It has been demonstrated that Thy-1.1(lo)Lin(-/lo)Sca-1+ cells are the only HSCs in C57BL/Ka-Thy-1.1 bone marrow. In this study, we separated C57/Ka-Thy-1.1 bone marrow cells by counterflow centrifugal elutriation (CCE) into four fractions and characterized Thy-1.1(lo)Lin(-/lo)Sca-1+ cells in each eluted fraction. The peak number of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells was highly enriched in one eluted fraction, which was also highly enriched for day-12 to -13 CFU-S. Activities for day-13 CFU-S, radioprotection, and long-term multilineage reconstitution correlated with, and could be generally predicted by determining, the frequency of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells in a given eluted fraction. However, the fraction that was highly enriched for blast cells and contained a low frequency of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells, only provided short-term but not long-term radioprotection, with a predicted cell number (100 cells) that should have protected > or = 95% (PD95) of hosts. Still, when 300 Thy-1.1(lo)Lin(-/lo)Sca-1+ cells from this fraction (3 X PD95 for unfractionated HSCs) were injected, mice were radioprotected and donor cells provided long-term multilineage reconstitution. We propose that such blast cells may contain two Thy-1.1(lo)Lin(-/lo)Sca-1+ subsets, one providing short-term and the other long-term multilineage sustained hematopoiesis, the latter presumably due to HSC self-renewal.

  View details for Web of Science ID A1996UE81400009

  View details for PubMedID 8605970

• From thymic lineages back to hematopoietic stem cells, sometimes using homing receptors JOURNAL OF IMMUNOLOGY Weissman, I. L. 1996; 156 (6): 2019-2025

  View details for Web of Science ID A1996TZ28100001

  View details for PubMedID 8690888

• Hematopoietic stem cells are not direct cytotoxic targets of natural killer cells BLOOD Aguila, H. L., Weissman, I. L. 1996; 87 (4): 1225-1231

  Abstract

  Bone marrow (BM) transplants from one individual to an irradiated histoincompatible individual of the same species are rejected. In mice, the primary host barrier cells that recognize bone marrow grafts bearing hematopoietic histocompatibility antigens bear surface markers of natural killer (NK) lymphocytes. Because of the innate ability of NK cells to kill susceptible targets, it has been proposed that the cytotoxic bone marrow graft rejection. To test this hypothesis, we purified hematopoietic stem cells from mice and incubated them with purified populations of actively cytotoxic allogeneic and semisyngeneic NK cells, followed by analysis of the ability of the treated hematopoietic stem cells (HSCs) to rescue lethally irradiated syngeneic animals. Such rescue was unimpaired. Also, HSC allografts were transplanted into transgenic mice deficient in NK and killer T-cell cytotoxicity generated by expressing diphtheria toxin A chain under the control of granzyme A promoter. Allogeneic HSCs were susceptible to allogeneic restriction in these mice, implying that the effector functions of NK marker-positive cells do not require NK cell cytotoxicity.

  View details for Web of Science ID A1996TV52300003

  View details for PubMedID 8608208

• Transplantation of purified hematopoietic stem cells: requirements for overcoming the barriers of allogeneic engraftment. Biology of blood and marrow transplantation Shizuru, J. A., Jerabek, L., Edwards, C. T., Weissman, I. L. 1996; 2 (1): 3-14

  Abstract

  Allogeneic bone marrow transplantation currently plays a critical role in the treatment of leukemias and inherited disorders of hematopoiesis, and it shows great promise for the treatment of numerous other diseases. The problems of graft-vs-host disease (GVHD) and failure to engraft, however, remain formidable obstacles to the widespread use of this therapy. Successful transplantation of purified populations of hematopoietic stem cells (HSCs) can theoretically avoid the problem of GVHD, since purified HSCs lack the mature elements that allow the graft to mount a response against the host. In previous studies from our laboratory, a population of purified HSCs (Thy-1loLin-/loSca-1+) was isolated from mouse bone marrow (BM). These cells represent approximately 0.05% of BM cells and are capable of self-renewal and long-term reconstitution of all blood lineages. Here we report long-term engraftment of these purified HSCs transplanted in mice across successively more difficult allogeneic-histocompatibility barriers. Transplantation of purified HSCs were quantitatively compared with whole bone marrow (WBM) grafts containing equivalent numbers of stem cells. The mouse strain combinations tested were parent transplanted into F1 (Hh disparate), minor histocompatibility complex (mHC), and major histocompatibility complex (MHC) plus mHC disparities. One of the recipient strains studied for MHC-disparate transplantations was that of spontaneously autoimmune diabetic mice. Recipient mice were administered lethal doses of whole-body irradiation in the presence or absence of antibodies directed against natural killer (NK) cell-associated determinants and/or monoclonal antibodies against the CD4+ T cell subset. We find that as the barrier to transplantation increases, greater numbers of HSCs are required for radioprotection and engraftment. In all cases, stable hematopoietic chimeras were generated with HSCs alone, but 10-60 times the number of HSCs was required for radioprotection of mice transplanted across allogeneic or semiallogeneic disparities as compared to Ly-5 congenic differences. Furthermore, we demonstrate a clear advantage of WBM vs HSCs with regard to tha ability to engraft [corrected]. Chimeric mice showed no symptoms of GVHD, and their T cells were unable to induce GVHD in neonatal mice expressing H-2 antigens of donor and host. These data confirm that a cell population resident in WBM and distinct from purified stem cells is important in facilitating hematopoietic engraftment, in this case, of purified allogeneic HSCs. The differences in engraftment between WBM and HSCs could be reduced significantly by the addition of antibodies directed against NK determinants to the host preparative regimen. Similarly, since antibodies directed against host NK-associated antigens can reduce the barrier to allogeneic HSC engraftment, an interaction between the facilitating population within donated WBM and a resistant host population with NK determinants is implied.

  View details for PubMedID 9078349

• Sequence and characterization of two HSP70 genes in the colonial protochordate Botryllus schlosseri IMMUNOGENETICS Fagan, M. B., Weissman, I. L. 1996; 44 (2): 134-142

  Abstract

  Two genes belonging to the heat shock protein 70 gene family have been cloned from the colonial protochordate Botryllus schlosseri. The two intronless genes (HSP70.1 and HSP70.2) exhibit 93.6% sequence identity within the predicted coding region, and 83.3% and 81.7% sequence identity in the 5' and 3' flanking regions, respectively. The predicted amino acid sequences are 95% identical and contain several signatures characteristic of cytoplasmic eukaryotic HSP70 genes (Gupta et al. 1994; Rensing and Maier 1994). Northern blotting and sequence analysis suggest that both genes are heat-inducible members of the HSP70 gene family. Given these characteristics, HSP70.1 and HSP70.2 appear to be good candidates for protochordate homologues of the major histocompatibility complex-linked HSP70 genes of human, mouse, and rat (Milner and Campbell 1990; Walter et al. 1994). Further experiments to determine whether there is functional evidence for such similarity are in progress.

  View details for Web of Science ID A1996UP88100008

  View details for PubMedID 8662076

• SIMILAR BUT NONIDENTICAL AMINO-ACID-RESIDUES ON VASCULAR CELL-ADHESION MOLECULE-1 ARE INVOLVED IN THE INTERACTION WITH ALPHA(4)BETA(1) AND ALPHA(4)BETA(7) UNDER DIFFERENT ACTIVITY STATES JOURNAL OF IMMUNOLOGY Chiu, H. H., Crowe, D. T., Renz, M. E., Presta, L. G., Jones, S., Weissman, I. L., Fong, S. 1995; 155 (11): 5257-5267

  Abstract

  The integrin receptors alpha 4 beta 1 and alpha 4 beta 7 both bind to vascular cell adhesion molecule-1 (VCAM-1). Here, we report that the amino acid residue requirements for murine VCAM-1 adhesion to murine alpha 4 beta 1 (WEHI 231) and alpha 4 beta 7 (38C13/beta 7-transfectant) positive cells are strikingly similar but nonidentical under multiple adhesion activity states. By site-directed mutagenesis of domain 1 of VCAM-1, the amino acid residues on the loop between beta strands C and D (R36, Q38, I39, D40, P42) and on the adjacent antiparallel beta strand F (L70 and T72) were required for basal level adhesion to both alpha 4 beta 1-positive and alpha 4 beta 7-positive cells. Mutation at two other sites, N44 (loop between beta strands C and D) and E66 (loop between beta strands E and F), specifically reduced alpha 4 beta 7-positive cell adhesion, but not alpha 4 beta 1-positive cell adhesion. Mutation H85A augmented alpha 4 beta 7 binding but not alpha 4 beta 1 binding. These apparent differences relate to the higher intrinsic activity state of alpha 4 beta 1 on WEHI 231 than on alpha 4 beta 7 (38C13/beta 7-transfectant). In contrast, under higher adhesion activity states induced by either MnCl2 or truncation of the beta 7 cytoplasmic tail, mutation of either amino acid residue D40 or L70 completely blocked cell adhesion without evidence of structural perturbation of VCAM-1. These results suggested that the two structurally discontinuous amino acid residues, the negatively charged D40 and the hydrophobic L70 adjacently located on domain 1 of VCAM-1, are essential for interaction under multiple activity states with both alpha 4 beta 1 and alpha 4 beta 7 integrin receptors.

  View details for Web of Science ID A1995TF68600023

  View details for PubMedID 7594538

• LIFE-HISTORY PLASTICITY IN CHIMERAS OF THE COLONIAL ASCIDIAN BOTRYLLUS-SCHLOSSERI PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES CHADWICKFURMAN, N. E., Weissman, I. L. 1995; 262 (1364): 157-162

  Abstract

  Colonies of the ascidian Botryllus schlosseri may fuse with kin to form chimaeras which vary their life histories depending on environmental conditions. We placed chimaeric colonies of this species in Monterey Bay, California, U.S.A., where they received planktonic food continuously. In the field, chimaeras grew rapidly, attained large sizes, and produced many eggs. They formed compact disc-shaped colonies in which genotypic composition remained stable throughout their lifespan. In most cases, genotypic partners in field chimaeras senesced and died synchronously. We also cultured genetically identical replicates of the same chimaeras under laboratory conditions, where they were fed once daily. In the laboratory environment, chimaeras grew slowly, shrank, and fragmented. Most genotypes in chimaeric colonies produced significantly fewer zooids and eggs in the laboratory than they did in the field. Somatic cell parasitism, in the form of resorption of tissues of one genotype by the other, occurred mainly in the laboratory environment, and not in the field. The phenomenon of resorption may thus be a dispensible strategy of fused genotypes depending on environmental conditions. Genotypes in field chimaeras may grow and reproduce rapidly because of the non-limiting food resources available. These data demonstrate that chimaeras of B. schlosseri have extremely plastic life histories, and employ different strategies depending on the environment.

  View details for Web of Science ID A1995TH71200007

  View details for PubMedID 8524910

• 2 PATHWAYS OF EXOCYTOSIS OF CYTOPLASMIC GRANULE CONTENTS AND TARGET-CELL KILLING BY CYTOKINE-INDUCED CD3(+)CD56(+) KILLER-CELLS BLOOD Mehta, B. A., SCHMIDTWOLF, G. H., Weissman, I. L., Negrin, R. S. 1995; 86 (9): 3493-3499

  Abstract

  Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell-induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3-mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP-sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be-identified target cell surface molecules.

  View details for Web of Science ID A1995TB16300028

  View details for PubMedID 7579455

• TRANSGENIC MICE CARRYING THE DIPHTHERIA-TOXIN-A CHAIN GENE UNDER THE CONTROL OF THE GRANZYME-A PROMOTER - EXPECTED DEPLETION OF CYTOTOXIC-CELLS AND UNEXPECTED DEPLETION OF CD8 T-CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Aguila, H. L., HERSHBERGER, R. J., Weissman, I. L. 1995; 92 (22): 10192-10196

  Abstract

  We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

  View details for Web of Science ID A1995TB46700053

  View details for PubMedID 7479752

• THE PURIFICATION AND CHARACTERIZATION OF FETAL LIVER HEMATOPOIETIC STEM-CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Morrison, S. J., Hemmati, H. D., WANDYCZ, A. M., Weissman, I. L. 1995; 92 (22): 10302-10306

  Abstract

  Thy-1loSca-1+Lin-Mac-1+CD4- cells have been isolated from the livers of C57BL-Thy-1.1 fetuses. This population appears to be an essentially pure population of hematopoietic stem cells (HSC), in that injection of only six cells into lethally irradiated adult recipients yields a limit dilution frequency of donor cell-reconstituted mice. Sixty-seven to 77% of clones in this population exhibit long-term multilineage progenitor activity. This population appears to include all long-term multilineage reconstituting progenitors in the fetal liver. A high proportion of cells are in cycle, and the absolute number of cells in this population doubles daily in the fetal liver until 14.5 days postcoitum. At 15.5 days postcoitum, the frequency of this population falls dramatically. Long-term reconstituting HSC clones from the fetal liver give rise to higher levels of reconstitution in lethally irradiated mice than long-term reconstituting HSC from the bone marrow. The precise phenotypic and functional characteristics of HSC vary according to tissue and time during ontogeny.

  View details for Web of Science ID A1995TB46700075

  View details for PubMedID 7479772

• EXTRATHYMIC MATURATION OF ALPHA-BETA T-CELLS FROM HEMATOPOIETIC STEM-CELLS JOURNAL OF IMMUNOLOGY DEJBAKHSHJONES, S., Jerabek, L., Weissman, I. L., Strober, S. 1995; 155 (7): 3338-3344

  Abstract

  The object of the study was to determine whether alpha beta T cells can develop from hemopoietic stem cells in the absence of the thymus. C57BL/6 (Ly-5.1 and Thy-1.2) mice were thymectomized or sham-thymectomized at 4 wk of age, and received lethal whole body irradiation 2 wk later. These mice were reconstituted with an i.v. injection of 500 highly purified hemopoietic stem cells (Mac-1-, B220-, TER-119-, CD3-, CD4-, CD8-, Thy 1low, SCA-1+) obtained from the bone marrow of C57BL/6 (Ly-5.2 and Thy-1.1) donors. A similar percentage of Ly-5.2+ alpha beta T cells (donor) was found in the marrow of thymectomized recipients, sham-thymectomized recipients, and normal donor mice at least 3 mo after stem cell transplantation. The percentage of Ly-5.2+ alpha beta T cells in the spleens of sham-thymectomized and normal donor mice was similar. The percentage in the spleens of thymectomized recipients was reduced by about 50%, and approximately one-half of the latter T cells expressed the CD4-CD8- alpha beta+ phenotype. A purified population of Ly-5.2+ alpha beta- cells obtained from the marrow of thymectomized recipients was incubated in vitro for 48 h without exogenous growth factors. After the incubation procedure a proportion of the marrow cells acquired alpha beta TCR surface receptors. The results show that alpha beta T cells can develop from hemopoietic stem cells in the absence of the thymus.

  View details for Web of Science ID A1995RV93900010

  View details for PubMedID 7561027

• LIFE-HISTORIES AND SENESCENCE OF BOTRYLLUS-SCHLOSSERI (CHORDATA, ASCIDIACEA) IN MONTEREY BAY BIOLOGICAL BULLETIN CHADWICKFURMAN, N. E., Weissman, I. L. 1995; 189 (1): 36-41

  Abstract

  The colonial ascidian Botryllus schlosseri is a model organism for research on invertebrate histocompatibility, development, and evolutionary biology. Nonetheless, the basic life history of Pacific Ocean populations of the species remains unknown. We determined field rates of growth, reproduction, and senescence in four cohorts of B. schlosseri colonies in Monterey Bay, California. Colonies grew exponentially as juveniles and reached sizes of up to 1400 zooids within 69 days. After a juvenile phase lasting at least 49 days, the colonies began to reproduce sexually. Each zooid produced up to 10 clutches, each with a maximum of 5 eggs, resulting in very high fecundity of up to 8000 eggs per colony. Following a short period (maximum 70 days) of continuous sexual reproduction, colonies abruptly senesced and died while still bearing a full clutch of eggs. Senescence progressed through four distinct stages over 1-2 weeks, and inevitably led to the simultaneous death of all zooids in the colony. Although senescence was the main cause of mortality, some colonies died as a result of predation or undermined causes. Certain life history traits varied significantly between cohorts that settled at different times of year. For example, lifespans in the field varied from about 3 months for spring to 8 months for fall-born colonies, but the lifetime fecundity of colonies did not vary between cohorts. The morphologies and life histories of colonies monitored in the field and reported here differed from those of colonies cultured previously in the laboratory.

  View details for Web of Science ID A1995RN59900006

  View details for PubMedID 7654845

• HETEROGENEITY OF HEMATOPOIETIC STEM-CELLS - IMPLICATIONS FOR CLINICAL-APPLICATIONS PROCEEDINGS OF THE ASSOCIATION OF AMERICAN PHYSICIANS Morrison, S. J., Weissman, I. L. 1995; 107 (2): 187-194

  View details for Web of Science ID A1995TF49800003

  View details for PubMedID 8624852

• The biology of hematopoietic stem cells ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Morrison, S. J., Uchida, N., Weissman, I. L. 1995; 11: 35-71

  Abstract

  Hematopoietic stem cells (HSC) are the only cells in the blood-forming tissues that can give rise to all blood cell types and that can self-renew to produce more HSC. In mouse and human, HSC represent up to 0.05% of cells in the bone marrow. HSC are almost entirely responsible for the radioprotective and short- and long-term reconstituting effects observed after bone marrow transplantation. The subsets of HSC that give rise to short-term vs long-term multilineage reconstitution can be separated by phenotype, demonstrating that the fates of HSC are intrinsically determined. Here we review the ontogeny and biology of HSC, their expression of fate-determining genes, and the clinical importance of HSC for transplantation and gene therapy.

  View details for Web of Science ID A1995TY44800003

  View details for PubMedID 8689561

• President's Message Amer Assoc Immunol Newsletter Weissman, I. 1995; May-Jun-Jul: 1-3
• Hematopoietic Stem Cells Clinical Oncology Fleming, W., Weissman, I. Churchill Livingstone. 1995: 127–134
• Phenotypic and functional analysis of hematopoietic stem cells in mouse and human Hematopoietic Stem Cells: Biology and Therapeutic Applications Tsukamoto, A., Weissman, I., et al Marcel Dekker. 1995: 85–124
• ALLOIMMUNE HIERARCHIES AND STRESS-INDUCED REVERSALS IN THE RESORPTION OF CHIMERIC PROTOCHORDATE COLONIES PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Rinkevich, B., Weissman, I. L., Shapira, M. 1994; 258 (1353): 215-220

  View details for Web of Science ID A1994QB14000002

• MOLECULAR EVOLUTION OF THE VERTEBRATE IMMUNE-SYSTEM PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bartl, S., Baltimore, D., Weissman, I. L. 1994; 91 (23): 10769-10770

  View details for Web of Science ID A1994PQ93800004

  View details for PubMedID 7971960

• THE LONG-TERM REPOPULATING SUBSET OF HEMATOPOIETIC STEM-CELLS IS DETERMINISTIC AND ISOLATABLE BY PHENOTYPE IMMUNITY Morrison, S. J., Weissman, I. L. 1994; 1 (8): 661-673

  Abstract

  The Thy-1.1loSca-1hiLin-/lo population, representing 0.05% of C57BL/Ka-Thy-1.1 bone marrow, is highly enriched for hematopoietic stem cells and includes all multipotent progenitors in this mouse strain; however, the functional reconstituting activity of this fraction is heterogeneous. Only around 25% of clonal reconstitutions by cells from this population are long term; remaining clones yield transient multilineage reconstitutions. By fractionating based on lineage marker expression, the Thy-1.1loSca-1hiLin-/lo population has been resolved into three subpopulations: Lin-Mac-1-CD4-; Lin-Mac-1loCD4-; and Mac-1loCD4lo. Of these, only the Lin-Mac-1-CD4- population is highly enriched for long-term reconstituting hematopoietic stem cells. A comparison of transient and long-term multipotent progenitors indicates that long-term progenitors have less CFU-S activity, are equally radioprotective, and are less frequently in cell cycle. The ability to predict the longevity of reconstitution based on lineage marker expression indicates that reconstitution potential is deterministic, not stochastic.

  View details for Web of Science ID A1994PT93000004

  View details for PubMedID 7541305

• STEM-CELLS, CLONAL PROGENITORS, AND COMMITMENT TO THE 3 LYMPHOCYTE LINEAGES - T-CELLS, B-CELLS, AND NK-CELLS IMMUNITY Weissman, I. L. 1994; 1 (7): 529-531

  View details for Web of Science ID A1994PN12700001

  View details for PubMedID 7600281

• The common gamma (gamma c) chain for multiple cytokine receptors. Japanese journal of cancer research Weissman, I. L. 1994; 85 (10): inside front cover

  View details for PubMedID 7961100

• CHARACTERIZATION OF PRENEOPLASTIC THYMOCYTES AND OF THEIR NEOPLASTIC PROGRESSION IN IRRADIATED C57BL/KA MICE JOURNAL OF IMMUNOLOGY SENMAJUMDAR, A., Guidos, C., Kina, T., Lieberman, M., Weissman, I. L. 1994; 153 (4): 1581-1592

  Abstract

  Mice that receive whole body split-dose irradiation develop thymic lymphomas after a long latent period. Before emergence of frank lymphomas, preneoplastic thymocytes, which are defined by their ability to progress to full malignancy on intrathymic transfer to congenic hosts, appear. A combination of mAb 1C11, which binds to a cell surface glycoprotein on lymphoma cells, and of Abs to the differentiation markers CD4 and CD8 (MHC co-receptors), and CD3 (TCR complex) was used to characterize the phenotypes of preneoplastic thymocytes and to place them within the scheme of normal T cell ontogeny. In the irradiated, preneoplastic thymus, the 1C11 molecule was found to be present on CD4-8-, CD4-8+, and CD4+8+, but not CD4+8-, cells. After intrathymic transfer to Thy-1 congenic recipients, 1C11highCD4-8- cells from irradiated mice showed the highest leukemogenic potential, followed by the 1C11highCD4-8+ and 1C11highCD4+8+ subsets. Within the 1C11highCD4-8- subset, CD3+ cells were more leukemogenic than CD3- cells. The resulting lymphomas were 1C11highCD3+4-8+ and 1C11highCD3+4+8+, phenotypes that are absent or very rare in the normal thymus, but similar to those of primary radiation-induced lymphomas. Examination of the TCR V beta repertoire in these lymphomas shows a highly significant bias, in that approximately 50% express the V beta 8 gene product. These results indicate, but do not prove, that the 1C11highCD3+4-8- cells, a very small subset of normal thymocytes, are either the target of neoplastic transformation after radiation or the earliest identifiable cell population after the transforming event. These results also suggest at least one possible pathway to define the process leading to overt lymphoma.

  View details for Web of Science ID A1994PB40600018

  View details for PubMedID 8046233

• Infection and multiple sclerosis: a possible role for superantigens? Trends in microbiology Brocke, S., VEROMAA, T., Weissman, I. L., Gijbels, K., Steinman, L. 1994; 2 (7): 250-254

  Abstract

  The association of infection with autoimmune diseases is enigmatic, partly because cause and effect are difficult to establish in chronic diseases. Microorganisms might initiate multiple sclerosis and trigger relapses of disease. Superantigens might be involved in autoimmunity through the (re)activation of T cells, including autoreactive cells, expressing certain T cell receptor beta chain variable regions.

  View details for PubMedID 8081652

• EXPRESSION OF GRANZYME-A IN SALIVARY-GLAND BIOPSIES FROM PATIENTS WITH PRIMARY SJOGRENS-SYNDROME ARTHRITIS AND RHEUMATISM Alpert, S., Kang, H. I., Weissman, I., FOX, R. I. 1994; 37 (7): 1046-1054

  Abstract

  To determine whether the destruction of salivary gland epithelial cells in Sjögren's syndrome (SS) could be mediated by granzyme A, a serine protease that is contained in the granules of activated lymphocytes.We used in situ hybridization and polymerase chain reaction amplification to determine the expression of granzyme A messenger RNA (mRNA) in salivary gland biopsy samples.Granzyme A mRNA expression was detected in salivary glands from SS patients, but not in those from normal controls. Granzyme A mRNA content was significantly correlated (P < 0.001) with the size of lymphocytic infiltrates in the salivary glands and with the clinical activity of the disease.Cells that express granzyme A mRNA may play a role in the destruction of the target organ (i.e., the salivary gland) in patients with SS. The strong association of granzyme A mRNA expression and larger lymphoid infiltrates in the patients' salivary glands suggests that granzyme A mRNA is expressed at a relatively late stage of the local inflammatory process. Therapies designed to modulate or block granzyme A induction and action should be investigated in SS patients.

  View details for Web of Science ID A1994NW27400010

  View details for PubMedID 8024614

• DEMONSTRATION THAT THY(LO) SUBSETS OF MOUSE BONE-MARROW THAT EXPRESS HIGH-LEVELS OF LINEAGE MARKERS ARE NOT SIGNIFICANT HEMATOPOIETIC PROGENITORS BLOOD Morrison, J., Lagasse, E., Weissman, I. L. 1994; 83 (12): 3480-3490

  Abstract

  We have been unable to reproduce experiments suggesting the existence of three lineage-restricted progenitor populations from mouse bone marrow. Thy1.1loMac-1+B220+ cells were reported to give rise to greatly expanded numbers of myeloid and lymphoid cells, while Thy1.1loMac-1+B220- and Thy1.1loMac-1-B220+ cells were reported to be highly proliferative myeloid and B-lineage-restricted progenitors, respectively. Both Mac-1+ cell types appear to be much less frequent than previously reported, and we observed no activity consistent with their characterization as committed progenitors of expanded numbers of cells. The original identification of these populations may have resulted from a failure to distinguish bonafide signals from autofluorescent background and nonspecific staining. The progenitor activities originally associated with these populations may have been due to hematopoietic stem cell contamination. This study shows that low levels of Mac-1 are expressed on cells with multipotent progenitor activity. Thy1.1loB220+Mac-1- cells can be purified from bone marrow, but in these experiments they do not give rise to detectable levels of progeny on injection into lethally irradiated mice. Thy1.1loB220+Mac-1- cells appear to be pro-B cells without significant proliferation potential in vivo. The finding that the described populations do not have the reported progenitor activities leaves the pathways of stem cell differentiation open to further study.

  View details for Web of Science ID A1994NR90500010

  View details for PubMedID 7515713

• RAPID AND SUSTAINED HEMATOPOIETIC RECOVERY IN LETHALLY IRRADIATED MICE TRANSPLANTED WITH PURIFIED THY-1.1(LO) LIN(-) SCA-1(+) HEMATOPOIETIC STEM-CELLS BLOOD Uchida, N., Aguila, H. L., Fleming, W. H., Jerabek, L., Weissman, I. L. 1994; 83 (12): 3758-3779

  Abstract

  Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca-1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

  View details for Web of Science ID A1994NR90500041

  View details for PubMedID 7911343

• AN ANALYSIS OF THE EXPRESSION OF CYCLOPHILIN-C REVEALS TISSUE RESTRICTION AND AN INTRIGUING PATTERN IN THE MOUSE KIDNEY AMERICAN JOURNAL OF PATHOLOGY Friedman, J., Weissman, I., Friedman, J., Alpert, S. 1994; 144 (6): 1247-1256

  Abstract

  Cyclophilin C (cyp C) is a cyclosporin A (CsA) binding protein originally isolated from a mouse bone marrow stromal cell line. We have compared the expression patterns of the mammalian cyclophilins A, B, and C in mouse tissues using in situ hybridization. These studies reveal that cyp C is expressed in a restricted subset of tissues including mouse ovary, testis, bone marrow, and kidney. Within the kidney, cyp C is highly expressed in a narrow zone in the outer medulla. Using monoclonal antibodies reactive against cyp C, we find that the kidney cells expressing cyp C correspond to the S3 segment of the nephron. The S3 segment has been shown to sustain histopathological damage from high dosages of CsA, raising the possibility that cyp C may be involved in mediating this damage.

  View details for Web of Science ID A1994NQ32400015

  View details for PubMedID 8203464

  View details for PubMedCentralID PMC1887451

• REGULATION OF THE AVIDITY OF INTEGRIN ALPHA(4)BETA(7) BY THE BETA(7) CYTOPLASMIC DOMAIN JOURNAL OF BIOLOGICAL CHEMISTRY Crowe, D. T., Chiu, H., Fong, S., Weissman, I. L. 1994; 269 (20): 14411-14418

  Abstract

  Integrins are cell-surface heterodimeric receptors with adhesive and transmembrane signaling properties. Their cytoplasmic domains can affect receptor avidity, cytoskeletal association, and post-receptor occupancy events. The alpha 4 beta 7 integrin mediates cell adhesion to Peyer's patch high walled endothelial venules (HEV), VCAM, and CS-1/fibronectin. To determine the role of the beta 7 cytoplasmic domain in these interactions, wild-type and truncated beta 7 subunits were stably expressed in the alpha 4+/beta 1-/beta 7- B cell lymphoma 38C13. The cell line delta 727 lacks the entire beta 7 cytoplasmic domain, delta 753 lacks the 34 C-terminal residues, and LXSN is vector-transfected 38C13. Cells expressing wild-type beta 7 bound Peyer's patch HEV, fibronectin, and immobilized VCAM constitutively and did not require prior activation with phorbol esters. Interestingly, delta 753 displayed no ligand binding activity, while delta 727 was constitutively active for all ligands and displayed greater avidity for fibronectin and Peyer's patch HEV than the wild-type beta 7. beta 7, delta 753, delta 727, and LXSN were also tested for the ability to bind soluble VCAM in the presence of various divalent cations. In the presence of Ca2+, but not Mg2+, delta 727 constitutively bound soluble VCAM, whereas beta 7, delta 753, and LXSN did not. beta 7 and delta 753 could bind soluble VCAM if first activated with Mn2+. The results suggest that: (i) alpha 4 beta 7 can be expressed in a constitutively active state, (ii) the beta 7 cytoplasmic domain regulates the avidity of alpha 4 beta 7, and (iii) 38C13 cell lines expressing wild-type and truncated beta 7 subunits define three stable activation states of alpha 4 beta 7: inactive (delta 753), partially active (beta 7), and fully active (delta 727) receptors.

  View details for Web of Science ID A1994NM06500019

  View details for PubMedID 8182047

• EXPRESSION OF THE INTEGRIN ALPHA-4-BETA-1 ON MELANOMA-CELLS CAN INHIBIT THE INVASIVE STAGE OF METASTASIS FORMATION CELL Qian, F., Vaux, D. L., Weissman, I. L. 1994; 77 (3): 335-347

  Abstract

  Among a series of adhesion molecules, expression of integrin alpha 4 beta 1 showed a unique inverse correlation with the invasive potential of B16 melanoma cell lines. When an alpha 4 cDNA was introduced into an alpha 4-beta 1+ highly invasive melanoma line, alpha 4 beta 1 heterodimers were expressed on the surface. Matrigel invasion by the alpha 4+ beta 1+ cells was reduced. Pulmonary metastasis was also suppressed when the transfectants were placed subcutaneously, but not when injected intravenously. Expression of alpha 4 beta 1 promoted homotypic intercellular adhesion. The homotypic adhesion was abrogated, and the alpha 4+ beta 1+ (less invasive cell lines) increased matrigel invasion following the anti-alpha 4 MAb treatment. These results suggest that integrin alpha 4 beta 1 could play a role in controlling melanoma cell metastasis at the invasive stage.

  View details for Web of Science ID A1994NK97000005

  View details for PubMedID 8181055

• BCL-2 INHIBITS APOPTOSIS OF NEUTROPHILS BUT NOT THEIR ENGULFMENT BY MACROPHAGES JOURNAL OF EXPERIMENTAL MEDICINE Lagasse, E., Weissman, I. L. 1994; 179 (3): 1047-1052

  Abstract

  Neutrophils, the most common inflammatory leukocytes, have the most limited life span of all blood cells. After they undergo apoptosis, they are recognized and engulfed by macrophages. bcl-2, a proto-oncogene rearranged and deregulated in B cell lymphomas bearing the t(14;18) translocation, is known to inhibit programmed death. bcl-2 expression is localized in early myeloid cells of the bone marrow but is absent in mature neutrophils. Transgenic mice that expressed bcl-2 in mature neutrophils showed that bcl-2 blocked neutrophil apoptosis. Despite this, homeostasis of neutrophil population is essentially unaffected. In fact, macrophage uptake of neutrophils expressing bcl-2 still occurred. This transgenic model indicates that the mechanism that triggers phagocytosis of aging neutrophils operates independently of the process of apoptosis regulated by bcl-2.

  View details for Web of Science ID A1994MY48400032

  View details for PubMedID 8113673

• IN-VITRO DEVELOPMENT OF B-CELLS AND MACROPHAGES FROM EARLY MOUSE FETAL THYMOCYTES EUROPEAN JOURNAL OF IMMUNOLOGY Peault, B., Khazaal, I., Weissman, I. L. 1994; 24 (3): 781-784

  Abstract

  The developmental potentialities of early mouse fetal thymocytes were analyzed by in vitro culturing cell suspensions obtained from 12-15-gestational day thymus in contact with a bone marrow stroma clonal cell line that supports pre-B and myeloid cell differentiation. B cell and macrophage development from fetal thymocytes was observed at all fetal stages tested, but limiting dilution analysis revealed that the frequency of cells forming colonies on bone marrow stroma is the highest in the fetal thymus at day 12, then dramatically decreases until day 15. These observations suggest that the thymus rudiment is seeded by multipotential precursor cells which are not immediately committed to T cell development in the thymic cellular environment.

  View details for Web of Science ID A1994NB71800044

  View details for PubMedID 8125146

• PCR PRIMERS CONTAINING AN INOSINE TRIPLET TO COMPLEMENT A VARIABLE CODON WITHIN A CONSERVED PROTEIN-CODING REGION BIOTECHNIQUES Bartl, S., Weissman, I. L. 1994; 16 (2): 246-?

  View details for Web of Science ID A1994MV76800018

  View details for PubMedID 8179887

• RADIATION LEUKEMIA VIRUS-INDUCED THYMIC LYMPHOMAS EXPRESS A RESTRICTED REPERTOIRE OF T-CELL RECEPTOR V-BETA GENE-PRODUCTS JOURNAL OF VIROLOGY SENMAJUMDAR, A., Weissman, I. L., Hansteen, G., Marian, J., Waller, E. K., Lieberman, M. 1994; 68 (2): 1165-1172

  Abstract

  We have investigated the phenotypic changes that take place during the process of neoplastic transformation in the thymocytes of C57BL/Ka mice infected by the radiation leukemia virus (RadLV). By the combined use of antibodies against the envelope glycoprotein gp70 of RadLV, the transformation-associated cell surface marker 1C11, and the CD3-T-cell receptor (TCR) complex, we found that in the RadLV-infected thymus, the earliest expression of viral gp70 is in 1C11hi cells; a small but significant percentage of these cells also express CD3. A first wave of viral replication, manifested by the expression of high levels of gp70 in thymocytes (over 70% positive), reaches a peak at 2 weeks; during this period, no significant changes are observed in the expression of 1C11 or CD3. The population of gp70+ cells is drastically reduced at 3 to 4 weeks after infection. However, a second cohort of gp70+ cells appears after 4 weeks, and these cells express high levels of 1C11 and TCR determinants as well. RadLV-induced lymphomas differ from normal thymocytes in their CD4 CD8 phenotype, with domination by one or more subsets. Characterization of TCR gene rearrangements in RadLV-induced lymphomas shows that most of these tumors are clonal or oligoclonal with respect to the J beta 2 TCR gene, while the J beta 1 TCR gene is rearranged in a minority (4 of 11) of lymphomas. TCR V beta repertoire analysis of 12 tumors reveals that 6 (50%) express exclusively the V beta 6 gene product, 2 (17%) are V beta 5+, and 1 (8%) each are V beta 8+ and V beta 9+. In normal C57BL/Ka mice, V beta 6 is expressed on 12%, V beta 5 is expressed on 9%, V beta 8 is expressed on 22%, and V beta 9 is expressed on 4% of TCRhi thymocytes. Thus, it appears that RadLV-induced thymic lymphomas are not randomly selected with respect to expressed TCR V beta type.

  View details for Web of Science ID A1994MW25200066

  View details for PubMedID 8289345

• DEVELOPMENTAL SWITCHES IN THE IMMUNE-SYSTEM CELL Weissman, I. L. 1994; 76 (2): 207-218

  View details for Web of Science ID A1994MU67800005

  View details for PubMedID 8293459

• ISOLATION AND CHARACTERIZATION OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-IIB GENES FROM THE NURSE SHARK PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bartl, S., Weissman, I. L. 1994; 91 (1): 262-266

  Abstract

  The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.

  View details for Web of Science ID A1994MQ47900054

  View details for PubMedID 8278377

• ISOLATION OF CD3(-), CD4(-), CD8(-), IL-2R(+) THYMOCYTE PRECURSORS BY PANNING JOURNAL OF IMMUNOLOGICAL METHODS Small, M., Majumdar, A. S., Lieberman, M., Weissman, I. 1994; 167 (1-2): 103-107

  Abstract

  We present a panning method for isolation of thymocytes that are CD3-, CD4-, CD8- and IL-2R+. These cells have been isolated by positive selection on dishes coated with 7D4 antibody followed by treatment with biotinylated 145-2C11, GK1.5, and 53-6.7 antibodies and negative selection on avidin coated dishes.

  View details for Web of Science ID A1994MU31400011

  View details for PubMedID 8308269

• THE ISOLATION OF PUTATIVE MAJOR HISTOCOMPATIBILITY COMPLEX GENE FRAGMENTS FROM DOGFISH AND NURSE SHARK Conference on Primordial Immunity: Foundations for the Vertebrate Immune System Bartl, S., Weissman, I. L. NEW YORK ACAD SCIENCES. 1994: 346–349

  View details for Web of Science ID A1994BA22R00035

  View details for PubMedID 8192346

• President's Message Amer Assoc Immunol Newsletter Weissman, I. 1994; May-June: 1-3,19
• President’s Message: Can We Retrieve NIH-sponsored Biotechnology Profits for NIH Research and Training? Amer Assoc Immunol Newsletter Weissman, I. 1994; July-Aug: 1-2
• President's Message Amer Assoc Immunol Newsletter Weissman, I. 1994; Nov-Dec: 1-2
• President’s Message: Results of July Poll In Amer Assoc Immunol Newsletter Weissman, I. 1994; Sep-Oct: 1-2
• CRYSTAL-STRUCTURE OF MURINE CYCLOPHILIN-C COMPLEXED WITH IMMUNOSUPPRESSIVE DRUG CYCLOSPORINE-A PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ke, H. M., Zhao, Y. D., Luo, F., Weissman, I., Friedman, J. 1993; 90 (24): 11850-11854

  Abstract

  Cyclophilin is a cellular receptor for the immunosuppressive drug cyclosporin A (CsA). Cyclophilin C (CyPC) is highly expressed in murine kidney, making it a potential mediator of the nephrotoxic effects of CsA. The structure of murine CyPC complexed with CsA has been solved and refined to an R factor of 0.197 at a 1.64-A resolution. Superposition of the CyPC-CsA structure with the unligated cyclophilin A (CyPA) revealed significant migration of three loops: Gln-179 to Thr-189, Asp-47 to Lys-49, and Met-170 to Ile-176. The proximity of the loop Gln-179 to Thr-189 to the CsA binding site may account for the unique binding of a 77-kDa glycoprotein, CyPC binding protein (CyCAP), to CyPC. The binding of CsA to CyPC is similar to that of CsA to human T-cell cyclophilin A (CyPA). However, the conformation of CsA when bound to CyPC is significantly different from that when bound to CyPA. These differences may reflect conformational variation of CsA when bound to different proteins. Alternatively, the previous CyPA-CsA structure at low resolution may not provide sufficient details for a comparison with the CyPC-CsA structure.

  View details for Web of Science ID A1993MM51500085

  View details for PubMedID 8265636

• PHENOTYPIC CHARACTERIZATION AND IDENTIFICATION OF EFFECTOR-CELLS INVOLVED IN TUMOR-CELL RECOGNITION OF CYTOKINE-INDUCED KILLER-CELLS EXPERIMENTAL HEMATOLOGY SCHMIDTWOLF, I. G., Lefterova, P., Mehta, B. A., Fernandez, L. P., HUHN, L. D., Blume, K. G., Weissman, I. L., Negrin, R. S. 1993; 21 (13): 1673-1679

  Abstract

  Cytokine-induced killer (CIK) cells are highly efficient cytotoxic effector cells capable of lysing tumor cell targets. Cultures of human CIK cells have been shown to have enhanced cytotoxicity and to proliferate more rapidly than lymphokine activated killer (LAK) cells by both in vitro and in vivo studies. In this report, we have further characterized the phenotype of CIK cells and explored the molecular structures involved in CIK-mediated cell lysis of tumor target cells. The dominant cell phenotype in CIK cell cultures expresses the alpha, beta T cell receptor (TCR-alpha/beta). In addition, CD56 is expressed on the main effector cell on a per-cell basis. Interestingly, the total number of CD56+ cells increases more than 1000-fold during the generation of CIK cells, mainly due to expansion of CD56+ cells coexpressing CD3. The higher lytic activity of CIK cells as compared to LAK cells is mainly due to the higher proliferation of CD3+CD56+ cells and to the cytotoxic activity of TCR-alpha/beta+ cells in CIK cell cultures. CIK-mediated cellular lysis is non-major histocompatibility antigen (MHC) restricted. The cytotoxic effect of CIK cells against tumor targets is blocked by antibodies directed against lymphocyte function-associated antigen (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1).

  View details for Web of Science ID A1993MW61900009

  View details for PubMedID 7694868

• AIDS - THE WHOLE-BODY VIEW CURRENT BIOLOGY Weissman, I. L. 1993; 3 (11): 766-769

  View details for Web of Science ID A1993MR82300008

  View details for PubMedID 15335842

• THE IN-VIVO EFFECTS OF STEEL FACTOR ON NATURAL-KILLER LINEAGE CELLS IN MURINE SPLEEN AND BONE-MARROW NATURAL IMMUNITY Miller, S. C., Fleming, W. H., Zsebo, K. M., Weissman, I. L. 1993; 12 (6): 293-301

  Abstract

  Steel factor (S1F), also known as stem cell factor, is a potent growth stimulator of hemopoietic progenitor cells. In the context of transplantation of hemopoietic cells to irradiated allogeneic hosts, natural killer (NK) cells exert restrictive control on hemopoietic cell proliferation, and are regularly found in elevated concentration in areas of intense hemopoiesis. The present study was designed to examine the effects with time of S1F in vivo on the numbers of NK cells, identified by the presence of the NK 1.1 surface molecule, in the spleen and bone marrow. Throughout the first 3 days of S1F exposure, NK cell numbers, in spite of rapid (1 day) and significant increases in the other hemopoietic cell lineages, did not change in either the spleen or the bone marrow. However, NK cells were increased 2-fold in both organs by 7 days of S1F exposure. At this time, immature granuloid and erythroid cells and the large lymphoid cells in the spleen had more than doubled their respective control numbers and in the bone marrow, immature granuloid cells increased by 47% of control levels. The presence of a late, but not early, influence of S1F on NK cells of the spleen and bone marrow suggests an indirect effect of S1F on this lineage, occurring only when S1F-stimulated hemopoiesis becomes sufficiently intense, providing, thus, an abundance of NK cell targets.

  View details for Web of Science ID A1993MF46400001

  View details for PubMedID 7505667

• NEITHER MACROMOLECULAR-SYNTHESIS NOR MYC IS REQUIRED FOR CELL-DEATH VIA THE MECHANISM THAT CAN BE CONTROLLED BY BCL-2 MOLECULAR AND CELLULAR BIOLOGY Vaux, D. L., Weissman, I. L. 1993; 13 (11): 7000-7005

  Abstract

  Expression of c-myc and macromolecular synthesis have been associated with physiological cell death. We have studied their requirement for the death of factor (interleukin-3)-dependent cells (FDC-P1) bearing an inducible bcl-2 expression construct. FDC-P1 cells expressing bcl-2 turned off expression of c-myc when deprived of interleukin-3 but remained viable as long as bcl-2 was maintained. A subsequent decline in Bcl-2 allowed the cells to undergo apoptosis directly from G0, in the absence of detectable c-myc expression. Thus c-myc expression may lead to apoptosis in some cases but is not directly involved in the mechanism of physiological cell death that can be controlled by Bcl-2. The macromolecular synthesis inhibitors actinomycin D and cycloheximide triggered rapid cell death of FDC-P1 cells in the presence of interleukin-3, but the cells could be protected by Bcl-2. Thus, the cell death machinery can exist in a quiescent state and can be activated by mechanisms that do not require synthesis of RNA or protein.

  View details for Web of Science ID A1993MC84400038

  View details for PubMedID 7692234

• HOW THE IMMUNE-SYSTEM DEVELOPS SCIENTIFIC AMERICAN Weissman, I. L., COOPER, M. D. 1993; 269 (3): 64-71

  View details for Web of Science ID A1993LR86900009

  View details for PubMedID 8211092

• FUNCTIONAL-HETEROGENEITY IS ASSOCIATED WITH THE CELL-CYCLE STATUS OF MURINE HEMATOPOIETIC STEM-CELLS JOURNAL OF CELL BIOLOGY Fleming, W. H., ALPERN, E. J., Uchida, N., Ikuta, K., Spangrude, G. J., Weissman, I. L. 1993; 122 (4): 897-902

  Abstract

  Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic cell lineages while retaining their capacity for self renewal. One of the predictions of this model is the existence of a heterogeneous pool of HSCs, some members of which are destined to become lineage restricted progenitor cells while others function to renew the stem cell pool. To test whether HSCs are heterogeneous with respect to cell cycle status, we determined the fraction of phenotypically defined murine HSCs (Thy1.1lo Lin-/lo Sca-1+) that contain > 2n amount of DNA as measured by propidium iodide staining, Hoechst dye uptake and [3H]thymidine labeling; that fraction is 18-22%. In contrast, in the developing fetal liver, 40% of HSCs are in the S/G2/M phases of the cell cycle. Those HSCs which exhibit a low level of staining with rhodamine 123 are almost exclusively in G0/G1 (97%) whereas only 70% of HSCs which stain brightly for rhodamine 123 are in G0/G1. The injection of 100 G0/G1 HSCs rescued 90% of lethally irradiated mice in contrast to 100 S/G2/M HSCs, which protected only 25% of lethally irradiated recipients. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of 100 G0/G1 HSCs compared to recipients of 100 S/G2/M cells. These data indicate that a significant proportion of HSCs are actively proliferating during steady state hematopoiesis and that this subpopulation of cells exhibits reduced stem cell activity.

  View details for Web of Science ID A1993LR62900013

  View details for PubMedID 8349737

  View details for PubMedCentralID PMC2119585

• ANALYSIS OF CANDIDATE HUMAN BLOOD STEM-CELLS IN HUMANIZED IMMUNE-DEFICIENCY SCID MICE LEUKEMIA Peault, B., Weissman, I., Baum, C. 1993; 7: S98-S101

  Abstract

  Immune deficient SCID mice support the hematopoietic development of surgically implanted human fetal thymus and bone. The usability of the resulting SCID-hu hematolymphoid chimaera as an assay system for human candidate blood stem cells was suggested by the observation that allogeneic CD34+ cells sorted from fetal liver and bone marrow can engraft and differentiate in the transplanted human thymus and bone marrow. The SCID-hu mouse has been further used, in combination with a mouse bone marrow stromal cell line that supports human myeloid and B lymphoid differentiation in vitro, to identify a minor subset of fetal CD34-expressing cells that exhibit multilineage hematopoietic potentialities.

  View details for Web of Science ID A1993MA47900023

  View details for PubMedID 7689676

• ALPHA-4-BETA-7-INTEGRIN MEDIATES LYMPHOCYTE BINDING TO THE MUCOSAL VASCULAR ADDRESSIN MADCAM-1 CELL Berlin, C., BERG, E. L., Briskin, M. J., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L., Hamann, A., BUTCHER, E. C. 1993; 74 (1): 185-195

  Abstract

  The mucosal vascular addressin, MAdCAM-1, is an immunoglobulin superfamily adhesion molecule for lymphocytes that is expressed by mucosal venules and helps direct lymphocyte traffic into Peyer's patches (PP) and the intestinal lamina propria. We demonstrate that the lymphocyte integrin alpha 4 beta 7, also implicated in homing to PP, is a receptor for MAdCAM-1. Certain antibodies to alpha 4 and beta 7 integrin chains but not to the beta 2 integrin LFA-1 inhibit lymphocyte binding to purified MAdCAM-1 and to MAdCAM-1 transfectants. Lymph node lymphocytes, alpha 4 beta 7+ TK1 lymphoma cells, and a beta 7-transfected variant of an alpha 4+ B cell line, 38C13, bind constitutively to MAdCAM-1. Binding is enhanced by Mn(++)-induced integrin activation. The related integrin alpha 4 beta 1 supports efficient binding to VCAM-1 but not to MAdCAM-1, even after integrin activation, indicating that MAdCAM-1 is a preferential ligand for alpha 4 beta 7. Alpha 4 beta 7 can also bind VCAM-1, but this requires greater integrin activation than binding to MAdCAM-1. The findings imply a selective role for the interaction of alpha 4 beta 7 and MAdCAM-1 lymphocyte in homing to mucosal sites.

  View details for PubMedID 7687523

• CLONING AND CHARACTERIZATION OF CYCLOPHILIN C-ASSOCIATED PROTEIN - A CANDIDATE NATURAL CELLULAR LIGAND FOR CYCLOPHILIN-C PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Friedman, J., Trahey, M., Weissman, I. 1993; 90 (14): 6815-6819

  Abstract

  We report the protein purification and the cloning and characterization of a cDNA encoding the proteins that bind with high affinity to cyclophilin C (Cyp-C) in the absence of cyclosporin A. Transfection of this cDNA into COS cells directs the production of a glycoprotein of 77 kDa that binds to Cyp-C in the absence, but not the presence, of cyclosporin A. Homology comparisons reveal that this protein and gene, termed CyCAP for Cyp-C-associated protein, possess a cysteine-rich domain (scavenger receptor cysteine-rich domain) found in a variety of cell-surface molecules; the rest of the sequence is apparently specific. This result raises the possibility that Cyp-C serves as a mediator or regulator of an as-yet-unidentified signal or cellular process initiated via the Cyp-C-associated protein.

  View details for Web of Science ID A1993LM68100090

  View details for PubMedID 8341703

• CHARACTERIZATION OF POSTTRANSPLANT LYMPHOMAS THAT EXPRESS T-CELL ASSOCIATED MARKERS - IMMUNOPHENOTYPES, MOLECULAR-GENETICS, CYTOGENETICS, AND HETEROTRANSPLANTATION IN SEVERE COMBINED IMMUNODEFICIENT MICE BLOOD Waller, E. K., ZIEMIANSKA, M., Bangs, C. D., Cleary, M., Weissman, I., Kamel, O. W. 1993; 82 (1): 247-261

  Abstract

  Immunosuppressed individuals are at high risk for the development of hematologic malignancies. The typical lymphomas arising in organ transplant recipients are B-cell non-Hodgkin's lymphomas that contain Epstein-Barr virus (EBV) DNA sequences. We investigated the characteristics of posttransplant lymphomas that lacked expression of the usual markers associated with EBV transformation. We describe four large-cell lymphomas seen recently at our institution. Two of these four cases were CD4+, one was CD8+, and in one staining for CD4 and CD8 expression was not performed. One CD4+ lymphoma was a CD30+, EBV- large-cell lymphoma from a 65-year-old kidney transplant recipient, the second was an EBV+ large-cell lymphoma from a 25-year-old heart transplant patient. Two T-cell lymphomas were EBV+ and had clonal T-cell receptor beta gene rearrangements. The other two lymphomas expressed T-cell markers CD4 and CD43, and lacked expression of B-cell markers CD19, CD20, CD21, CD22, CD23, and surface Ig. Both CD4+ lymphomas were tumorigenic after their heterotransplantation into severe combined immunodeficient (SCID) mice. Cytogenetics, immunophenotyping, and genotyping of the secondary tumors from SCID mice showed their clonality and identity with the patients' primary tumors. Novel CD4+ lymphoma cell lines, LH521/4 and LK418/4, were established from tumors that had been passaged in SCID mice. An immunodeficient environment may facilitate the growth of these T-cell or biphenotypic lymphomas; the etiology of their genesis can include transformation with EBV and other, as yet unidentified mechanisms.

  View details for Web of Science ID A1993LL36600033

  View details for PubMedID 8100721

• DIFFERENTIATION OF CD3-4-8- HUMAN FETAL THYMOCYTES INVIVO - CHARACTERIZATION OF A CD3-4+8- INTERMEDIATE JOURNAL OF EXPERIMENTAL MEDICINE Kraft, D. L., Weissman, I. L., Waller, E. K. 1993; 178 (1): 265-277

  Abstract

  Human thymocyte differentiation was examined by injecting fetal thymic progenitor populations into human thymic xenografts in SCID-hu mice. Thymic progenitors were fluorescently labeled with the lipophilic dye PKH2. The phenotypes of their progeny could be identified by flow cytometric analysis of cells with a very high fluorescent PKH2 signal. Intrathymic injection of purified triple negative (TN) CD3-4-8- thymocytes resulted in the sequential appearance of CD3-4+8-, CD3-4+8+, and CD3+4+8+ cells, with the subsequent appearance of small numbers of phenotypically mature CD3+4+8- and CD3+4-8+ cells over a 4-d period. Sorted CD3-4+8- thymocytes injected intrathymically rapidly differentiated to CD4+8+ cells. CD4+8+ fetal thymocytes in cell cycle differentiated into phenotypically mature CD3+4+8- and CD3+4-8+ populations, whereas nondividing CD4+8+ cells failed to differentiate after intrathymic transfer. The number of cell divisions that occurred between the injection of TN thymocytes and their progeny at different time points was estimated based on the decrease in the intensity of the PKH2 label. The average length of the cell cycle for the TN population was calculated to be 24 h. The SCID-hu model thus provides a useful tool for studying the kinetics of cell division and differentiation of human thymocytes in vivo.

  View details for Web of Science ID A1993LH54900024

  View details for PubMedID 8315382

• LYAR, A NOVEL NUCLEOLAR PROTEIN WITH ZINC-FINGER DNA-BINDING MOTIFS, IS INVOLVED IN CELL-GROWTH REGULATION GENES & DEVELOPMENT Su, L. S., HERSHBERGER, R. J., Weissman, I. L. 1993; 7 (5): 735-748

  Abstract

  A cDNA encoding a novel zinc finger protein has been isolated from a mouse T-cell leukemia line on the basis of its expression of a Ly-1 epitope in a lambda gt11 library. The putative gene was mapped on mouse chromosome 1, closely linked to Idh-1, but not linked to the Ly-1 (CD5) gene. The cDNA is therefore named Ly-1 antibody reactive clone (LYAR). The putative polypeptide encoded by the cDNA consists of 388 amino acids with a zinc finger motif and three copies of nuclear localization signals. Antibodies raised against a LYAR fusion protein reacted with a protein of 45 kD on Western blots and by immunoprecipitation. Immunolocalization indicated that LYAR was present predominantly in the nucleoli. The LYAR mRNA was not detected in brain, thymus, bone marrow, liver, heart, and muscle. Low levels of LYAR mRNA were detected in kidney and spleen. However, the LYAR gene was expressed at very high levels in immature spermatocytes in testis. The LYAR mRNA is present at high levels in early embryos and preferentially in fetal liver and fetal thymus. A number of B- and T-cell leukemic lines expressed LYAR at high levels, although it was not detectable in bone marrow and thymus. During radiation-induced T-cell leukemogenesis, high levels of LYAR were expressed in preleukemic thymocytes and in acute T leukemia cells. Fibroblast cells overexpressing the LYAR cDNA from a retrovirus vector, though not phenotypically transformed in vitro, had increased ability to form tumors in nu/nu mice. Therefore, LYAR may function as a novel nucleolar oncoprotein to regulate cell growth.

  View details for Web of Science ID A1993LC49800002

  View details for PubMedID 8491376

• STEEL FACTOR INFLUENCES THE DISTRIBUTION AND ACTIVITY OF MURINE HEMATOPOIETIC STEM-CELLS INVIVO PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fleming, W. H., ALPERN, E. J., Uchida, N., Ikuta, K., Weissman, I. L. 1993; 90 (8): 3760-3764

  Abstract

  To determine the effects of steel factor (SIF) on the number and distribution of phenotypically defined hematopoietic stem cells in vivo, mice were treated with continuous s.c. infusions of SIF for up to 7 days. The bone marrow demonstrated a transient 5-fold increase in the number of c-kit-positive lineage-negative/low cells with no change in cellularity. The radioprotective capacity of bone marrow cells was significantly reduced, and a 30% decrease in Thylo Lin-/lo Sca-1+ stem cells (Sca+ cells) was observed. In marked contrast, in the spleen a 2-fold increase in cellularity was accompanied by a 24-fold increase in c-kit-positive lineage-negative/low cells. SIF-treated spleen cells provided increased radioprotection and a corresponding 4-fold increase in the number of Sca+ cells. In the peripheral blood, an increase in both neutrophils and lymphocytes resulted; however, the number of c-kit-positive lineage-negative/low cells remained < 1%. SIF produced a 25-fold increase in radioprotection capacity and a 20-fold increase in the number of Sca+ cells in the peripheral blood. The increased radioprotection capacity of both the spleen cells and peripheral blood cells was associated with donor-derived, long-term multilineage reconstitution of recipient mice. The total number of Sca+ cells isolated per mouse after SIF treatment was not significantly increased. These results show that exogenous SIF treatment causes a redistribution of Sca+ cells and stem cell activity while having little effect on the total number of stem cells in the mouse.

  View details for Web of Science ID A1993KX81600133

  View details for PubMedID 7682717

• HETEROGENEITY OF HEMATOPOIETIC STEM-CELLS CURRENT OPINION IN IMMUNOLOGY Uchida, N., Fleming, W. H., ALPERN, E. J., Weissman, I. L. 1993; 5 (2): 177-184

  Abstract

  Hematopoietic stem cells are capable of multi-lineage differentiation to all blood cell types as well as self-renewal and radioprotection. Thy-1.1lo Lin-/lo Sca-1+ cells are a heterogeneous mixture of quiescent and self-renewing hematopoietic stem cells as well as multi-lineage expanding cells.

  View details for Web of Science ID A1993KY90900002

  View details for PubMedID 8099486

• A MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF PROGRAMMED CELL-DEATH IN BOTRYLLUS-SCHLOSSERI (TUNICATA, ASCIDIACEA) CELL AND TISSUE RESEARCH Lauzon, R. J., Patton, C. W., Weissman, I. L. 1993; 272 (1): 115-127

  Abstract

  The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction.

  View details for Web of Science ID A1993KT71600014

  View details for PubMedID 8386984

• A COLONIAL INVERTEBRATE SPECIES THAT DISPLAYS A HIERARCHY OF ALLORECOGNITION RESPONSES BIOLOGICAL BULLETIN Rinkevich, B., Saito, Y., Weissman, I. L. 1993; 184 (1): 79-86

  View details for Web of Science ID A1993KM31600008

• THY-1 EXPRESSING CD34+ HUMAN-CELLS EXPRESS MULTIPLE HEMATOPOIETIC POTENTIALITIES INVITRO AND IN SCID-HU MICE NOUVELLE REVUE FRANCAISE D HEMATOLOGIE Peault, B., Weissman, I. L., Buckle, A. M., Tsukamoto, A., Baum, C. 1993; 35 (1): 91-93

  Abstract

  We have detected a minor subset of fetal liver and bone marrow CD34+ cells that coexpress the Thy-1 antigen at their surface. In immune-deficient SCID mice engrafted with human fetal hematopoietic tissues, CD34+ Thy-1+ reconstitute the full range of thymic lymphocytes and yield a progeny of myeloid and B cells in the human bone marrow. The frequency of cells initiating long-term human myeloid and B lymphocytes in vitro cultures at the contact of the Sys-1 stromal cell line was also found to be 10-15 times higher among CD34+ Thy-1+ cells than in the Thy-1- counterpart population. We propose that CD34+ Thy-1+ cells include candidate human hematopoietic stem cells.

  View details for Web of Science ID A1993KW53500023

  View details for PubMedID 7685521

• The Peyer's patch homing receptor. Current topics in microbiology and immunology Hu, M. C., Holzmann, B., Crowe, D. T., Neuhaus, H., Weissman, I. L. 1993; 184: 125-138

  View details for PubMedID 8313716

• The granzyme a gene: a marker for cytolytic lymphocytes in vivo Cytotoxic Cells Griffiths, G., Alpert, S., Herschberger, R., Su, L., Weissman, I. Birkhauser. 1993: 273–277
• T lymphocyte development from fetal hematopoietic stem cells Sem Dev Biol Ikuta, K., Weissman, I. 1993; 4: 371-378
• Sequential occurrence of positive and negative selection during T lymphocyte maturation Biomedical Sciences and Symposia on Molecular Mechanisms of Immunological Self-Recognition Guidos, C., Weissman, I. Academic Press. 1993: 137–147
• Isolation and characterization of hematopoietic progenitor and stem cells Bone Marrow Transplantation Baum, C., Uchida, N., Peault, B., Weissman, I. Blackwell Scientific. 1993: 53–71
• Characterization and purification of candidate mouse and human hematopoietic stem cells Application of Basic Science to Hematopoiesis and Treatment of Disease Peault, B., Baum, C., Weissman, I. Raven Press. 1993
• THE USE OF GRANZYME A AS A MARKER OF HEART-TRANSPLANT REJECTION IN CYCLOSPORINE OR ANTI-CD4 MONOCLONAL ANTIBODY-TREATED RATS TRANSPLANTATION CHEN, R. H., IVENS, K. W., Alpert, S., Billingham, M. E., Fathman, C. G., Flavin, T. F., Shizuru, J. A., Starnes, V. A., Weissman, I. L., Griffiths, G. M. 1993; 55 (1): 146-153

  Abstract

  Granzyme A is a serine protease expressed by populations of human and mouse natural killer cells and activated CD4+ and CD8+ cytotoxic lymphocytes; its expression marks a subset of inflammatory cells in allografts, autoimmune diabetes, and a number of other inflammatory lesions. In order to describe more completely the correlation between granzyme A expression and the presence of in vivo cytolytic effects, we grafted allogeneic rat hearts with vascular anastomoses in a heterotopic location, and treated the hosts with either cyclosporine, anti-CD4 monoclonal antibody (MRC OX38), or no therapy. The grafts were evaluated by palpation for cardiac functions, by immunohistochemistry for CD4/CD8 expression, by hematoxylin-and-eosin staining for inflammatory infiltration, and by in situ hybridization for granzyme A expression. The appearance of granzyme A+ cells in untreated allografts preceded both functional and standard histopathological and immunohistochemical evidence of graft rejection by two days. In donor-recipient combinations where cyclosporine and anti-CD4 treatments allowed indefinite allograft survival, the allografts showed minimal numbers of granzyme A+ cells, whether cellular infiltrates developed or not. The number of granzyme A+ cells present in the cardiac allografts in treated and untreated animals correlated with either current or impending episodes of rejection. The early time course of granzyme A expression suggests that it can be used as an early and reliable marker of graft rejection.

  View details for Web of Science ID A1993KH66000027

  View details for PubMedID 8420039

• PREVENTION OF PROGRAMMED CELL-DEATH IN CAENORHABDITIS-ELEGANS BY HUMAN BCL-2 SCIENCE Vaux, D. L., Weissman, I. L., Kim, S. K. 1992; 258 (5090): 1955-1957

  Abstract

  Programmed cell death is a physiological process that eliminates unwanted cells. The bcl-2 gene regulates programmed cell death in mammalian cells, but the way it functions is not known. Expression of the human bcl-2 gene in the nematode Caenorhabditis elegans reduced the number of programmed cell deaths, suggesting that the mechanism of programmed cell death controlled by bcl-2 in humans is the same as that in nematodes.

  View details for Web of Science ID A1992KD08800038

  View details for PubMedID 1470921

• GENOMIC ORGANIZATION OF THE MOUSE GRANZYME-A GENE - 2 MESSENGER-RNAS ENCODE THE SAME MATURE GRANZYME-A WITH DIFFERENT LEADER PEPTIDES JOURNAL OF BIOLOGICAL CHEMISTRY HERSHBERGER, R. J., Gershenfeld, H. K., Weissman, I. L., Su, L. S. 1992; 267 (35): 25488-25493

  Abstract

  Granzyme A is a serine protease that, together with the other granular components of cytotoxic T lymphocyte (CTL) cells, has been implicated in the cytolysis process. We report here two different messages and the genomic organization of the mouse granzyme A gene. The granzyme A gene is composed of six exons spanning 7 kilobases. Alternative splicing of the second exon results in the two transcripts. The two mRNA species encode the same mature granzyme A protein but with different leader sequences. The first (HF1) encodes a typical leader signal sequence similar to other granzymes, but the second (HF2) putative leader sequence is different and less hydrophobic. Both messages are present in cultured CTL cell lines and in normal lymphoid tissues. They are both induced when CTL cells are activated in vitro or in vivo. Both messages can be translated in vitro, although the HF1 message appears to be much more efficient as a template. The putative 5' promoter region of the HF gene sequenced (500 base pairs of upstream sequences) contains no well defined promoter sequences aside from the TATA box. The results suggest that (a) granzyme A may be produced with putative different leader sequences from two different mRNAs; (b) this may provide a model system for studying alternate splicing and the evolution of a complex enzymatic system in an organelle; and (c) the genomic DNA reported will be useful for studying transcription regulations involved in controlling the specific expression pattern of this gene.

  View details for Web of Science ID A1992KB60300087

  View details for PubMedID 1460043

• TRANSFECTION OF MOUSE CYTOTOXIC LYMPHOCYTE-T WITH AN ANTISENSE GRANZYME-A VECTOR REDUCES LYTIC ACTIVITY JOURNAL OF IMMUNOLOGY Talento, A., Nguyen, M., Law, S., Wu, J. K., Poe, M., Blake, J. T., Patel, M., Wu, T. J., MANYAK, C. L., SILBERKLANG, M., Mark, G., Springer, M., Sigal, N. H., Weissman, I. L., Bleackley, R. C., Podack, E. R., Tykocinski, M. L., Koo, G. C. 1992; 149 (12): 4009-4015

  Abstract

  Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.

  View details for Web of Science ID A1992KB98200032

• HUMAN T-CELL DEVELOPMENT IN SCID-HU MICE - STAPHYLOCOCCAL ENTEROTOXINS INDUCE SPECIFIC CLONAL DELETIONS, PROLIFERATION, AND ANERGY BLOOD Waller, E. K., SENMAJUMDAR, A., Kamel, O. W., HANSTEEN, G. A., Schick, M. R., Weissman, I. L. 1992; 80 (12): 3144-3156

  Abstract

  SCID-hu mice provide an in vivo model for studying the events of normal intrathymic human T-cell development and differentiation. We injected SCID-hu mice with staphylococcal enterotoxins (SE) and determined their effects on the development and responsiveness of human T-cell populations defined by their expression of CD4 and CD8, and the type of V beta molecule in their T-cell receptors. After single intraperitoneal injections of SEB or SEE, we observed specific effects on thymic T cells expressing a cognate V beta T-cell receptor (TCR) (V beta 12.1 in the case of SEB-treated SCID-hu mice and V beta 8.1 in the case of SEE-treated mice) using both immunohistochemical staining of thymic frozen sections and flow cytometric analyses. An injection of SEB resulted in a 32% decrease in the total percentages of V beta 12.1+ cells in thymic sections after 2 days, with the greatest effect seen in the medulla, without a demonstrable effect on V beta 5.2/5.3+ or V beta 8.1+ cells. Fluorescence-activated cell sorter analysis demonstrated that TCRhi thymocytes expressing a cognate V beta TCR declined transiently by 35% to 45% 1 to 2 days after the injection of SE. Analysis of thymic subpopulations showed decreases in the TCRhi CD4+8- and CD4-8+ cells and an increase in TCRlo CD4-8+ cells. Multiple injections of SE resulted in 50% to 60% decreases in cognate V beta TCR+ CD4+8- populations. Thymocytes prepared from SE-treated SCID-hu mice demonstrated specific anergy to the SE to which they had previously been exposed in vivo, but had a normal proliferative response to other superantigens in an in vitro assay. In contrast to the effects on thymic T cells, single injections of SE resulted in a twofold increase in the total numbers of circulating CD4+8- and CD4-8+ human T cells and a fourfold to eightfold increase in T cells expressing a cognate V beta TCR. Using SE as superantigens in SCID-hu mice, we have been able to induce antigen-specific clonal deletions, anergy, and proliferation of human T cells.

  View details for Web of Science ID A1992KC83800023

  View details for PubMedID 1467521

• ALLOGENEIC RESPONSES BETWEEN 3 REMOTE POPULATIONS OF THE COSMOPOLITAN ASCIDIAN BOTRYLLUS-SCHLOSSER ZOOLOGICAL SCIENCE Rinkevich, B., Shapira, M., Weissman, I. L., Saito, Y. 1992; 9 (5): 989-994

  View details for Web of Science ID A1992JX97500008

• CLONING AND EXPRESSION OF MOUSE INTEGRIN-BETA(P)(BETA(7)) - A FUNCTIONAL-ROLE IN PEYER PATCH-SPECIFIC LYMPHOCYTE HOMING PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hu, M. C., Crowe, D. T., Weissman, I. L., Holzmann, B. 1992; 89 (17): 8254-8258

  Abstract

  Lymphocytes express integrin receptors, termed lymphocyte Peyer's patch high endothelial venule (HEV) adhesion molecules (LPAMs), that mediate their organ-specific adhesion to specialized HEVs found in mucosal lymphoid organs (Peyer's patches). LPAM-1 consists of a murine integrin alpha 4 noncovalently associated with integrin beta p. Here, we describe the cloning and expression of a mouse cDNA encoding beta p, which is an 806-amino acid transmembrane glycoprotein. The genomic Southern blot analysis indicates that beta p is the murine homologue of human beta 7. The function of alpha 4 beta 7 as a Peyer's patch-specific adhesion molecule was tested directly by expression of the murine beta 7 cDNA in an alpha 4+ beta 7-B-cell line or coexpression of the alpha 4 and beta 7 cDNAs in an alpha 4-beta 7-T-cell line. The transfected cells exhibited a new Peyer's patch-specific adhesive phenotype that could be specifically blocked by monoclonal antibodies against alpha 4 and beta 7. Moreover, an anti-beta 7 monoclonal antibody specifically blocked binding of normal lymphocytes to Peyer's patch HEV but did not inhibit their binding to peripheral lymph node HEVs, indicating that beta 7 is a unique component of the Peyer's patch-specific homing receptor.

  View details for Web of Science ID A1992JL61400085

  View details for PubMedID 1518854

• UNEXPECTED EFFECTS OF THE SEVERE COMBINED IMMUNODEFICIENCY MUTATION ON MURINE LYMPHOMAGENESIS JOURNAL OF EXPERIMENTAL MEDICINE Lieberman, M., HANSTEEN, G. A., Waller, E. K., Weissman, I. L., SENMAJUMDAR, A. 1992; 176 (2): 399-405

  Abstract

  Strain C.B17 scid/scid (SCID) mice, which lack functional T and B lymphocytes, show heightened susceptibility to the induction of thymic lymphomas by x-irradiation. Susceptibility is highest in thymus-chimeric SCID-BL mice (thymectomized SCID mice bearing a C57BL thymus graft). All SCID-BL lymphomas originate in the cells of the thymic graft (C57BL type) and lack murine leukemia virus expression. Both SCID and SCID-BL lymphomas are phenotypically CD4-8+ and/or CD4+8+, but only the SCID-BL tumors express CD3. Injection of C57BL or BALB/c bone marrow into irradiated SCID-BL mice prevents lymphoma development, but SCID marrow is completely ineffective. The results suggest that the scid condition enhances the activity of a putative lymphomagenic agent induced in the bone marrow by x-irradiation and that C57BL thymic cells are highly sensitive targets. Moreover, the failure of SCID bone marrow to protect against lymphomagenesis vs. the efficacy of marrow from immunocompetent donors points to involvement of T or B lineage cells in this process.

  View details for Web of Science ID A1992JF80300009

  View details for PubMedID 1500852

• INCIDENTS OF REJECTION AND INDIFFERENCE IN FU/HC INCOMPATIBLE PROTOCHORDATE COLONIES JOURNAL OF EXPERIMENTAL ZOOLOGY Rinkevich, B., Weissman, I. L. 1992; 263 (1): 105-111

  Abstract

  We test here for the existence of specific alloimmune memory in the rejection responses of the colonial tunicate Botryllus schlosseri. Colony specificity in these organisms is controlled by the Fu/HC locus. Rejection occurs only between colonies that do not share any allelic determinant at this locus. Two sets of experiments were conducted: 1) Forty-nine pairs of nonfusible oozooids were interacted naturally along a period of several months. They rejected, disconnected, and in 20% of the cases, interacted again. 2) Repeated colony allorecognition assays were done on 15 pairs of interacting subclones (up to 5 consecutive tests/pair). Major results indicate: 1) Not all ampulla-ampulla interactions resulted in necrotic areas, points of rejection (PORs). 2) A full repertoire of PORs was attained within the first 10 days. Thereafter, no more PORs were added. 3) The outcome of indifference (cases where ampulla-ampulla contacts did not result in rejection) was repeatedly recorded in multiple tests, and its frequency increased in the secondary and tertiary tests along a set of 5 consecutive tests. It is concluded that allospecific memory, as measured by an accelerated production of PORs and amplification in their number, was not characteristic of the Botryllus rejection phenomenon, which, however, reveals the qualities of a low responder. These results are discussed in the light of some aspects of tolerance in the mammalian system.

  View details for Web of Science ID A1992JF66000010

  View details for PubMedID 1645117

• DIFFERENTIATION OF CD3-4-8- THYMOCYTES IN SHORT-TERM THYMIC STROMAL CELL-CULTURE JOURNAL OF EXPERIMENTAL MEDICINE SENMAJUMDAR, A., Lieberman, M., Alpert, S., Weissman, I. L., Small, M. 1992; 176 (2): 543-551

  Abstract

  We have investigated the ability of a heterogeneous thymic stromal cell (HTSC) culture system to promote in vitro differentiation of CD3-4-8- thymocytes. Culture of purified murine CD3-4-8- thymocytes on HTSC for 1 d resulted in the appearance of CD4+8+ cells, which did not occur when the sorted cells were maintained in medium alone. It is remarkable that when the culture period was extended to 2 d, CD3-4-8- progenitors differentiated further to CD4+8- and CD4-8+ cells, which also expressed high levels of TCR-CD3. This rapid differentiation on stroma in vitro appears to outpace parallel development in vivo. The differentiation potential of a subset of CD3-4-8- thymocytes that express high levels of a marker of normal and neoplastic thymic progenitors, the 1C11 antigen, was examined next. 1C11hiCD3-4-8- cells also gave rise to CD4-8+ and CD4+8+ populations after 1 d of culture on HTSC. Extending the culture period to 2 d resulted in a significant percentage of CD3-expressing cells that were CD4+8+, CD4+8- and CD4-8+ cells. These results suggest that in the in vitro HTSC culture system, various subsets of immature thymocytes can differentiate into all the mature phenotypes of cells normally found in the adult mouse thymus. This may provide a novel and rapid assay for thymic progenitors.

  View details for Web of Science ID A1992JF80300023

  View details for PubMedID 1386875

• BCL-2 PREVENTS DEATH OF FACTOR-DEPRIVED CELLS BUT FAILS TO PREVENT APOPTOSIS IN TARGETS OF CELL-MEDIATED KILLING INTERNATIONAL IMMUNOLOGY Vaux, D. L., Aguila, H. L., Weissman, I. L. 1992; 4 (7): 821-824

  Abstract

  'Programmed cell death' has been used to describe the death of cells killed by cytotoxic T cells or growth factor deprivation. Although bcl-2 can prevent death of cells deprived of growth factor, it failed to protect cells against T cell killing. In spite of bcl-2 expression, the DNA of targeted cells was degraded into nucleosome-sized fragments. Therefore the early steps in apoptosis induced by factor deprivation differ from those triggered by cytotoxic T cells, although they share a common final pathway featuring degradation of the DNA and loss of cytoplasmic membrane integrity.

  View details for Web of Science ID A1992JE65500014

  View details for PubMedID 1498090

• ALLOGENEIC RESORPTION IN COLONIAL PROTOCHORDATES - CONSEQUENCES OF NONSELF RECOGNITION DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Rinkevich, B., Weissman, I. L. 1992; 16 (4): 275-286

  Abstract

  Colonial tunicates (protochordates of the subfamily Botryllinae) have the property of forming natural chimeras in the wild or in the laboratory with other members of the same species if both individuals share at least one allele at a single highly polymorphic locus, termed the fusibility/histocompatibility (Fu/HC) locus. Laboratory studies revealed that after the establishment of a common blood circulatory system between a fusible pair one of the colonies in the chimera is usually resorbed, a phenomenon that occurs after an interval of 1 week or extends to up to 8 months. In the present article, we review this allogeneic resorption in Botryllus schlosseri, a cosmopolitan protochordate. The studies on allogeneic resorption in B. schlosseri revealed 13 typical characteristics. However, several processes interrupt successful resorption, such as the retreat growth phenomenon, chimeric death, and others. It was also found that allogeneic resorption is not only genetically controlled but is also controlled by a polymorphic hierarchial phenomenon, including the Fu/HC locus and additional unrelated loci. One basic rule for this genetic system is that colonies heterozygotic on the resorption elements will resorb more homozygotic partners. This colony resorption, expressed by a morphological elimination of one partner, is probably manifested by cellular elements circulating in the tunicate blood system. However, it was also shown that some blood-borne cells of the inferior partner, such as the stem cells, may escape resorption, a phenomenon that raises the threat of germ/somatic cell parasitism.

  View details for Web of Science ID A1992JB85500002

  View details for PubMedID 1505688

• A CYCLICAL, DEVELOPMENTALLY-REGULATED DEATH PHENOMENON IN A COLONIAL UROCHORDATE DEVELOPMENTAL DYNAMICS Lauzon, R. J., Ishizuka, K. J., Weissman, I. L. 1992; 194 (1): 71-83

  Abstract

  Botryllus schlosseri is a colonial ascidian whose asexually derived, clonally modular systems of zooids exhibit developmental synchrony. The blastogenic cycle culminates in a phase of programmed cell and zooid death called takeover, in which all functional zooids die over a 30 hr period, and are replaced by a new generation of individuals. Because of the weekly recurrence and magnitude of visceral death in this model organism, we have begun to characterize the mechanisms that govern takeover. Here we describe a monoclonal antibody (B3F12.9) that recognizes a novel 57 Kd polypeptide (under reducing conditions) localized to the perivisceral extracellular matrix (PVEM) of buds and zooids, as well as blood cells of Botryllus by immunofluorescence and immunogold labeling of tissue sections. During their active feeding phase, zooids exhibited a uniform labeling pattern of PVEM along their anteroposterior (A-P) axis. At the onset of takeover (T = 3 hr), B3F12.9 immunostaining became diffuse or absent at the anterior end, which paralleled the axis of contraction of the dying zooid, whereas the posterior end retained its labeling integrity. During mid (T = 15 hr) to late (T = 28 hr) takeover, issue damage was extensive, large blood macrophages and other B3F12.9 immunoreactive blood cells invaded the peribranchial cavity, whereas PVEM labeling gradually disappeared along the entire A-P axis. These findings indicate that takeover is a dynamic process in which extracellular matrix breakdown proceeds in a polarized fashion, beginning at the anterior end of each zooid and gradually propagating toward the posterior end.

  View details for Web of Science ID A1992JN15200008

  View details for PubMedID 1421521

• MOUSE MRP8 AND MRP14, 2 INTRACELLULAR CALCIUM-BINDING PROTEINS ASSOCIATED WITH THE DEVELOPMENT OF THE MYELOID LINEAGE BLOOD Lagasse, E., Weissman, I. L. 1992; 79 (8): 1907-1915

  Abstract

  MRP8 and MRP14 are two S100-like calcium-binding proteins of unknown function, associated with numbers of human inflammatory disorders. Both molecules have been described as L1 complex, cystic fibrosis antigen, or p8 and p14. We report here the cloning of mouse MRP8 and MRP14 and their pattern of expression during hematopoiesis. Mouse MRP8 and MRP14 proteins share 59% identity with their human counterparts, but they are more divergent than the other members of the S100 protein family. Mouse MRP proteins are coexpressed in fetal myeloid progenitors, where they are detected as early as day 11 of gestation. In fetal liver and yolk sac, MRP+ cell populations increased in number, in association with the development of the myeloid lineage. In adult mouse, we identified MRP8 and MRP14 proteins in immature myeloid cells of the bone marrow, myeloid cells in the splenic red pulp and marginal zone, in addition to monocytes and blood neutrophils. However, MRP expression is lost as cells terminally differentiate into tissue macrophages. In addition, using thioglycollate-induced peritoneal inflammatory exudates, we showed that MRP8 and MRP14 proteins are highly expressed in recruited neutrophils and monocytes.

  View details for Web of Science ID A1992HW41600002

  View details for PubMedID 1373330

• EVIDENCE FOR A PROGRAMMED LIFE-SPAN IN A COLONIAL PROTOCHORDATE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rinkevich, B., Lauzon, R. J., Brown, B. W., Weissman, I. L. 1992; 89 (8): 3546-3550

  Abstract

  The variety of theories that have attempted to define the mechanisms of aging and life span can be broadly divided into two alternative but nonexclusive viewpoints. The first stipulates that random changes of cellular and molecular structures lead to death following progressive "wear and tear." The second argues that life span is, at least in part, genetically programmed, and therefore aging may also result from time-dependent intrinsic processes. Here we demonstrate that ramets (clonal replicates) experimentally separated from colonies of the acidian protochordate Botryllus schlosseri died months after their separation, almost simultaneously with their parent colony and sibling ramets. In addition, in experimentally joined chimeras between ramets of senescent and nonsenescent colonies, elements from different parent colonies displayed parent-colony-specific timing of mortality. Thus, the senescent phenotype was simultaneously expressed both in chimeras and in unfused ramets of the parent colony that was undergoing senescence, whereas control ramets from the other partner survived. These findings provide experimental evidence for a heritable basis underlying mortality in protochordates, unlinked to reproductive effort and other life history traits of this species.

  View details for Web of Science ID A1992HP04300080

  View details for PubMedID 1565651

• ISOLATION OF A CANDIDATE HUMAN HEMATOPOIETIC STEM-CELL POPULATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Baum, C. M., Weissman, I. L., Tsukamoto, A. S., Buckle, A. M., Peault, B. 1992; 89 (7): 2804-2808

  Abstract

  We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains multipotent hematopoietic precursors. The population of human precursor cells that express Thy-1 and CD34 but no known lineage markers is enriched for clonogenic activity that establishes long-term, multilineage (myelomonocytic and B lymphoid) cultures on mouse marrow stromal lines. Further, the Thy-1+CD34+ subset that takes up little of the fluorescent mitochondrial dye rhodamine 123 contains virtually all the cells that establish long-term cultures. In human fetal thymus transplanted into SCID (severe combined immunodeficiency) mice, Thy-1+CD34+ fetal bone marrow cells differentiate into T lymphocytes. In two of nine cases, allogeneic Thy-1+CD34+ cells could engraft intact human fetal bone marrow grown in SCID mice, resulting in donor-derived myeloid and B cells. By extrapolation, the rare human Thy-1+Lin-CD34+ cell population contains pluripotent hematopoietic progenitors; we propose that it is highly enriched for candidate hematopoietic stem cells.

  View details for Web of Science ID A1992HL81600061

  View details for PubMedID 1372992

• EVIDENCE THAT HEMATOPOIETIC STEM-CELLS EXPRESS MOUSE C-KIT BUT DO NOT DEPEND ON STEEL FACTOR FOR THEIR GENERATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ikuta, K., Weissman, I. L. 1992; 89 (4): 1502-1506

  Abstract

  The interaction of the mouse c-kit receptor, designated Kit receptor, and steel factor promotes the proliferation and differentiation of hematopoietic progenitor cells. Monoclonal antibodies against the extracellular portion of the mouse Kit receptor were established. Five percent to 10% of total bone marrow cells expressed the Kit receptor, and half of them lack the expression of lineage markers. The Kit receptor was expressed on 70-80% of Thy-1.1lo Lin-Sca-1+ cells, which express Thy-1.1 antigen at a low level and constitute approximately 0.05% of adult bone marrow and fetal liver; by previous studies, these cells have been shown to be highly enriched for multipotent hematopoietic stem cells (HSCs) and are the only hematopoietic cell subset with this activity. Spleen colony formation and long-term multilineage reconstitution activities were contained in the Kit+ but not in the Kit- subpopulations of Thy-1lo Lin-Sca-1+ cells from adult bone marrow, suggesting that the Kit receptor is expressed on HSCs from the earliest stage-i.e., pluripotent HSCs. The role of steel factor in the development and self-renewal of HSCs was tested with Sl/Sl homozygote fetuses, which lack genes to encode functional steel factor. They were shown to have 30-40% of the number of HSCs on days 13-15 when compared with normal litermates. However, the absolute number of HSCs increased during fetal development in the Sl/Sl mice. The results suggest that the Kit receptor-steel factor interaction may not be essential for the initiation of hematopoiesis and the self-renewal of (at least) fetal HSCs.

  View details for Web of Science ID A1992HE60600074

  View details for PubMedID 1371359

• CHIMERAS VS GENETICALLY HOMOGENEOUS INDIVIDUALS - POTENTIAL FITNESS COSTS AND BENEFITS OIKOS Rinkevich, B., Weissman, I. L. 1992; 63 (1): 119-124

  View details for Web of Science ID A1992HK90600012

• PERFORIN AND GRANZYME-A EXPRESSION IDENTIFYING CYTOLYTIC LYMPHOCYTES IN RHEUMATOID-ARTHRITIS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Griffiths, G. M., Alpert, S., Lambert, E., McGuire, J., Weissman, I. L. 1992; 89 (2): 549-553

  Abstract

  Lymphocytes from the synovial fluid of patients with rheumatoid arthritis were examined for the expression of granzyme A and perforin. Previous studies have demonstrated that the expression of these proteins, which are implicated as mediators of cytotoxicity, can be used to identify putative cytolytic lymphocytes in vivo. Twenty-two synovial fluid samples were analyzed by in situ hybridization and immunohistochemistry. In six patients receiving low doses of immunosuppressant, a population of granzyme A- and perforin-expressing lymphocytes could be identified. In contrast, lymphocytes from patients who were receiving high doses of immunosuppressant did not contain any granzyme A- or perforin-expressing lymphocytes. Synovial fluid lymphocytes from patients with osteoarthritis did not express either marker. The expression of these markers demonstrates the presence of potentially functional cytolytic lymphocytes, expressing proteins required to mediate killing, in the synovial fluid of patients with rheumatoid arthritis. This suggests that cytolytic lymphocytes may be involved in the pathogenesis of rheumatoid arthritis.

  View details for Web of Science ID A1992GZ69600021

  View details for PubMedID 1731326

• CHARACTERIZATION OF SEVERAL CLASSES OF MOUSE HEMATOPOIETIC PROGENITOR CELLS CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY Heimfeld, S., Weissman, I. L. 1992; 177: 95-105

  View details for Web of Science ID A1992JD12400008

  View details for PubMedID 1353436

• DEVELOPMENT OF GAMMA-DELTA T-CELL SUBSETS FROM FETAL HEMATOPOIETIC STEM-CELLS CONF ON CD5 B-CELLS IN DEVELOPMENT AND DISEASE Ikuta, K., Kina, T., MacNeil, I., Uchida, N., Peault, B., Chien, Y. H., Weissman, I. L. NEW YORK ACAD SCIENCES. 1992: 21–32

  View details for Web of Science ID A1992BW41S00003

• Hematopoietic stem cells and lymphopoiesis Curr Opin Oncol Weissman, I. 1992; 4(suppl 1): S8-S10
• Hematopoietic stem cells in normal and malignant states Marrow Transplantation Rev Negrin, R., Weissman, I. 1992; 2: 23-26
• Development of gamma delta T-cell subsets from fetal hematopoietic stem cells Ann NY Acad Sci Ikuta, K., Kina, T., MacNeil, I., Uchida, N., Peault, B., Chien, Y., Weissman, I. 1992; 4 (651): 21-32
• LYMPHOCYTE DEVELOPMENT FROM STEM-CELLS ANNUAL REVIEW OF IMMUNOLOGY Ikuta, K., Uchida, N., Friedman, J., Weissman, I. L. 1992; 10: 759-783

  Abstract

  Highly enriched pluripotent and multipotent hematopoietic stem cells (HSCs) are isolated from bone marrow and fetal liver as Thy-1loLin-Sca-1+ cells. Pluripotent HSCs express c-kit receptor on their surface, but the generation and proliferation of early fetal HSCs take place in the absence of steel factor. T precursor cells migrate into the fetal thymus by chemotactic mechanism. CD4lo precursors represent a newly defined phase of T-cell development in the thymus between the bone marrow-derived stem cells and the CD4-8- intrathymic precursors. Only fetal, but not adult, HSCs have the capacity to differentiate into V gamma 3+ and V gamma 4+ T cells under the fetal thymic microenvironment, and HSC themselves may lose some of their developmental potential during ontogeny. It is postulated that HSCs are the locus of a complicated but precise developmental clock that may determine both the time-dependent closure of some gene loci (e.g. V gamma 3 and V gamma 4 T cell receptor, and embryonic and fetal globin) and the activation of others (e.g. the N nucleotide insertion machinery).

  View details for Web of Science ID A1992HN01200027

  View details for PubMedID 1375474

• LYMPHOCYTE HOMING RECEPTORS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Hu, M. C., Siegelman, M. H., Holzmann, B., Crowe, D. T., Weissman, I. L. 1992; 57: 291-308

  View details for Web of Science ID A1992LV41600032

  View details for PubMedID 1339666

• SEARCHING FOR HEMATOPOIETIC STEM-CELLS - EVIDENCE THAT THY-1.1(LO) LIN- SCA-1+ CELLS ARE THE ONLY STEM-CELLS IN C57BL/KA-THY-1.1 BONE-MARROW JOURNAL OF EXPERIMENTAL MEDICINE Uchida, N., Weissman, I. L. 1992; 175 (1): 175-184

  Abstract

  Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

  View details for Web of Science ID A1992GY43600022

  View details for PubMedID 1346154

• INTERPOPULATIONAL ALLOGENEIC REACTIONS IN THE COLONIAL PROTOCHORDATE BOTRYLLUS-SCHLOSSERI INTERNATIONAL IMMUNOLOGY Rinkevich, B., Weissman, I. L. 1991; 3 (12): 1265-1272

  Abstract

  Botryllus schlosseri is a cosmopolitan encrusting colonial tunicate which undergoes a natural transplantation reaction. When the growing edges of two colonies come into direct contact by interaction between their extracorporeal blood vessel termini, the ampullae, they either reject each other or fuse. This phenomenon is controlled by a single gene locus (Fu/HC) with multiple codominantly expressed alleles. Rejecting colonies share no alleles. Here we analyze allogeneic responses of Monterey (Mon), California, versus Woods Hole (WH), Massachusetts, colonies. Of 42 Mon x WH pairs tested, allogeneic rejection reactions occurred in all. Necrotic lesions (points of rejection, PORs) were produced and developed only by Woods Hole ampullae, either within the Woods Hole tunic, in the borderline between the paired colonies, or within the Monterey tunic. Four types of PORs were characterized. All types involved reactions of blood cells and vessels, including infiltration, hemorrhage formation, retraction and ampullae amputation. These findings were observed in single WH x Mon pairs, in multiple subclones of WH x Mon from two parental colonies (seven independent colony pairs were assayed), and on multiple repeats of interactions from pairs that had already undergone a rejection reaction. In all cases, the range of reaction types, the location of PORs, and the timing of the responses could be found in primary as well as repeat reactions.

  View details for Web of Science ID A1991GV85100008

  View details for PubMedID 1777421

• GROWTH OF PRIMARY T-CELL NON-HODGKINS-LYMPHOMAS IN SCID-HU MICE - REQUIREMENT FOR A HUMAN LYMPHOID MICROENVIRONMENT BLOOD Waller, E. K., Kamel, O. W., Cleary, M. L., Majumdar, A. S., Schick, M. R., Lieberman, M., Weissman, I. L. 1991; 78 (10): 2650-2665

  Abstract

  We reasoned that the SCID-hu mouse could provide an appropriate lymphoid or stromal microenvironment to support the growth of primary human lymphoma. Heterotransplantation of nine cases of primary T-cell non-Hodgkin's lymphoma (NHL) into untreated SCID mice and SCID mice reconstituted with human fetal thymus, spleen, and liver (SCID-hu) resulted in the development of lymphoid tumors in five (56%) cases. Two clonal T-cell NHL grew after a mean of 90 days after injection of primary lymphoma cell suspensions into the thymus xenografts in SCID-hu mice and failed to grow in a variety of sites in SCID mice, except for small tumors that developed after a long (157-day) latency period after intracranial injection of tumor cell suspensions into weanling SCID mice. Successful serial transplantation of NHL in SCID and SCID-hu mice required the presence of a human lymphoid or tumor microenvironment, and was enhanced by pretreating the SCID mice with 175 rad radiation and antiasialo antisera. Analysis of the primary and transplanted T-cell tumors showed identical patterns of T-cell surface markers by flow cytometry and immunophenotyping of fixed tissue sections, and, in one case, reactivity with a specific monoclonal antibody to V beta 5.1. Genotyping of the transplanted tumors showed T-cell receptor gene rearrangements identical to those present in the primary tumors. In one case, the presence of Epstein-Barr virus-positive B cells in association with the primary tumor resulted in the growth of a lymphoblastoid B-cell neoplasm in addition to the malignant T-cell lymphoma after transplantation of tumor fragments to SCID mice. The data support the hypothesis that a human lymphoid microenvironment enhances the growth of T-cell NHL in SCID mice. The SCID-hu thymus graft provides an apparently unique microenvironment that supports the growth of primary T-cell NHL, and can be used to study the interaction between lymphoma cells, nontransformed lymphoid cells, and the surrounding stromal microenvironment in vivo.

  View details for Web of Science ID A1991GP88700024

  View details for PubMedID 1824259

• CLONING AND EXPRESSION OF CDNAS FOR THE ALPHA-SUBUNIT OF THE MURINE LYMPHOCYTE-PEYERS PATCH ADHESION MOLECULE JOURNAL OF CELL BIOLOGY Neuhaus, H., Hu, M. C., Hemler, M. E., Takada, Y., Holzmann, B., Weissman, I. L. 1991; 115 (4): 1149-1158

  Abstract

  cDNA clones encoding the alpha chain of the murine lymphocyte-Peyer's patch adhesion molecule (LPAM), which is associated with lymphocyte homing, have been isolated by screening with the human VLA-4 (alpha 4h) probe. Several alpha 4 antigenic determinants were identified on COS-7 cells after transfection. From overlapping clones, approximately 5 kb of contiguous nucleotide sequence have been determined, encoding a protein sequence of 1039 amino acids for the LPAM alpha chain (alpha 4m). LPAM is a member of the integrin family of cell-surface heterodimers, and alpha 4m is the murine homologue of the human alpha 4 h chain. The two proteins have a total sequence similarity of 84%, with an almost perfect conservation (31/32 amino acids) in the cytoplasmic domain. Like alpha 4h, alpha 4m is distinct from other integrin alpha chains because it has neither an I-domain nor a COOH-terminal cleavage site. The positions of the characteristic Cysteine residues are conserved, and a putative protease cleavage site is located near the middle of the protein sequence. The NH2-terminal part of the protein contains seven homologous repeats, and three of them include putative divalent cation-binding sites. These sites are among the most conserved between the alpha 4m sequence and other alpha chains, and may therefore be involved in the binding of integrin alpha and beta chains. An additional cDNA clone was isolated which shares a sequence of perfect homology with the alpha 4m encoding cDNAs, but has a unique 3' poly-A end. This observation correlates with the fact that three discrete murine RNA bands are observed in Northern blot experiments using alpha 4m as a probe, whereas only two human RNA species are described for alpha 4h, indicating a higher complexity for murine than for human sequences.

  View details for Web of Science ID A1991GP80200024

  View details for PubMedID 1840602

• LYMPHOID RECONSTITUTION OF THE HUMAN FETAL THYMUS IN SCID MICE WITH CD34+ PRECURSOR CELLS JOURNAL OF EXPERIMENTAL MEDICINE Peault, B., Weissman, I. L., Baum, C., McCune, J. M., Tsukamoto, A. 1991; 174 (5): 1283-1286

  Abstract

  The search for human hematopoietic stem cells has been hampered by the lack of appropriate assay systems. Demonstration of the ability of precursor cell candidates to give rise to T cells is of significant difficulty since dissociated in vitro cultured thymus stroma cells lose their ability to sustain thymocyte maturation. To define further the differentiative capacities of the rare human fetal liver and bone marrow cells that express the CD34 surface antigen and exhibit in vitro myeloid and pre-B cell activities, we have microinjected them into HLA-mismatched fetal thymus fragments, partially depleted of hematopoietic cells by low temperature culture. In vitro colonized thymuses have then been allowed to develop upon engraftment into immunodeficient SCID mice. Using this modification of the SCID-hu system, we show that low numbers of fetal CD34+ progenitor cells can repopulate the lymphoid compartment in the human thymus.

  View details for Web of Science ID A1991GM80300039

  View details for PubMedID 1719121

• THE INVITRO RESPONSE OF PHENOTYPICALLY DEFINED MOUSE STEM-CELLS AND MYELOERYTHROID PROGENITORS TO SINGLE OR MULTIPLE GROWTH-FACTORS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Heimfeld, S., Hudak, S., Weissman, I., Rennick, D. 1991; 88 (21): 9902-9906

  Abstract

  Pluripotential stem cells (Thylo Lin- Sca+; referred to as Sca+) and primitive myeloerythroid progenitor cells (Thylo Lin- Sca-; referred to as Sca-), defined by their in vivo repopulating properties, have been purified from mouse bone marrow. In this study, the growth factor requirements of these two subsets were compared in colony-forming assays. Sca- progenitor cells grew well in interleukin (IL) 3 alone and showed maximum growth when two factors, IL-3 plus IL-1 or IL-3 plus IL-6, were combined. In contrast, Sca+ stem cells were generally not responsive to any single factor tested. Some colony formation was found when IL-3 was paired with either IL-1 or IL-6, and this was significantly enhanced as additional factors were included. A remarkable frequency of as much as 1 colony per 1.7 input Sca+ cells was achieved when IL-1, IL-3, IL-6, and colony-stimulating factors were used together. These differences in factor requirements presumably reflect the need for multiple factor signaling in the more primitive stem cell population. In most other aspects of colony formation, Sca+ and Sca- cells were very similar. They generated colonies that had equivalent distributions in size and cellular composition. One notable difference was found in the kinetics of their response. Whereas nearly all Sca- cells formed colonies within 7 days, a significant fraction of Sca+ cells delayed colony formation for greater than 1 week. During this quiescent period, cell survival was absolutely dependent on the presence of factors in the medium.

  View details for Web of Science ID A1991GM78100111

  View details for PubMedID 1946416

• THE JUNCTIONAL MODIFICATIONS OF A T-CELL RECEPTOR GAMMA-CHAIN ARE DETERMINED AT THE LEVEL OF THYMIC PRECURSORS JOURNAL OF EXPERIMENTAL MEDICINE Ikuta, K., Weissman, I. L. 1991; 174 (5): 1279-1282

  Abstract

  T precursors from fetal liver and adult bone marrow were compared for their ability to give rise to V gamma 4+ T cell development. Fetal thymic lobes were repopulated with fetal liver or adult bone marrow cells, and the organ-cultured thymocytes were analyzed for their T cell receptor expression by the polymerase chain reaction (PCR). Both day 14 fetal liver and adult bone marrow cells gave rise to thymocytes with V gamma 4-J gamma 1 transcripts. However, the average size of the PCR products derived from adult precursors was slightly larger than that from fetal precursors. DNA sequence analysis of the V gamma 4-J gamma 1 transcripts showed that early fetal liver precursors predominantly gave rise to thymocytes with the V gamma 4-J gamma 1 transcripts without N nucleotide insertion, while late fetal liver and adult marrow precursors predominantly gave rise to thymocytes with modified V gamma 4-J gamma 1 junctions. These results suggest the possibility that the level of the N nucleotide insertion is programmed at the level of thymic precursors. This study also supported the model presented previously that the developmental potential of hematopoietic stem cells may change during ontogeny.

  View details for Web of Science ID A1991GM80300038

  View details for PubMedID 1834763

• MOUSE HEMATOPOIETIC STEM-CELLS BLOOD Spangrude, G. J., Smith, L., Uchida, N., Ikuta, K., Heimfeld, S., Friedman, J., Weissman, I. L. 1991; 78 (6): 1395-1402

  View details for Web of Science ID A1991GF44400001

  View details for PubMedID 1884012

• STEM-CELLS NATURE Weissman, I., Spangrude, G., Heimfeld, S., Smith, L., Uchida, N. 1991; 353 (6339): 26-26

  View details for Web of Science ID A1991GD80500041

  View details for PubMedID 1881443

• MOUSE HEMATOPOIETIC STEM-CELLS AND THE INTERACTION OF C-KIT RECEPTOR AND STEEL FACTOR INTERNATIONAL JOURNAL OF CELL CLONING Ikuta, K., INGOLIA, D. E., Friedman, J., Heimfeld, S., Weissman, I. L. 1991; 9 (5): 451-460

  Abstract

  Hematopoietic stem cells (HSCs) are distinguished from other hematopoietic progenitors in bone marrow by their unique ability to undergo multilineage differentiation and self-renewal. Two mouse mutations, dominant spotting (W) and steel (Sl), have pleiotropic effects on hematopoiesis, gametogenesis, and melanoblast development. These two mutations have been shown to be intrinsic (W) and microenvironmental (Sl) defects. Recently, molecular studies revealed that the W and Sl loci encode the c-kit receptor and steel factor (SLF), respectively. The c-kit receptor is expressed on HSCs and hematopoietic progenitors, while SLF is produced by stromal cells. SLF acts on hematopoietic progenitors synergistically with other growth factors. Here we review the effect of these mutations on mouse hematopoiesis, and show that SLF acts on HSCs and other myeloerythroid progenitors, but that it, in our hands, does not play a critical role in HSC generation or self-renewal. Rather, SLF is the most potent co-mitogen (with IL-1, IL-3, IL-6, G-CSF, GM-CSF, or M-CSF) found that acts on these cells, but the effect of such treatments is the rather specific and massive expansion of myeloerythropoiesis, not lymphopoiesis, and perhaps at the expense of HSC self-renewal.

  View details for Web of Science ID A1991GH26000002

  View details for PubMedID 1720154

• 2 CYTOPLASMIC CANDIDATES FOR IMMUNOPHILIN ACTION ARE REVEALED BY AFFINITY FOR A NEW CYCLOPHILIN - ONE IN THE PRESENCE AND ONE IN THE ABSENCE OF CSA CELL Friedman, J., Weissman, I. 1991; 66 (4): 799-806

  Abstract

  We report the cloning and characterization of a new binding protein for the immunosuppressive drug cyclosporin A (CsA). This new cyclophilin, cyclophilin C (cyp C), shows extensive homology with all previously identified cyclophilins. Cyp C mRNA is expressed in a restricted subset of tissues relative to cyclophilins A and B, but is present in those tissues reported to be most affected by CsA therapy. A cyp C fusion protein has peptidyl-prolyl isomerase activity, and CsA inhibits this activity. Using the cyp C fusion protein as an affinity ligand to probe cellular extracts, we find that the cyp C fusion protein binds specifically to a 77 kd protein in the absence of CsA, while in the presence of CsA it instead binds specifically to a 55 kd protein. We propose that the p77 is involved in cyp C native function and that the p55 is involved in signal transduction events blocked by treatment with immunosuppressive levels of CsA.

  View details for Web of Science ID A1991GC74500018

  View details for PubMedID 1652374

• CALCINEURIN IS A COMMON TARGET OF CYCLOPHILIN-CYCLOSPORINE-A AND FKBP-FK506 COMPLEXES CELL Liu, J., Farmer, J. D., Lane, W. S., Friedman, J., Weissman, I., Schreiber, S. L. 1991; 66 (4): 807-815

  Abstract

  Although the immediate receptors (immunophilins) of the immunosuppressants cyclosporin A (CsA) and FK506 are distinct, their similar mechanisms of inhibition of cell signaling suggest that their associated immunophilin complexes interact with a common target. We report here that the complexes cyclophilin-CsA and FKBP-FK506 (but not cyclophilin, FKBP, FKBP-rapamycin, or FKBP-506BD) competitively bind to and inhibit the Ca(2+)- and calmodulin-dependent phosphatase calcineurin, although the binding and inhibition of calcineurin do not require calmodulin. These results suggest that calcineurin is involved in a common step associated with T cell receptor and IgE receptor signaling pathways and that cyclophilin and FKBP mediate the actions of CsA and FK506, respectively, by forming drug-dependent complexes with and altering the activity of calcineurin-calmodulin.

  View details for Web of Science ID A1991GC74500019

  View details for PubMedID 1715244

• CULTURE OF PHENOTYPICALLY DEFINED HEMATOPOIETIC STEM-CELLS AND OTHER PROGENITORS AT LIMITING DILUTION ON DEXTER MONOLAYERS BLOOD Weilbaecher, K., Weissman, I., Blume, K., Heimfeld, S. 1991; 78 (4): 945-952

  Abstract

  Highly enriched, phenotypically defined hematopoietic stem, Thy-1loLin-Sca-1+, and progenitor cell populations from mouse bone marrow (BM) were tested at limiting dilution for their ability to reconstitute Dexter monolayers. Several classes of BM cells can reconstitute Dexter cultures, first forming discrete "cobblestone" areas which then mature into colonies consisting primarily of maturing myeloid and erythroid cells. Most such colonies have a limited lifespan in culture. Only the Thy-1loLin-Sca-1+ cell fraction gives rise to colonies that survive longer than 3 weeks, which suggests that a limiting-dilution analysis for long-term reconstitution of Dexter cultures can serve as a quantitative measure of stem cell activity. Additional experiments were performed to assess the formation of new progenitor cells in reconstituted Dexter cultures. Again, only cultures seeded with the stem cell-enriched fraction contained expanded numbers of replatable WEHI-3 CM responsive colony-forming cells (CFU-GM). Quantitative analysis indicates that 97% of the replatable CFU-GM of whole BM is contributed by the Thy-1loLin-Sca-1+ cell fraction, again suggesting a potential stem cell-specific assay. Such quantitative in vitro assays might prove useful in characterization and isolation of human stem cells where in vivo assays are lacking.

  View details for Web of Science ID A1991GB20800011

  View details for PubMedID 1678290

• USE OF A SCID MOUSE HUMAN LYMPHOMA MODEL TO EVALUATE CYTOKINE-INDUCED KILLER-CELLS WITH POTENT ANTITUMOR CELL-ACTIVITY JOURNAL OF EXPERIMENTAL MEDICINE SCHMIDTWOLF, I. G., Negrin, R. S., Kiem, H. P., Blume, K. G., Weissman, I. L. 1991; 174 (1): 139-149

  Abstract

  C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti-CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL-4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before infusion. The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. The cytokine-induced killer cells generated as described here could have an important impact on bone marrow purging for autologous bone marrow transplantation as well as for adoptive immunotherapy.

  View details for Web of Science ID A1991FU89700018

  View details for PubMedID 1711560

• MOST CD8+ CELLS IN SKIN-LESIONS OF CD3+CD4+ MYCOSIS-FUNGOIDES ARE CD3+ T-CELLS THAT LACK CD11B, CD16, CD56, CD57, AND HUMAN HANUKAH FACTOR MESSENGER-RNA AMERICAN JOURNAL OF PATHOLOGY Wood, G. S., DUBIEL, C., Mueller, C., Abel, E. A., Hoppe, R. T., Edinger, A., Weissman, I., Warnke, R. A. 1991; 138 (6): 1545-1552

  Abstract

  To define further the characteristics of CD8+ cells in skin lesions of CD3+ CD4+ mycosis fungoides (MF), the authors used single- and double-label immunohistologic techniques and in situ hybridization to detect antigens and transcripts associated with certain types of cytotoxic or suppressor function. The cytotoxic markers included CD16, CD56, CD57, and an anti-sense probe for human Hanukah factor (HuHf) mRNA. Analysis of 23 cases demonstrated that lesional CD8+ cells were CD3+ T cells that generally lacked expression of any of the cytotoxic markers studied. Analysis of another 10 cases confirmed the CD3+ T-cell lineage of lesional CD8+ cells and demonstrated that these cells also lacked expression of the suppressor-associated marker, CD11b. In aggregate, these results indicate that most CD8+ cells in CD3+ CD4+ MF skin lesions are of T-cell rather than NK-cell differentiation. Their overall phenotype suggests that they may be major histocompatibility complex (MHC)-restricted cytotoxic T cells lacking appreciable levels of HuHF serine protease. Because the induction of CD8+ suppressor T cells is mediated by CD4+ T cells expressing the CD45RA+ RO- phenotype, CD45 epitope expression was studied in 15 MF cases. The vast majority (13/15) contained CD3+ CD4+ tumor cells that were CD45+ RA- RB+ RO+ 2B11+. This phenotype is consistent with memory T cells rather than suppressor-inducer T cells, and correlates with the paucity of phenotypically defined suppressor T cells in CD3+ CD4+ MF skin lesions.

  View details for Web of Science ID A1991FR74600026

  View details for PubMedID 1828937

• CLONAL ANALYSIS OF HEMATOPOIETIC STEM-CELL DIFFERENTIATION INVIVO PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Smith, L. G., Weissman, I. L., Heimfeld, S. 1991; 88 (7): 2788-2792

  Abstract

  Previous work has shown that the 0.02-0.05% of adult mouse bone marrow cells that bear the cell surface phenotype Thy-1loLin-Sca-1+ are enriched 1000- to 2000-fold for hematopoietic stem-cell activity in a variety of assays. When 50-100 cells of this phenotype are injected into an irradiated animal, they can permanently repopulate the entire hematopoietic system. In the present study, limiting-dilution and single-cell experiments were used to address the question of how individual Thy-1loLin-Sca-1+ stem cells contribute to repopulation of the hematopoietic system following irradiation. We calculated that 1 of 13 Thy-1loLin-Sca-1+ cells formed a clone comprising greater than 1% of peripheral white blood cells 3-7 weeks after injection. The majority of these clones included both lymphoid and myeloid lineages. Approximately one-third of the clones continued to produce new blood cells for 9 weeks or more, but the remainder disappeared earlier, including many that were multilineage. Thus, while the majority of Thy-1loLin-Sca-1+ bone marrow cells whose progeny are detected in the in vivo repopulation assay are pluripotential, only a subset undergo long-term self-renewal in vivo. Repopulation appears to be oligoclonal when limiting numbers of Thy-1loLin-Sca-1+ cells are injected. However, the number of clones contributing to hematopoiesis increases in proportion to the number of Thy-1loLin-Sca-1+ cells injected, bringing into question the notion that steady-state hematopoiesis in normal individuals is oligoclonal.

  View details for Web of Science ID A1991FE86400037

  View details for PubMedID 1672767

• GRANZYME-A AND PERFORIN AS MARKERS FOR REJECTION IN CARDIAC TRANSPLANTATION EUROPEAN JOURNAL OF IMMUNOLOGY Griffiths, G. M., Namikawa, R., Mueller, C., Liu, C. C., Young, J. D., Billingham, M., Weissman, I. 1991; 21 (3): 687-692

  Abstract

  The use of granzyme A and perforin as markers for rejection after cardiac transplantation has been investigated. Using in situ hybridization we have detected lymphocytes expressing granzyme A and perforin RNA that are infiltrating the donor heart after transplantation. A total of 29 different biopsies from 17 different patients who had undergone cardiac transplantation were examined. Twelve biopsies classified by conventional histological criteria as showing evidence of rejection were found to contain lymphocytes expressing granzyme A and perforin. Seven biopsies classified as showing no histological evidence of rejection infiltrating lymphocytes were found not to be expressing granzyme A or perforin. However, in 10 other biopsies from 5 different patients that had been classified as showing no evidence of rejection by the conventional grading system, lymphocytes expressing granzyme A and perforin were detected. In six of these cases the patient was found to have undergone a subsequent rejection episode. In the other four cases the biopsies were either taken at a very early stage after transplantation and the high doses of immunosuppression used routinely at that stage are likely to have averted any rejection episodes, or it was not possible to follow subsequent rejection episodes. These results, which are statistically significant (p = 0.06), demonstrate that granzyme A- and perforin-expressing lymphocytes can be identified in rejecting biopsies before histological damage is seen. The identification of perforin and granzyme A expression in vivo suggest a possible role for these proteins in the cytolysis that occurs during transplantation rejection. Furthermore, the data presented here suggest that it may be possible to use granzyme A and perforin as early predictive markers of transplantation rejection.

  View details for Web of Science ID A1991FF29300021

  View details for PubMedID 2009911

• EVIDENCE FOR A ROLE OF THE INTEGRIN VLA-4 IN LYMPHO-HEMATOPOIESIS JOURNAL OF EXPERIMENTAL MEDICINE Miyake, K., Weissman, I. L., Greenberger, J. S., Kincade, P. W. 1991; 173 (3): 599-607

  Abstract

  Adhesion molecules are probably required for retention of maturing lymphocyte precursors in bone marrow, where they closely interact with and are dependent on stromal cells. Lymphomyeloid cell lines avidly adhere to cloned stromal cell lines in culture and screening pairs of these resulted in a selection strategy for a new monoclonal antibody to a leukocyte adhesion molecule. Immunoprecipitation analyses and comparison to a previously described antibody showed that it recognizes the alpha 4 chain of the integrin, VLA-4. This antibody totally inhibited lymphopoiesis and retarded myelopoiesis in long-term bone marrow cultures. A similar selection strategy resulted in two additional antibodies which define a single 100-kD species on stromal cells. This stromal cell adhesion molecule is a potential counter-receptor/ligand for VLA-4 on murine lympho-myeloid cells. Our findings suggest a new role for VLA-4 in lymphoid progenitor-microenvironment interactions. Recognition molecules that function in cell migration and inflammation in peripheral tissues may be important for steady-state lymphopoiesis within bone marrow.

  View details for Web of Science ID A1991EZ66300009

  View details for PubMedID 1997648

• IDENTIFICATION OF A 107-KD GLYCOPROTEIN THAT MEDIATES ADHESION BETWEEN STROMAL CELLS AND HEMATOLYMPHOID CELLS JOURNAL OF EXPERIMENTAL MEDICINE Kina, T., SENMAJUMDAR, A., Heimfeld, S., Kaneshima, H., Holzmann, B., Katsura, Y., Weissman, I. L. 1991; 173 (2): 373-381

  Abstract

  The mechanism of cell complex formation between lymphocytes and stromal cells was investigated. We found that lymphoid lines of both T and B lineages could form cell complexes with stromal cells from the thymus as well as bone marrow but not with macrophages or typical fibroblast lines. Formation of these cell complexes is temperature dependent and requires the presence of Mg2+, active cellular metabolism, and microfilament assembly of cytoskeleton. We raised an antiserum against a thymic stromal cell clone (BATE-2) in rats and found that, after absorption, this serum could effectively block cell complex formation between lymphocytes and stromal cells from both thymus and bone marrow. An efficient blocking was obtained only when the antiserum was added at the initial stage of cell interaction. From the blocking experiments and the SDS-PAGE analysis of immunoprecipitated materials from the stromal cell surface, we identified a unique 107-kD glycoprotein on the stromal cells as a molecule for mediating stromal cell-lymphocyte interaction. This is further supported by the findings that an antiserum raised in hamsters against the excised gel band corresponding to 107 kD, which specifically immunoprecipitated the 107-kD molecule, effectively blocked the lymphocyte-stromal cell interaction. The possible function of this molecule in hematolymphoid development is discussed.

  View details for Web of Science ID A1991EV17500012

  View details for PubMedID 1988540

• EXPRESSION OF THE PROTEASE GENE HF AS A MARKER IN REJECTING ALLOGENEIC MURINE HEART-TRANSPLANTS TRANSPLANTATION Mueller, C., Shelby, J., Weissman, I. L., PERINATFREY, T., Eichwald, E. J. 1991; 51 (2): 514-517

  Abstract

  Phenotypic characterization of the inflammatory infiltrate often provides very little information about the fate of a transplant. Therefore we decided to look for functional markers, characteristic for activated effector cells involved in the rejection of primarily vascularized MHC-mismatched heart transplants in mice. Infiltrating cells in the interstitium of eventually rejected allogeneic heart transplants were found to express the gene for the serine esterase HF (granzyme A) in high numbers already on day 2 following transplantation, whereas HF mRNA positive cells were absent, or only rarely found, in syngeneic nonrejecting transplants throughout the entire observation period of 10 days after transplantation. These findings could also become of importance for the prediction of the outcome of organ transplants in humans.

  View details for Web of Science ID A1991EY48300049

  View details for PubMedID 1994548

• DEVELOPMENT OF MOUSE HEMATOPOIETIC LINEAGES CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY Heimfeld, S., Weissman, I. L. 1991; 25: 155-175

  View details for Web of Science ID A1991GV02100007

  View details for PubMedID 1743054

• The peripheral lymph node homing receptor: tandem structural and functional domains in a novel class of adhesion molecule Vascular Adhesion Molecules Siegelman, M., Weissman, I. 1991: 61–89
• Abelson leukemia virus tumorigenesis: cellular genes that regulate growth and invasiveness Origins of Human Cancer: a Comprehensive Review Weissman, I., Shih, C., Sherwood, P. Cold Spring Harbor Laboratory Press. 1991
• The peyer's patch homing receptor: a novel member of the integrin family Vascular Adhesion Molecules Hu, M., Holzman, B., Neuhaus, H., Weissman, I. 1991: 91–110
• THE SCID-HU MOUSE - A SMALL ANIMAL-MODEL FOR HIV-INFECTION AND PATHOGENESIS ANNUAL REVIEW OF IMMUNOLOGY McCune, J., Kaneshima, H., Krowka, J., Namikawa, R., Outzen, H., Peault, B., Rabin, L., Shih, C. C., Yee, E., Lieberman, M., Weissman, I., Shultz, L. 1991; 9: 399-429

  Abstract

  The SCID-hu mouse is a heterochimeric small animal model designed to support hematopoietic differentiation and function in vivo. Multiple organs of the human hematolymphoid system have been successfully engrafted into the immunodeficient C.B-17 scid scid mouse, including fetal liver, thymus, lymph node, and skin. Co-implantation of human fetal liver and human fetal thymus results in long-term, multilineage human hematopoiesis in vivo. Mature human lymphocytes within the SCID-hu mouse are phenotypically and functionally normal. HIV infection of the SCID-hu mouse reflects a tropism similar to that found in humans: only human organs with CD4+ cells are infected. Viral replication can thereafter be monitored with assays that are safe, reproducible, and quantitative. Given this small animal model, it is now possible to study systematically the infective process of HIV and to address questions about the efficacy of novel antiviral compounds or vaccines in vivo.

  View details for Web of Science ID A1991FF99800015

  View details for PubMedID 1910684

• IGH ENHANCER DEREGULATED EXPRESSION OF L-MYC - ABNORMAL LYMPHOCYTE-T DEVELOPMENT AND T-CELL LYMPHOMAGENESIS EMBO JOURNAL Moroy, T., Fisher, P., Guidos, C., Ma, A., Zimmerman, K., Tesfaye, A., DePinho, R., Weissman, I., Alt, F. W. 1990; 9 (11): 3659-3666

  Abstract

  Transgenic constructs containing the murine L-myc gene under the control of the immunoglobulin transcriptional enhancer element (Emu) are expressed at unexpectedly high levels in thymocytes and proliferating T cells compared with cells from bone marrow and proliferating B cells. In contrast, double transgenic animals bearing constructs containing the L- and N-myc genes similarly linked to the Emu element maintain preferential L-myc expression in T cells but express the N-myc transgene preferentially in B cells. These results indicate that the L-myc gene contains elements that act in concert with the Emu element to allow preferential expression in T lineage cells. In correspondence to the expression pattern, Emu-L-myc transgenic mice show expanded thymic cortices and irregularly formed splenic follicles with expanded T cell areas. Moreover, the percentage of thymocytes positive for the surface marker 1C11, which defines thymic progenitor cells, activated T cells and preleukemic T cells, is dramatically raised in transgenic mice compared with normal littermates. Emu-L-myc transgenic animals are predisposed to clonal lymphoid tumors, most of which are T cell lymphomas. The relative incidence, latency period, and degree of malignancy of Emu-L-myc tumors compared with Emu-N- or c-myc tumors is consistent with a lower oncogenic potential of the L-myc gene. However, the Emu-L-myc tumors do not express detectable levels of endogenous myc family genes indicating that the L-myc protein can substitute for c- or N-myc in the generation and growth of lymphoid neoplasms.

  View details for Web of Science ID A1990ED92200029

  View details for PubMedID 2120050

• A DEVELOPMENTAL SWITCH IN THYMIC LYMPHOCYTE MATURATION POTENTIAL OCCURS AT THE LEVEL OF HEMATOPOIETIC STEM-CELLS CELL Ikuta, K., Kina, T., MacNeil, I., Uchida, N., Peault, B., Chien, Y. H., Weissman, I. L. 1990; 62 (5): 863-874

  Abstract

  Hematopoietic stem cells (HSCs) isolated from mouse fetal liver, like adult HSCs, are Thy-1lo Lin- Sca-1+. Donor-derived V gamma 3+ T cells were detected in fetal thymic lobes repopulated in vitro with fetal liver HSCs, but not in those with adult bone marrow HSCs. Single clonogenic fetal HSCs gave rise to thymic progeny that include V gamma 3+, other gamma delta+, and alpha beta+ T cells. No V gamma 3+ T cells were detected in adult thymus injected intrathymically with either fetal or adult HSCs. These results support the hypothesis that only fetal HSCs have the capacity to differentiate into V gamma 3+ T cells in the fetal thymic microenvironment and that the developmental potential of HSCs may change during ontogeny.

  View details for Web of Science ID A1990DY10000006

  View details for PubMedID 1975515

• T-CELL RECEPTOR-MEDIATED NEGATIVE SELECTION OF AUTOREACTIVE LYMPHOCYTE-T PRECURSORS OCCURS AFTER COMMITMENT TO THE CD4 OR CD8 LINEAGES JOURNAL OF EXPERIMENTAL MEDICINE Guidos, C. J., Danska, J. S., Fathman, C. G., Weissman, I. L. 1990; 172 (3): 835-845

  Abstract

  To identify the maturational stage(s) during which T cell receptor (TCR)-mediated positive and negative selection occurs, we followed the development of CD4+8- and CD4-8+ T cells from TCRlo CD4+8+ thymic blasts in the presence of different positive and negative selecting (major histocompatibility complex or Mls) elements. We describe novel lineage-committed transitional intermediates that are TCRmed CD4+8lo or TCRmed CD4lo8+, and that show evidence of having been positively selected. Furthermore, negative selection is not evident until after cells have attained one of the TCRmed transitional phenotypes. Accordingly, we propose that negative selection in normal mice occurs only after TCRlo CD4+8+ precursors have been positively selected into either the CD4 or CD8 lineage.

  View details for Web of Science ID A1990DW49100018

  View details for PubMedID 2143774

• BIOSYNTHESIS PATHWAY OF GP90MEL-14, THE MOUSE LYMPH NODE-SPECIFIC HOMING RECEPTOR JOURNAL OF IMMUNOLOGY VANDERIJN, M., Weissman, I. L., Siegelman, M. 1990; 145 (5): 1477-1482

  Abstract

  The mouse lymph node specific homing receptor gp90MEL-14 is a 95-kDa molecular mass ubiquitinated cell surface molecule involved in the binding of lymphocytes to high endothelial venules in peripheral lymph nodes. The molecule is thought to consist of a core protein to which ubiquitin side chains are covalently bound. Recently we cloned the cDNA encoding the core protein; this cDNA clone encodes for a polypeptide with an estimated molecular mass of 37 kDa. We have studied the biosynthesis of gp90MEL-14 in an effort to explain the difference in molecular mass between the core protein and the 95-kDa mature molecule. Pulse labeling experiments show a rapid synthesis of a 70-kDa precursor form that contains high-mannose N-linked oligosaccharides. On processing of the high-mannose oligosaccharides into complex N-linked oligosaccharides, the precursor matures in a single step into the 95-kDa form. Experiments using deglycosylating enzymes and inhibitors of N-linked glycosylation demonstrate that the molecular mass of deglycosylated gp90MEL-14 is 45 kDa; extensive N-linked glycosylation is responsible for the difference in molecular mass with the mature 95-kDa form. The core protein molecular weight of in vitro transcribed and translated gp90MEL-14 cDNA is consistent with the estimated molecular mass of 37 kDa, calculated from the cDNA sequence of the core protein, and 8 to 10 kDa less than the protein molecular mass of gp90MEL-14 translated in vivo in the presence of tunicamycin (45 kDa). Inasmuch as we have ruled out glycosylation as accounting for this discrepancy, this is consistent with the addition of one ubiquitin moiety to the core protein during biosynthesis. Limited proteolysis confirms the similarity between in vitro transcribed gp90MEL-14 cDNA and the tunicamycin form of gp90MEL-14.

  View details for Web of Science ID A1990DV27900028

  View details for PubMedID 2166761

• FAILURE TO FIND ALLOIMMUNE MEMORY IN THE RESORPTION PHENOMENON OF BOTRYLLUS CYTOMICTICAL CHIMERA EUROPEAN JOURNAL OF IMMUNOLOGY Rinkevich, B., Weissman, I. L. 1990; 20 (8): 1775-1779

  Abstract

  It has been previously shown that mixed-cell chimeras may be established between different colonies of the compound tunicate Botryllus schlosseri given that they share one allele in common on the fusibility locus. However, one of the partners in each chimera is often resorbed. Here we tested for the existence of a memory component to this response, as measured by the accelerated interval to resorption of a second set of semiallogeneic colonies. Eight genetically unrelated colonies gave rise to eighteen chimeras. After a complete resorption of the "inferior" partners in these chimeras, secondary and tertiary sets of chimeras were established with some of them by fusion of the "superior" with naive subclones of the "inferior" partners. It is shown here that (a) when multiple chimeras are prepared using the same pair of colonies, subclones of only one of the partners are resorbed; (b) high variation in the time for resorption is recorded when several chimeras are prepared from any specific pair of colonies, and (c) the time for resorption is not related to the number of vessels connecting between the partners in the chimera, nor to their relative body sizes. Allospecific memory is not documented here and reasons for its absence are discussed.

  View details for Web of Science ID A1990EA22300022

  View details for PubMedID 2209689

• Genomic organization of the selectin family of leukocyte adhesion molecules on human and mouse chromosome 1. journal of experimental medicine Watson, M. L., Kingsmore, S. F., Johnston, G. I., Siegelman, M. H., Le Beau, M. M., Lemons, R. S., Bora, N. S., Howard, T. A., Weissman, I. L., McEver, R. P. 1990; 172 (1): 263-272

  Abstract

  A structurally and functionally related group of genes, lymph node homing receptor (LHR), granule membrane protein 140 (GMP-140), and endothelial leukocyte adhesion molecule 1 (ELAM-1) are shown to constitute a gene cluster on mouse and human chromosome 1. In situ hybridization mapped GMP-140 to human chromosome 1 bands 21-24 consistent with chromosomal localization of LHR. Gene linkage analysis in the mouse indicated that these genes and serum coagulation factor V (FV) all map to a region of distal mouse chromosome 1 that is syntenic with human chromosome 1, with no crossovers identified between these four genes in 428 meiotic events. Moreover, long range restriction site mapping demonstrated that these genes map to within 300 kb in both the human and mouse genomes. These data suggest that LHR, ELAM-1, and GMP-140 comprise an adhesion protein family, the selectins, that arose by multiple gene duplication events before divergence of mouse and human. Furthermore, the location of these genes on mouse and human chromosome 1 is consistent with a close evolutionary relationship to the complement receptor-related genes, which also are positioned on the same chromosomes in both species and with which these genes share a region of sequence homology. These data characterize the organization of a genomic region that may be critical for intercellular communication within the immune system.

  View details for PubMedID 1694218

• GENOMIC ORGANIZATION OF THE SELECTION FAMILY OF LEUKOCYTE ADHESION MOLECULES ON HUMAN AND MOUSE CHROMOSOME-1 JOURNAL OF EXPERIMENTAL MEDICINE Watson, M. L., Kingsmore, S. F., Johnston, G. I., Siegelman, M. H., LEBEAU, M. M., Lemons, R. S., Bora, N. S., Howard, T. A., Weissman, I. L., McEver, R. P., Seldin, M. F. 1990; 172 (1): 263-272

  View details for Web of Science ID A1990DL09100031

• MORPHOLOGICAL AND GENETIC VERIFICATION THAT MONTEREY BOTRYLLUS AND WOODS HOLE BOTRYLLUS ARE THE SAME SPECIES BIOLOGICAL BULLETIN BOYD, H. C., Weissman, I. L., Saito, Y. 1990; 178 (3): 239-250

  View details for Web of Science ID A1990DL11200008

• THE MOUSE LYMPH-NODE HOMING RECEPTOR IS IDENTICAL WITH THE LYMPHOCYTE CELL-SURFACE MARKER LY-22 - ROLE OF THE EGF DOMAIN IN ENDOTHELIAL BINDING CELL Siegelman, M. H., Cheng, I. C., Weissman, I. L., Wakeland, E. K. 1990; 61 (4): 611-622

  Abstract

  The lymph node homing receptor core polypeptide (mLHRc) is composed of a tandem collection of domains: a lectin domain, an epidermal growth factor (EGF) domain, and two repeats common in complement regulatory proteins. Here we demonstrate localization of mLHRc to chromosome 1, the portion syntenic with chromosome 1 in man. This locus is inseparable in mouse strains from the murine lymphocyte cell surface marker Ly-22. The data indicate that Ly-22 is an allelic determinant on the LHR resulting from a single amino acid interchange within the EGF domain. Cross-blocking experiments demonstrate that anti-Ly-22 and MEL-14 recognize independent epitopes and that Ly-22 is distinct from the carbohydrate binding region. Application of anti-Ly-22 in the in vitro binding assay shows inhibition of binding of lymphocytes to high endothelial venules (HEVs). The localization of the Ly-22 epitope in this novel chimeric protein suggests direct participation of the EGF domain in the adhesion of lymphocytes to HEV.

  View details for Web of Science ID A1990DE90400007

  View details for PubMedID 1693096

• CHARACTERIZATION OF A RAT MONOCLONAL-ANTIBODY SPECIFIC FOR A DETERMINANT ENCODED BY THE V-BETA-7 GENE SEGMENT - DEPLETION OF V-BETA-7+ T-CELLS IN MICE WITH MLS-1A HAPLOTYPE JOURNAL OF IMMUNOLOGY Okada, C. Y., Holzmann, B., Guidos, C., PALMER, E., Weissman, I. L. 1990; 144 (9): 3473-3477

  Abstract

  We have generated a rat mAb, TR310, which recognizes a determinant encoded by the murine V beta 7 gene segment of the TCR. TR310 immunoprecipitates TCR from cell lysates, co-modulates with CD3, and can be used for immunofluorescence staining of T cells. By using this antibody, we found that the average percentage of V beta 7+ peripheral T cells in Mls-1b mice was 3.8%, but only 0.8% in Mls-1a mice. A similar difference was also observed in the mature TCRhi thymocyte subsets, suggesting that V beta 7+ T cells are deleted during intrathymic maturation in Mls-1a mice. TR310 should prove to be a valuable reagent in further studies of the TCR repertoire and the analysis of factors which alter it.

  View details for Web of Science ID A1990DA76500031

  View details for PubMedID 1691759

• THE GROWTH-FACTOR IL-7 INDUCES EXPRESSION OF A TRANSFORMATION-ASSOCIATED ANTIGEN IN NORMAL PRE-B-CELLS INTERNATIONAL IMMUNOLOGY Sherwood, P. J., Weissman, I. L. 1990; 2 (5): 399-406

  Abstract

  The B lineage antigen 6C3Ag is expressed by a number of cell types involved in lymphopoiesis. We have investigated whether there is a relationship between activation of normal pre-B cells by the growth factor IL-7 and the expression of 6C3Ag. Sorted sub-populations of bone marrow pre-B cells were cultured with IL-7 and their expression of surface antigens measured. There is a rapid induction of 6C3Ag expression after exposure to IL-7 by pre-B cells but not mature B cells. In particular, the 6C3lo pre-B cells respond very strongly by increasing 6C3Ag expression. The expression of 6C3Ag coincides with increased DNA synthesis and formation of lymphoblasts by the responding cells. The responses seen were specific to IL-7, and to a lesser degree IL-4. We conclude from this that IL-7 specifically induces the expression of 6C3Ag.

  View details for Web of Science ID A1990DK02300003

  View details for PubMedID 2085485

• B-CELL INFILTRATION OF THE THYMIC MEDULLA IN NEW-ZEALAND BLACK, NEW-ZEALAND WHITE, AND (NEW-ZEALAND BLACK X NEW-ZEALAND WHITE) F1-MICE - EFFECT OF TOTAL LYMPHOID IRRADIATION ARTHRITIS AND RHEUMATISM Farinas, M. C., Adkins, B., STALL, A. M., Weissman, I., Strober, S. 1990; 33 (5): 702-710

  Abstract

  Thymuses from female (New Zealand black x New Zealand white)F1 [( NZB x NZW]F1), New Zealand black, and New Zealand white mice of different ages were examined by immunohistochemical and flow cytometric analysis. Two-and-a-half-month-old (NZB x NZW)F1 mice showed infiltration of the thymus with B cells, and by 6-8 months of age, showed a disruption of the entire medullary area. More than 80% of the thymic B cells had the phenotypic characteristics of conventional B cells (IgM+, IgD+, Ly-1-). Total lymphoid irradiation induced a marked depletion of medullary B cells and a restoration of the thymic architecture.

  View details for Web of Science ID A1990DE73800012

  View details for PubMedID 2346525

• GENES ENCODING TUMOR NECROSIS FACTOR-ALPHA AND GRANZYME-A ARE EXPRESSED DURING DEVELOPMENT OF AUTOIMMUNE DIABETES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Held, W., MacDonald, H. R., Weissman, I. L., Hess, M. W., Mueller, C. 1990; 87 (6): 2239-2243

  Abstract

  Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. & Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.

  View details for Web of Science ID A1990CU85200040

  View details for PubMedID 2179951

• ALLORECOGNITION HISTOCOMPATIBILITY IN A PROTOCHORDATE SPECIES - IS THE RELATIONSHIP TO MHC SOMATIC OR STRUCTURAL IMMUNOLOGICAL REVIEWS Weissman, I. L., Saito, Y., Rinkevich, B. 1990; 113: 227-241

  Abstract

  Colonial tunicates are complex marine invertebrates (in fact protochordates) that undergo a variety of histocompatibility reactions in their intraspecific competition for feeding surfaces. By means of these reactions colonies fuse with kin, extend domination over a feeding surface, while isolating unrelated conspecifics. The primary determinant of fusion (with kin) or rejection (of non-kin) is a single, highly polymorphic, histocompatibility gene locus (or haplotype), called Fu/HC. Following fusion with nonidentical kin sharing 1 or more Fu/HC allele(s), the fused pair expands both chimeric partners via an asexual budding process, further extending domination over a feeding surface. However, at some later time point an intense set of histoincompatibility reactions occurs between fused kin, resulting in the destruction of all individuals of one of the genotypes, ending the chimeric state. In this review we describe what is known of the genetics and several biological properties encoded by the Fu/HC, and the several independent gene loci that control the colony resorption phenomena that return the colony to the province of a single genotypic individual.

  View details for Web of Science ID A1990CT61100010

  View details for PubMedID 2180808

• BOTRYLLUS-SCHLOSSERI (TUNICATA) WHOLE COLONY IRRADIATION - DO SENESCENT ZOOID RESORPTION AND IMMUNOLOGICAL RESORPTION INVOLVE SIMILAR RECOGNITION EVENTS JOURNAL OF EXPERIMENTAL ZOOLOGY Rinkevich, B., Weissman, I. L. 1990; 253 (2): 189-201

  Abstract

  The colonial tunicate Botryllus schlosseri undergoes cyclic blastogenesis where feeding zooids are senescened and resorbed and a new generation of zooids takes over the colony. When non-identical colonies come into direct contact, they either reject each other or fuse. Fusion is usually followed by the resorption of one of the partners in the chimera (immunological resorption). The striking morphological similarities between the two resorption phenomena suggest that both may involve tissue destruction following self-nonself recognition events. Here we attempt to modify these two events by whole colony gamma irradiation assays. Three sets of experiments were performed: 1) different doses of whole colony irradiation for determination of irradiation effects (110 colonies, up to 8,000 rads); 2) pairs of irradiated-nonirradiated isografts of clonal replicates for the potential of reconstruction of the irradiated partners (23 pairs); 3) chimeras of irradiated-nonirradiated partners for analysis of resorption hierarchy. Mortality increased with the irradiation dose. All colonies exposed to more than 5,000 rads died within 19 days, while no colony died below 2,000 rads. The average mortality periods, in days, for doses of 6,000-8,000, 5,000, and 2,500-4,000 rads were 14.4 +/- 3.1 (n = 24), 19.8 +/- 6.0 (n = 15), and 19.6 + 5.1 (n = 22), respectively. Younger colonies (3-6 months old) may survive radiation better than older ones (more than 13 months). Many morphological alterations were recorded in irradiated colonies: ampullar contraction and/or dilation, accumulation of pigment cells within ampullae, abnormal bleeding from blood vessels, sluggish blood circulation, necrotic zones, reduction in bud number, and irregularities in zooid and system structures. With doses of 3,000-4,000 rads and above, irradiation arrested the formation of new buds and interrupted normal takeover, turning the colony into a chaotic bulk of vessels, buds, and zooid segments. Death supervened after a period of up to 1 month of poor condition, which was also characterized by loss of organization in systems. In isografts of irradiated-nonirradiated parts, the normal subclone resorbed all zooids and buds of the irradiated one within less than 1 week, even if it was up to 13 times smaller, without showing any sign of harmful effects. Thus, the irradiated subclone is not reconstituted by sharing blood circulation with a syngeneic part. Under 2,000 rads some of the irradiated zooids within this type of union started to regenerate, and at 1,000 rads no resorption was recorded, even though the number of zooids decreased in the irradiated part.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1990CP93600008

  View details for PubMedID 2313247

• Structural similarity between a primitive chordate membrane heterodimer and lymphocyte antigen receptors. International immunology Danska, J. S., MCINTYRE, B. W., McDevitt, H. O., Weissman, I. L. 1990; 2 (9): 795-802

  Abstract

  Botryllus schlosseri is a colonial tunicate that shared a common ancestor with the lineage leading to mammals about 450 million years ago, and flourishes today along the California coast. Prior studies of Botryllus populations have demonstrated the presence of a co-dominantly expressed, highly polymorphic histocompatibility locus (Fu/HC) controlling the acceptance (fusion) or rejection of new individuals into a parabiotic colony. Intercolonial blood cell contact, and recognition of self/not self, precedes both fusion and rejection reactions. Efforts to understand the evolution of the immune system necessitate study of cell surface molecules involved in cell-cell recognition events in primitive species. In mammals, birds, amphibians, and fishes clonally distributed lymphocyte surface molecules that are responsible for antigen recognition (B cell immunoglobulins and T cell receptors) can be distinguished by the disulfide linkage that pairs two or more polypeptides containing constant and variable regions. We have identified a disulfide-linked, heterodimeric (alpha beta) cell surface molecule in Botryllus with biochemical resemblance to mammalian lymphocyte antigen receptors. Observed charge variants of constituent chains of the tunicate protein described here do not correlate with Fu/HC allelic diversity. Both chains of this heterodimer can be resolved into several isoforms which are not based upon post-translational carbohydrate or phosphate additions. Comparisons of iodinated tryptic peptides from two beta chain isomorphs reveal one distinct and several common peptides.

  View details for PubMedID 2278999

• The paternal centrosome directs the polarity of early pattern formation in the fertilized ascidia ceratodes egg Advances in Invertebrate Reproduction 5 Lauzon, R., Weissman, I. Elsevier. 1990: 131–138
• DNA generated by polymerase chain reaction using taq DNA polymerase has non-template nucleotide additions: implications for cloning PCR products A Forum for PCR Users Denney, D., Weissman, I. 1990; 4: 25-26
• Morphologic and genetic verification that Monterey Botryllus and Woods Hole Botryllus are the same species Biol Bull Boyd, H., Weissman, I., Saito, Y. 1990; 178: 239-250
• Hemopoietic stem cells and early hematolymphoid differentiation in mouse-man Molecular Aspects of Immune Response and Infectious Diseases Weissman, I., et al 1990
• The SCID-hu mouse as a model system for hiv infection Human Retroviruses McCune, M., Namikawa, R., Lieberman, M., Weissman, I., Kaneshima, H. 1990
• AN IMMUNODOMINANT MURINE LYMPHOMA CELL-SURFACE HETERODIMER MARKS THYMIC PROGENITOR SUBSETS JOURNAL OF IMMUNOLOGY SENMAJUMDAR, A., Guidos, C., Kaneshima, H., White, J. H., Marian, J., Lieberman, M., Weissman, I. L. 1990; 144 (1): 111-121

  Abstract

  mAb 1C11 was raised against the cells of retrovirus-negative, radiation-induced thymomas of C57BL/Ka mice. MAb 1C11 binds to radiation- and RadLV-induced C57BL/Ka lymphomas, to lymphomas of other mouse strains and to B-lineage tumors. The 1C11 Ag is expressed on a subpopulation of normal thymocytes that is enriched in immature cells. After fractionated x-irradiation, this percentage increases gradually during the preleukemic period, hence mAb 1C11 appears to identify a transformation-related cell surface molecule. This conclusion is supported by experiments demonstrating that flow microfluorimetry-sorted, 1C11-expressing preleukemic thymocytes progress rapidly to full neoplasia following intrathymic injection, whereas nonexpressing cells do not. Most of day-14 fetal thymocytes are as strongly positive as thymic lymphomas for the 1C11 Ag whereas Ag-activated T cell lines express moderate levels. Multiparameter flow microfluorimetry analysis shows that 1C11 is expressed predominantly on CD3-/lo thymic blast cells of three phenotypically defined subsets: CD4-8-, CD4-8+, and CD4+8+, all of which contain thymic progenitors. By immunohistochemical staining, the Ag is also found in association with epithelial cells on a variety of normal, nonlymphoid tissue, but is not detectable on heart tissue. The 1C11 antibody immunoprecipitates a disulfide-linked heterodimeric protein of 85/37 kDa and the antigenic determinant is located on the H chain of the molecule. When analyzed by SDS-PAGE under nonreducing conditions, the molecule exists as a 130-kDa protein. Enzymatic digestion of the heterodimer indicates that the H chain, but not the L chain, has at least three N-linked glycosylation sites. We propose that this novel cell surface glycoprotein may be associated with processes of differentiation and lymphomagenesis.

  View details for Web of Science ID A1990CG62300015

  View details for PubMedID 2404061

• THE EARLY B-LINEAGE ANTIGEN BP-1 AND THE TRANSFORMATION-ASSOCIATED ANTIGEN 6C3 ARE ON THE SAME MOLECULE JOURNAL OF IMMUNOLOGY Wu, Q., Tidmarsh, G. F., Welch, P. A., Pierce, J. H., Weissman, I. L., COOPER, M. D. 1989; 143 (10): 3303-3308

  Abstract

  Biochemical similarities and cellular distribution patterns of the early B lineage-specific BP-1 alloantigen and the B lineage transformation-associated 6C3 Ag prompted this comparative study of the reactivities of the BP-1 and 6C3 mAb. Both Ag were found to be expressed on the same cells in normal tissues, and on the same cell lines when a large panel was analyzed. The Ag are both phosphorylated and have identical m.w., which may vary in different cell types because of differences in glycosylation. Immunoprecipitation of pre-B cell lysates with the BP-1 antibody removed the 6C3-reactive molecules and vice versa. However, the 6C3 antibody did not inhibit binding of the BP-1 antibody to viable cells and, in fact, enhanced immunofluorescence staining was observed when both antibodies were added together. These results indicate that the BP-1 and 6C3 antibodies react with different epitopes on the same molecule that is expressed in relatively low levels on normal early B lineage cells, and in relatively high levels on most neoplastic pre-B cells, pre-B cells in long term bone marrow cultures and certain stromal cell lines.

  View details for Web of Science ID A1989AZ28800027

  View details for PubMedID 2809203

• INTRATHYMIC MATURATION OF MURINE LYMPHOCYTES-T FROM CD8+ PRECURSORS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Guidos, C. J., Weissman, I. L., Adkins, B. 1989; 86 (19): 7542-7546

  Abstract

  The CD4-8- thymocyte subset contains immature precursors for phenotypically and functionally mature CD4+8- and CD4-8+ thymocytes and peripheral T cells, as well as nonmature CD4+8+ thymocytes, most of which die in situ. The intrathymic death of most thymocytes is probably related to selective influences that ensure that only those precursors bearing self-major histocompatibility complex (MHC)-restricted and self-tolerant T-cell antigen receptors (TCR) survive to complete the maturation process. Interactions between surface molecules on thymocytes (TCR, CD4, and CD8) and thymic stromal cells (MHC proteins) are critical to repertoire selection. To understand this process, the lineage relationships among immature, nonmature, and mature thymocytes must be defined. We have examined directly the precursor-progeny relationships among CD4+8-, CD4-8+, and CD4+8+ murine thymocyte subsets by assessing their short-term (less than 5 days) developmental potentials following intrathymic injection into Thy-1 congenic, unirradiated host mice. Our results identify TCR-/lo CD4-8+ and TCRlo CD4+8+ blast cells as sequential intermediates in the development of mature TCRhi CD4+8- and TCRhi CD4-8+ thymocytes from CD4-8- precursors, thus defining at least one intrathymic maturation pathway for T lymphocytes.

  View details for Web of Science ID A1989AT78200054

  View details for PubMedID 2508090

• VARIATION IN THE OUTCOMES FOLLOWING CHIMERA FORMATION IN THE COLONIAL TUNICATE BOTRYLLUS-SCHLOSSERI BULLETIN OF MARINE SCIENCE Rinkevich, B., Weissman, I. L. 1989; 45 (2): 213-227

  View details for Web of Science ID A1989CD94200003

• MEASUREMENT OF BINDING-SPECIFICITY BETWEEN T-CELL LYMPHOMAS AND MURINE LEUKEMIA VIRUSES JOURNAL OF IMMUNOLOGICAL METHODS ONEILL, H. C., Weissman, I. L. 1989; 122 (1): 79-90

  Abstract

  We have previously reported the presence of receptors on radiation leukemia virus (RadLV)-induced thymomas and malignant thymocytes from AKR mice which specifically bind retrovirus produced by these T cell clones. These receptors have been shown to have specificity for virus reminiscent of an immune-specific receptor. Previous studies on T cell lymphoma binding to retroviruses have involved measurement of the interaction of labelled virus with cells using fluorescence-activated cell sorter (FACS) analysis (McGrath et al., J. Virol. (1978) 25, 923; McGrath and Weissman, Cell (1979) 17, 65; Weissman and McGrath, Curr. Top. Microbiol. Immunol. (1982) 98, 103). Here we report development of an assay for measuring lymphoma binding to virus, prepared as an immunoabsorbent adhered to a microtiter plate. Using this assay, we have shown that only T and not B cell lymphomas can bind to T cell-tropic viruses, and some cell lines have greatest specificity for homologous virus. The AKR-derived T cell lymphomas, SL3 and KKT-2, show greater specificity for leukemogenic AKR viruses, than for an AKR xenotropic virus or the recombinant AKR virus, MCF247. The RadLV-induced T cell lines, C6VL/1 and BL/VL3, have been found to bind cross-reactively to several different murine leukemia viruses (MuLVs). RadLV-induced T cell lymphomas do have greater specificity for their cognate retroviruses since free, homologous retrovirus can best block the interaction between cells and virus adhered to the wells of a microtiter plate. Cross-reactive interactions are more easily demonstrated by this assay, probably because low avidity interactions are stabilized as a result of the mode of virus presentation. Binding specificity for retroviral envelope determinants has been demonstrated using a rat anti-retroviral antiserum prepared as an F(ab)1 fragment. This antiserum can inhibit the interaction between the C6VL/1 thymoma and its RadLV virus. Specificity of this antibody for a gp70-like protein was confirmed by NaDodSO4-polyacrylamide gel electrophoresis (PAGE) and by loss of this activity after absorption of antibody on virus. Antibodies specific for RadLV/VL3 gp70 determinants can inhibit the interaction of C6VL/1 with RadLV/VL3 suggesting that cross-reactive binding to heterologous virus is also specific for a gp70 viral env determinant.

  View details for Web of Science ID A1989AK71900011

  View details for PubMedID 2547874

• HUMAN HOMOLOG OF MOUSE LYMPH-NODE HOMING RECEPTOR - EVOLUTIONARY CONSERVATION AT TANDEM CELL-INTERACTION DOMAINS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Siegelman, M. H., Weissman, I. L. 1989; 86 (14): 5562-5566

  Abstract

  A cDNA clone homologous to the mouse lymph node homing receptor core protein (mLHRc) was isolated from a cDNA library derived from stimulated human peripheral blood lymphocytes. Human RNA blot analysis shows a tissue and cell-line distribution of transcript expression generally parallel to that seen in the mouse, with expression confined to lymphoid tissues and some cell lines. Genomic DNA analysis suggests a low-copy gene under high-stringency conditions. The complete nucleotide sequence predicts a mature protein of 334 amino acids, identical in length to mLHRc. The protein shows striking conservation globally between human and mouse sequences. In particular, all three genre of protein interaction domains identified in the mouse--an animal lectin domain, an epidermal growth factor (EGF)-like domain, and two homologous repeat units preserving the motif of complement regulatory proteins (CRP)--are present in the human protein (hLHRc), and maintain the same tandem arrangement. The lectin and EGF-like regions are the most homologous, while the CRP domains are less conserved between species. The two CRP units in hLHRc are distinct from those in mLHRc in that they are homologous to one another rather than identical, suggesting strong pressure for maintenance of two repeats in this molecule. hLHRc is distinct from other kinds of lymphocyte adhesion molecules represented by VLA-4 (integrin) or CD44/gp90Hermes and, together with mLHRc and two other recently described molecules having a similar domain motif, defines a novel class of adhesion molecules exhibiting distinct evolutionary features. We propose that hLHRc likely represents the protein core of the human homologue of mLHRc functionally as well as structurally.

  View details for Web of Science ID A1989AG35900072

  View details for PubMedID 2664786

• HEMATOPOIETIC STEM-CELL PURIFICATION IMMUNOLOGY TODAY Weissman, I. L., Heimfeld, S., Spangrude, G. 1989; 10 (6): 184-184

  View details for Web of Science ID A1989AB45100003

  View details for PubMedID 2751826

• PEYERS PATCH-SPECIFIC LYMPHOCYTE HOMING RECEPTORS CONSIST OF A VLA-4-LIKE ALPHA-CHAIN ASSOCIATED WITH EITHER OF 2 INTEGRIN BETA-CHAINS, ONE OF WHICH IS NOVEL EMBO JOURNAL Holzmann, B., Weissman, I. L. 1989; 8 (6): 1735-1741

  Abstract

  Lymphocytes home to various lymphoid organs by adhering to and migrating through specialized high endothelial venules (HEV). The murine cell surface heterodimer LPAM-1 is involved in the homing of lymphocytes to mucosal sites (Peyer's patches). LPAM-1 has an alpha subunit (alpha 4m) analogous to the alpha chain of the human integrin molecule VLA-4. Here we show that the LPAM-1 beta subunit (beta p) is immunochemically and biochemically distinct from previously defined integrin beta subunits, suggesting that beta p represents a novel integrin beta subunit. Depending on the cellular source two alternative beta subunits, beta p and integrin beta 1, can be isolated in association with alpha 4m. Therefore, alpha 4m is the common subunit of the unique integrin LPAM-1 (alpha 4m beta p) and of the heterodimer LPAM-2 (alpha 4m beta 1), which is analogous to VLA-4. Antibody-blocking experiments suggest that, in addition to LPAM-1, LPAM-2 is also involved in the organ-specific adhesion of lymphocytes to Peyer's patch HEV.

  View details for Web of Science ID A1989U846900014

  View details for PubMedID 2670559

• DEVELOPMENTAL POTENTIAL OF CD4-8- THYMOCYTES - PERIPHERAL PROGENY INCLUDE MATURE CD4-8- T-CELLS BEARING ALPHA-BETA T-CELL RECEPTOR JOURNAL OF IMMUNOLOGY Guidos, C. J., Weissman, I. L., Adkins, B. 1989; 142 (11): 3773-3780

  Abstract

  We have used the intra-thymic transfer system to investigate the population dynamics of thymocyte and mature T cell subsets in the absence of continuing precursor input from the bone marrow. We have followed the development and life span of CD4+ and CD8+ thymocyte subsets and mature peripheral T cells from intra-thymically injected adult or fetal CD4-8- thymic precursors. Both precursor types proliferated, differentiated, and exported to peripheral lymphoid tissues alpha beta-TCR+CD4+8- and CD4-8+ progeny which formed a stable, long-lived component of the peripheral T cell pool. The production of phenotypically mature thymocytes and peripheral T cells occurred more rapidly from fetal CD4-8- precursors. CD4+8-:CD4-8+ ratios among peripheral progeny of intra-thymically-injected CD4-8- precursors were initially normal, but they steadily declined among progeny of the fetal precursors. Thus, there appear to be differences in the life span and/or proliferative capacity of mature T cells derived from embryonic vs adult progenitors. In addition to the predominant CD4+8- and CD4-8+ subsets of peripheral T cells, a minor (1 to 20%) population of Thy-1+CD3+4-8- T cells was identified among peripheral progeny of intra-thymically-injected CD4-8- thymocytes, as well as in lymph nodes of unmanipulated animals. A total of 20 to 34% of this subset expressed V beta 8+ TCR and the majority were CD5hi, Pgp-1+, and J11d-. The function and specificity of this newly identified population of thymically derived peripheral T cells remains to be investigated.

  View details for Web of Science ID A1989U688300006

  View details for PubMedID 2785564

• ROLE OF INTERLEUKIN-4 IN T-CELL ONTOGENY - CHANGES IN CELL-SURFACE PHENOTYPE AND LYMPHOKINE PRODUCTION OF IMMATURE THYMOCYTES AFTER CULTURE WITH INTERLEUKIN-4 AND PHORBOL ESTER JOURNAL OF AUTOIMMUNITY Guidos, C., Ransom, J., Fischer, M., Weissman, I., Zlotnik, A. 1989; 2: 141-153

  Abstract

  We have analyzed the phenotype and functional capabilities of adult and fetal CD4 8 thymocytes after 4 d of culture in IL-4 and PMA. Both adult and day 14 fetal CD4 8 thymocytes failed to acquire CD4 or CD8 antigens following culture. However, changes in expression of other antigens typical of immature thymocytes were observed. For example, the frequency with which cells expressed high levels of J11d or IL-2-R was greatly decreased following culture, whereas the frequency with which high levels of MEL-14, the lymph node homing receptor were expressed were greatly increased. This phenomenon may be due to direct induction by IL-4 and PMA of MEL-14 expression on purified MEL-14lo CD4-8- thymocytes. The frequency of cells expressing CD3, Ly-1 and Pgp-1 changed only slightly. Functionally, the cultured cells produced large amounts of interferon gamma but very little IL-2 or IL-4, although freshly isolated CD4-8- thymocytes produced all three lymphokines. These results suggest that in addition to a proliferative stimulus, culture in IL-4/PMA alters the expression of several early thymocyte antigens, the functional capabilities of CD4-8- progenitor thymocytes, and may act as a selective differentiation stimulus to MEL-14lo CD4-8- thymocytes.

  View details for Web of Science ID A1989AE29900015

  View details for PubMedID 2505789

• MOUSE HEMATOPOIETIC STEM-CELL ANTIGEN SCA-1 IS A MEMBER OF THE LY-6 ANTIGEN FAMILY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA VANDERIJN, M., Heimfeld, S., Spangrude, G. J., Weissman, I. L. 1989; 86 (12): 4634-4638

  Abstract

  Recently, hematopoietic stem cells were purified to homogeneity from mouse bone marrow. The protein structure of Sca-1, the cell surface antigen used in the isolation of hematopoietic stem cells, is described here. It is shown that the Sca-1 antigen is a member of the Ly-6 antigen family. The anti-Sca-1 antibody was used in immunohistochemistry experiments to define the structures in several tissues that had previously been shown to contain Ly-6 antigens. In thymus, spleen, and kidney, specific staining of parenchymal cells can be demonstrated, whereas only vasculature reacts with anti-Sca-1 in brain, heart, and liver and possibly in lung.

  View details for Web of Science ID A1989AB24900063

  View details for PubMedID 2660142

  View details for PubMedCentralID PMC287325

• IDENTIFICATION OF A NOVEL BONE MARROW-DERIVED B-CELL PROGENITOR POPULATION THAT COEXPRESSES B220 AND THY-1 AND IS HIGHLY ENRICHED FOR ABELSON LEUKEMIA-VIRUS TARGETS MOLECULAR AND CELLULAR BIOLOGY Tidmarsh, G. F., Heimfeld, S., WHITLOCK, C. A., Weissman, I. L., MULLERSIEBURG, C. E. 1989; 9 (6): 2665-2671

  Abstract

  A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells.

  View details for Web of Science ID A1989U757700039

  View details for PubMedID 2474759

• IL-2 INDUCES EXPRESSION OF SERINE PROTEASE ENZYMES AND GENES IN NATURAL-KILLER AND NONSPECIFIC T-KILLER CELLS JOURNAL OF IMMUNOLOGY MANYAK, C. L., Norton, G. P., Lobe, C. G., Bleackley, R. C., Gershenfeld, H. K., Weissman, I. L., Kumar, V., Sigal, N. H., Koo, G. C. 1989; 142 (10): 3707-3713

  Abstract

  The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.

  View details for Web of Science ID A1989U545800052

  View details for PubMedID 2785561

• RELATIVE V-BETA TRANSCRIPT LEVELS IN THYMUS AND PERIPHERAL LYMPHOID-TISSUES FROM VARIOUS MOUSE STRAINS - INVERSE CORRELATION OF I-E AND MLS EXPRESSION WITH RELATIVE ABUNDANCE OF SEVERAL V-BETA TRANSCRIPTS IN PERIPHERAL LYMPHOID-TISSUES JOURNAL OF EXPERIMENTAL MEDICINE Okada, C. Y., Weissman, I. L. 1989; 169 (5): 1703-1719

  Abstract

  We have measured the relative levels of transcripts for 15 of the 22 known V beta gene segments. The level of transcripts for the highest and lowest expressed V beta gene segment differed by greater than 20-fold in the thymus and an even larger difference was observed in the periphery. The levels of expressions were unrelated to the order of the V beta genes on the chromosome. For most of the V beta gene segments, the relative transcript levels were the same in the thymus and periphery, suggesting that thymic selection in general does not act solely upon the V beta gene segment. One V beta gene segment in the BALB and B10 mice strains was an exception to this rule. V beta 5.2 expression in the periphery of BALB and B10 mice inversely correlated with the expression of the MHC class II molecule I-E. Five V beta gene segments had reduced transcript levels in the periphery of Mls-1a mice compared with their thymic levels or to the levels found in Mls-1b mice. The peripheral level of V beta 3 transcripts vary with MHC and Mls-2 haplotypes. The observation that certain V beta transcript levels are reduced in the periphery when compared with the thymus favors the hypothesis that self tolerance at the T cell level results in the elimination of self-reactive T cells, rather than paralysis by a block at some post-transcriptional step. Finally, the wide variability of V beta gene segment expression in the thymus suggests mechanisms exist to import an early bias to the repertoire. Whether this bias results from differential V beta segment rearrangement rates, differential V beta expression rates, or events occurring after TCR-alpha/beta expression on immature/nonmature thymocyte cell surfaces is yet to be determined.

  View details for Web of Science ID A1989U366600016

  View details for PubMedID 2497226

• ANALYSIS OF NATURALLY-OCCURRING DELAYED-TYPE HYPERSENSITIVITY REACTIONS IN LEPROSY BY INSITU HYBRIDIZATION JOURNAL OF EXPERIMENTAL MEDICINE Cooper, C. L., Mueller, C., SINCHAISRI, T. A., Pirmez, C., Chan, J., Kaplan, G., Young, S. M., Weissman, I. L., Bloom, B. R., Rea, T. H., Modlin, R. L. 1989; 169 (5): 1565-1581

  Abstract

  Analysis of tissue lesions of the major reactional states of leprosy was undertaken to study the immune mechanisms underlying regulation of cell-mediated immunity and delayed-type hypersensitivity (DTH) in man. In situ hybridization hybridization of reversal reaction biopsy specimens for INF-gamma mRNA expression revealed a 10-fold increase in specific mRNA-containing cells over that observed in unresponsive lepromatous patients. Expression of huHF serine esterase, a marker for T cytotoxic cells, were fourfold increased in reversal reaction and tuberculoid lesions above that detected in unresponsive lepromatous individuals. Immunohistology of reversal reactions confirmed a selective increase of Th and T cytotoxic cells in the cellular immune response. Of interest, the microanatomic location of these serine esterase mRNA-containing cells was identical to the distribution of CD4+ cells. Analysis of erythema nodosum leprosum (ENL) lesions revealed differences in the underlying immune processes in comparison with reversal reaction lesions. Although phenotypic Th cells predominated in ENL lesions, IFN-gamma and serine esterase gene expression were markedly reduced. We suggest that reversal reactions represent a hyperimmune DTH response characterized by a selective increase of CD4+ IFN-gamma producing cells and T cytotoxic cells, which result in the clearing of bacilli and concomitant tissue damage. In contrast, ENL reactions may be viewed as a transient diminution of Ts cells and activity leading to a partial and transient augmentation in cell-mediated immunity, perhaps sufficient to result in antibody and immune complex formation, but insufficient to clear bacilli from lesions.

  View details for Web of Science ID A1989U366600005

  View details for PubMedID 2523952

• INTEGRIN MOLECULES INVOLVED IN LYMPHOCYTE HOMING TO PEYER PATCHES IMMUNOLOGICAL REVIEWS Holzmann, B., Weissman, I. L. 1989; 108: 45-61

  Abstract

  This review summarizes experiments designed to analyze lymphocyte receptors mediating recognition of and adhesion to HEV in mucosal lymphoid organs. A monoclonal antibody (R1-2) was selected which inhibits the adhesion of murine lymphocytes to Peyer's patch HEV. Antibody R1-2 recognizes the alpha chain (alpha 4m) of the murine lymphocyte cell-surface alpha beta heterodimer LPAM-1. The association of LPAM-1 alpha and beta chains requires the presence of Ca++ ions. Two proteins of Mr 84,000 and 62,000 which are also precipitated by antibody R1-2 most likely represent fragments of alpha 4m. The cross-reactivity of a monospecific rabbit anti-serum indicated that alpha 4m is analogous to the alpha chain of the human integrin molecule VLA-4. In addition, a cDNA clone encoding the human VLA-4 alpha chain hybridized with RNA from alpha 4m+ but not alpha 4m- cell lines. However, the LPAM-1 beta subunit (beta p) was shown to be immunochemically and biochemically distinct from integrin beta 1, beta 2, and beta 3, indicating that beta p represents a unique integrin beta chain. When the beta subunits associated with alpha 4m on a panel of lymphoma cell lines were analyzed, it was found that, depending on the cellular source, alpha 4m can associate with either of two beta chains: beta p or integrin beta 1. Therefore alpha 4m appears to be the common subunit of the two lymphocyte cell surface heterodimers LPAM-1 (alpha 4m/beta p) and LPAM-2 (alpha 4m/beta 1). LPAM-2 is analogous to the human VLA-4 molecule, whereas LPAM-1 represents a unique integrin heterodimer. Antibody R1-2 inhibited Peyer's patch HEV-adhesion of normal mouse lymphocytes and every lymphoma cell line tested including LPAM-1 and LPAM-2 single-positive cell lines. We also showed that the binding capacity of variants of a clonal lymphoma cell line to Peyer's patch HEV correlates with the level of LPAM-1 expression. It therefore appears that both heterodimers are involved in lymphocyte-Peyer's patch HEV interactions and that the adhesion of lymphocytes to Peyer's patch HEV is generally LPAM-1- or LPAM-2-dependent. We further investigated whether VLA-4, the human analog of LPAM-2, can mediate adhesion of human lymphocytes to HEV in mucosal lymphoid organs.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1989U687100003

  View details for PubMedID 2670742

• MOUSE LYMPH-NODE HOMING RECEPTOR CDNA CLONE ENCODES A GLYCOPROTEIN REVEALING TANDEM INTERACTION DOMAINS SCIENCE Siegelman, M. H., VANDERIJN, M., Weissman, I. L. 1989; 243 (4895): 1165-1172

  Abstract

  Isolation of a clone encoding the mouse lymph node homing receptor reveals a deduced protein with an unusual protein mosaic architecture, containing a separate carbohydrate-binding (lectin) domain, an epidermal growth factor-like (EGF) domain, and an extracellular precisely duplicated repeat unit, which preserves the motif seen in the homologous repeat structure of complement regulatory proteins and other proteins. The receptor molecule is potentially highly glycosylated, and contains an apparent transmembrane region. Analysis of messenger RNA transcripts reveals a predominantly lymphoid distribution in direct relation to the cell surface expression of the MEL-14 determinant, and the cDNA clone is shown to confer the MEL-14 epitope in heterologous cells. The many novel features, including ubiquitination, embodied in this single receptor molecule form the basis for numerous approaches to the study of cell-cell interactions.

  View details for Web of Science ID A1989T473200027

  View details for PubMedID 2646713

• 2 MONOCLONAL-ANTIBODIES IDENTIFY THYMIC-REPOPULATING CELLS IN MOUSE BONE-MARROW JOURNAL OF IMMUNOLOGY Spangrude, G. J., Klein, J., Heimfeld, S., Aihara, Y., Weissman, I. L. 1989; 142 (2): 425-430

  Abstract

  The progenitor cells in the bone marrow that home to and repopulate the thymus have been incompletely characterized. In particular, it is not clear whether thymocytes differentiate directly from pluripotent hemopoietic stem cells that seed to the thymus, or whether T lymphoid-committed stem cells (prothymocytes) arise in the bone marrow before the thymic migration. In order to resolve this question, we have used mAb specific for cell-surface Ag to identify the bone marrow cells which can seed to and repopulate the thymus of irradiated mice. We report here that the majority of thymic-repopulating cells in mouse bone marrow express low levels of the Thy-1 Ag (Thy-1lo) plus high levels of a newly described Ag termed stem cell Ag (Sca-1). Two distinct populations of thymic-repopulating Thy-1loSca-1+ cells in mouse bone marrow can be discriminated based on expression of any of a number of hemolymphoid lineage-specific (Lin) markers. Thus, Thy-1loLin-Sca-1+ and Thy-1loLin+Sca-1+ fractions of bone marrow contain thymic-repopulating cells. A second Ag, stem cell Ag-2 (Sca-2), is expressed by Thy-1loLin+Sca-1+ cells but not by Thy-1loLin-Sca-1+ cells. The Thy-1loLin-Sca-1+ fraction expresses intermediate levels of the phagocyte glycoprotein-1 Ag, and comprises 30% of the Thy-1loLin- bone marrow cells, which have previously been shown to be highly enriched in pluripotent hemopoietic stem cells. By facilitating the isolation of highly purified subpopulations of bone marrow cells that can repopulate the thymus, Sca-1 and Sca-2 should provide an experimental tool for describing the developmental potential of such bone marrow subsets.

  View details for Web of Science ID A1989R647800009

  View details for PubMedID 2562963

• IDENTIFICATION OF A MURINE PEYERS PATCH SPECIFIC LYMPHOCYTE HOMING RECEPTOR AS AN INTEGRIN MOLECULE WITH AN ALPHA-CHAIN HOMOLOGOUS TO HUMAN VLA-4-ALPHA CELL Holzmann, B., MCINTYRE, B. W., Weissman, I. L. 1989; 56 (1): 37-46

  Abstract

  Lymphocyte homing is controlled by organ-specific interactions of lymphocytes and high endothelial venules (HEV). Adhesion of lymphocytes to Peyer's patch HEV, but not to peripheral node HEV, is inhibited by an antibody recognizing the murine lymphocyte antigen LPAM-1. Lymphoma cell variants were selected on the FACS for differences in LPAM-1 expression: the binding capacity of these variants to Peyer's patch HEV directly correlates with the level of LPAM-1 expression. The anti-LPAM-1 antibody recognizes the alpha subunit of an Mr 160,000/130,000 cell surface alpha beta heterodimer. The association of LPAM-1 alpha and beta chains requires the presence of Ca2+ ions. Proteins of Mr 84,000 and Mr 62,000 present in LPAM-1 immunoprecipitates appear to be products of the proteolytic processing of alpha chains. The structure of LPAM-1 is virtually identical to that of the human integrin receptor VLA-4. The cross-reactivity of a monospecific rabbit antiserum demonstrated the similarity between the human VLA-4 alpha chain and the alpha subunit of LPAM-1.

  View details for Web of Science ID A1989R859200007

  View details for PubMedID 2463092

• DEVELOPMENTAL ANALYSIS OF THE MOUSE HEMATOLYMPHOID SYSTEM COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Heimfeld, S., Guidos, C. J., Holzmann, B., Siegelman, M. H., Weissman, I. L. 1989; 54: 75-85

  View details for Web of Science ID A1989JX74500008

  View details for PubMedID 2700949

• Tolerance, the thymus, and H-Y Realm of Tolerance Jerabek, L., Greenspan, S., Okada, C., Weissman, I. Springer-Verlag. 1989: 50–59
• Lymphoid tissues and organs Fundamental Immunology Butcher, E., Weissman, I. Raven Press. 1989; 2: 117–138
• Some observations on the life history of lymphocytes Weissman, I. 1989: 43–69
• Allorecognition in colonial tunicates parallels with and tangents from vertebrate immunity Progress in Immunology, 7th International Congress of Immunology Weissman, I., Saito, Y., Rinkevich, B. 1989
• Developing ethical human models for experimental medicine Stanford Med Weissman, I. 1989: 17-32
• THE SCID-HU MOUSE - CURRENT STATUS AND POTENTIAL APPLICATIONS CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY McCune, J. M., Kaneshima, H., Lieberman, M., Weissman, I. L., Namikawa, R. 1989; 152: 183-193

  View details for Web of Science ID A1989CW74300022

  View details for PubMedID 2680299

• A SERINE PROTEASE-ENCODING GENE THAT MARKS ACTIVATED CYTO-TOXIC T-CELLS INVIVO AND INVITRO CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY HERSHBERGER, R. J., Mueller, C., Gershenfeld, H. K., Weissman, I. L. 1989; 140: 81-92

  View details for Web of Science ID A1989T869200007

  View details for PubMedID 2644077

• Maturation of hematolymphoid cells that express Thy-1. Immunology series Müller-Sieburg, C. E., Tidmarsh, G. F., Weissman, I. L., Spangrude, G. J. 1989; 45: 289-316

  View details for PubMedID 2577321

• Some observation on the life history of lymphocytes. Harvey lectures Weissman, I. L. 1989; 85: 43-69

  View details for PubMedID 2519150

• INFECTION OF THE SCID-HU MOUSE BY HIV-1 SCIENCE Namikawa, R., Kaneshima, H., Lieberman, M., Weissman, I. L., McCune, J. M. 1988; 242 (4886): 1684-1686

  Abstract

  SCID-hu mice with human fetal thymic or lymph node implants were inoculated with the cloned human immunodeficiency virus-1 isolate, HIV-1JR-CSF. In a time- and dose-dependent fashion, viral replication spread within the human lymphoid organs. Combination immunohistochemistry and in situ hybridization revealed only viral RNA transcripts in most infected cells, but some cells had both detectable viral transcripts and viral protein. Infected cells were always more apparent in the medulla than in the cortex of the thymus. These studies demonstrate that an acute infection of human lymphoid organs with HIV-1 can be followed in the SCID-hu mouse.

  View details for Web of Science ID A1988R492900039

  View details for PubMedID 3201256

• DOWN-REGULATION OF HOMING RECEPTORS AFTER T-CELL ACTIVATION JOURNAL OF IMMUNOLOGY Jung, T. M., Gallatin, W. M., Weissman, I. L., Dailey, M. O. 1988; 141 (12): 4110-4117

  Abstract

  The specific pattern of lymphocyte localization and recirculation is important for the induction and expression of normal immune responses. In order to home to lymph nodes (LN), lymphocytes must first recognize and bind to specific high endothelial venules (HEV) in the LN. Binding to LN HEV is mediated by specific lymphocyte receptors, termed homing receptors, which are recognized by the mAb MEL-14. We examined the changes that occur in homing receptor expression after activation of murine T lymphocytes in vitro. Cells activated in MLC or by Con A undergo a 75% loss in their ability to recognize HEV, as demonstrated by a decrease in binding to HEV in vitro. Large, activated cells isolated from a primary MLC by elutriator centrifugation were completely unable to recognize HEV, whereas the small cells in the same culture continued to bind well. Flow cytometric analysis with MEL-14 showed that the activated fraction had lost expression of gp90MEL-14, the homing receptor Ag, whereas the inactivated cells remained MEL-14+. Concomitant with the loss of homing receptor expression, most of the activated cells became strongly peanut agglutinin (PNA)-positive, demonstrating a marked change in surface glycosylation. Thus, these MLC consist of two major populations of T cells--small, inactivated lymphocytes that are MEL-14+PNAlo and large, activated blast cells that are MEL-14-PNAhi. Purified MEL-14+ T cells activated by Con A gave rise to MEL-14- progeny, showing that gp90MEL-14 is lost from gp90MEL-14-positive precursors, rather than from the selective growth of MEL-14- cells. Furthermore, the loss of Ag expression on at least some activated cells is reversible in resting culture, with almost half of the cells reverting to MEL-14+ after the cessation of stimulation. These experiments show that activation of T cells results in down-regulation of surface homing receptors, resulting in their inability to recognize and bind to the endothelial surface of HEV. This suggests that the activation of T cells in vivo would result in a dramatic and physiologically significant change in their migration and localization properties which would be important during a normal immune response.

  View details for Web of Science ID A1988R303000005

  View details for PubMedID 3058798

• THE STEM-CELL ANTIGENS SCA-1 AND SCA-2 SUBDIVIDE THYMIC AND PERIPHERAL LYMPHOCYTE-T INTO UNIQUE SUBSETS JOURNAL OF IMMUNOLOGY Spangrude, G. J., Aihara, Y., Weissman, I. L., Klein, J. 1988; 141 (11): 3697-3707

  Abstract

  Stem cell Ag 1 and 2 (Sca-1 and Sca-2), so named due to their expression by mouse bone marrow stem cells, were evaluated for expression by populations of cells within the thymus. Immunohistochemical analysis demonstrated that Sca-1 was expressed by cells in the thymic medulla and by some subcapsular blast cells, as well as by the thymic blood vessels and capsule. Sca-2 expression, which was limited to the thymic cortex, could be associated with large cycling thymic blast cells. Both Sca-1 and Sca-2 were expressed on a sub-population of CD4-CD8- thymocytes, and this subpopulation was entirely contained within the Ly-1lo progenitor fraction of cells. Sca-1 expression by a phenotypically mature subset of CD4+CD8- thymocytes was also noted. Conversely, Sca-2 expression was observed on a phenotypically immature or nonmature subpopulation of CD4-CD8- thymocytes. MEL-14, an antibody that defines functional expression of a lymphocyte homing molecule, identified a small population of thymocytes that contained all four major thymic subsets. Sca-2 split the MEL-14hi thymocyte subset into two Sca-2+ non-mature/immature phenotype fractions and two Sca-2- mature phenotype fractions. In peripheral lymphoid organs, Sca-1 identified a sub-population of mature T lymphocytes that is predominantly CD4+CD8-, in agreement with the thymic distribution of Sca-1. Peripheral T cells of the CD4-CD8+ phenotype were predominantly Sca-1-. In contrast, Sca-2 did not appear to stain peripheral T lymphocytes, but recognized only a subset of B lymphocytes which could be localized by immunohistochemistry to germinal centers. Thus, expression of Sca-1 is observed throughout T cell ontogeny, whereas Sca-2 is expressed by some subsets of thymocytes, including at least one half of thymic blasts, but not by mature peripheral T lymphocytes.

  View details for Web of Science ID A1988R004700002

  View details for PubMedID 2460547

• INSITU LOCALIZATION OF HUHF SERINE PROTEASE MESSENGER-RNA AND CYTO-TOXIC CELL-ASSOCIATED ANTIGENS IN HUMAN DERMATOSES - A NOVEL METHOD FOR THE DETECTION OF CYTO-TOXIC CELLS IN HUMAN-TISSUES AMERICAN JOURNAL OF PATHOLOGY Wood, G. S., Mueller, C., Warnke, R. A., Weissman, I. L. 1988; 133 (2): 218-225

  Abstract

  Human Hanukah Factor (HuHF) is a trypsinlike serine protease associated with cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Employing a radiolabeled RNA probe for the HuHF gene, cells containing HuHF mRNA in situ were detected in skin lesions from patients with a variety of reactive and neoplastic dermatoses including positive allergic contact dermatitis patch tests, lichen planus, erythrodermic psoriasis, Sezary syndrome, and poikilodermatous mycosis fungoides. The results were correlated with in situ studies of CTL/NK subsets as defined immunohistologically by a panel of monoclonal antibodies applied to sermiserial sections of the same tissue blocks used for the HuHF hybridizations. The results suggest that cytotoxic cells are present in each of these dermatoses, that they may be situated within either the epidermis or the dermis, and that they belong predominantly to the CTL subset because Leu-7+ or CD16+ cells (NK cells) were typically rare or absent. A variable proportion of cells expressed Leu-19 antigen (a marker for non-MHC-restricted cytotoxic cells); however, its rarity in several cases suggests that most of the HuHF+ cells identified in them belonged to the MHC-restricted, Leu-19- CTL subset. It is concluded that the correlation of molecular biologic and immunohistologic data will be a useful method for the further characterization of cytotoxic cell subsets in human dermatoses.

  View details for Web of Science ID A1988Q993500004

  View details for PubMedID 2461088

• THE SCID-HU MOUSE - MURINE MODEL FOR THE ANALYSIS OF HUMAN HEMATOLYMPHOID DIFFERENTIATION AND FUNCTION SCIENCE McCune, J. M., Namikawa, R., Kaneshima, H., Shultz, L. D., Lieberman, M., Weissman, I. L. 1988; 241 (4873): 1632-1639

  Abstract

  The study of human hematopoietic cells and the human immune system is hampered by the lack of a suitable experimental model. Experimental data are presented showing that human fetal liver hematopoietic cells, human fetal thymus, and human fetal lymph node support the differentiation of mature human T cells and B cells after engraftment into mice with genetically determined severe combined immunodeficiency. The resultant SCID-hu mice are found to have a transient wave of human CD4+ and CD8+ T cells and human IgG (immunoglobulin G) in the peripheral circulation. The functional status of the human immune system within this mouse model is not yet known.

  View details for Web of Science ID A1988Q137200029

  View details for PubMedID 2971269

• MATURE T-CELLS GENERATED FROM SINGLE THYMIC CLONES ARE PHENOTYPICALLY AND FUNCTIONALLY HETEROGENEOUS JOURNAL OF IMMUNOLOGY Spangrude, G. J., Weissman, I. L. 1988; 141 (6): 1877-1890

  Abstract

  A limiting dilution system for cloning thymic CFU (CFUt) from murine bone marrow has been critically evaluated to test the clonal origin of the thymic colonies. Simultaneous limiting dilution transfer of three populations of bone marrow, each expressing a unique allelic cell surface determinant, resulted in independent segregation of donor-derived thymocyte populations within groups of recipient mice. Statistical analysis of the data allowed an estimate of 1 CFUt/3.3 x 10(4) i.v. transferred bone marrow cells. A pulse-chase experiment was utilized to establish whether CFUt seed directly to the thymus, or whether thymic seeding is secondary to extra-thymic engraftment. The results supported the conclusion that bone marrow CFUt utilize a specific interaction with thymic blood vessel endothelial cells to recognize and enter the thymus, and that this seeding occurs within 4 h of i.v. infusion. A kinetic analysis of emigration of the CFUt progeny into the peripheral blood revealed that, in most cases, an early wave of predominantly CD4+ CD8- lymphocytes emerges from the thymus approximately 4 wk after radiation and reconstitution. In a few cases, the first progeny of CFUt to emerge from the thymus were predominantly CD4- CD8+. Commitment of CFUt to TCR beta-chain rearrangements was assessed by quantitating expression of the V beta 8 family of TCR V region genes. Although some clones expressed a significantly higher or lower percentage of V beta 8+ cells, these differences were not stable with time. Thus, CFUt do not undergo absolute commitment to cell surface phenotype of TCR rearrangement, as reflected by the phenotypes of their progeny. Clones of mature peripheral progeny of CFUt could be expanded in culture in the presence of mitogen and growth factors; approximately 30 to 50% of proliferating clones could mediate cytotoxicity in a lectin-dependent assay, further indicating that CFUt are not absolutely committed to a particular T cell function.

  View details for Web of Science ID A1988Q102200013

  View details for PubMedID 3262643

• AUTOREACTIVE BLOOD-CELLS AND PROGRAMMED CELL-DEATH IN GROWTH AND DEVELOPMENT OF PROTOCHORDATES JOURNAL OF EXPERIMENTAL ZOOLOGY Harp, J. A., TSUCHIDA, C. B., Weissman, I. L., Scofield, V. L. 1988; 247 (3): 257-262

  Abstract

  The tunicate Botryllus is a marine protochordate whose clonal colonies undergo regulated natural transplantations when they come into contact in nature. The outcome of these transplantations (fusion or rejection) is controlled by genes of a highly polymorphic histocompatibility system that resembles in many respects the mammalian major histocompatibility complex (MHC). While fusion or rejection reactions are often completed within 24 hr after transplantation, resorption of one partner of a pair of fused semiallogeneic colonies may occur days to weeks after initial contact. The latter process is similar to the degeneration of old individuals, or zooids, that precedes maturation of each new generation of asexual buds. Here we describe comparisons of in vitro reactions of a) mixtures of cells from allogeneic animals and b) cells taken from animals at the zooid-resorption ("takeover") stage of colony development. In vitro autoreactivity of cells from resorbing colonies may reflect in vivo responses to senescent cells, which in turn may be related to allorecognition events that govern fusion or rejection between colonies.

  View details for Web of Science ID A1988Q239900008

  View details for PubMedID 3183596

• A T-CELL-SPECIFIC AND NATURAL-KILLER CELL-SPECIFIC, TRYPSIN-LIKE SERINE PROTEASE - IMPLICATIONS OF A CYTOLYTIC CASCADE ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Gershenfeld, H. K., HERSHBERGER, R. J., Mueller, C., Weissman, I. L. 1988; 532: 367-379

  Abstract

  A new trypsin-like serine protease was cloned from both a murine cytotoxic T lymphocyte and a human PHA-stimulated peripheral blood lymphocyte cDNA library. In both the mouse and human system, this transcript had a T cell- and NK-specific distribution, being detected in cytotoxic T lymphocytes (CTL), some T-helper clones, and NK, but not in a variety of normal tissues. T-cell activation with Con A plus IL-2 induced mouse spleen cells to express this gene with kinetics correlating with the acquisition of cytolytic capacity. Both the mouse and human nucleotide sequences of this gene encoded an amino acid sequence with 25-40% identity to members of the serine protease family. The active-site "charge-relay" residues (His-57, Asp-102, and Ser-195 of the chymotrypsin numbering system) are conserved, as well as the trypsin-specific Asp (position 189 in trypsin). We reviewed the evidence of this serine protease's role in lymphocyte lysis and proposed a "lytic cascade." We discussed the biological and clinical implications of a cascade, proposing these enzymes as markers for cytolytic cells and as targets for rational drug therapy. Genetic and acquired deficits in the lethal hit-delivery system are considered as a basis for approaching some immunodeficiency states, including severe EBV infections, T-gamma leukemias, and T8+ lymphocytosis syndromes.

  View details for Web of Science ID A1988T985000035

  View details for PubMedID 3052212

• Was the MHC made for the immune system, or did immunity take advantage of an ancient polymorphic gene family encoding cell surface interaction molecules? A speculative essay. International reviews of immunology Weissman, I. L. 1988; 3 (4): 397-416

  View details for PubMedID 3246573

• MEMORY B-CELLS EXPRESS A PHENOTYPE CONSISTENT WITH MIGRATORY COMPETENCE AFTER SECONDARY BUT NOT SHORT-TERM PRIMARY IMMUNIZATION CELLULAR IMMUNOLOGY Kraal, G., Weissman, I. L., BUTCHER, E. C. 1988; 115 (1): 78-87

  Abstract

  The cell surface phenotype of dinitrophenol (DNP)-specific memory B cells, defined by their capacity to transfer IgG responses into syngeneic irradiated recipients, was assessed using two markers of relevance to lymphocyte migratory properties: (i) peanut agglutinin, which binds to terminal galactosyl residues expressed at high levels by several nonmigrating lymphocyte subsets and, among lymph node B cells, is highly specific for germinal center cells; and (ii) MEL-14, a monoclonal antibody specific for lymphocyte surface receptors required for migration from the blood into peripheral lymph nodes. At various times after primary or secondary immunization with DNP-keyhole limpet hemocyananin (KLH), lymph node B cells were separated by fluorescence-activated cell sorting on the basis of staining with PNA and/or MEL-14, and the presence of B-memory cells in each fraction was assessed by adoptive transfer with antigen (DNP-KLH) and helper T cells. One week after immunization, most of the memory sorted in the PNAhi population, confirming a previous report by R. F. Coico, B. S. Bhogal, and G. J. Thorbecke (J. Immunol. 131, 2254, 1983) that early memory B cells or their precursors are contained within the germinal center cell population, a population which is known to be MEL-14- and migratory-incompetent. Six weeks after primary stimulation, however, the bulk of memory cells, unlike germinal center cells, were MEL-14hi. After secondary immunization, memory was still predominantly MEL-14+ and PNAlo, although in some experiments adoptive responses were transferred by all sorted fractions. The results are consistent with the hypothesis that antigen-specific B cells initially undergo local (sessile) differentiation and proliferation in germinal centers, where they develop the capacity for adoptive transfer of antigen-specific secondary responses, but that with continued development their long-lived memory-containing progeny express a phenotype permitting their reentry into the recirculating lymphocyte pool.

  View details for Web of Science ID A1988P804100007

  View details for PubMedID 3261207

• PURIFICATION AND CHARACTERIZATION OF MOUSE HEMATOPOIETIC STEM-CELLS SCIENCE Spangrude, G. J., Heimfeld, S., Weissman, I. L. 1988; 241 (4861): 58-62

  Abstract

  Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors.

  View details for Web of Science ID A1988P049100025

  View details for PubMedID 2898810

• MOLECULAR TOOLS FOR INACTIVATING A YEAST ENZYME INVIVO MOLECULAR AND CELLULAR BIOLOGY Carlson, J. R., Weissman, I. L. 1988; 8 (6): 2647-2650

  Abstract

  As part of an effort to develop a new means of inducibly inactivating cellular proteins in vivo, three monoclonal antibodies which neutralize yeast alcohol dehydrogenase (ADH) activity were isolated and characterized with respect to criteria important for the inactivation strategy. The significance of these criteria is considered, and a general means of generating appropriate antibodies is suggested. All three antibodies described here were specific for ADH I; they did not recognize the closely related isozyme ADH II in a plate-binding assay and did not immunoprecipitate molecules other than ADH from a Saccharomyces cerevisiae extract. Neutralization occurred in a yeast extract and, for two antibodies, was blocked by high concentrations of the coenzyme NAD+. This finding suggests that the antibodies may block enzyme activity by stabilizing an inactive form of ADH lacking bound NAD+. These results provide a foundation for the use of these antibodies to inactivate ADH in vivo.

  View details for Web of Science ID A1988N633800046

  View details for PubMedID 3043187

• PROLIFERATION AND DIFFERENTIATION OF HIGHLY ENRICHED MOUSE HEMATOPOIETIC STEM-CELLS AND PROGENITOR CELLS IN RESPONSE TO DEFINED GROWTH-FACTORS JOURNAL OF EXPERIMENTAL MEDICINE MULLERSIEBURG, C. E., Townsend, K., Weissman, I. L., Rennick, D. 1988; 167 (6): 1825-1840

  Abstract

  Three distinct hematopoietic populations derived from normal bone marrow were analyzed for their response to defined growth factors. The Thy-1loT- B- G- M-population, composing 0.2% of bone marrow, is 370-fold enriched for pluripotent hematopoietic stem cells. The two other populations, the Thy-1- T- B- G- M- and the predominantly mature Thy-1+ T+ B+ G+ M+ cells, lack stem cells. Thy-1loT- B- G- M- cells respond with a frequency of one in seven cells to IL-3 in an in vitro CFU-C assay, and give rise to many mixed colonies as expected from an early multipotent or pluripotent progenitor. The Thy-1- T- B- G- M- population also contains progenitor cells which responded to IL-3. However, colonies derived from Thy-1- T- B- G- M- cells are almost exclusively restricted to the macrophage/granulocyte lineages. This indicates that IL-3 can stimulate at least two distinct clonogenic early progenitor cells in normal bone marrow: multipotent Thy-1loT- B- G- M- cells and restricted Thy-1- T- B- G- M- cells. Thy-1loT- B- G- M-cells could not be stimulated by macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF) or IL-5 (Eosinophil-CSF). The hematopoietic precursors that react to these factors are enriched in the Thy-1- T- G- B- M- population. Thus, multipotent and restricted progenitors can be separated on the basis of the expression of the cell surface antigen Thy-1.

  View details for Web of Science ID A1988P187300007

  View details for PubMedID 3260264

• TOTAL LYMPHOID IRRADIATION LEADS TO TRANSIENT DEPLETION OF THE MOUSE THYMIC MEDULLA AND PERSISTENT ABNORMALITIES AMONG MEDULLARY STROMAL CELLS JOURNAL OF IMMUNOLOGY Adkins, B., GANDOUR, D., Strober, S., Weissman, I. 1988; 140 (10): 3373-3379

  Abstract

  Mice given multiple doses of sublethal irradiation to both the thymus and the peripheral lymphoid tissues showed major transient, and some persistent disruptions in general thymic architecture and in thymic stromal components. At 2 wk after total lymphoid irradiation (TLI), the thymus lacked identifiable medullary regions by immunohistochemical analyses. Medullary stromal cells expression MHC Ag or a medullary epithelial cell Ag, as well as medullary macrophages, were undetectable. Instead, the processes of cortical epithelial cells were observed throughout the entire thymus. Strikingly, thymocyte subsets with mature phenotypes (CD4+CD8- and CD4-CD8+) were present in the apparent absence of a medulla. This early, gross effect was rapidly reversed such that by 1 to 2 mo after TLI, medullary areas with MHC Ag-positive cells were evident. However, abnormalities in a subset of medullary stromal cells appeared to be more persistent. Medullary epithelial cells, identified by the MD1 mAb, were greatly reduced in number and abnormally organized for at least 4 mo after TLI. In addition, macrophages containing endogenous peroxidase activity, normally abundant in medullary regions, were undetectable at all times examined after TLI. Therefore, this irradiation regimen induced both transient and long term effects in the thymus, primarily in medullary regions. These results suggest that TLI may be used as an experimental tool for studying the impact of selective depletion of medullary stromal cells on the development of specific T cell functions.

  View details for Web of Science ID A1988N240500015

  View details for PubMedID 2452185

• 2 RARE POPULATIONS OF MOUSE THY-110 BONE-MARROW CELLS REPOPULATE THE THYMUS JOURNAL OF EXPERIMENTAL MEDICINE Spangrude, G. J., MULLERSIEBURG, C. E., Heimfeld, S., Weissman, I. L. 1988; 167 (5): 1671-1683

  Abstract

  Two-color FACS analysis of mouse bone marrow reveals a rare population, comprising 0.1-0.3% of the total, that expresses low levels of the Thy-1 antigen but does not express any of five surface markers that characterize differentiated hematolymphoid cells. We demonstrate here that this fraction of mouse bone marrow is enormously enriched in cells that can home to the thymus and differentiate into mature T lymphocytes, subsequently migrating to peripheral lymphoid organs. Only a subset of the FACS-isolated fraction (1/90 after intrathymic injection) is capable of responding to the thymic microenvironment with a productive commitment to the T cell lineage. A second fraction of mouse bone marrow, which expresses how levels of Thy-1 but is also positive for at least one of five hematolymphoid lineage-specific markers, also contains cells that home to the thymus and establish colonies of thymocytes. The two fractions each contribute approximately equal amounts of thymic colony-forming units (CFUt) to the bone marrow, and together can account for at least half of the CFUt in whole bone marrow.

  View details for Web of Science ID A1988N306900013

  View details for PubMedID 2896758

• ENDOPROTEOLYTIC CLEAVAGE OF GP160 IS REQUIRED FOR THE ACTIVATION OF HUMAN IMMUNODEFICIENCY VIRUS CELL McCune, J. M., RABIN, L. B., Feinberg, M. B., Lieberman, M., Kosek, J. C., Reyes, G. R., Weissman, I. L. 1988; 53 (1): 55-67

  Abstract

  The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.

  View details for Web of Science ID A1988M922100008

  View details for PubMedID 2450679

• EXPRESSION OF 2 SERINE ESTERASE GENES DURING AN ALLOGRAFT-REJECTION IN THE MOUSE TRANSPLANTATION PROCEEDINGS Mueller, C., Gershenfeld, H. K., Lobe, C. G., Okada, C. Y., Bleackley, R. C., Weissman, I. L. 1988; 20 (2): 251-253

  View details for Web of Science ID A1988N228200035

  View details for PubMedID 3363633

• NORMAL THYMIC CORTICAL EPITHELIAL-CELLS DEVELOPMENTALLY REGULATE THE EXPRESSION OF A B-LINEAGE TRANSFORMATION-ASSOCIATED ANTIGEN IMMUNOGENETICS Adkins, B., Tidmarsh, G. F., Weissman, I. L. 1988; 27 (3): 180-186

  Abstract

  Nonlymphoid, stromal cells in the mouse thymus are believed to be important in T cell maturation and have been proposed to play a central role in the acquisition of major histocompatibility complex (MHC) restriction and self-tolerance by maturing thymocytes. Both cortical and medullary epithelial cells in the thymus express high levels of class II (A) major histocompatibility antigens (MHC Ags). We show here that a specific subset of these A+ epithelial cells express a transformation-associated antigen (6C3Ag) found previously on the surfaces of Abelson murine leukemia virus-transformed pre-B cells and on those bone marrow-derived stromal cell clones which support normal and preneoplastic pre-B cell proliferation. Among solid lymphoid organs, only the thymus contains 6C3Ag+ cells and within the thymus, this antigen is found exclusively on A+ epithelial cells in cortical regions. It is striking that the expression of the 6C3Ag on thymic epithelium is developmentally regulated, suggesting a role for this lymphostromal antigen in the maturation of the thymic microenvironment.

  View details for Web of Science ID A1988L714500004

  View details for PubMedID 3257459

• ACTIVATION OF CTL-SPECIFIC GENES DURING CELL-MEDIATED CYTOLYSIS INVIVO - EXPRESSION OF THE HF GENE ANALYZED BY INSITU HYBRIDIZATION IMMUNOLOGICAL REVIEWS Mueller, C., Gershenfeld, H. K., Weissman, I. L. 1988; 103: 73-85

  View details for Web of Science ID A1988N935500005

  View details for PubMedID 3292397

• APPROACHES TO AN UNDERSTANDING OF PATHOGENETIC MECHANISMS IN AIDS REVIEWS OF INFECTIOUS DISEASES Weissman, I. 1988; 10 (2): 385-398

  Abstract

  AIDS, presumably caused by the human retrovirus, human immunodeficiency virus (HIV), is a disease with multiple pathologies, most of which are the consequence of a profound immunodeficiency. The first two sections of this review focus primarily on the normal development and function of the cells of the immune system and the known abnormalities that occur in this system in AIDS patients. Very little is known of the pathogenesis, in humans, of the four major clinical manifestations of AIDS--immunodeficiency, encephalopathy, Kaposi's sarcoma, and lymphoma. Because most pathologic studies derive from autopsy findings in terminal AIDS patients, it has been difficult to track the course of HIV infection from the time of initial contact with the virus through the evolution of the disease. Therefore, the final section of this review focuses on actual and potential animal models of AIDS and how such models might be valuable for studies on the pathogenesis of the disease, the development of relevant vaccines, and the testing of potential therapies.

  View details for Web of Science ID A1988M696500012

  View details for PubMedID 3287566

• A HIGH PROPORTION OF LYMPHOCYTES-T THAT INFILTRATE INCOMPATIBLE-H-2 HEART ALLOGRAFTS INVIVO EXPRESS GENES ENCODING CYTO-TOXIC CELL-SPECIFIC SERINE PROTEASES, BUT DO NOT EXPRESS THE MEL-14-DEFINED LYMPH-NODE HOMING RECEPTOR JOURNAL OF EXPERIMENTAL MEDICINE Mueller, C., Gershenfeld, H. K., Lobe, C. G., Okada, C. Y., Bleackley, R. C., Weissman, I. L. 1988; 167 (3): 1124-1136

  Abstract

  The role of cytotoxic cells in in vivo immune functions such as allograft rejection is unknown. To begin to assess the function of cytolytic cells in vivo we have begun with cytolytic cell-specific functional molecules: we have isolated and characterized cytolytic cell-specific cDNA clones from cytolytic T cell clones, both encoding distinct serine esterases. The HF gene encodes a trypsin-like enzyme while the C11 gene encodes an enzyme with likely specificity for acidic residues. Here we demonstrate, using in situ hybridization with RNA probe, that both genes are expressed selectively in a subset of T lymphocytes that have infiltrated cardiac allografts. The phenotype of these cells is consistent with the most frequent phenotype of active CTL raised in vitro: they are predominantly CD4-, CD8+, MEL-14- T cell blasts. Thus the expression of these genes, each of which encodes serine esterase found in killer cell granules in vitro, is a valid marker for these cells in vivo as well. The kinetics of their accumulation is consistent with, but not proof of, a putative role in allograft rejection. It is likely that HF and C11 gene expression will be of diagnostic value.

  View details for Web of Science ID A1988M691400028

  View details for PubMedID 3280725

• CLONING AND CHROMOSOMAL ASSIGNMENT OF A HUMAN CDNA-ENCODING A T-CELL-SPECIFIC AND NATURAL-KILLER CELL-SPECIFIC TRYPSIN-LIKE SERINE PROTEASE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gershenfeld, H. K., HERSHBERGER, R. J., SHOWS, T. B., Weissman, I. L. 1988; 85 (4): 1184-1188

  Abstract

  A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage lambda gt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16+ natural killer cells and CD3+, CD16- T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The predicted protein has a 22-amino acid presegment, a 6-amino acid prosegment, and an active enzyme of 234 amino acids with a calculated unglycosylated molecular weight of 25,820. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. We propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells.

  View details for Web of Science ID A1988M196200050

  View details for PubMedID 3257574

• HOMING RECEPTORS AND METASTASIS ADVANCES IN CANCER RESEARCH SHER, B. T., BARGATZE, R., Holzmann, B., Gallatin, W. M., MATTHEWS, D., Wu, N., PICKER, L., BUTCHER, E. C., Weissman, I. L. 1988; 51: 361-390

  Abstract

  As discussed in the preceding sections, there are several indications that the lymphocyte homing receptors involved in the normal process of lymphocyte recirculation are also relevant to the behavior of metastatic cells. Cell fusion experiments indicate that previously nonmetastatic cells can acquire metastatic capacity from fusion with normal lymphocytes. Murine T lymphomas that bear high levels of functional homing receptors can metastasize to peripheral lymphoid organs, whereas those lymphomas lacking homing receptors cannot. Virtually all lymph node metastases of lymphomas contain a high proportion of MEL-14hi cells, even if the primary tumor has been selected to be relatively deficient in these cells. Further investigations of the biology of lymphocyte homing receptors will reveal whether or not there are additional lymphocyte homing receptors and will clarify the role of lymphocyte homing receptors in metastasis. Antibodies against three lymphocyte homing receptors could therefore be useful for diagnosis and treatment of metastatic disease.

  View details for Web of Science ID A1988Q449700007

  View details for PubMedID 3066147

• Lymphocyte homing receptors, ubiquitin, and cell surface proteins Ubiquitin Siegelman, M., Weissman, I. Plenum Publishing Corp.. 1988
• Thymic lymphocyte maturation Thy Lymphocyte, Structure and Function Scollay, R., et al Marcel Dekker. 1988: 143–170
• Retreat growth in the ascidian Botryllus schlosseri: a consequence of nonself recognition Invertebrate HIstorecognition Rinkevich, B., Weissman, I. Plenum. 1988: 93–109
• Speculations on the relationships of two botryllus allorecognition reactions - colony specificity and resorption - to vertebrate histocompatibility Invertebrate Historecognition Weissman, I., Scofield, V., Saito, Y., Boyd, H., Rinkevich, B. Plenum. 1988: 67–78
• Biosynthesis of gpMel-14 the murine lymph-node-specific homing receptor Current Communications in Molecular Biology: the Ubiquitin System van de Rijn, M., Weissman, I. 1988: 171–177
• COMPARATIVE MOLECULAR-MODEL BUILDING OF 2 SERINE PROTEINASES FROM CYTO-TOXIC LYMPHOCYTES-T PROTEINS-STRUCTURE FUNCTION AND GENETICS Murphy, M. E., Moult, J., Bleackley, R. C., Gershenfeld, H., Weissman, I. L., James, M. N. 1988; 4 (3): 190-204

  Abstract

  Two genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three-dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has provided useful structural information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X-ray structures of four serine proteinases to provide a structurally based sequence alignment: alpha-chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat mast cell protease 2 (RMCP2). The root mean square differences in alpha-carbon atom positions among these four structures when compared in a pairwise fashion range from 0.79 to 0.97 A for structurally equivalent residues. The sequences of the two lymphocyte enzymes were then aligned to these proteinases using chemical criteria and the superimposed X-ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. With RMCP2 as a template for CCP1 and the two enzymes RMCP2 and BT as templates for HF, the molecular models were constructed. Intramolecular steric clashes that resulted from the replacement of amino acid side chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angles in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginine residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 position of the substrate. Both CCP1 and HF have a free cysteine in the segment of polypeptide 88 to 93.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1988R339000005

  View details for PubMedID 3237717

• INDIRECT INDUCTION OF RADIATION LYMPHOMAS IN MICE - EVIDENCE FOR A NOVEL, TRANSMISSIBLE LEUKEMOGEN JOURNAL OF EXPERIMENTAL MEDICINE Lieberman, M., HANSTEEN, G. A., McCune, J. M., Scott, M. L., White, J. H., Weissman, I. L. 1987; 166 (6): 1883-1893

  Abstract

  The transmission of a lymphomagenic agent(s) from the bone marrow of irradiated mice to thymic target cells has been demonstrated by: (a) the induction of T cell lymphomas in nonirradiated thymic grafts implanted in irradiated, Thy-l-congenic mice, (b) the induction of T cell lymphomas of host origin in mice infused with bone marrow from irradiated, Thy-l-congenic donors. The latter procedure also yields an appreciable number of pre-B cell lymphomas of uncertain origin. The results confirm Kaplan's theory that radiation induces thymic lymphomas in mice by an indirect mechanism. However, the previously described radiation leukemia virus is clearly not involved in the majority of transferred lymphomas. We propose that the mediating agent in radiation lymphomagenesis is a novel, transmissible agent induced in the bone marrow, but exerting its transforming activity on cells in the thymus. The nature and mode of action of the agent are under investigation.

  View details for Web of Science ID A1987L063000020

  View details for PubMedID 3316475

• A LONG-TERM STUDY ON FUSED SUBCLONES IN THE ASCIDIAN BOTRYLLUS-SCHLOSSERI - THE RESORPTION PHENOMENON (PROTOCHORDATA, TUNICATA) JOURNAL OF ZOOLOGY Rinkevich, B., Weissman, I. L. 1987; 213: 717-733

  View details for Web of Science ID A1987L623100010

• THE FATE OF BOTRYLLUS (ASCIDIACEA) LARVAE COSETTLED WITH PARENTAL COLONIES - BENEFICIAL OR DELETERIOUS CONSEQUENCES BIOLOGICAL BULLETIN Rinkevich, B., Weissman, I. L. 1987; 173 (3): 474-488

  View details for Web of Science ID A1987M034000003

• THE PHENOTYPE OF THYMOCYTES DERIVED FROM A SINGLE CLONOGENIC PRECURSOR JOURNAL OF IMMUNOLOGY Ezine, S., Jerabek, L., Weissman, I. 1987; 139 (7): 2195-2199

  Abstract

  Clonogenic repopulation of the thymus of thymus-homing bone marrow cells leads to intrathymic populations representing all four major Lyt-2- and L3T4-defined phenotypes. Although all four phenotypes may be represented in a single clone, quite often a striking bias in the proportion of L3T4 single positive to Lyt-2 single positive cells may exist within a clone, but not in the host thymocytes in general. Because at any one time these clones may be located in specific subregions of the thymus (specifically cortex only, medulla only, or cortex and medulla), we propose the hypothesis that different microenvironments in the thymus might, in fact, be responsible for the predominant maturation of different single positive mature thymic subsets.

  View details for Web of Science ID A1987K235800011

  View details for PubMedID 2888822

• HIGH ENDOTHELIAL VENULE BINDING AS A PREDICTOR OF THE DISSEMINATION OF PASSAGED MURINE LYMPHOMAS JOURNAL OF EXPERIMENTAL MEDICINE BARGATZE, R. F., WU, N. W., Weissman, I. L., BUTCHER, E. C. 1987; 166 (4): 1125-1131

  Abstract

  It has long been postulated that normal lymphocyte homing mechanisms help determine the metastatic spread of lymphoid neoplasms. The traffic of normal lymphocytes is controlled in part by the regulated expression of surface receptors for high endothelial venules (HEV), specialized venules that mediate the extravasation of circulating lymphocytes from the blood into lymphoid organs and sites of chronic inflammation. Here we have compared the in vivo growth patterns of HEV-binding vs. nonbinding murine lymphomas passaged intramuscularly into syngeneic recipients. We report that lymphomas that bind well to HEV (as assessed in a quantitative in vitro assay) disseminate widely via the blood, involving all lymph node groups symmetrically. Although both HEV-binding and nonbinding lymphomas gain access to the blood, gross involvement of lymph nodes by nonbinding lymphomas is limited to nodes draining local tumor at the site of injection, a prominent feature of these lymphomas; distant lymph nodes are not enlarged. The results suggest that the expression of functional receptors for HEV either controls the hematogenous dissemination of malignant lymphocyte populations to HEV-bearing organs, or is coregulated with factors determining this metastatic behavior. The findings support the concept that normal lymphocyte homing mechanisms are important to the spread of leukemias and lymphomas.

  View details for Web of Science ID A1987K464000024

  View details for PubMedID 3655655

• UNUSUAL IMMUNOGLOBULIN DNA-SEQUENCES FROM THE NONEXPRESSED CHROMOSOME OF MOUSE NORMAL LYMPHOCYTES-B - IMPLICATIONS FOR ALLELIC EXCLUSION AND THE DNA REARRANGEMENT PROCESS JOURNAL OF IMMUNOLOGY Nottenburg, C., STJOHN, T., Weissman, I. L. 1987; 139 (5): 1718-1726

  Abstract

  Allelic exclusion of immunoglobulin gene products results in the expression of only one of two possible alleles in normal B lineage cells. Attempts to define the role of heavy chain gene rearrangements in this process have revealed the nonexpressed allele to be rarely in its germline context, but rather incompletely rearranged (D/J rearrangements) or completely rearranged (V/D/J rearrangements) and in a context that cannot be expressed as a protein. Nearly all DNA sequences of heavy chain genes from the nonexpressed allele have originated from plasmacytomas, virus-induced pre-B leukemias, and hybridomas--all clonal cell lines perhaps altered by the neoplastic process. In this communication, we present the first examples of isolation and DNA sequence analysis of heavy chain gene rearrangements from the nonexpressed allele of normal B lymphocytes. Splenic B lymphocytes from allotype heterozygous (BALB/c X C57BL/6J)F1 mice expressing the BALB/c IgD allotype were detected with a monoclonal antibody, H10-4.22, to the BALB/c delta chain genetic marker and were isolated with a FACS (fluorescence-activated cell sorter). lambda phage clones containing the JH gene region from the sorted cells were isolated, and those containing sequences derived from the C57BL/6J nonexpressed chromosome were identified by a restriction fragment length polymorphism. DNA sequences of seven rearranged clones from the nonexpressed allele are presented. Five of these rearrangements have unexpected compositions. Four of the five clones contain V/D/J rearrangements with no obvious impediments to expression generated by the rearrangements. The novel variable region gene in one of these V/D/J rearrangements is a member of a newly described VH gene family. The fifth clone has a 3.5 kb deletion removing all of the JH gene segments. The remaining two clones contain D/J rearrangements that are typical of the initial stage of variable region assembly. These findings suggest that the mechanisms that generate nontranslatable heavy chains do not exclusively account for allelic exclusion. Rather, several different mechanisms may contribute to the establishment of allelic exclusion in normal B lymphocytes.

  View details for Web of Science ID A1987J763900053

  View details for PubMedID 3114375

• A SUBSET OF T-CELL RECEPTORS ASSOCIATED WITH L3T4 MOLECULES MEDIATES C6VL LEUKEMIA-CELL BINDING OF ITS COGNATE RETROVIRUS CELL ONEILL, H. C., McGrath, M. S., Allison, J. P., Weissman, I. L. 1987; 49 (1): 143-151

  Abstract

  We show here that the interaction of a radiation leukemia virus-induced thymoma, C6VL, with its cognate retroviruses occurs in the vicinity of the T cell receptor (TCR). While an anti-clonotypic antibody completely inhibits this interaction, antibodies specific for another T cell receptor complex determinant, L3T4, only partially inhibit the cell-retrovirus interaction. Several antibodies to more abundant cell-surface determinants (T200, Ly15, H-2Db) do not inhibit this interaction. Under capping conditions, either of the antibodies to L3T4 or TCR epitopes modulate virus binding receptors from the surface of C6VL/1 cells. L3T4 and the TCR do not comodulate significantly on the surface of C6VL/1 cells. These experimental findings implicate the existence of rare TCR-L3T4 complexes on C6VL/1 cells, and the involvement of these complexes in the binding of C6VL/1 to its cognate retrovirus. In addition, the clonotypic anti-TCR antibody inhibits C6VL/1 cell proliferation at concentrations that block its binding to its produced retroviruses.

  View details for Web of Science ID A1987G882600017

  View details for PubMedID 2435413

• BONE-MARROW STROMAL CELL-LINES WITH LYMPHOPOIETIC ACTIVITY EXPRESS HIGH-LEVELS OF A PRE-B NEOPLASIA-ASSOCIATED MOLECULE CELL WHITLOCK, C. A., Tidmarsh, G. F., MULLERSIEBURG, C., Weissman, I. L. 1987; 48 (6): 1009-1021

  Abstract

  Bone marrow stromal cell lines have been isolated that directly support B lymphopoiesis in vitro. Single B-lineage precursors proliferate and differentiate on certain of these stromal cell lines to establish long-term B-lineage cultures. These lymphopoietic stromal cells produce novel soluble factors that support proliferation of in vitro established pre-B cell populations. Lymphoid populations established on lymphopoietic stromal cell lines lack surface Ig-bearing cells, but give rise to surface Ig+ cells when transferred to mixed bone marrow feeder layers. Several stromal lines expressed a B-lineage neoplasia marker detected by the monoclonal antibody MAb6C3. Remarkably, only the 6C3Aghi stromal lines supported long-term proliferation of B-lineage cells. We propose that the 6C3 antigen-bearing molecule may play a role in stromal cell-dependent, pre-B cell proliferation, as well as in neoplastic proliferation of pre-B leukemias.

  View details for Web of Science ID A1987G694600011

  View details for PubMedID 3493849

• EXPRESSION OF THE 6C3 ANTIGEN ON MURINE HEMATOPOIETIC NEOPLASMS - ASSOCIATION WITH EXPRESSION OF ABL, RAS, FES, SRC, ERBB, AND CAS NS-1 ONCOGENES BUT NOT WITH MYC JOURNAL OF EXPERIMENTAL MEDICINE Morse, H. C., Tidmarsh, G. F., Holmes, K. L., FREDERICKSON, T. F., HARTLEY, J. N., Pierce, J. H., Langdon, W. Y., Dailey, M. O., Weissman, I. L. 1987; 165 (3): 920-925

  Abstract

  The monoclonal antibody 6C3 was used to test a wide variety of murine hematopoietic neoplasms for cell surface expression of a 160 kD glycoprotein (gp160(6C3)) previously shown to be expressed by neoplastic pre-B and some B lymphocytes transformed by Abelson murine leukemia virus (A-MuLV). This antigen was expressed on many pre-B and B cell lymphomas, but not on A-MuLV-transformed fibroblasts, T cell lymphomas, or myelomonocytic leukemias, gp160(6C3) was expressed by most early B-lineage spontaneous tumors, and early B tumors induced by replication-defective MuLV-containing oncogenes the products of which are associated with the cytoplasmic aspect of the plasma membrane, i.e., fes, abl, H-ras, bas, src, erbB, and Cas NS-1. By comparison, none of the early B lineage lymphomas induced by the "nuclear" oncogene avian v-myc MuLV, or arising in mice transgenic for a murine c-myc gene, or later B cell lineage stages bearing translocations of the c-myc locus expressed this antigen.

  View details for Web of Science ID A1987G292200026

  View details for PubMedID 3493323

• THE CORRELATION OF LECTIN-STIMULATED PROLIFERATION AND CYTOTOXICITY IN MURINE THYMOCYTES WITH EXPRESSION OF THE MEL-14-DEFINED HOMING RECEPTOR JOURNAL OF IMMUNOLOGY Wilson, A., Scollay, R., REICHERT, R. A., BUTCHER, E. C., Weissman, I. L., Shortman, K. 1987; 138 (2): 352-357

  Abstract

  The relationship between the expression of the MEL-14-defined lymphocyte homing receptor and the proliferation and functional differentiation of thymocytes in response to lectin stimulation was examined. Two-color fluorescent staining with MEL-14 in various combinations with PNA and anti-Ly-2 and anti-L3T4 was used to separate thymocyte populations for functional analysis. A high cloning-efficiency limit-dilution culture system was used to determine the frequency of all cells responsive to concanavalin A (PTL-p) or of all precursors of lectin-enhanced cytolytic lymphocytes (CTL-p). As expected from earlier studies, PTL-p and CTL-p were concentrated in the PNA- thymocytes, PTL-p were in both the Ly-2- L3T4+ and the Ly-2+ L3T4- subpopulations, and CTL-p were predominantely in the Ly-2+ L3T4- subpopulation. Within the PNA- thymocytes, two distinct peaks of PTL-p were found in cells stained with MEL-14, corresponding to MEL-14- and MEL-14medium-to-high cells, whereas the CTL-p frequency increased in fractions showing increasing expression of the MEL-14-defined antigen. Within the Ly-2- L3T4+ subpopulation, two distinct peaks of PTL-p were found corresponding to groups of MEL-14- and MEL-14medium-to-high cells, with the intermediate fraction of MEL-14low cells displaying a very low PTL-p frequency. The Ly-2+ L3T4- subpopulation included fewer MEL-14- cells and more MEL-14high cells than the Ly-2- L3T4+ subpopulation. Within the Ly-2+ L3T4- subpopulation, the few MEL-14- cells expressed a relatively low but definite frequency of CTL-p and PTL-p. The more numerous MEL-14+, Ly-2+ L3T4- cells included a high frequency of CTL-p and PTL-p, which did not vary over the medium-to-high MEL-14 expression range. These results indicate that the correlation of MEL-14 expression with CTL-p frequency among thymocytes is largely a consequence of the relative frequency of Ly-2+ L3T4- cells in the separated fractions, rather than a direct link between MEL-14 expression and function. Nevertheless, MEL-14 does define significant heterogeneity in both the Ly-2+ L3T4- and the Ly-2- L3T4+ subpopulations. In particular, there is a reduced functional response among the small subgroup of Ly-2+ L3T4- MEL-14- cells, suggesting this population includes either immature cells or cells of a different functional type.

  View details for Web of Science ID A1987F617900003

  View details for PubMedID 3491850

• MOLECULES RECOGNIZED BY ANTIIDIOTYPIC MONOCLONAL-ANTIBODIES TO THE B-CELL LYMPHOMA, BCL1 JOURNAL OF MOLECULAR AND CELLULAR IMMUNOLOGY TAMURA, G. S., McGrath, M. S., Weissman, I. L. 1987; 3 (4): 243-253

  Abstract

  Monoclonal and polyclonal antibodies to the variable portions of antigen receptors (anti-idiotypes and anti-idiotopes) are often employed to study the molecular nature and the biological role of these antigen receptors. Such antibodies are operationally defined as those antibodies which bind to a particular immunoglobulin but not to other immunoglobulins of the same class in a radioimmunoassay or ELISA. The monoclonal antibodies 32D1 and 31D1 were initially defined as anti-idiotypic as they recognized an immunoglobulin preparation from the murine B cell lymphoma BCL1, but not other immunoglobulins of the same isotype as assessed by a radioimmunoassay. A potential artifact in defining anti-idiotypic antibodies in this way is the possibility of copurification of antigen and antibody, resulting in the tentative identification of anti-antigen as anti-idiotype. Previous studies have demonstrated that BCL1-IgM is involved in binding of murine leukemia virus (MuLV), and BCL1 immunoglobulin and MuLV-gp70 apparently co-purified as an immune complex. Disruption of the immune complexes with SDS and sucrose gradient purification of the immunoglobulin was adequate to prepare BCL1 immunoglobulin free of gp70 as assessed by radioimmunoassay with the monoclonal anti-gp70 RA3-4A3. This preparation of immunoglobulin was used to show that 31D1 does not bind to BCL1 immunoglobulin, but to the contaminating gp70 in the BCL1 immunoglobulin preparation. However, MAb 32D1 was definitively proven to be anti-idiotypic as it recognized SDS sucrose density gradient purified IgM and immunoisolated heavy chain and light chain from BCL1 immunoglobulin. Several other lymphomas were recognized by mAb 32D1, including the T cell lymphoma UNC1 and the B cell lymphoma Balenlm17. To determine whether mAb 32D1 recognized immunospecific receptors on these lymphoma cell lines immunoprecipitation studies were performed. Immunoisolation and molecular analysis revealed that mAb 32D1 did not recognize the antigen receptor on these two cells, but instead recognized a cell-specific gp70. This observation demonstrates that monoclonal antibodies to known antigens (in this case an anti-idiotype) can crossreact with apparently unrelated molecules. The potential significance of this cross reaction to the antigens recognized by B cell lymphomas is discussed.

  View details for Web of Science ID A1987K403900005

  View details for PubMedID 3509925

• Normal and neoplastic early lymphocyte maturation Molecular Mechanisms in the Regulation of Cell Behavior; Fourth Decennial Review Conference of the Tissue Culture Association Weissman, I., et al 1987
• RECEPTOR MEDIATED LEUKEMOGENESIS - MURINE LEUKEMIA-VIRUS INTERACTS WITH BCL1 LYMPHOMA CELL-SURFACE IGM JOURNAL OF MOLECULAR AND CELLULAR IMMUNOLOGY McGrath, M. S., Tamura, G., Weissman, I. L. 1987; 3 (4): 227-242

  Abstract

  Murine leukemia virus (MuLV) induced T-lymphomas bear surface receptors specific for the leukemogenic retroviruses they produce. We have proposed that such virus receptors on lymphoid tumors are the antigen-specific receptors present on their normal lymphocyte counterparts. To determine the relationship between immune receptors and virus receptors on malignant lymphocytes, a spontaneous B cell lymphoma, BCL1, was investigated. BCL1-lymphoma cells from an in vivo passaged BCL1-cell line grew in vitro only in contact with splenic stromal cells. These stromal cells produced a retrovirus, termed BCL1-V, which was lymphotropic but not leukemogenic. BCL1 cells bound BCL1-V, whereas normal spleen cells did not. Isolated BCL1-IgM bound BCL1-V, whereas three other IgM myeloma proteins, MOPC-104E, CBPC-112, and HPC-76, did not. Rat anti-BCL1-IgM monoclonal antibodies recognizing mu chain isotypic determinants and BCL1-specific idiotypic specificities, blocked BCL1-V binding to BCL1 IgM. These data support the receptor mediated leukemogenesis hypothesis, suggest a role for virus:cell surface immunoglobulin interactions in the development of B cell lymphoma, and implicate an antigen presenting cell population in the lymphomagenic process.

  View details for Web of Science ID A1987K403900004

  View details for PubMedID 2855408

• CHIMERAS IN COLONIAL INVERTEBRATES - A SYNERGISTIC SYMBIOSIS OR SOMATIC-CELL AND GERM-CELL PARASITISM SYMBIOSIS Rinkevich, B., Weissman, I. L. 1987; 4 (1-3): 117-134

  View details for Web of Science ID A1987L547400010

• EARLY EVENTS IN T-CELL MATURATION ANNUAL REVIEW OF IMMUNOLOGY Adkins, B., Mueller, C., Okada, C. Y., REICHERT, R. A., Weissman, I. L., Spangrude, G. J. 1987; 5: 325-365

  View details for Web of Science ID A1987G957300015

  View details for PubMedID 3109456

• AN IMMUNODOMINANT DOMAIN IN ADENOVIRUS TYPE-2 EARLY REGION-1A PROTEINS JOURNAL OF VIROLOGY Tsukamoto, A. S., Ferguson, B., Rosenberg, M., Weissman, I. L., Berk, A. J. 1986; 60 (1): 312-316

  Abstract

  Six independent rat hybridoma cell lines producing monoclonal antibodies to human subgroup C adenovirus early region 1A (E1A) proteins were isolated. Competition binding experiments revealed that each of the monoclonal antibodies was directed against the same epitope or overlapping cluster of epitopes on the E1A proteins. Viral E1A deletion mutants and deleted forms of E1A proteins expressed in Escherichia coli were used to localize the antibody recognition sites to sequences between amino acids 23 and 120, encoded within the first exon of the E1A gene. Similarly, polyclonal antisera raised against the trpE-E1A fusion protein, as well as against the native, biologically active E1A protein, were also directed primarily against this immunodominant region.

  View details for Web of Science ID A1986E073800039

  View details for PubMedID 2427749

• EXPRESSION OF A MONOCLONAL ANTIBODY-DEFINED, B-LINEAGE TRANSFORMATION ANTIGEN SPECIFICALLY IDENTIFIES ABELSON-DISEASED ANIMALS - GENETICALLY-DETERMINED RESISTANCE TO ABELSON MURINE LEUKEMIA-VIRUS ACTS BEFORE INDUCTION OF GP1606C3 JOURNAL OF EXPERIMENTAL MEDICINE Tidmarsh, G. F., Dailey, M. O., Weissman, I. L. 1986; 164 (4): 1356-1361

  Abstract

  Mice genetically susceptible or genetically resistant to the leukemogenic effects of A-MuLV(Mo) were tested for their expression of the B-lineage neoplastic transformation-associated antigen, 6C3Ag. Genetically resistant inbred strains and recombinant inbred lines developed neither cells expressing high levels of 6C3Ag (6C3Aghi) in their hematolymphoid tissues nor Abelson leukemias. Genetically susceptible inbred strains and recombinant inbred lines developed high percentages of 6C3Aghi hematolymphoid cells concomitant with development of Abelson leukemias and lymphomas. Thus the genetically-determined resistance to A-MuLV(Mo) leukemogenesis appears to act at some step(s) after virus infection but before the stage of malignant progression, which is marked by 6C3Ag expression.

  View details for Web of Science ID A1986E228000031

  View details for PubMedID 3489809

• LYMPHOCYTE HOMING RECEPTORS AND THE IMMUNE-RESPONSE INVIVO BIOESSAYS Weissman, I. L. 1986; 5 (3): 112-116

  View details for Web of Science ID A1986E034000004

  View details for PubMedID 3551934

• ANTIGEN-INDUCED CHANGES IN B-CELL SUBSETS IN LYMPH-NODES - ANALYSIS BY DUAL FLUORESCENCE FLOW CYTOFLUOROMETRY EUROPEAN JOURNAL OF IMMUNOLOGY Kraal, G., Hardy, R. R., Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. 1986; 16 (7): 829-834

  Abstract

  Changes in the representation and surface phenotype of defined B cell subsets in murine lymph nodes stimulated with keyhole limpet hemocyanin or sheep red blood cells have been analyzed by two-color immunofluorescence fluorocytometric analysis. Shortly after immunization with either antigen there is a dramatic increase in both the frequency and absolute number of IgM+, IgD+ B cells, which is followed by the formation of germinal centers. Germinal center cells, as soon as they appear on day 3 after primary immunization, bind high levels of peanut agglutinin, bear low levels of surface IgM but no detectable surface IgD, and are characterized by lack of staining with MEL-14, a monoclonal antibody which recognizes a lymphocyte surface receptor involved in lymphocyte homing. The level of I-A and H-2K region-encoded surface antigens on early germinal center cells is higher than on PNAlo B cells. During the first 7 days of the germinal centers there is a progressive decrease in the average level of H-2K but not of Ia antigens. A similar decrease was observed for ThB. It is confirmed that the germinal center cell population contains the majority of antigen-binding cells in the stimulated lymph node. These findings indicate that B cells are recruited nonspecifically to antigen-stimulated lymph nodes, and that the antigen-specific cells then selectively participate in the formation of germinal centers where they undergo specific differentiation events.

  View details for Web of Science ID A1986D318200017

  View details for PubMedID 2873041

• NURSING THE THYMUS - EDITORIAL LABORATORY INVESTIGATION Weissman, I. L. 1986; 55 (1): 1-4

  View details for Web of Science ID A1986D243200001

  View details for PubMedID 3487677

• REARRANGEMENT AND EXPRESSION OF T-CELL ANTIGEN RECEPTOR AND GAMMA-GENES DURING THYMIC DEVELOPMENT JOURNAL OF EXPERIMENTAL MEDICINE Haars, R., Kronenberg, M., Gallatin, W. M., Weissman, I. L., OWEN, F. L., Hood, L. 1986; 164 (1): 1-24

  Abstract

  Rearrangement and expression of the T cell antigen receptor and the gamma genes during T cell ontogeny is a regulated process; the gamma genes are rearranged and expressed first, followed by the beta and then the alpha genes. Expression of both functional alpha and beta gene RNA first occurs at day 17 of gestation, along with the expression of T3 delta chain RNA. T cell antigen receptor gene rearrangements occur primarily or exclusively in the thymus, although some gamma gene rearrangements occur outside the thymus in fetal liver cells that may be committed T cell progenitors. There is no gross difference in the extent of beta and gamma gene rearrangements in the adult thymocyte subpopulations that were analyzed, despite the fact that some of these populations cannot respond to antigen and never emigrate from the thymus. Quantitative analysis of rearrangements in total adult thymocyte DNA shows that beta gene rearrangements generally occur on both chromosomal homologs, and that rearrangements occur preferentially to the J beta 2 gene segment cluster.

  View details for Web of Science ID A1986C991900001

  View details for PubMedID 3487610

• CLONING OF A CDNA FOR A-T CELL-SPECIFIC SERINE PROTEASE FROM A CYTOTOXIC LYMPHOCYTE-T SCIENCE Gershenfeld, H. K., Weissman, I. L. 1986; 232 (4752): 854-858

  Abstract

  A new serine protease was encoded by a clone isolated from a murine cytotoxic T-lymphocyte complementary DNA library by an RNA-hybridization competition protocol. Complementary transcripts were detected in cytotoxic T lymphocytes, spleen cells from nude mice, a rat natural killer cell leukemia, and in two of eight T-helper clones (both cytotoxic), but not in normal mouse kidney, liver, spleen, or thymus, nor in several tested T- and B-cell tumors. T-cell activation with concanavalin A plus interleukin-2 induced spleen cells to express this gene with kinetics correlating with the acquisition of cytolytic capacity. The nucleotide sequence of this gene encoded an amino acid sequence of approximately 25,700 daltons, with 25 to 35 percent identity to members of the serine protease family. The active site "charge-relay" residues (His57, Asp102, and Ser195 of the chymotrypsin numbering system) are conserved, as well as the trypsin-specific Asp (position 189 in trypsin). A Southern blot analysis indicated that this gene is conserved in humans, mouse, and chicken. This serine protease may have a role in lymphocyte lysis and a "lytic cascade."

  View details for Web of Science ID A1986C223200030

  View details for PubMedID 2422755

• ONTOGENY OF LYMPHOCYTE HOMING RECEPTOR EXPRESSION IN THE MOUSE THYMUS JOURNAL OF IMMUNOLOGY REICHERT, R. A., Jerabek, L., Gallatin, W. M., BUTCHER, E. C., Weissman, I. L. 1986; 136 (10): 3535-3542

  Abstract

  The monoclonal antibody MEL-14 has been used in conjunction with immunohistology and multiparameter immunofluorescence to identify and characterize homing receptor-bearing thymocytes at various stages of embryonic and neonatal development. MEL-14hi thymocytes first appear at day 14 of gestation and come to represent about 40% of day 15 fetal thymocytes. Thereafter, the proportion of MEL-14hi thymocytes rapidly declines such that by birth (usually the 20th day of embryonic development) only about 2% of thymocytes are MEL-14hi. Although newborn thymocytes resemble adult thymocytes in this respect, the phenotypic characteristics of fetal and neonatal MEL-14hi thymocytes suggest that this unique subset undergoes a gradual transition from containing exclusively phenotypically immature cells in early gestation to containing predominantly phenotypically mature cells by young adulthood. Thus, virtually none of day 15 MEL-14hi fetal thymocytes are peanut agglutinin (PNA)lo, Ly-1hi, or either Lyt-2-/L3T4+ or Lyt-2+/L3T4-, whereas in the weeks that follow a steadily greater proportion of MEL-14hi thymocytes come to express this mature pattern (roughly 70% at 4 wk of age). Most day 15 MEL-14hi fetal thymocytes appear to express the functional homing receptor molecule, since day 15 fetal thymocytes bind to peripheral lymph node high endothelial venules about 40 to 50% as well as do adult mesenteric node lymphocytes, whereas adult thymocytes bind only about 5% as well. We have also identified a population of outer cortical MEL-14hi Lyt-2-/L3T4- lymphoblasts that appears during thymus regeneration 5 to 6 days after the administration of hydrocortisone. These lymphoblasts express the same phenotype as cells that constitute 40% of the day 15 fetal thymus and only 0.4% of normal adult thymocytes, implying that this particular subset may make up a significant fraction of thymocytes whenever there is a requirement for rapid expansion of the intrathymic and/or peripheral T cell pools. Taken together, these results are consistent with the notion that expression of the MEL-14-defined homing receptor may be closely linked to important intrathymic events that may occur early in T cell development and yet still have an overriding impact on the selection of those thymocytes that will serve as precursors of thymus emigrants.

  View details for Web of Science ID A1986C223100005

  View details for PubMedID 3084634

• DUAL IMMUNOFLUORESCENCE STUDIES OF CORTISONE-INDUCED THYMIC INVOLUTION - EVIDENCE FOR A MAJOR CORTICAL COMPONENT TO CORTISONE-RESISTANT THYMOCYTES JOURNAL OF IMMUNOLOGY REICHERT, R. A., Weissman, I. L., BUTCHER, E. C. 1986; 136 (10): 3529-3534

  Abstract

  Cortisone-resistant thymocytes (CRT) have been used as the experimental equivalent of medullary thymocytes for the past 15 yr. Studies with CRT have provided evidence that the medullary population is similar to mature T cells in phenotype and function and may therefore be the major source of thymus emigrants. However, we have recently demonstrated that CRT differ from medullary thymocytes in their expression of the homing receptor molecule recognized by the monoclonal antibody MEL-14. Thus, many CRT express high levels of the MEL-14-defined homing receptor, whereas medullary thymocytes are MEL-14- to MEL-14lo. In normal adult mice, only 1 to 3% of thymocytes are MEL-14hi; these cells are located exclusively in the cortex and many are phenotypically and functionally mature. In this study we have used dual immunofluorescence techniques to further characterize those thymocytes resistant to cortisone treatment. Aside from being of mature phenotype with respect to expression of peanut agglutinin binding sites and the cell surface molecules H-2K, Ly-1, Lyt-2, and L3T4, CRT can be divided into MEL-14lo and MEL-14hi subpopulations, suggesting that they may actually be derived from both the medullary and the MEL-14hi cortical thymocyte subsets.

  View details for Web of Science ID A1986C223100004

  View details for PubMedID 3084633

• PHENOTYPIC ANALYSIS OF THYMOCYTES THAT EXPRESS HOMING RECEPTORS FOR PERIPHERAL LYMPH-NODES JOURNAL OF IMMUNOLOGY REICHERT, R. A., Weissman, I. L., BUTCHER, E. C. 1986; 136 (10): 3521-3528

  Abstract

  Thymocytes that express high levels of homing receptors for peripheral lymph nodes can be detected with the monoclonal antibody MEL-14. We have shown that in adult mice these rare MEL-14hi thymocytes a) are cortical in location and typically constitute 1 to 3% of the total thymocyte population, b) may be a major source of thymus emigrants, and c) contain a high frequency of precursors of alloreactive cytotoxic T lymphocytes. In this study we have analyzed the phenotype of the MEL-14hi thymocyte subset. Most normal adult MEL-14hi thymocytes are midsize and express the mature phenotype typical of thymus emigrants, medullary thymocytes, and peripheral T cells: they are predominantly PNAlo, H-2K+, Thy-1+, Ly-1hi, and either Lyt-2-/L3T4+ or Lyt-2+/L3T4-. These findings argue strongly for the presence of rare MEL-14hi immunocompetent cortical thymocytes that, aside from their homing receptor expression, are phenotypically indistinguishable from medullary thymocytes. However, a minority (20 to 30%) of MEL-14hi thymocytes are large and phenotypically nonmature: they express intermediate to high levels of PNA binding sites, and are H-2K- to H-2Klo, Thy-1hi, Ly-1+, and either Lyt-2+/L3T4+ or Lyt-2-/L3T4-. Through a technique that selectively labels outer cortical cells, phenotypically nonmature MEL-14hi thymocytes have been shown to be concentrated in the subcapsular blast region of the outer cortex. Although we have no direct evidence of a precursor-product relationship, we consider it likely that the phenotypically nonmature outer cortical MEL-14hi lymphoblasts give rise to phenotypically mature MEL-14hi cells located deeper in the cortex. These results are consistent with our previous proposal that MEL-14hi thymocytes are a major source of thymus emigrants, and indicate that expression of high levels of MEL-14-defined homing receptors may be closely linked to the intrathymic selection process.

  View details for Web of Science ID A1986C223100003

  View details for PubMedID 3084632

• INSITU IDENTIFICATION OF IDIOTYPE-POSITIVE CELLS PARTICIPATING IN THE IMMUNE-RESPONSE TO PHOSPHORYLCHOLINE EUROPEAN JOURNAL OF IMMUNOLOGY RAZZECA, K. J., Pillemer, E., Weissman, I. L., Rouse, R. V. 1986; 16 (4): 393-399

  Abstract

  The phosphorylcholine idiotype (Id)/anti-Id system has been used to study the role of antigen-specific cells in antigen-induced microenvironmental changes. Anti-Id staining of lymph nodes following PC immunization shows the presence of Id on follicular dendritic cells at 12 h and in plasma cells beginning at day 3. Germinal centers began to form at day 3, peaking in size and number at days 8-10. Scattered Id-positive small lymphocytes are present in germinal centers but with rare exceptions over 98% of germinal center cells are Id-negative. Idiotype-positive small lymphocytes are depleted from primary follicles adjacent to germinal centers but not from distant, unstimulated nodes. These results extend previous studies showing architectural alterations in lymph nodes following antigenic stimulation and demonstrate antigen-specific cells are a prominent component of these antigen-induced microenvironmental changes.

  View details for Web of Science ID A1986C242800013

  View details for PubMedID 3486128

• LYMPHOCYTE HOMING RECEPTORS CELL Gallatin, M., STJOHN, T. P., Siegelman, M., Reichert, R., BUTCHER, E. C., Weissman, I. L. 1986; 44 (5): 673-680

  View details for Web of Science ID A1986A542800001

  View details for PubMedID 3004743

• ISOLATION OF 2 EARLY LYMPHOCYTE-B PROGENITORS FROM MOUSE MARROW - A COMMITTED PRE-PRE-B-CELL AND A CLONOGENIC THY-1LO HEMATOPOIETIC STEM-CELL CELL MULLERSIEBURG, C. E., WHITLOCK, C. A., Weissman, I. L. 1986; 44 (4): 653-662

  Abstract

  Two novel early B lymphocyte precursor populations have been identified by their capacity to differentiate in Whitlock-Witte bone marrow cultures. Cells expressing neither the B lineage antigen B220 nor Thy-1 contain committed B cell precursors which differentiate in short-term culture into pre-B and B cells. The other population expresses low levels of Thy-1, and lacks B220 as well as the T cell markers L3T4 and Lyt-2. The Thy-1+ cells which initiate long-term B cell cultures contain clonogenic B cell precursors at a frequency of 1 in 11, a 100-fold enrichment over unseparated bone marrow. Thy-1+ cells are also highly enriched for myeloid-erythroid precursors (CFU-S). Thy-1+ cells allow long-term survival of lethally irradiated mice and fully reconstitute the hematopoietic system, including T and B lymphocyte compartments. These results indicate that this population (approximately 0.1% of bone marrow) may contain the pluripotent hematopoietic stem cell.

  View details for Web of Science ID A1986A397100014

  View details for PubMedID 2868799

• CELL-SURFACE MOLECULE ASSOCIATED WITH LYMPHOCYTE HOMING IS A UBIQUITINATED BRANCHED-CHAIN GLYCOPROTEIN SCIENCE Siegelman, M., Bond, M. W., Gallatin, W. M., STJOHN, T., Smith, H. T., Fried, V. A., Weissman, I. L. 1986; 231 (4740): 823-829

  Abstract

  Partial amino acid sequence analysis of a purified lymphocyte homing receptor demonstrates the presence of two amino termini, one of which corresponds precisely to the amino terminus of ubiquitin. This observation extends the province of this conserved polypeptide to the cell surface and leads to a proposed model of the receptor complex as a core polypeptide modified by glycosylation and ubiquitination. Independent antibodies to ubiquitin serve to identify additional cell surface species, an indication that ubiquitination of cell surface proteins may be more general. It is proposed that functional binding of lymphocytes to lymph node high endothelial venules might involve the ubiquitinated region of the receptor; if true, cell surface ubiquitin could play a more general role in cell-cell interaction and adhesion.

  View details for Web of Science ID A1986AZH9100022

  View details for PubMedID 3003913

• EXPRESSION CLONING OF A LYMPHOCYTE HOMING RECEPTOR CDNA - UBIQUITIN IS THE REACTIVE SPECIES SCIENCE STJOHN, T., Gallatin, W. M., Siegelman, M., Smith, H. T., Fried, V. A., Weissman, I. L. 1986; 231 (4740): 845-850

  Abstract

  The lymphocyte cell surface receptor for the high endothelial venules (HEV's) of peripheral lymph nodes is specifically recognized by the monoclonal antibody MEL-14. Three independent complementary DNA (cDNA) clones, each of which encodes the protein ubiquitin, were detected by virtue of the expression of the MEL-14 antigenic determinant on cDNA-beta-galactosidase bacterial fusion proteins. The antigenic determinant defined by MEL-14 resides in the carboxyl terminal 13-amino-acid proteolytic peptide of ubiquitin, but is undetected in intact undenatured ubiquitin and other cellular ubiquitinated proteins. Antisera and monoclonal antibodies to ubiquitin determinants bind to the surface of both HEV-receptor positive and negative cell lines. The MEL-14-identified cDNA clones hydridize to RNA transcripts that encode tandemly repeated ubiquitins. Sequence analysis of these polyubiquitin cDNA's does not identify a leader sequence for export to the cell surface. The expression of the MEL-14 epitope of ubiquitin depends upon its local environment. The steady-state levels of expression of the ubiquitin messenger RNA's do not correlate with either the tissue derivation of the RNA or the expression of the lymphocyte HEV receptor. Regulation of the expression of the HEV receptor is not likely to reflect the transcriptional control of ubiquitin genes, but rather to reflect control of the expression of the HEV core polypeptide or its level or form of ubiquitination.

  View details for Web of Science ID A1986AZH9100028

  View details for PubMedID 3003914

• GROWTH AND SEXUAL-MATURATION OF LABORATORY-CULTURED MONTEREY BOTRYLLUS-SCHLOSSERI BIOLOGICAL BULLETIN BOYD, H. C., Brown, S. K., Harp, J. A., Weissman, I. L. 1986; 170 (1): 91-109

  View details for Web of Science ID A1986A340500008

• MONOCLONAL-ANTIBODY TO THE AMINO-TERMINAL-L SEQUENCE OF MURINE LEUKEMIA-VIRUS GLYCOSYLATED GAG POLYPROTEINS DEMONSTRATES THEIR UNUSUAL ORIENTATION IN THE CELL-MEMBRANE JOURNAL OF VIROLOGY Pillemer, E. A., KOOISTRA, D. A., Witte, O. N., Weissman, I. L. 1986; 57 (2): 413-421

  Abstract

  To analyze cell surface murine leukemia virus gag protein expression, we have prepared monoclonal antibodies against the spontaneous AKR T lymphoma KKT-2. One of these antibodies, 43-13, detects an AKR-specific viral p12 determinant. A second monoclonal antibody, 43-17, detects a novel murine leukemia virus-related antigen found on glycosylated gag polyproteins (gp95gag, gp85gag, and gp55gag) on the surface of cells infected with and producing ecotropic endogenous viruses, but does not detect antigens within these virions. The 43-17 antibody immunoprecipitates the precursor of the cell surface gag protein whether in its glycosylated or unglycosylated state, but does not detect the cytoplasmic precursor of the virion gag proteins (Pr65gag). Based on these findings, we have localized the 43-17 determinant to the unique amino-terminal part of the glycosylated gag polyprotein (the L domain). We have determined that gp95gag contains L-p15-p12-p30-p10 determinants, whereas gp85gag lacks the carboxyterminal p10 determinant, and gp55gag lacks both p30 and p10 carboxy terminal determinants. Analysis of cell surface gag expression with the 43-17 antibody leads us to propose that the L domain plays a crucial role in (i) the insertion and orientation of murine leukemia virus gag polyproteins in the cell membrane and (ii) the relative abundance of expression of AKR leukemia virus versus Moloney murine leukemia virus glycosylated gag polyproteins in infected cells.

  View details for Web of Science ID A1986AXZ1900001

  View details for PubMedID 2418213

• HOMING RECEPTORS AND THE CONTROL OF LYMPHOCYTE MIGRATION IMMUNOLOGICAL REVIEWS JALKANEN, S., REICHERT, R. A., Gallatin, W. M., BARGATZE, R. F., Weissman, I. L., BUTCHER, E. C. 1986; 91: 39-60

  Abstract

  The traffic of lymphocytes is controlled in part by the selective interaction of circulating lymphocytes with specialized high endothelial venule (HEV) cells at sites of lymphocyte exit from the blood. At least three independent receptor systems are responsible for controlling lymphocyte traffic to different lymphoid organs or to sites of inflammation: one mediates lymphocyte interaction with HEV in peripheral lymph nodes, another in mucosa-associated lymphoid tissues, and a third in inflamed synovium. The receptors mediating lymphocyte recognition of HEV in different organs appear to be structurally related yet antigenically and functionally distinct 90 kD glycoproteins. Receptors for lymph node HEV can function as mammalian lectins, and probably interact with specific carbohydrate ligands on high endothelial cells. Mouse and human homing receptors share both antigenic and structural features, indicating a high conservation of lymphocyte-endothelial recognition systems during evolution. They play an essential part in the immune process by controlling lymphocyte traffic during B- and T-cell differentiation, and by segregating effector cells derived from stimulation in different tissues, thus simultaneously increasing the efficiency of organ-specific immune responses and decreasing possibilities for autoimmune crossreactions. Homing receptors are also expressed by many mouse and human lymphoid neoplasms, and appear to play a role in lymphoma metastasis. Related if not identical receptors are expressed by other leukocyte types, including polymorphonuclear leukocytes, monocytes, and large granular lymphocytes (natural killer cells). Thus lymphocyte homing receptors are members of a family of glycoprotein receptors for endothelium that control the extravasation of lymphocytes as well as other leukocytes, and help regulate both non-specific and specific immune responses in vivo.

  View details for Web of Science ID A1986D137400002

  View details for PubMedID 2426181

• Mature and immature thymocytes: surface phenotype, immune function and intrathymic location Leukocytes and Host Defense; 17th Meeting of the International Leukocyte Culture Conference Held Jointly with the 22nd National Meeting of the Reticuloendothelial Society Shortman, K., Scollay, R., Wilson, A., Andrews, P., Boyd, R., Butcher, E., Weissman, I. 1986: 3–10
• THE INVIVO BEHAVIOR OF T-CELL CLONES - ALTERED MIGRATION DUE TO LOSS OF THE LYMPHOCYTE SURFACE HOMING RECEPTOR JOURNAL OF MOLECULAR AND CELLULAR IMMUNOLOGY Dailey, M. O., Gallatin, W. M., Weissman, I. L. 1985; 2 (1): 27-36

  Abstract

  Although cloned lines of T lymphocytes have been valuable in defining the in vitro functions of well-defined cell types, they have often demonstrated relatively poor activity in vivo. One striking property of T cells clones which might affect their in vivo activity is their unusual inability to localized in lymphoid tissue as do most normal T cells. Normal lymphocyte recirculation and localization requires that lymphocytes recognize and pass through the walls of specialized high endothelial venules (HEV) as they enter into lymph nodes. We previously showed that murine T cell clones are unable to home into the peripheral lymphoid organs-lymph nodes and Peyer's patches. The inability of these cells to recognize lymph node HEV in an in vitro frozen section adherence assay suggested that the lack of lymphoid homing was due to the loss of a normal lymphocyte surface receptor for HEV. The present experiments were designed to determine: 1) the molecular mechanism responsible for the loss of normal lymphocyte migration, and 2) whether these migration and homing characteristics are irreversible features of T cell clones. Flow cytometric analysis of helper and cytolytic clones using a monoclonal antibody (MEL-14) specific for the lymphocyte homing receptor showed that they lack this surface receptor. This lack of receptor expression was confirmed by the inability to detect the antigen in detergent-solubilized extracts of surface-radiolabeled cells. Thus, the lack of homing to lymph nodes appears to be due to the loss of expression of the surface receptor which mediates the interaction between lymphocytes and HEV. When clones were rested in vitro in a nonproliferative state without stimulation by antigen or growth factors, they did not regain expression of the surface homing receptor or the ability to migrate to lymph nodes in vivo. The lack of receptor expression, therefore, is not merely associated with a rapidly proliferating state, but rather seems to be an irreversible feature of T cell clones, at least under in vitro culture conditions. T cell clones, both rested and recently restimulated, share certain features characteristic of activated T cells, as shown by recent results with MLC-stimulated T cell blasts. Both populations are large, brightly PNA-positive lymphocytes which lack expression of the MEL-14 receptor and do not home to peripheral lymphoid tissue. We propose that this PNA-high, MEL-14- nonrecirculating phenotype may represent a normal phase of T cell differentiation through which many T cells pass after being activated by specific antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1985AEC2300004

  View details for PubMedID 2978224

• Lymphocyte and lymphoma receptors utilized in differentiation, in homing, and in lymphomagenesis. Haematology and blood transfusion Weissman, I. L., McGrath, M. S., REICHERT, R. A., Gallatin, W. M., Ezine, S., Fink, P., BUTCHER, E. C., Marian, J., O'Neill, H. C. 1985; 29: 449-454

  View details for PubMedID 4029738

• THYMIC LYMPHOCYTE DIFFERENTIATION AND THYMIC LEUKEMOGENESIS INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Weissman, I. L. 1985; 11 (1): 57-64

  Abstract

  Henry Kaplan helped establish the fields of lymphocyte biology and viral leukemogenesis by his early and continuing studies on radiation leukemogenesis. As one of Henry's students I carried on these dual preoccupations with thymic lymphocytopoiesis and thymic lymphomagenesis. This communication demonstrates that thymic lymphocytes are derived from bone marrow precursors which lack any T cell markers; these bone marrow cells (or their clonogenic subsets) can give rise to either thymic cortical plus medullary progeny, or medullary progeny alone; thymic lymphocytes mature in contact with 3-5 classes of nonlymphoid cells (thymic nurse cells, cortical dendritic epithelial cells, medullary epithelial cells, dendritic reticular cells, and macrophages), and one of these subsets, cortical dendritic epithelial cells, express an unusual distribution of MHC antigen (perhaps utilized in the maturation of T cell MHC restriction); the population of cells which are poised to emigrate from the thymus are a unique subset of cortical cells which possess peripheral lymphoid organ homing receptors; and the thymic target cells for retrovirus lymphomagenesis express highly specific retrovirus receptors that are analogous (and perhaps synonymous) with antigen-specific T cell receptors.

  View details for Web of Science ID A1985ABW5500007

  View details for PubMedID 3871434

• The receptor-mediated leukemogenesis hypothesis: a model of retroviral oncogenesis by viral stimulation of cell-surface receptors Leukemia. Dahlem KOnferenzen Weissman, I., McGrath, M., Tamura, G. Verlag. 1985: 235–249
• HOMING RECEPTOR-BEARING THYMOCYTES, AN IMMUNOCOMPETENT CORTICAL SUBPOPULATION NATURE Fink, P. J., Gallatin, W. M., REICHERT, R. A., BUTCHER, E. C., Weissman, I. L. 1985; 313 (5999): 233-235

  Abstract

  Much of the differentiation of murine T cells takes place in the thymus, perhaps influenced by the operation of stringent selection mechanisms whose existence has been inferred from the high rate of thymocyte turnover in the absence of extensive emigration. The origin of those 1% of total thymocytes which leave the thymus and seed the peripheral lymphoid organs is obscure. Recent thymic emigrants are functionally and phenotypically mature, and the purported greater maturity of medullary relative to cortical thymocytes is often cited a evidence for the medullary origin of thymic emigrants, a suggestion not without its critics. To approach this question, we have now isolated a a subpopulation of thymocytes expressing high levels of a receptor that mediates the homing of blood-borne lymphocytes into peripheral lymph nodes. Surprisingly, this population of cells (1-3% of total thymocytes) is both cortical and immunocompetent, containing approximately half of all thymic cytolytic T-lymphocyte precursors. The combination of homing receptor expression and immunocompetence makes this cortical population ideally suited for emigration to peripheral lymphoid organs.

  View details for Web of Science ID A1985AAB1100056

  View details for PubMedID 3871507

• GERMINAL CENTER CELLS - ANTIGEN-SPECIFICITY, HEAVY-CHAIN CLASS EXPRESSION AND EVIDENCE OF MEMORY ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Kraal, G., Weissman, I. L., BUTCHER, E. C. 1985; 186: 145-151

  View details for Web of Science ID A1985APB7200018

  View details for PubMedID 3876700

• TRANSFORMED LYMPHOCYTES FROM ABELSON-DISEASED MICE EXPRESS LEVELS OF A-B-LINEAGE TRANSFORMATION-ASSOCIATED ANTIGEN ELEVATED FROM THAT FOUND ON NORMAL LYMPHOCYTES JOURNAL OF EXPERIMENTAL MEDICINE Tidmarsh, G. F., Dailey, M. O., WHITLOCK, C. A., Pillemer, E., Weissman, I. L. 1985; 162 (5): 1421-1434

  Abstract

  Animals injected with Abelson murine leukemia virus (A-MuLV) rapidly develop fatal bone marrow-derived lymphosarcomas. In all such diseased animals tested, a subpopulation of bone marrow cells expressed a monoclonal antibody-defined, B lineage transformation-associated antigen (6C3 Ag) at levels increased from that detected on normal lymphocytes. Cells bearing a high level of this antigen were found to be transformed as measured by clonal growth in agar, and they expressed surface antigen markers characteristic of early pre-B cells. High-level antigen-expressing cells were found in the bone marrow, lymph nodes, and spleen, but never in the thymus of diseased animals. This distribution agrees with the published pathology of Abelson disease.

  View details for Web of Science ID A1985ATL1600002

  View details for PubMedID 2997360

• THE MURINE T-CELL RECEPTOR USES A LIMITED REPERTOIRE OF EXPRESSED V-BETA GENE SEGMENTS NATURE Barth, R. K., Kim, B. S., Lan, N. C., Hunkapiller, T., SOBIECK, N., Winoto, A., Gershenfeld, H., Okada, C., HANSBURG, D., Weissman, I. L., Hood, L. 1985; 316 (6028): 517-523

  Abstract

  Only 10 different V beta gene segments were found when the sequences of 15 variable (V beta) genes of the mouse T-cell receptor were examined. From this analysis we calculate that the total number of expressed V beta gene segments may be 21 or fewer, which makes the expressed germline V beta repertoire much smaller than that of immunoglobulin heavy-chain or light-chain genes. We suggest that beta-chain somatic diversification is concentrated at the V beta-D beta-J beta junctions.

  View details for Web of Science ID A1985ANN2200049

  View details for PubMedID 2412120

• THYMUS HOMING CLONOGENIC BONE-MARROW CELLS ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Ezine, S., Weissman, I. L., Rouse, R. V. 1985; 186: 223-227

  View details for Web of Science ID A1985APB7200027

  View details for PubMedID 2864801

• EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGENS IN THE THYMUSES OF CHIMERIC MICE TRANSPLANTATION Rouse, R. V., Ezine, S., Weissman, I. L. 1985; 40 (4): 422-426

  Abstract

  Thymuses of various types of bone-marrow-chimeric mice have been examined by tissue section immunologic staining for the presence and distribution of major histocompatibility complex (MHC) antigens. Cortical and medullary thymic epithelial cells continue to express thymus genotype I-A and H-2K/D antigens for at least 6 months posttransplantation. The appearance of bone-marrow-type MHC antigens is limited to low levels of H-2K/D on cortical and medullary lymphocytes, and to dendritic cells in the medulla; the medullary dendritic cells express high levels of donor-type I-A antigens as soon as 3 weeks posttransplantation. The observed patterns support the concept that I-A antigens are synthesized by thymic epithelial cells but are acquired by thymocytes. The findings are of relevance to the understanding of the role of the thymus in the generation of MHC restriction.

  View details for Web of Science ID A1985ASJ4000015

  View details for PubMedID 2864757

• T-CELL CLONES - A MODEL OF A NON-RECIRCULATING PHASE OF T-CELL DIFFERENTIATION ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Dailey, M. O., Gallatin, W. M., Kraal, G., Butcher, E., Weissman, I. 1985; 186: 621-627

  View details for Web of Science ID A1985APB7200076

  View details for PubMedID 2996315

• A HOMING RECEPTOR-BEARING CORTICAL THYMOCYTE SUBSET - IMPLICATIONS FOR THYMUS-CELL MIGRATION AND THE NATURE OF CORTISONE-RESISTANT THYMOCYTES CELL REICHERT, R. A., Gallatin, W. M., BUTCHER, E. C., Weissman, I. L. 1984; 38 (1): 89-99

  Abstract

  The thymus exports a selected subset of virgin T lymphocytes to the peripheral lymphoid organs. The mature phenotype of these thymus emigrants is similar to that of medullary thymocytes and has been cited as supporting a medullary rather than cortical exit site. Using the monoclonal antibody MEL-14, we identify a 1%-3% subpopulation of thymocytes that expresses high levels of a receptor molecule involved in lymphocyte homing to peripheral lymph nodes. We present evidence that these rare MEL-14hi thymocytes are predominantly of mature phenotype and represent the major source of thymus emigrants. Surprisingly, MEL-14hi thymocytes are exclusively cortical in location, although their mature phenotype may allow them to masquerade as medullary cells in conventional studies. We also demonstrate that unlike medullary thymocytes, many cortisone-resistant thymocytes (CRT) are MEL-14hi. Thus, in contrast to current dogma, CRT do not represent a sample of medullary thymocytes as they are found in situ and their level of immunocompetence does not necessarily reflect that of the medullary population. Our findings refute the hypothesis that phenotypically and functionally mature cells are restricted to the medulla, and support our proposition that most thymus emigrants are derived from the MEL-14hi cortical subset.

  View details for Web of Science ID A1984TF99100012

  View details for PubMedID 6432340

• CLONING OF TERMINAL TRANSFERASE CDNA BY ANTIBODY SCREENING PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Landau, N. R., STJOHN, T. P., Weissman, I. L., Wolf, S. C., SILVERSTONE, A. E., Baltimore, D. 1984; 81 (18): 5836-5840

  Abstract

  A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the lambda phage vector lambda gt11. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had beta-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody. The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome.

  View details for Web of Science ID A1984TM40300045

  View details for PubMedID 6091113

• Immunolofy Hood, L., Weissman, I., Wood, B., Wilson, J. Addison-Wesley. 1984
• The role of immunospecific receptors in retrovirus-induced lymphomagenesis Concepts in Viral Pathogenesis McGrath, M., Weissman, I. Springer-Verlag. 1984: 216–224
• THE IMMUNOCOMPETENCE OF MURINE STROMAL CELL-ASSOCIATED THYMOCYTES JOURNAL OF IMMUNOLOGY Fink, P. J., Weissman, I. L., KAPLAN, H. S., KYEWSKI, B. A. 1984; 132 (5): 2266-2272

  Abstract

  Thymocyte subpopulations that associate in vivo with distinct nonlymphoid cells of the thymus have been isolated, and their immunocompetence was analyzed. Previous studies have indicated that greater than 95% of such cells bear a surface antigen phenotype representative of immature thymocytes, and are among the earliest thymic compartments repopulated by bone marrow-derived cells after lethal and sub-lethal irradiation. Stromal cell-associated thymocytes may be activated in vivo because they proliferate well in vitro with no additional stimulus, and show little increase in proliferation with the addition of T cell mitogens or allogeneic spleen cells. Stromal cell-associated T cells contain cytotoxic T lymphocyte (CTL) precursors that are indistinguishable from mature peripheral T cells by the parameters of self tolerance, alloreactivity, H-2 restriction, and stringency of self H-2 preference. CTL precursor frequencies and the cytotoxic activity of cells further separated on the basis of high levels of Thy-1 expression argue against the possibility that stromal cell-associated CTL activity is due solely to contaminating mature lymphocytes. Our data suggest that stromal cell-associated thymocytes represent an intermediate subpopulation of thymocytes that is functionally mature and that expresses an immature surface phenotype. Furthermore, the imposition of self tolerance and MHC restriction specificity appears to be tightly associated with the acquisition of immunocompetence in these thymocyte subpopulations.

  View details for Web of Science ID A1984SN37200019

  View details for PubMedID 6609194

• Lymphoid tissues and organs Fundamental Immunology Butcher, E., Weissman, I. Raven Press. 1984: 109–127
• THYMIC CYTO-TOXIC LYMPHOCYTES-T ARE PRIMED INVIVO TO MINOR HISTOCOMPATIBILITY ANTIGENS JOURNAL OF EXPERIMENTAL MEDICINE Fink, P. J., Bevan, M. J., Weissman, I. L. 1984; 159 (2): 436-451

  Abstract

  Potent cytotoxic T lymphocyte (CTL) activity can be derived from cultures of thymocyte responders and minor H different spleen cell stimulators. As is the case of the spleen cell response previously reported, this cytotoxic activity requires in vivo priming. We performed several experiments designed to determine whether the in vivo priming effect is due to the in situ priming of the thymocyte CTL precursors, to contamination of thymus cell preparations with cells of neighboring lymph nodes, or to the appearance in the thymus of antigen-reactive peripheral T cells. We show by depletion of peripheral cells with antilymphocyte serum and part body irradiation that recent thymic immigrants derived from the bone marrow contribute to the primed thymic response. Thymic CTL were primed in animals in which peripheral T cell responses were completely eliminated by repeated treatment in vivo with monoclonal anti-Thy-1 reagents. Primed, antigen-activated lymph node cells were also demonstrated to contribute to the thymus-derived CTL response. Thus, the minor H-specific thymic CTL response is due both to in situ priming and the immigration of activated peripheral T cells. We discuss the possible significance for models of T cell differentiation of the presence within the thymus of antigen and antigen-reactive mature T cells.

  View details for Web of Science ID A1984SH34900008

  View details for PubMedID 6607314

• Improved lysis of fusion-expressing bacteria upon removal of NaCl from buffer Biotechniques Gershenfeld, H., Weissman, I. 1984; 2 (4)
• Receptor-mediated leukemogenesis: analysis of avian and human lymphomas for retrovirus-binding specificities Human T-Cell Leukemia Lymphoma Virus: the Family of Human T-Lymphotropic Retroviruses: Their Role in Malignancies and Association with AIDS McGrath, M., Weissman, I. 1984: 205–216
• TRANSFORMATION-ASSOCIATED PROTEINS IN MURINE B-CELL LYMPHOMAS THAT ARE DISTINCT FROM ABELSON VIRUS GENE-PRODUCTS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Pillemer, E., Whitlock, C., Weissman, I. L. 1984; 81 (14): 4434-4438

  Abstract

  In an effort to identify cellular proteins that may be involved in the Abelson murine leukemia virus (A-MuLV) transformation process, we have isolated a hybridoma antibody (6C3) that detects a tumor-associated antigen in all A-MuLV-induced pre-B-cell lymphomas. The 6C3 antibody immunoprecipitates two molecules of Mr 160,000 and Mr 125,000 from metabolically labeled A-MuLV tumors. The two proteins recognized by the 6C3 antibody are distinct from the A-MuLV-transforming protein in that they lack viral gag determinants and are neither phosphoproteins nor protein kinases. The 6C3 proteins can be detected in all A-MuLV pre-B-cell lymphomas and some nonviral B lymphomas but are not detected on any other tumor or normal cell, including A-MuLV-transformed fibroblast lines. Thus, the 6C3 proteins may represent the products of novel cellular genes whose expression is induced, stabilized, or amplified in B-cell tumors of both viral and nonviral origin. Further evidence in support of this hypothesis is provided by the finding that 6C3 antigen expression correlates with autonomous cell growth and the transformed phenotype in both normal bone marrow cultures and those infected with A-MuLV.

  View details for Web of Science ID A1984TD00100042

  View details for PubMedID 6611551

• TOLERANCE AND THE H-Y-ANTIGEN - REQUIREMENT FOR MALE T-CELLS, BUT NOT B-CELLS, TO INDUCE TOLERANCE IN NEONATAL FEMALE MICE TRANSPLANTATION Weissman, I. L., Jerabek, L., Greenspan, S. 1984; 37 (1): 3-6

  Abstract

  Transplantation tolerance of the H-Y antigen may be induced and transferred by lymphoid cells, but not by nonlymphoid cells. In this study we have demonstrated that amongst lymphoid cells, T cells--but not B cells or macrophages--are capable of inducing transplantation tolerance. In contrast, all three populations express sufficient amounts of immunogenic H-Y antigen to sensitize adult female mice to a subsequent male skin graft, as demonstrated by the second-set reaction.

  View details for Web of Science ID A1984RZ82600003

  View details for PubMedID 6364485

• ALLELIC EXCLUSION IN RAT KAPPA-IMMUNOGLOBULIN CHAINS - EXTENT OF JK REARRANGEMENT IN NORMAL LYMPHOCYTE-B EMBO JOURNAL Tsukamoto, A., Weissman, I. L., Hunt, S. V. 1984; 3 (5): 975-981

  Abstract

  The frequency of normal rat peripheral B lymphocytes stained for surface immunoglobulin kappa allotypes a and b in (a X b) F1 heterozygotes was assessed by two-colour immunofluorescence on a fluorescence-activated cell sorter. The upper limit to the frequency of double- stainers was 8% among all kappa-positive cells, though it was not resolved how far cytophilic antibody accounted for these. Cells expressing each allotype singly were isolated and the extent of rearrangement of the genes encoding the joining-kappa segment on the expressed and non-expressed chromosome were independently assessed. The expressed allele was found to be virtually completely rearranged while the non-expressed allele showed approximately 45-60% rearrangement. The implications of this substantial non-productive rearrangement for models of allelic exclusion are discussed.

  View details for Web of Science ID A1984SR31600008

  View details for PubMedID 6428882

• MONOCLONAL-ANTIBODIES DEVELOPED AGAINST BOTRYLLUS BLOOD-CELL ANTIGENS BIND TO CELLS OF DISTINCT LINEAGES DURING EMBRYONIC-DEVELOPMENT JOURNAL OF EXPERIMENTAL ZOOLOGY SCHLUMPBERGER, J. M., Weissman, I. L., Scofield, V. L. 1984; 229 (2): 205-213

  View details for Web of Science ID A1984SD99800004

• SEPARATION AND LABELING OF SPECIFIC SUBPOPULATIONS OF BOTRYLLUS BLOOD-CELLS JOURNAL OF EXPERIMENTAL ZOOLOGY SCHLUMPBERGER, J. M., Weissman, I. L., Scofield, V. L. 1984; 229 (3): 401-411

  Abstract

  Blood cells of the colonial tunicate Botryllus were separated by density gradient centrifugation in Percoll. Unseparated blood cells were used to immunize mice for development of hybridoma cell lines producing anti-Botryllus monoclonal antibodies. These antibodies identify specific subpopulations of blood cells, indicating possible divisions of these cells into defined subgroups sharing particular differentiation antigens. Additional studies utilizing fluorescein- conjugated lectins also revealed differential binding to density- and monoclonal antibody-defined blood cell fractions. These methods allow separation of the different Botryllus blood cell types for functional studies.

  View details for Web of Science ID A1984SJ90100007

  View details for PubMedID 6368739

• BONE-MARROW CELLS GIVE RISE TO DISTINCT CELL CLONES WITHIN THE THYMUS NATURE Ezine, S., Weissman, I. L., Rouse, R. V. 1984; 309 (5969): 629-631

  Abstract

  The thymus is the major, if not the sole site of maturation of T lymphocytes from their haematopoietic precursors. During embryonic life (at a few well-defined intervals, at least in birds) the thymus receives thymus-homing haematopoietic precursors that give rise to antigen-specific functional T lymphocytes. Although the number and thymic location of distinct T-cell lineages destined to form the peripheral T-cell pool are not yet well defined, at least two independent pathways have been proposed. First, thymic subcapsular lymphoblasts divide and differentiate to give rise to small deep cortical thymic lymphocytes, medullary lymphocytes and thymus emigrants (I.W., unpublished data) and second, the medulla contains an independent self-renewing population that contains the precursors of the peripheral T-cell pool. Following irradiation the thymus may be repopulated by injected haematopoietic cells presumably related to the thymus-homing haematopoietic cells of the embryo. Here we have reconstituted irradiated mice with limiting numbers of bone marrow cells from Thy-1 congeneic donors and have found distinct clones of cells within the thymus. The pattern of reconstitution by the precursor cells indicates that two independent thymus lineages exist: cortex plus medulla, and medulla alone.

  View details for Web of Science ID A1984SV60500051

  View details for PubMedID 6374470

• LOCALIZATION OF LYMPHOCYTE SUBPOPULATIONS IN PERIPHERAL LYMPHOID ORGANS - DIRECTED LYMPHOCYTE MIGRATION AND SEGREGATION INTO SPECIFIC MICROENVIRONMENTS AMERICAN JOURNAL OF ANATOMY Rouse, R. V., REICHERT, R. A., Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. 1984; 170 (3): 391-405

  Abstract

  The distribution of lymphocytes in the peripheral lymphoid organs is controlled by recirculatory and microenvironmental factors. Specific interactions between recirculating lymphocytes and high endothelial venules in various lymphoid organs determine the presence and proportions of the various lymphoid sets and subsets in those organs. Separate endothelial determinants on peripheral node and Peyer's patch endothelium along with complementary lymphocyte receptors mediate this organ specificity. B and T cells also exhibit nonrandom organization within lymphoid tissues; after entry via high endothelial venules they segregate into their respective domains, which appear to be determined by distinct types of nonlymphoid stromal cells. Antigenic stimulation results in changes in lymphocyte phenotype as well as in the lymphoid microenvironment. The response to most complex antigens is the formation of germinal centers (GC) composed primarily of proliferating B cells; the phenotype of the few T cells therein is supportive of the GC as a site of B-T interaction. The phenotype of the B cells in GCs suggest a role for GCs in immunoglobulin class switching and the determination of subsequent homing specificity.

  View details for Web of Science ID A1984TC39700012

  View details for PubMedID 6383006

• ISOLATION OF MOLECULES RECOGNIZED BY MONOCLONAL-ANTIBODIES AND ANTISERA - THE SOLID-PHASE IMMUNOISOLATION TECHNIQUE ANALYTICAL BIOCHEMISTRY TAMURA, G. S., Dailey, M. O., Gallatin, W. M., McGrath, M. S., Weissman, I. L., Pillemer, E. A. 1984; 136 (2): 458-464

  Abstract

  A simple technique for the isolation of antigens recognized by antisera and monoclonal antibodies has been developed. This method, the solid-phase immunoisolation technique, employs the protein-binding properties of polyvinylchloride microtiter plates. Antibodies are adsorbed to the plates either directly or via an anti-immunoglobulin reagent. Antigen is then placed in the wells, and allowed to adsorb to the antibody. The well is washed, and the antigen is then eluted with a denaturing electrophoresis sample buffer for one- or two-dimensional analysis. The solid-phase immunoisolation technique has been used to isolate a variety of cell membrane antigens with high signals and low backgrounds. The ease of the procedure and the high signal-to-noise ratio make this method preferable to the use of a staphylococcal adsorbent for many applications.

  View details for Web of Science ID A1984SE47000028

  View details for PubMedID 6721145

• EVOLUTION OF A MULTIGENE FAMILY OF V-KAPPA GERM LINE GENES EMBO JOURNAL Joho, R., Gershenfeld, H., Weissman, I. L. 1984; 3 (1): 185-191

  Abstract

  We have isolated a series of related V kappa germ line genes from a BALB/c sperm DNA library. DNA sequence analysis of four members of this V kappa 24 multigene family implies that three V kappa genes are functional whereas the fourth one (psi V kappa 24) is a pseudogene. The prototype gene (V kappa 24) encodes the variable region gene segment expressed in an immune response against phosphorylcholine. The other two functional genes (V kappa 24A and V kappa 24B) may be expressed against streptococcal group A carbohydrate. The time of divergence of the four genes was estimated by the rate of synonymous nucleotide changes. This implies that an ancestral gene has duplicated approximately 33-35 million years ago and a subsequent gene duplication event has occurred approximately 23 million years ago.

  View details for Web of Science ID A1984RZ48900029

  View details for PubMedID 6546718

• IMMUNOGLOBULIN GENE REARRANGEMENT DURING PRE-B CELL-DIFFERENTIATION JOURNAL OF MOLECULAR AND CELLULAR IMMUNOLOGY Coffman, R. L., Weissman, I. L. 1983; 1 (1): 31-38

  Abstract

  The experiments reported here were designed to answer two questions: (1) At what stage in normal pre-B cell development do immunoglobulin gene rearrangements occur?; and (2) Do heavy chain and kappa light chain genes rearrange in concert, or in an ordered sequence? To answer these questions, we studied immunoglobulin gene rearrangements in pre-B cell populations purified on the fluorescence-activated cell sorter (FACS). Gene rearrangement was assessed by measuring the loss of germ-line joining (J) segment-containing restriction fragments in B cells and two populations of pre-B cells. Large pre-B cells, the earliest identifiable cells in the B lineage, have rearrangements at both JH loci but do not have rearrangements at the kappa chain loci. Thus heavy chain rearrangement occurs concurrently with or prior to the expression of the surface marker B220, which we use to identify and isolate pre-B cells. Small pre-B cells, which include the immediate precursors of B cells, likewise have rearrangements at both JH loci, but may also have J kappa rearrangements. Approximately 1/3 of the J kappa loci are rearranged in small pre-B cells compared to 2/3 in kappa chain-expressing B cells. This suggests that the small pre-B cell population is actively undergoing kappa chain gene rearrangement. The striking asynchrony in heavy and light chain gene rearrangement is reflected at the level of gene expression; both pre-B cell populations synthesize mu chains but not kappa light chains. Heavy chain rearrangement is therefore a very early event in B lineage development and may begin in a cell not yet fully committed to the B lineage, whereas kappa rearrangement occurs just prior to the expression of surface immunoglobulin (sIg) and may be a rate-limiting step in the transition from pre-B to B cells.

  View details for Web of Science ID A1983SX35900005

  View details for PubMedID 6336590

• Surface phenotype and migration properties of activated lymphocytes and T cell clones ntercellular Communication in Leukocyte Function. Proceedings of the 15th Leukocyte Culture Conference Dailey, M., Gallatin, M., Weissman, I., Butcher, E. John Wiley and Sons. 1983: 641–644
• Lymphocyte recirculation: a molecular basis for the recognition of vascular endothelium Intercellular Communication in Leukocyte Function. 15th Leukocyte Culture Conference Gallatin, M., Weissman, I., Butcher, E. 1983: 645–648
• HAPLOTYPE-SPECIFIC SUPPRESSION OF CYTO-TOXIC T-CELL INDUCTION BY ANTIGEN INAPPROPRIATELY PRESENTED ON T-CELLS JOURNAL OF EXPERIMENTAL MEDICINE Fink, P. J., Weissman, I. L., Bevan, M. J. 1983; 157 (1): 141-154

  Abstract

  To detect a strong cytotoxic T lymphocyte (CTL) response to minor histocompatibility (H) antigens in a 5-d mixed lymphocyte culture, it is necessary to use a responder that has been primed in vivo with antigen-bearing cells. It has previously been shown that minor-H-specific CTL can be primed in vivo both directly by foreign spleen cells and by presentation of foreign minor H antigens on host antigen-presenting cells. This latter route is evident in the phenomenon of cross-priming, in which H-2 heterozygous (A x B)F1 mice injected 2 wk previously with minor H-different H-2A (A') spleen cells generate both H-2A- and H-2B-restricted minor-H-specific CTL. In a study of the kinetics of direct- vs. cross-priming to minors in F1 mice, we have found that minor H-different T cells actually suppress the induction of virgin CTL capable of recognizing them. CTL activity measured from F1 mice 3-6 d after injection with viable A' spleen cells is largely H-2B restricted. The H-2A-restricted response recovers such that roughly equal A- and B-restricted activity is detected in mice as early as 8-10 d postinjection. This temporary hyporeactivity does not result from generalized immunosuppression--it is specific for those CTL that recognize the foreign minor H antigen in the context of the H-2 antigens on the injected spleen cells. The injected spleen cells that mediate this suppression are radiosensitive T cells; Lyt-2+ T cells are highly efficient at suppressing the induction of CTL in vivo. No graft vs. host reaction by the injected T cells appears to be required, as suppression of direct primed CTL can be mediated by spleen cells that are wholly tolerant of both host H-2 and minor H antigens. Suppression cannot be demonstrated by in vitro mixing experiments. Several possible mechanisms for haplotype-specific suppression are discussed, including inactivation of responding CTL by veto cells and in vivo sequestration of responding CTL by the injected spleen cells.

  View details for Web of Science ID A1983PX64900012

  View details for PubMedID 6217276

• GENETIC-CONTROL OF T-CELL SUBSET REPRESENTATION IN INBRED MICE IMMUNOGENETICS Kraal, G., Weissman, I. L., BUTCHER, E. C. 1983; 18 (6): 585-592

  Abstract

  Lyt-2+ T cells constitute a significantly greater proportion of the total peripheral T-cell population in C57BL mice than in BALB/c and other mouse strains. The inheritance of this differential representation of Lyt-2- vs. Lyt-2+ T cells was studied by two-color immunofluorescence analysis of peripheral T cell subsets in BALB/c, C57BL, F1 and F2 generations, and in CXB recombinant inbred strains. It was shown that the C57BL phenotype (low Lyt-2-/Lyt-2+ ratio) is a dominant Mendelian character. Studies of subpopulations of thymocytes and of early thymus emigrants indicate that the representation of mature Lyt-2- and Lyt-2+ T cells is influenced by mechanisms of selection or differential turnover in the peripheral lymphoid organs, but that thymic and prethymic influences may also play a role.

  View details for Web of Science ID A1983RW97000003

  View details for PubMedID 6606619

• CHARACTERISTICS OF THYMUS-HOMING BONE-MARROW CELLS JOURNAL OF IMMUNOLOGY Lepault, F., Coffman, R. L., Weissman, I. L. 1983; 131 (1): 64-69

  Abstract

  Using a short-term in vivo assay, we studied bone marrow cells capable of homing to the thymus of lethally irradiated recipients. In the bone marrow, these cells lack the T cell markers Thy-1 and Lyt. Soon after their homing into the thymus, however, the immigrants begin to express Thy-1 and Lyt antigens, but not TL antigen. Unexpectedly, Lyt antigens appear to be expressed before Thy-1. These thymus-homing bone marrow cells seem to be already separated from the B cell lineage. Most of the homing cells are engaged in the cell cycle.

  View details for Web of Science ID A1983QX45700014

  View details for PubMedID 6134770

• MIMICKING THE ALLOANTIGENICITY OF PROTEINS WITH CHEMICALLY SYNTHESIZED PEPTIDES DIFFERING IN SINGLE AMINO-ACIDS NATURE Alexander, H., Johnson, D. A., ROSEN, J., Jerabek, L., Green, N., Weissman, I. L., Lerner, R. A. 1983; 306 (5944): 697-699

  Abstract

  Recent studies have shown that short chemically synthesized peptides very often induce antibodies which react with the cognate sequence in the intact folded protein. Since such antibodies react with known regions of proteins, they are of predetermined specificity and offer a precision not previously possible with immunological probes. A basic concept emerging from the use of such antibodies in viral systems is that the differential immunogenicity of closely related proteins can be mimicked by short peptides which span the regions of sequence variation. To generalize this concept, we have studied the two Thy-1 proteins which vary by only a single amino acid. Chemically synthesized peptides differing in only one out of 19 amino acids were able to induce allospecific antisera. Thus, single amino acid changes have similar effects on the immunogenicity of proteins and small peptides, even though the latter are free from constraints provided by neighbouring structures in the tertiary configuration of the intact folded proteins.

  View details for Web of Science ID A1983RU75800050

  View details for PubMedID 6656870

• A CELL-SURFACE MOLECULE INVOLVED IN ORGAN-SPECIFIC HOMING OF LYMPHOCYTES NATURE Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. 1983; 304 (5921): 30-34

  Abstract

  Lymphocytes migrate from the bloodstream by recognizing and binding to specialized endothelial cells lining the high endothelial venules (HEV) in lymph nodes and Peyer's patches. We describe here a monoclonal antibody, MEL-14, specific for a lymphocyte surface molecule that appears to mediate recognition of lymph node HEV, and to be required for lymphocyte homing into lymph nodes in vivo.

  View details for Web of Science ID A1983QX44800035

  View details for PubMedID 6866086

• DIFFERENCES IN INVIVO DISTRIBUTION AND HOMING OF T-CELL SUBSETS TO MUCOSAL VS NONMUCOSAL LYMPHOID ORGANS JOURNAL OF IMMUNOLOGY Kraal, G., Weissman, I. L., BUTCHER, E. C. 1983; 130 (3): 1097-1102

  Abstract

  The migratory properties of Lyt-2- and Lyt-2+ T cells in the mouse have been investigated. In short-term in vivo homing studies, Lyt-2- T cells localized consistently more efficiently than Lyt-2+ T cells in Peyer's patches (about 1.5 times as well), whereas both populations localized roughly equivalently in peripheral lymph nodes. These homing characteristics of Lyt-2- and Lyt-2+ subsets are largely independent of their organ source. The specificity of migration appears to be determined by selective recognition of organ-specific determinants on the endothelial cells of high endothelial venules (HEV), specialized venules that mediate the exit of migrating lymphocytes from the blood: In an in vitro assay of lymphocyte binding to HEV in lymphoid organ frozen sections, Lyt-2- cells constituted a significantly and consistently greater proportion of T cells binding to Peyer's patch HEV than of those binding to peripheral node HEV. The homing and HEV recognition preferences of the Lyt subsets are reflected in differences in their in situ representation in mucosal vs nonmucosal lymphoid organs, which suggests that the selective migration of these populations may be an important factor in determining the character of local immune responses.

  View details for Web of Science ID A1983QC72100018

  View details for PubMedID 6600472

• IMMUNOGLOBULIN GENE REARRANGEMENT AND EXPRESSION DURING LYMPHOCYTE DEVELOPMENT CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY Joho, R., Nottenburg, C., Coffman, R. L., Weissman, I. L. 1983; 18: 15-58

  View details for Web of Science ID A1983QE66000003

  View details for PubMedID 6404604

• GERMINAL CENTER B-CELLS LACK HOMING RECEPTORS NECESSARY FOR NORMAL LYMPHOCYTE RECIRCULATION JOURNAL OF EXPERIMENTAL MEDICINE REICHERT, R. A., Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. 1983; 157 (3): 813-827

  Abstract

  Germinal center B cells (GCLC) are a discrete population of antigen-activated lymphoblasts that lack surface IgD and express abundant cell surface binding sites for peanut agglutinin (PNA). These phenotypic features render GCLC easily distinguishable from nearly all plasma cells, T cells, and unstimulated B cells, and have enabled us to identify and isolate GCLC from antigen-stimulated murine lymphoid organs. We have examined the migratory properties of these lymphoblasts in (a) short-term in vivo homing studies, and (b) an in vitro assay of lymphocyte binding to post-capillary, high endothelial venules (HEV) in frozen sections of Peyer's patches and peripheral lymph nodes. In the in vivo experiments, intravenously injected GCLC failed to migrate in significant numbers to peripheral lymphoid organs in comparison with T cells or IgD+ B cells. In the in vitro binding assay, GCLC did not adhere to HEV in either Peyer's patch or peripheral node sections. A variety of factors, such as preferential sequestration in the liver, may operate in vivo to influence the localization of these cells. However, their nearly total failure to migrate into lymphoid organs can best be explained by their inability to recognize and adhere to the specialized HEV which normally mediate the emigration of recirculating lymphocytes from the blood into these sites. The concept that GCLC fail to express functional homing receptors for HEV has been further supported by studies using MEL-14, a monoclonal antibody that appears to recognize the lymphocyte surface receptor for peripheral node HEV: In contrast to most peripheral lymphocytes, GCLC fail to bind MEL-14. These migratory and endothelial-recognition properties of GCLC, when viewed in the context of the possible role of these cells as precursors of plasma cells and/or memory B cells, have led us to propose that the inability of GCLC to recognize HEV may be transient and related to a phase of sessile B cell differentiation.

  View details for Web of Science ID A1983QH26700001

  View details for PubMedID 6339668

  View details for PubMedCentralID PMC2186964

• ABNORMAL MIGRATION OF LYMPHOCYTE-T CLONES JOURNAL OF IMMUNOLOGY Dailey, M. O., Fathman, C. G., BUTCHER, E. C., Pillemer, E., Weissman, I. 1982; 128 (5): 2134-2136

  Abstract

  Several in vitro T cell clones were markedly deficient in their ability to home to peripheral lymphoid tissue. This was found for an alloreactive noncytolytic clone, a soluble antigen- (KLH)specific line, and cytotoxic clones specific for allogeneic cells and for Abelson virus-induced lymphoma cells. This abnormal circulation pattern was probably caused by the lack of the receptors of the lymphocytes for high endothelial venules (HEV), as implied by the lack of binding of these T cells to HEV in frozen sections of mouse lymph node and Peyer's patches. The loss of surface receptors that are necessary for normal lymphocyte migration may thereby alter the in vivo function of adoptively transferred T cells.

  View details for Web of Science ID A1982NL17700035

  View details for PubMedID 6460817

• Functional considerations of normal and neoplastic T lymphocytes Immune Regulation, Evolutionary and Biological Significance. Symposium on the Biological Significance to Immune Regulation Weissman, I., Scofield, V., Schlumpberger, J., Pillemer, E., Hollander, N., Rouse, R., Stevens, S., Butcher, E. Marcel Dekker. 1982: 99–113
• Tumor antigen antibody interactions in murine lymphomas possible implications for human lymphomas Malignant Lymphomas: Etiology, Immunology, Pathology, Treatment Weissman, I., Pillemer, E., Kooistra, D., Tsukamoto, A., Jerabek, L., Humphrey, D., Coffman, R., McGrath, M., Nord, S., Ellis, R. edited by Rosenberg, S., Kaplan, H. 1982: 131–154
• IMMUNOGLOBULIN GENE REARRANGEMENTS IN NORMAL MOUSE-B CELLS MOLECULAR AND CELLULAR BIOLOGY Early, P., Nottenburg, C., Weissman, I., Hood, L. 1982; 2 (7): 829-836

  Abstract

  We have analyzed the structure of rearranged mu heavy-chain genes obtained from the genomic DNA of normal BALB/c mouse spleen cells expressing surface immunoglobulin M. Examples were found of two types of nonproductive rearrangements, which may be responsible for allelic exclusion in normal B cells. In one of these rearrangements, a germ line D gene segment has joined to the JH4 gene segment but no V/D joining has occurred. We present evidence that D gene segments lie as a cluster between V and J gene segments in the germ line. A comparison of conserved sequences in V and D gene segments suggests that the D gene segments, which are found only in the heavy-chain gene family, may have evolved from V gene segments similar to the Vk family.

  View details for Web of Science ID A1982NV68400012

  View details for PubMedID 6821506

• Receptor–mediated leukemogenesis: retrovirus receptors on B and T lymphomas share idiotypic determinants Exp Hematol Today McGrath, M., Jerabek, L., Weissman, I. 1982: 93-100
• A recognition function of endothelial cells: directing lymphocyte traffic Pathobiology of the Endothelial Cell Butcher, E., Kraal, G., Stevens, S., Weissman, I. Academic Press. 1982: 408–424
• Lymphocyte-endothelial cell recognition in lymphocyte migration and the segregation of mucosal and nonmucosal immunity Recent Advances in Mucosal Immunity. Workshop on Mechanisms in Muscosal Immunity Butcher, E., Stevens, S., Reichert, R., Scollay, R., Weissman, I. Raven Press. 1982: 3–23
• EXPRESSION OF HLA ANTIGENS BY HUMAN THYMIC EPITHELIAL-CELLS HUMAN IMMUNOLOGY Rouse, R. V., Parham, P., GRUMET, F. C., Weissman, I. L. 1982; 5 (1): 21-34

  Abstract

  Human thymuses were examined by tissue section staining with antibodies specific for monomorphic and polymorphic HLA-A, B, C, and DR determinants. The principal cell type expressing high levels of HLA antigens has the distribution of epithelial cells. Immunoelectron microscopy confirmed their epithelial nature. As in the mouse, both medullary and cortical epithelial cells express high levels of class II (DR) antigens, a finding that is remarkable in that these antigens were originally thought to be restricted to lymphoid and accessory cells. Class I (A, B, and C) antigens are also present on thymic epithelial cells. They are easily detectable on medullary epithelial cells, but two distinct patterns of cortical staining were observed. One group of antibodies produced intense dendritic staining throughout the cortex; the other group produced only faint or no cortical dendritic staining at all. These different staining patterns do not correlate with known properties of the antibodies and thus appear to be due to intrinsic properties of the different A, B, and C antigens.

  View details for Web of Science ID A1982PH53300002

  View details for PubMedID 6956563

• Thymic lymphocyte maturation in the thymic microenvironment Behring Institute Report. International Workshop on the Influence of the Thymus on the Generation of the T Cell Repertoire Weissman, I., Rouse, R., Kyewski, B., Lepault, F., Butcher, E., Kaplan, H., Scollay, R. 1982
• ANTIGEN-SPECIFICITY OF GERMINAL CENTER CELLS AND CHANGES IN ISOTYPE EXPRESSION Kraal, G., Weissman, I. L., BUTCHER, E. C. FEDERATION AMER SOC EXP BIOL. 1982: 418–18

  View details for Web of Science ID A1982NG28200898

• MOUSE LYMPH-NODE GERMINAL-CENTERS CONTAIN A SELECTED SUBSET OF T-CELLS - THE HELPER PHENOTYPE JOURNAL OF IMMUNOLOGY Rouse, R. V., LEDBETTER, J. A., Weissman, I. L. 1982; 128 (5): 2243-2246

  Abstract

  Cells staining for Lyt-1 are more frequent than cells staining for Lyt-2 in both primary follicles and the cuffs of secondary follicles; there is an even more striking predominance of cells bearing only Lyt-1 in germinal centers. In addition, there is an increase in the total percentage of cells bearing T cell antigens in germinal centers compared to primary follicles. These differences in phenotype and distribution of T cell populations indicate the T cells in B cell areas, and especially in germinal centers, are not randomly selected, but rather represent a specific subpopulation of T cells enriched for the helper phenotype (Lyt-1+2-), perhaps involved in the development and/or function of germinal centers.

  View details for Web of Science ID A1982NL17700054

  View details for PubMedID 6120978

• RETROVIRUS LYMPHOMAGENESIS - RELATIONSHIP OF NORMAL IMMUNE RECEPTORS TO MALIGNANT-CELL PROLIFERATION CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY Weissman, I. L., McGrath, M. S. 1982; 98: 103-112

  View details for Web of Science ID A1982NF17900009

  View details for PubMedID 6979463

• PROTECTION AGAINST SYNGENEIC LYMPHOMA BY A LONG-LIVED CYTO-TOXIC T-CELL CLONE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Dailey, M. O., Pillemer, E., Weissman, I. L. 1982; 79 (17): 5384-5387

  Abstract

  The effect of a cloned T-cell line on the in vivo growth of syngeneic lymphoma cells was studied. 1E4 is an H-2-restricted cytotoxic T-cell clone that efficiently kills Abelson virus-induced lymphoma target cells (L1-2) at low effector/target ratios, as measured by in vitro cytotoxicity assays. In addition, it is long lived in vitro in the absence of stimulation and survives for more than 1 wk in vivo in the absence of exogenous antigen or growth factors. Mice injected intraperitoneally with lethal doses of L1-2 and then treated with 1E4 survived longer than animals treated with saline or with a control T-cell clone. Multiple weekly injections of effector cells, or a single injection in animals given a low dose of tumor cells, resulted in 50-80% long-term survivors. The observation that intravenous injection of killer cells was less effective than intraperitoneal treatment, coupled with the previous demonstration of markedly abnormal circulatory patterns for T-cell clones, suggests that those animals succumbing to progressively growing neoplasm die because the effector cells are unable to home into peripheral sites of tumor deposition. Thus, although this cytotoxic T-cell clone does have useful in vivo activity, its function may be partially limited by a generalized defect in migration.

  View details for Web of Science ID A1982PE86500057

  View details for PubMedID 6982472

• SELECTIVE MIGRATION OF MURINE LYMPHOCYTES AND LYMPHOBLAST POPULATIONS AND THE ROLE OF ENDOTHELIAL-CELL RECOGNITION ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY BUTCHER, E. C., Kraal, G., Stevens, S. K., Weissman, I. L. 1982; 149: 199-206

  View details for Web of Science ID A1982PL44700028

  View details for PubMedID 6983214

• DIFFERENCES IN THE MIGRATION OF LYMPHOCYTE-B AND LYMPHOCYTE-T - ORGAN-SELECTIVE LOCALIZATION INVIVO AND THE ROLE OF LYMPHOCYTE-ENDOTHELIAL CELL RECOGNITION JOURNAL OF IMMUNOLOGY Stevens, S. K., Weissman, I. L., BUTCHER, E. C. 1982; 128 (2): 844-851

  Abstract

  The migration of B and T lymphocytes in the mouse has been studied by using 1) short-term in vivo homing studies, and 2) an in vitro assay of lymphocyte binding to specialized lymphoid organ venules (post-capillary, high endothelial venules (HEV)) in frozen sections of lymph nodes and Peyer's patches. The homing characteristics of B and T cell populations are largely independent of their organ of origin. B cells from any source distribute preferentially to Peyer's patches, whereas T cells home preferentially to peripheral lymph nodes. This organ specificity of migration appears to be determined at the site of lymphocyte exit from the blood by selective recognition of organ-specific determinants on the endothelial cells of HEV. In addition, the in vivo tendency of B cells to migrate preferentially to the spleen, and of T cells to localize better in lymph nodes is confirmed. The results indicate that, in a hypothetical situation in which an equal number of B and T lymphocytes localized in peripheral lymph nodes (or bound in vitro to peripheral node HEV), there would be about 2.5 B cells for every T cell in the mesenteric node, four to six B cells per T cell in Peyer's patches, and seven to nine B cells per T cell in the spleen. Comparison of these homing preferences with the distribution of B and T lymphocyte populations in situ suggests that selective lymphocyte migration may help determine the proportions of functionally distinct lymphocyte classes in particular lymphoid organs or sites of chronic inflammation, and thus may serve to influence the character of local immune responses.

  View details for Web of Science ID A1982MZ49700059

  View details for PubMedID 6976385

• COLONY SPECIFICITY IN THE COLONIAL TUNICATE BOTRYLLUS AND THE ORIGINS OF VERTEBRATE IMMUNITY AMERICAN ZOOLOGIST Scofield, V. L., SCHLUMPBERGER, J. M., Weissman, I. L. 1982; 22 (4): 783-794

  View details for Web of Science ID A1982PT53700006

• SURFACE PHENOTYPE AND MIGRATORY CAPABILITY OF PEYERS PATCH GERMINAL CENTER CELLS ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY BUTCHER, E. C., REICHERT, R. A., Coffman, R. L., Nottenburg, C., Weissman, I. L. 1982; 149: 765-772

  Abstract

  Peanut agglutinin (PNA) binds selectively to germinal center cells in mouse peripheral lymphoid organs. Using PNA as a marker, we have determined that Peyer's patch germinal center cells are B cells with a unique phenotype---they express a low level of surface immunoglobulin (about 85% Ig+), predominantly of the IgA class (70% alpha+), with only 10% bearing surface IgM, and few if any expressing IgD. This phenotype identifies murine Peyer's patch germinal center cells as fairly late cells in B cell differentiation, and suggests that they may be precursors of IgA-secreting plasma cells in the gut wall. In addition, we have described a means of purifying PNA+ Peyer's patch lymphocytes, and have demonstrated that these cells lack functional receptors for high endothelial venules and fail to migrate to lymphoid organs in vivo. It is speculated that PNA may be a general marker for nonmigratory lymphocyte populations undergoing local differentiation.

  View details for Web of Science ID A1982PL44700107

  View details for PubMedID 6983239

• GERMINAL CENTER B-CELLS - ANTIGEN-SPECIFICITY AND CHANGES IN HEAVY-CHAIN CLASS EXPRESSION NATURE Kraal, G., Weissman, I. L., BUTCHER, E. C. 1982; 298 (5872): 377-379

  Abstract

  Germinal centres are histologically defined aggregates of blast cells that occur in B-cell areas of lymphoid tissues after antigenic stimulation. They are believed to be associated with the development of B-cell memory and plasma cell (especially secondary, IgG and IgA) responses. Recent studies of murine lymphoid tissues have defined cell-surface markers that distinguish germinal centre B cells from other mature B cells, permitting their identification and characterization in cell suspensions. Here we have used these markers to define and study germinal centre cells in lympho nodes, and have found that they constitute a unique population of B cells which (1) arises in response to antigenic stimulation, (2) contains nearly all of the demonstrably antigen-specific B cells in the stimulated organ, (3) bears surface IgM after primary stimulation and (4) as a population, demonstrates isotype switching to a predominant population, demonstrates isotype switching to a predominant surface IgG phenotype after secondary stimulation with specific surface IgG phenotype after secondary stimulation with specific antigen. These findings demonstrate that germinal centres are a major site of proliferation and differentiation of antigen-specific B cells in vivo, and suggest that the germinal centre microenvironment may have an important role in heavy chain class switching during B-cell responses.

  View details for Web of Science ID A1982NY37400044

  View details for PubMedID 6806671

• PROTOCHORDATE ALLORECOGNITION IS CONTROLLED BY A MHC-LIKE GENE SYSTEM NATURE Scofield, V. L., SCHLUMPBERGER, J. M., West, L. A., Weissman, I. L. 1982; 295 (5849): 499-502

  View details for Web of Science ID A1982NA87700028

  View details for PubMedID 7057909

• GERMINAL CENTER B-CELLS ARE LYMPHOCYTES UNDERGOING A SESSILE PHASE OF DIFFERENTIATION REICHERT, R. A., Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. ELSEVIER GMBH, URBAN & FISCHER VERLAG. 1982: 248–49

  View details for Web of Science ID A1982PR60800225

• SURFACE PHENOTYPE OF PEYERS PATCH GERMINAL CENTER CELLS - IMPLICATIONS FOR THE ROLE OF GERMINAL-CENTERS IN B-CELL DIFFERENTIATION JOURNAL OF IMMUNOLOGY BUTCHER, E. C., Rouse, R. V., Coffman, R. L., Nottenburg, C. N., Hardy, R. R., Weissman, I. L. 1982; 129 (6): 2698-2707

  Abstract

  The surface phenotype of Peyer's patch germinal center lymphoid cells in the mouse is described. It is confirmed that most germinal center lymphocytes bind high levels of peanut agglutination (PNA), a lectin with specificity for terminal galactosyl residues. It is shown that germinal center lymphocytes can be identified in cell suspensions as a discrete PNAhi population distinct from other B cells, plasma cells, and most T cells, which bind only low levels of PNA. Using fluorescence-labeled PNA as a marker in dual fluorescence studies, we found that the majority of Peyer's patch germinal center cells are B lymphocytes: PNAhi Peyer's patch cells express B220, the B lineage-specific form of the T200 family of molecules, as well as low levels of surface Ig. They do not express the T cell-lineage antigens Thy-1, Lyt-1, or Lyt-2 (only 1 to 3% positive). They bear lower levels of H2-K than PNAlo B cells, but two to three times the level of surface I-A-encoded determinants. A discrete but variable subpopulation of PNAhi Peyer's patch cells bear ThB in AKR/c mice, but BALB/c PNAhi lymphocytes are ThB-. About 10 to 30% bear surface IgM or IgG, but in contrast to essentially all PNAlo B lymphocytes in this site, they express no detectable surface IgD. The majority of Peyer's patch germinal center cells bear surface IgA, and this IgA is allelically excluded in F1 mice, indicating it is synthesized by the germinal center cells themselves. In fact, germinal centers contain most of the IgA-bearing cells in Peyer's patches (70 to 85%). These findings lend considerable support to the concept that germinal centers in Peyer's patches are the site of generation of precursors of the IgA-secreting plasma cells that characterize mucosal immune responses, and also suggest that germinal centers may play an important role in the process of heavy chain class switching.

  View details for Web of Science ID A1982PR36900070

  View details for PubMedID 6982940

• THE PRINCIPAL CELLS IN THE THYMUS EXPRESSING MHC-ANTIGENS ARE EPITHELIAL ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Rouse, R. V., Weissman, I. L. 1982; 149: 401-405

  View details for Web of Science ID A1982PL44700056

  View details for PubMedID 7148567

• EXPRESSION OF T-CELL ANTIGENS BY CELLS IN MOUSE AND HUMAN PRIMARY AND SECONDARY FOLLICLES ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Rouse, R. V., Weissman, I. L., LEDBETTER, J. A., Warnke, R. A. 1982; 149: 751-756

  View details for Web of Science ID A1982PL44700105

  View details for PubMedID 6128883

• EFFECTS OF LYT ANTIBODIES ON T-CELL FUNCTIONS - AUGMENTATION BY ANTI-LYT-1 AS OPPOSED TO INHIBITION BY ANTI-LYT-2 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Hollander, N., Pillemer, E., Weissman, I. L. 1981; 78 (2): 1148-1151

  Abstract

  Monoclonal anti-Lyt-1 (alpha Lyt-1) and alpha Lyt-2 manifest inverse effects on allogeneic mixed lymphocyte reactions when added to the reaction mixtures without complement. alpha Lyt-1 augments cell proliferation and generation of cytotoxic cells but has no effect on cell-mediated cytolysis, whereas alpha Lyt-2 blocks cell proliferation, generation of killer cells, and cytolytic activity of killer cells. The augmenting effect of alpha Lyt-1 cannot be attributed to a direct mitogenic effect on T cells. Both inhibition by alpha Lyt-2 and potentiated by alpha Lyt-1 require interaction of responder cells with the antibodies during the first 24 hr of the mixed lymphocyte reaction, indicating that early stages of the reaction are sensitive to Lyt antibodies. The enhancing effect of alpha Lyt-1 on alloantigen-induced T-cell proliferation is associated with augmented production of T-cell growth factors. When alpha Lyt-1 is present in mixed lymphocyte cultures, the supernatant media collected after 24 or 48 hr of culture induce higher proliferation of activated T cells compared to media of mixed lymphocyte cultures incubated in the absence of antibodies or in the presence of alpha Lyt-2 which has no effect on secretion of growth factors. The differences in the effects of alpha Lyt-1 and alpha Lyt-2 could not be attributed to differences in heavy chain constant region functions because both were of the same lambda 2A immunoglobulin class and were used at the same concentration. The data suggest a possible role for Lyt-1 molecules in early activation and mitogenesis processes such as production of growth factors.

  View details for Web of Science ID A1981LF67000094

  View details for PubMedID 6972039

• ANALYSIS OF BOTRYLLUS BLOOD AND TISSUE-CELLS WITH MONOCLONAL-ANTIBODIES SCHLUMPBERGER, J. M., Weissman, I. L., Scofield, V. L. SOC INTEGRATIVE COMPARATIVE BIOLOGY. 1981: 982–82

  View details for Web of Science ID A1981MT25700291

• Allorecognition in biological systems Dev Comp Immunol Scofield, V., Weissman, I. 1981; 5: 23-28
• Microanatomy of the thymus: its relationship to T cell differentiation. Ciba Foundation symposium Rouse, R. V., Weissman, I. L. 1981; 84: 161-177

  Abstract

  The structure of the thymus can be determined by study at the light and electron microscopic levels, but relating it to the current knowledge of the thymus's function requires an approach that combines immunological and anatomical methods. The framework of the thymus consists of epithelial cells with interconnecting processes. Lymphocytes fill the spaces between the epithelial cells. In both the mouse and human thymus, immunological staining of tissue sections demonstrates that the principal cell bearing major histocompatibility complex (MHC) antigens is the epithelial cell. Differences are noted between I-A (HLA-DR) and H-2K/D (HLA-A, B) allotypic specificities in both species. Immunoelectron microscopy confirms the epithelial nature of these cells in both species. The continued expression of thymus-type MHC antigens in the thymuses of irradiated, bone marrow-reconstituted mice strongly suggests the synthesis of these antigens by the epithelial cells. Bone marrow-derived MHC antigens are largely confined to the medulla of the thymus.

  View details for PubMedID 7023868

• EXPRESSION OF IGD MAY USE BOTH DNA REARRANGEMENT AND RNA SPLICING MECHANISMS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Moore, K. W., Rogers, J., Hunkapiller, T., Early, P., Nottenburg, C., Weissman, I., Bazin, H., Wall, R., HOOD, L. E. 1981; 78 (3): 1800-1804

  Abstract

  From a library of mouse sperm DNA, we have isolated two overlapping clones which contain the C(delta) gene. One of these clones also contains the C(mu) gene. The C(delta) gene is separated from the C(mu) membrane exons by approximately 2 kilobases (kb) of DAN. The C(delta) gene was identified by (a) hybridization to poly(A)(+)RNA prepared from the IgD-producing rat plasma cell tumor IR731, and (b) homology of a translated nucleotide sequence to the amino acid sequence of the human delta chain. The C(delta) gene spans 8 kb of DNA in the germ line. Plasmid subclones of the C(delta) gene were used as probes in Southern and RNA blot experiments. RNA blot analysis of cytoplasmic poly(A)(+)RNA from IR731 and a mu(+)delta(+) B-cell hybridoma revealed 1.6- and 2.7-kb delta mRNA species with different 3' ends, which presumably encode the secreted and membrane-bound forms, respectively, of the delta chain. Southern blot analysis of DNA from two mu(+)delta(+) lymphomas revealed that the C(delta) gene is in the germ-line configuration in each case. Restriction map analysis of C(mu) and C(delta) genomic clones isolated from a library of normal mu(+)delta(+) B-cell DNA also gave no evidence for DNA rearrangement in the region between the C(mu) and C(delta) genes. Taken together, these data suggest that IgD expression in mu(+)delta(+) B cells does not involve a V(H)-to-C(delta) DNA switch rearrangement. We propose that simultaneous expression of C(delta) and C(delta) with a single V(H) gene is mediated by two alternative routes of RNA processing of a primary nuclear transcript which contains the V(H), C(mu), and C(delta) genes. In contrast, analogous experiments with myeloma IR731 DNA revealed that the C(mu) gene has been deleted from the myeloma DNA and that the C(delta) gene has undergone DNA rearrangement, presumably including a switch recombination of the V(H) gene from the C(mu) to the C(delta) gene. These results indicate that two alternative mechanisms may be used in the expression of IgD molecules-RNA splicing in B cells and DNA rearrangement in plasma cells.

  View details for Web of Science ID A1981LJ93300099

  View details for PubMedID 6262826

• C-MU-GENE REARRANGEMENT OF MOUSE IMMUNOGLOBULIN GENES IN NORMAL B-CELLS OCCURS ON BOTH THE EXPRESSED AND NONEXPRESSED CHROMOSOMES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Nottenburg, C., Weissman, I. L. 1981; 78 (1): 484-488

  Abstract

  We have examined the organization of heavy-chain immunoglobulin genes on both the expressed and nonexpressed chromosomes of normal B lymphocytes from allotype heterozygous (BALB/c X C57BL/J)F1 mice. The C mu genes of BALB/c mice are on 12.4-kilobase EcoRI and 13.1-kilobase Kpn I restriction fragments, whereas those of C57BL/J mice are on 13.6-kilobase EcoRI and 14.3-kilobase Kpn I restriction fragments, allowing the examination of rearrangements on each chromosome independently. B lymphocytes from spleen and Peyer's patches expressing both IgD and IgM of the BALB/c allotype were isolated with a fluorescence-activated cell sorter. EcoRI and Kpn I restriction digests were hybridized with a C mu gene-containing probe. The C mu gene is present on both chromosomes. DNA rearrangements occur on both the expressed and nonexpressed chromosome within the 3.6-kilobase Kpn I/EcoRI restriction fragment containing the joining (JH) gene locus. We conclude that allelic exclusion of heavy-chain immunoglobulin gene expression is not mediated by JH-region DNA rearrangement of the expressed chromosome only. In contrast, analysis of the C kappa gene region from the same sorted B-cell DNA reveals a substantial quantity of germ-line context DNA. We also demonstrate that the deletions observed on the Eco RI fragment containing the C mu gene in myeloma cells and in C mu gene-containing recombinant DNAs do not usually occur in normally differentiating B lymphocytes and are likely to be confined to myeloma tumor cells.

  View details for Web of Science ID A1981LA96300094

  View details for PubMedID 6264446

• The inhibitory effect of anti Lyt-2 antibodies on binding of cytotoxic T lymphocytes to target cells Mech Lymphocyte Activation Hollander, N., Pillemer, E., Weissman, I. 1981: 450-453
• Receptor mediated murine leukemogenesis: monoclonal antibody induced lymphoma cell growth arrest. Haematology and blood transfusion McGrath, M. S., Jerabek, L., Pillemer, E., Steinberg, R. A., Weissman, I. L. 1981; 26: 360-364

  View details for PubMedID 6172320

• IMMUNOSUPPRESSIVE AND TOLEROGENIC EFFECTS OF WHOLE-BODY, TOTAL LYMPHOID, AND REGIONAL IRRADIATION Immunosuppressive Therapy Strober, S., Weissman, I. MTP Press (Springer). 1981: 19–53
• AN INVIVO ASSAY FOR THYMUS-HOMING BONE-MARROW CELLS NATURE Lepault, F., Weissman, I. L. 1981; 293 (5828): 151-154

  View details for Web of Science ID A1981MF12500040

  View details for PubMedID 6115320

• A MONOCLONAL-ANTIBODY THAT DETECTS A VK-TEPC15 IDIOTYPIC DETERMINANT CROSS-REACTIVE WITH A THY-1 DETERMINANT JOURNAL OF EXPERIMENTAL MEDICINE Pillemer, E., Weissman, I. L. 1981; 153 (5): 1068-1079

  Abstract

  To identify T lymphocyte antigens with immunoglobulin-like determinants, we prepared rat anti-mouse T cell monoclonal antibodies and screened them against a panel of purified mouse myeloma proteins representing all isotypes of immunoglobulin. One hybridoma, designated 42-21, was found to detect a novel antigenic determinant shared by V kappa-TEPC15 and the Thy-1 molecule on all T lymphocytes. Although several explanations for this unusual phenomenon exist, it may imply some role for the Thy-1 molecule in antigen and/or mitogen recognition. In any event, future studies of idiotypes on T lymphocytes must consider the possibility that anti-idiotypic sera detect cell surface molecules unrelated to classical immunoglobulin.

  View details for Web of Science ID A1981LP66700004

  View details for PubMedID 6166711

• A MONOCLONAL-ANTIBODY THAT RECOGNIZES B-CELLS AND B-CELL PRECURSORS IN MICE JOURNAL OF EXPERIMENTAL MEDICINE Coffman, R. L., Weissman, I. L. 1981; 153 (2): 269-279

  Abstract

  The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

  View details for Web of Science ID A1981LB84500005

  View details for PubMedID 6787164

• SOMATIC DIVERSIFICATION IS REQUIRED TO GENERATE THE V-KAPPA GENES OF MOPC 511 AND MOPC 167 MYELOMA PROTEINS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Gershenfeld, H. K., Tsukamoto, A., Weissman, I. L., Joho, R. 1981; 78 (12): 7674-7678

  Abstract

  The immune response to phosphocholine in BALB/c mice involves one group of heavy chain variable region (VH) genes and at least three groups of light chain variable region (V kappa) genes, represented by the gene products of the myelomas TEPC 15, MOPC 603, and MOPC 167/MOPC 511. The amino acid sequences of BALB/c myeloma kappa chains MOPC 167 and MOPC 511 are known, and they differ by six amino acids. We have isolated several closely related V region genes of immunoglobulin light chains from a mouse sperm DNA phage library, selecting clones that cross-hybridize with a cDNA plasmid probe encoding the light chain of MOPC 167. We identified six strongly hybridizing clones, representing three separate cloning events. We determined the sequence of the coding and immediate flanking regions of three clones, representing the three separate cloning events, and they proved to be identical. This germ-line sequence encoded the amino acid sequence of neither MOPC 167 nor MOPC 511, but required four base pair changes to generate the V kappa M167 cDNA sequence and five base pair changes to generate the V kappa M511 gene. By Southern hybridization experiments, we demonstrated that neither MOPC 511 nor MOPC 167 germ-line genes exist. We conclude that the V kappa M167 and V kappa M511 genes are created somatically.

  View details for Web of Science ID A1981MV92700088

  View details for PubMedID 6801657

• B220 - A B-CELL-SPECIFIC MEMBER OF THE T200 GLYCOPROTEIN FAMILY NATURE Coffman, R. L., Weissman, I. L. 1981; 289 (5799): 681-683

  Abstract

  T200, a major cell-surface glycoprotein on lymphoid cells, exists in several forms with different electrophoretic mobilities. These forms have been correlated with different classes of lymphoid cell. The smaller forms, with molecular weights (MWs) of congruent to 170,000 and 180,000 are found predominantly on T cells while the 220,000 MW form is associated with B cells. The polypeptide portions of each molecule may be identical or closely related as all three forms share the same allelic variations, and all reported herologous antisera and monoclonal antibodies to T200 precipitate all three forms. We report here a monoclonal antibody specific for the 220,000 MW form of T200 and show that it is expressed only on B cells and a subset of bone marrow cells which includes B cell precursors. We suggest that this form of the molecule be designated provisionally B220.

  View details for Web of Science ID A1981LC53800044

  View details for PubMedID 6970340

• NORMAL AND NEOPLASTIC LYMPHOCYTE MATURATION JOURNAL OF SUPRAMOLECULAR STRUCTURE AND CELLULAR BIOCHEMISTRY Weissman, I. L., McGrath, M. S., Pillemer, E., Hollander, N., Rouse, R. V., Jerabek, L., Stevens, S. K., SCOLLAY, R. G., BUTCHER, E. C. 1981; 15 (3): 303-314

  View details for Web of Science ID A1981MA98800006

  View details for PubMedID 6790720

• PRODUCTION OF ALLOREACTIVE T-CELL LYMPHOMAS NATURE Fathman, C. G., Weissman, I. L. 1980; 283 (5745): 404-406

  View details for Web of Science ID A1980JC08100058

  View details for PubMedID 6965424

• Cellular, genetic, and evolutionary aspects of lymphocyte interactions with high-endothelia venules. Ciba Foundation symposium BUTCHER, E. C., Weissman, I. L. 1980; 71: 265-286

  View details for PubMedID 6899991

• Tumor immunology, T cell maturation, and T cell neoplasia. Progress in experimental tumor research Weissman, I. L. 1980; 25: 193-218

  View details for PubMedID 6986632

• Summing Up Ciba Foundation Symposium Weissman, I. 1980: 343–347
• BLOCKING EFFECT OF LYT-2 ANTIBODIES ON T-CELL FUNCTIONS Hollander, N., Pillemer, E., Weissman, I. L. FEDERATION AMER SOC EXP BIOL. 1980: 1194–94

  View details for Web of Science ID A1980JG86401858

• T-CELL MATURATION - THYMOCYTE AND THYMUS MIGRANT SUB-POPULATIONS DEFINED WITH MONOCLONAL-ANTIBODIES TO THE ANTIGENS LYT-1, LYT-2, AND THB JOURNAL OF IMMUNOLOGY Scollay, R., Weissman, I. L. 1980; 124 (6): 2841-2844

  Abstract

  The phenotypes of thymus cells for the antigens Lyt-1, Lyt-2 and ThB have been analyzed by using immunofluorescence techniques. Cells throughout the intrathymic maturation sequence have been tested, including the primitive subcapsular lymphoblasts, all size classes of cortical and medullary thymocytes, and thymus cell emigrants. ThB antigen is not detectable on migrant cells, but all subpopulations in the thymus are subdivided into two categories, bright and dull. Thus, it is possible that the bright and dull phenotypes represent a lineage specific rather than a stage-specific marker, at least inside the thymus. The Lyt-defined thymocyte subclasses Lyt1+2+-(Lyt-1) and Lyt 1+2+ (lyt-1,2) are also both represented in all subpopulations, including subcapsular lymphoblasts. This suggests that they may represent two separate lineages, and that the Lyt-12(3) class is not a precursor of the Lyt-1 class, although the possibility of a very early Lyt-(12(3) cell precursor common to both lines cannot be ruled out.

  View details for Web of Science ID A1980JT75200050

  View details for PubMedID 6154738

• Cell-cell interactions in the establishment and maintenance of lymphoid tissue architecture Strategies of Immune Regulation: Regulatory Features of the Immune System Weissman, I., Butcher, E., Rouse, R., Scollay, R. Academic Press. 1980: 77–94
• The role of MuLV receptors on T-lymphoma cells in lymphoma cell proliferation. Contemporary topics in immunobiology McGrath, M. S., Pillemer, E., Kooistra, D., Weissman, I. L. 1980; 11: 157-184

  View details for PubMedID 6160948

• MURINE LEUKEMOGENESIS - MONOCLONAL-ANTIBODIES TO T-CELL DETERMINANTS ARREST T-LYMPHOMA CELL-PROLIFERATION NATURE McGrath, M. S., Pillemer, E., Weissman, I. L. 1980; 285 (5762): 259-261

  Abstract

  We have proposed a receptor-mediated leukaemogenesis hypothesis wherein T lymphomas would be clones of T cells bearing mitogen-linked surface receptors specific for the envelope determinants of the inducing MuLV. A prediction of the hypothesis is that T-lymphoma proliferation is dependent on continued presentation of MuLV envelope determinants to these cell-surface receptors, and that substances which interfere with receptor-virus interactions should inhibit T-lymphoma proliferation. Rat monoclonal antibodies were raised to the AKR mouse T lymphoma KKT-2, and these antibodies were screened independently for blockade of virus-binding and for cytostatic activity on KKT-2 cells. We report here that those monoclonal antibodies which block virus binding inhibit growth of KKT-2 cells in vitro, whereas monoclonal antibodies which bind to these cells but do not block virus binding are not cytostatic. Three of the four cytostatic antibodies detect determinants on the Thy-1 molecule, while none of the other (noncytostatic) antibodies detect Thy-1. Antibody inhibition of KKT-2 cell growth is precluded by saturation of KKT-2 virus receptors with the inducing leukaemia virus.

  View details for Web of Science ID A1980JS91900055

  View details for PubMedID 6246448

• ORGAN SPECIFICITY OF LYMPHOCYTE MIGRATION - MEDIATION BY HIGHLY SELECTIVE LYMPHOCYTE INTERACTION WITH ORGAN-SPECIFIC DETERMINANTS ON HIGH ENDOTHELIAL VENULES EUROPEAN JOURNAL OF IMMUNOLOGY BUTCHER, E. C., SCOLLAY, R. G., Weissman, I. L. 1980; 10 (7): 556-561

  Abstract

  Evidence is presented that the organ specificity of lymphocyte migration is determined by selective interaction of lymphocytes with specialized endothelial cells. Mouse Peyer's patch and lymph node lymphocytes bind preferentially to high endothelial venules (HEV) in frozen sections of Peyer's patches and peripheral nodes, respectively, and this in vitro binding preference accurately predicts their differential segregation in vivo 30 min after i.v. injection. Both in vivo and in vitro, about 1.4 times as many as many Peyer's patch as lymph node lymphocytes bind HEV in Peyer's patches, and, conversely, twice as many lymph node cells interact with HEV in nonmesenteric lymph nodes. Even greater specificity is shown by certain homogeneous lymphocyte populations, i.e. thymic lymphomas. Some lymphomas bind with remarkable selectivity to HEV in Peyer's patches, and others interact almost exclusively with those in lymph nodes indicating that the mechanisms mediating selective recognition of HEV are capable of nearly absolute discrimination. Mesenteric node HEV are unique in that they allow both Peyer's patch- and lymph node-specific cells to bind. It is proposed that lymphocyte surface receptors specific for organ-restricted endothelial cell determinants mediate the antigen-independent organ specificity of lymphocyte migration. According to this model, there are at least 2 sets of complementary lymphocyte and endothelial cell receptors, one mediating lymphocyte-HEV adherence in Peyer's patches, the other in lymph nodes.

  View details for Web of Science ID A1980KG04100012

  View details for PubMedID 6157544

• THYMUS-CELL MIGRATION QUANTITATIVE ASPECTS OF CELLULAR TRAFFIC FROM THE THYMUS TO THE PERIPHERY IN MICE EUROPEAN JOURNAL OF IMMUNOLOGY SCOLLAY, R. G., BUTCHER, E. C., Weissman, I. L. 1980; 10 (3): 210-218

  Abstract

  We have used intrathymic injection of fluorescein isothiocyanate to label thymocytes in situ. The method gives random labeling of the thymocyte population and so can be used to quantitate the extent of migration of cells from the thymus to the periphery. Migrant cells can be visualized in frozen sections or cell suspensions of peripheral organs by their fluorescence. Our data show that in young adults, about 1% of thymocytes leave the thymus per day. Since the bulk of thymocytes turn over every 5 to 7 days, this indicates that the vast majority (95%) of thymocytes die within the thymus. Cells that do leave the thymus, go mainly to the T areas of lymph nodes, spleen and Peyer's patches. Migrants are extremely rare in bone marrow, gut and liver. Migration is about the same in neonates as in adults relative to the size of the thymus, but is considerably lower in older animals where it is only about 0.1% of thymocytes per day at the age of six months.

  View details for Web of Science ID A1980JQ94600009

  View details for PubMedID 7379836

• DIRECT FLUORESCENT LABELING OF CELLS WITH FLUORESCEIN OR RHODAMINE ISOTHIOCYANATE .2. POTENTIAL APPLICATION TO STUDIES OF LYMPHOCYTE MIGRATION AND MATURATION JOURNAL OF IMMUNOLOGICAL METHODS BUTCHER, E. C., SCOLLAY, R. G., Weissman, I. L. 1980; 37 (2): 109-121

  Abstract

  The effect of direct cell labeling with fluorescein or tetramethyl rhodamine isothiocyanate on lymphocyte migration is examined. In vitro conditions of labeling are defined which (1) do not significantly affect immediate or long term viability of lymphocytes (up to 2 weeks after transfer in vivo), (2) do not alter normal lymphocyte migration, (3) do not affect expression or detectability of surface antigens, and (4) permit direct visualization and counter-staining with fluorescent antibody reagents for days after intravenous injection. The potential application of this method to studies of lymphocyte migration and maturation is discussed.

  View details for Web of Science ID A1980KM67800002

  View details for PubMedID 6777427

• DIRECT FLUORESCENT LABELING OF CELLS WITH FLUORESCEIN OR RHODAMINE ISOTHIOCYANATE .1. TECHNICAL ASPECTS JOURNAL OF IMMUNOLOGICAL METHODS BUTCHER, E. C., Weissman, I. L. 1980; 37 (2): 97-108

  Abstract

  A rapid and simple method of cell labeling by stable conjugation with fluorescein or rhodamine is described. Viable cells are incubated under benign conditions (near physiologic pH in normal media) with free fluorescein or tetramethyl rhodamine isothiocyanate, and are adequately separated from unreacted fluorochrome by washing or centrifugation through fetal calf serum. The effects of the pH, the time and temperature of incubation, and the concentration of cells, fluorochrome, and free protein in the media are described. The method labels all cell types, although to different degrees. Fluorescence microscopy reveals fluorescence throughout the cell, although chromatin appears relatively spared. Cellular fluorescence is fairly stable at 4 and 25 degrees C, decays rapidly at 37 degrees C, but is nonetheless visible for days even at this temperature. In the case of lymphocytes, intense fluorescence is obtained without affecting cell viability, and without alteration of the ability to mount a graft versus host response.

  View details for Web of Science ID A1980KM67800001

  View details for PubMedID 7003013

• BLOCKING EFFECT OF LYT-2 ANTIBODIES ON T-CELL FUNCTIONS JOURNAL OF EXPERIMENTAL MEDICINE Hollander, N., Pillemer, E., Weissman, I. L. 1980; 152 (3): 674-687

  Abstract

  Monoclonal anti-Lyt-2 antibodies blocked effector function of cytotoxic thymus-derived (T) cells in the absence of added complement. Cytolysis of both allogeneic cells and syngeneic lymphoma or sarcoma target cells was inhibited at the level of the effector lymphocytes. Anti-Lyt-1 and anti-Thy-1 antibodies did not block killer cells. Proliferation of T cells in mixed lymphocyte culture was also inhibited by anti-Lyt-2, but not affected by anti-Lyt-1 or anti-Thy-1 antibodies. Although Lyt-1+ lymphocytes were required in the mixed lymphocyte reaction as helper cells for proliferation of Lyt-2+ lymphocytes, their helper function was not affected by the presence of Lyt-1 antibodies. Thus, although anti-Lyt-1, anti-Lyt-2 and anti-Thy-1 were of the same gamma 2A immunoglobulin class, had high titers, and interacted with T cells to the same extent, only anti-Lyt-2 blocked T cell functions. Polyclonal activation of T lymphocytes by concanavalin A, in contrast to activation by alloantigens, was not inhibited by Lyt-2 antibodies, suggesting that Lyt-2 antibodies interfere with T cell function at the level of the T cell antigen-receptor. The role which Lyt-2 molecules may play in T cell function is discussed.

  View details for Web of Science ID A1980KG38900016

  View details for PubMedID 6967947

• T-CELL MATURATION - THYMOCYTE AND THYMUS MIGRANT SUB-POPULATIONS DEFINED WITH MONOCLONAL-ANTIBODIES TO MHC REGION ANTIGENS JOURNAL OF IMMUNOLOGY Scollay, R., Jacobs, S., Jerabek, L., Butcher, E., Weissman, I. 1980; 124 (6): 2845-2853

  Abstract

  The maturation sequences of thymocytes is known to some extent: A generative layer of subcapsular large lymphoblasts gives rise to a major population of small cortical thymocytes and a minor population of midsize medullary thymocytes. The relative contribution of these three populations to the peripheral T cell populations is not yet known. In this study, subcapsular lymphoblasts, cortical small cells, medullary cells, and thymic emigrant cells have all been analyzed by immunofluorescence for expression of the antigens H-2D, I-A, H-2K, and TL. H-2D is expressed brightly on all subcapsular large cells, dimly on cortical small cells, and brightly on all migrants, cortisone-resistant thymocytes (CRT), and peripheral T cells. I-A can be detected at low levels on 30 to 50% of cells in all the thymic subpopulations, and on 30 to 50% of migrants and peripheral T cells. Fifty to 80% of small cortical cells do not express detectable H-2K, but all the other subpopulations, both inside and outside the thymus, stain uniformly quite brightly. TL3 is expressed on 70 to 80% of subcapsular and cortical thymocytes, 30 to 40% of CRT, is undetectable on migrants but can be seen at low levels on 10 to 20% of spleen and lymph node T cells. The possibility that some or all of these antigens represent stable markers of separate lineages rather than unstable, stage-specific markers is discussed.

  View details for Web of Science ID A1980JT75200051

  View details for PubMedID 6154739

• DISTRIBUTION OF H-2 MICRO-ENVIRONMENTS IN THE MOUSE THYMUS - IMMUNOELECTRON MICROSCOPIC IDENTIFICATION OF I-A AND H-2K BEARING CELLS JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY VANEWIJK, W., Rouse, R. V., Weissman, I. L. 1980; 28 (10): 1089-1099

  Abstract

  Antigens coded for by the major histocompatibility complex (MHC) are differentially expressed in the mouse thymus. Immunoperoxidase studies of frozen thymus sections incubated with monoclonal (hybridoma) anti-I-Ak antibodies revealed a dendritic straining pattern in the cortex and a confluent staining pattern in the medulla. Serial sections incubated with monoclonal anti-H-2Kk antibodies showed that H-2Kk antigens were only present at detectable levels in the medulla. Microenvironments expressing H-2Kk antigens also expressed I-Ak antigens. In cortico-medullary regions, relatively large MHC-negative areas were found. These areas appeared to connect to perivascular spaces surrounding blood vessels. Using a new postfixation labeling method for the detection of cell surface associated antigens on cells of the lymphoid system in situ, we have characterized the nature of MHC positive cell types at the ultrastructural level. These studies show that epithelial-reticular cells are the major MHC positive elements in the thymus. Lymphocytes in the medulla and in cortico-medullary bounderies are also MHC positive, however, lymphocytes in the cortex were not detectably labeled. These findings support the contention that epithelial-reticular cells are involved in the H2-restriction process during T cell maturation.

  View details for Web of Science ID A1980KG73500007

  View details for PubMedID 6999083

• V-J JOINING OF IMMUNOGLOBULIN-K GENES ONLY OCCURS ON ONE HOMOLOGOUS CHROMOSOME NATURE Joho, R., Weissman, I. L. 1980; 284 (5752): 179-181

  Abstract

  In general, heterozygous animal cells express both alleles at a particular locus. The only exceptions are cells of XX genotype after inactivation of one X chromosome, and immunoglobulin-producing cells; in each case only one of the two alleles is expressed in differentiated cells and their progeny. This phenomenon, termed allelic exclusion, has been described for several mammalian species including man and mouse. It has been shown that the variable (V) and constant (C) region genes of immunoglobulins undergo a rearrangement during ontogeny. We wished to test whether allelic exclusion in B cells could be the consequence of V- and C-region rearrangement on one of the two homologous chromosomes only. For that reason we chose to analyse the rearrangement of immunoglobulin light chain genes in normal B lymphocytes isolated on the fluorescence-activated cell sorter. We now present evidence that during normal B-lymphocyte differentiation V-C rearrangement occurs only on one chromosome.

  View details for Web of Science ID A1980JJ08900046

  View details for PubMedID 6244500

• LYMPHOMA-T RETROVIRAL RECEPTORS AND CONTROL OF LYMPHOMA-T CELL-PROLIFERATION COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY McGrath, M. S., Pillemer, E., KOOISTRA, D. A., Jacobs, S., Jerabek, L., Weissman, I. L. 1980; 44: 1297-1304

  View details for Web of Science ID A1979KQ93700066

  View details for PubMedID 6253204

• IMMUNOELECTRON MICROSCOPY OF CELL-SURFACE ANTIGENS - A QUANTITATIVE-ANALYSIS OF ANTIBODY-BINDING AFTER DIFFERENT FIXATION PROTOCOLS HISTOCHEMICAL JOURNAL VANEWIJK, W., COFFMAN, R. C., Weissman, I. L. 1980; 12 (3): 349-361

  Abstract

  The effect of different fixation solutions on the denaturation of membrane-associated antigens in murine lymphoid cells was determined quantitatively using microfluorometric analysis and a radioimmunoassay. Paraformaldehyde and periodate-lysine-paraformaldehyde solutions preserved the antigenicity of cell surface-associated immunoglobulin (S-Ig) antigens when used in concentrations ranging from 0.01 to 4%. However, glutaraldehyde destroyed the antigenicity of S-Ig and Thy 1.2 molecules at concentrations higher than 0.1%. Electron microscopic analysis of the different fixed cell suspensions, after labelling of the cells with a rabbit anti-mouse immunoglobulin-horseradish peroxidase conjugate (RaM-Ig-HRP) showed that prefixation of the sample with 0.1% glutaraldehyde was optimal for immunoelectron microscopical studies, since this concentration preserved both the antigenicity of membrane-associated antigens as well as the ultrastructure of the cells under study. Prolonged fixation periods affected antibody binding. However, S-Ig molecules denatured at a slower rate than Thy 1.2 molecules. A preparation method for the immunoelectron microscopical localization of lymphoid and non-lymphoid cell types in lymphoid organs is reported.

  View details for Web of Science ID A1980JX35200005

  View details for PubMedID 6969247

• ORGANIZATION OF KAPPA-LIGHT CHAIN GENES IN GERM-LINE AND SOMATIC TISSUE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Joho, R., Weissman, I. L., Early, P., Cole, J., Hood, L. 1980; 77 (2): 1106-1110

  Abstract

  We studied the organization of the kappa light chain genes in germ-line (sperm) and somatic (embryo) tissues. We constructed a plasmid containing a DNA insert coding for the kappa chain MOPC 167 and used the Southern blotting technique to determine the organization of kappa variable and constant region genes. In the haploid genome of the mouse there is only one constant region gene detectable and it has the same organization in sperm and embryo DNAs. There are several variable region genes in sperm and embryo that are related to the Vk167 gene. The organization of the V genes in sperm and embryo DNAs is identical. These results show that there is no rearrangement of variable region genes (or "minigenes") during early embryogenesis.

  View details for Web of Science ID A1980JL81300081

  View details for PubMedID 6244580

• IMMUNOGLOBULIN HEAVY-CHAIN GENE IS FORMED BY AT LEAST 2 RECOMBINATIONAL EVENTS NATURE DAVIS, M. M., Calame, K., EARLY, P. W., Livant, D. L., Joho, R., Weissman, I. L., Hood, L. 1980; 283 (5749): 733-739

  Abstract

  The events of B-cell differentiation can be reconstructed in part through an analysis of the organisation of heavy-chain gene segments in differentiated B cells. A mouse immunoglobulin alpha heavy-chain gene is composed of at least three noncontiguous germ-line DNA segments--a VH gene segment, a JH gene segment associated with the Cmu gene segment, and the C alpha gene segment. These gene segments are joined together by two distinct types of DNA rearrangements--a V-J joining and a CH switch.

  View details for Web of Science ID A1980JF60300036

  View details for PubMedID 6766532

• PATHOLOGY AND HOMING OF A TRANSPLANTABLE MURINE B CELL LEUKEMIA (BCL1) JOURNAL OF IMMUNOLOGY Warnke, R. A., Slavin, S., Coffman, R. L., BUTCHER, E. C., Knapp, M. R., Strober, S., Weissman, I. L. 1979; 123 (3): 1181-1188

  Abstract

  The pathology and homing characteristics of a murine B cell leukemia are described. Experiments utilizing autoradiography to determine the early homing pattern of the leukemic cells revealed a pronounced localization of the labeled cells to the spleen. The cells that were seen in the white pulp showed preferential localization to the follicles or B cell domains. Tissue section immunofluorescence with antibodies to kappa- and lambda-light chains was used to study the initial mouse with this disease as well as to study the mice that were injected with in vivo passaged cells. These mice also showed predominant involvement of the spleen. Although the initial mouse with this disease had 200,000 lambda-bearing B lymphocytes per mm3 in the peripheral blood and closely resembled a human chronic lymphocytic leukemia patient, the studies described suggest that this murine B cell neoplasm is a lymphoma with a striking predilection for splenic involvement. The other organs including the bone marrow as well as the peripheral blood appeared to be involved secondarily. This unusual spontaneously occurring murine B cell disease provides a useful model for the investigation of certain commonly occurring human lymphomas and leukemias.

  View details for Web of Science ID A1979HH67000037

  View details for PubMedID 381519

• MHC ANTIGENS ON THYMIC EPITHELIAL-CELLS Rouse, R. V., VANEWIJK, W., Weissman, I. L. WILEY-LISS. 1979: 325–325

  View details for Web of Science ID A1979GN48200858

• Lymphocyte-high endothelial venule interactions: examination of species specificity. Advances in experimental medicine and biology Butcher, E., Scollay, R., Weissman, I. 1979; 114: 65-72

  View details for PubMedID 313685

• ALLOGENEIC CYTOLYSIS OF RECONSTITUTED MEMBRANE-VESICLES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hollander, N., Mehdi, S. Q., Weissman, I. L., McConnell, H. M., KRISS, J. P. 1979; 76 (8): 4042-4045

  Abstract

  The successful use of lipid bilayer model membranes as targets for cytotoxic lymphocytes is described. Lipid vesicles were made from a mixture of dipalmitoyl lecithin, dimyristoyl lecithin, and cholesterol. Membrane proteins of LSTRA or EL4 tumor cells (as source of H-2 antigens), human eye muscle membrane proteins (as supporting proteins), and 51Cr marker were inserted into the lipid vesicles. Incubation of the reconstituted vesicles with lymphocytes sensitized in mixed lymphocyte cultures against allogeneic cells resulted in the specific release of intravesicular 51Cr. Vesicle damage was mediated by thymus-derived lymphocytes. H-2 antigens could be incorporated into vesicles without eye muscle proteins. However, immune damage of the vesicles could not be demonstrated when vesicles inserted with H-2 antigens in the absence of eye muscle proteins were used as targets.

  View details for Web of Science ID A1979HJ25800101

  View details for PubMedID 315069

• The derivation of the LEW.RI-1a (DA)/STA rat line Rat Newsletter Rozing, J., Jerabek, L., Gutman, G., Weissman, I. 1979: 5
• SEROLOGIC COMPARISON OF MOLONEY LYMPHOMA CELL-SURFACE AND MOLONEY ONCORNAVIRUS ANTIGENS JOURNAL OF IMMUNOLOGY Humphrey, D., TSUKAMOTOADEY, A., WITTE, O. M., Fox, R., Jerabek, L., Weissman, I. L. 1979; 123 (1): 412-418

  Abstract

  Moloney lymphomas and Moloney sarcomas share strong tumor antigens. In this report we analyze the cell-surface antigens on a Balb/c Moloney lymphoma, LSTRA, using hyperimmune sarcoma regressor sera (alphaMo) as a primary reagent. We also use heterologous anti-viral p30 and gp70 sera for a direct analysis of virion protein antigens on the LSTRA surface. Using radiolabeled alphaMo-binding assays, we demonstrate that LSTRA tumor antigens detected by these sera are all Moloney viral antigens; approximately 1/3 of these antigenic determinants are expressed on the intact virus, and the other determinants are revealed by detergent lysis of the virus. The major viral antigens expressed on the LSTRA cell surface are viral env gene products, whereas gag gene products are only sparsely represented. We conclude that alphaMo sera detect almost exclusively viral antigens on LSTRA cells, and these antigens are almost exclusively virion env gene products.

  View details for Web of Science ID A1979HB56900065

  View details for PubMedID 87479

• EXPRESSION OF MHC ANTIGENS BY MOUSE THYMIC DENDRITIC CELLS JOURNAL OF IMMUNOLOGY Rouse, R. V., VANEWIJK, W., Jones, P. P., Weissman, I. L. 1979; 122 (6): 2508-2515

  Abstract

  Thymic epithelial cells express MHC antigens in several different patterns. I-A is present throughout the thymic cortex on dendritic cells. The remainder of the I region and H-2K/D are expressed on dendritic cells apparently only variably in the cortex (at least in some haplotypes). All MHC antigens tested are present in the medulla on epithelial cells; expression on medullary lymphocytes cannot be evaluated. Monoclonal anti-MHC antibodies confirm these results. The significance of these findings to T cell maturation is discussed.

  View details for Web of Science ID A1979GX20000058

  View details for PubMedID 376735

• EVIDENCE OF CONTINUOUS EVOLUTIONARY CHANGE IN STRUCTURES MEDIATING ADHERENCE OF LYMPHOCYTES TO SPECIALIZED VENULES NATURE Butcher, E., Scollay, R., Weissman, I. 1979; 280 (5722): 496-498

  View details for Web of Science ID A1979HF85100050

  View details for PubMedID 460429

• AKR LEUKEMOGENESIS - IDENTIFICATION AND BIOLOGICAL SIGNIFICANCE OF THYMIC LYMPHOMA RECEPTORS FOR AKR RETROVIRUSES CELL McGrath, M. S., Weissman, I. L. 1979; 17 (1): 65-75

  Abstract

  We have previously demonstrated that in vitro cell lines of mouse thymic lymphomas express surface receptors specific for the retrovirus that induced them. This study extends these observations to an analysis of receptor-bearing cells in the preleukemic and leukemic phases of spontaneous AKR thymic lymphomagenesis. AKR mice regularly begin expressing N-tropic retroviruses (as assayed on NIH fibroblasts by the XC plaque assay) in several tissues early in life; thymic lymphocytes also express these viruses, but are not autonomously transformed. Later thymic lymphomas emerge which are capable of metastasizing in the host of origin or transplanting leukemias into syngeneic hosts. Just prior to the appearance of thymic lymphomas, these mice also begin producing xenotropic retroviruses [as assayed in xenogeneic (For example, mink) fibroblasts], and concomitant with the appearance of the leukemias is the appearance of "recombinant" retroviruses which cause mink fibroblast foci (MCF); these viruses express elements of both N- and X-tropic virus envelopes and N-tropic viral gene products in their cores. Spontaneous AKR leukemias also produce other retroviruses which do not cause XC plaques or mink fibroblast foci; these are called SL viruses. The subject of this study was to test whether in vivo thymocytes in the preleukemic and leukemic periods also bear receptors specific for N-tropic, recombinant MCF and SL AKR retroviruses. We demonstrated that each spontaneous thymic lymphoma does bear receptors that bind viruses produced by the lymphomas and MCF-247 to a high degree and that bind N-ecotropic AKR retroviruses less well. Thymic lymphocytes predominating in the preleukemic period do not express detectable levels of receptors for either of the viruses. In some mice, receptor-positive cells co-exist with receptor-negative cells; only the receptor-positive cells are capable of transplanting leukemia to syngeneic hosts. We conclude that the presence of specific cell surface receptors for lymphoma cell-produced and recombinant AKR retroviruses is a marker for leukemia in these hosts.

  View details for Web of Science ID A1979GV91300007

  View details for PubMedID 222476

• MOLONEY VIRUS-INDUCED CELL-SURFACE ANTIGENS AND HISTOCOMPATIBILITY ANTIGENS ARE LOCATED ON DISTINCT MOLECULES JOURNAL OF IMMUNOLOGY FOX, R. I., Weissman, I. L. 1979; 122 (5): 1697-1704

  View details for Web of Science ID A1979GU71100013

  View details for PubMedID 87439

• LYMPHOCYTE ADHERENCE TO HIGH ENDOTHELIAL VENULES - CHARACTERIZATION OF A MODIFIED INVITRO ASSAY, AND EXAMINATION OF THE BINDING OF SYNGENEIC AND ALLOGENEIC LYMPHOCYTE POPULATIONS JOURNAL OF IMMUNOLOGY BUTCHER, E. C., SCOLLAY, R. G., Weissman, I. L. 1979; 123 (5): 1996-2003

  View details for Web of Science ID A1979HT92000015

  View details for PubMedID 314954

• ABSENCE OF UNEXPECTED H-2 ALLOANTIGENS ON A MURINE LYMPHOMA JOURNAL OF IMMUNOLOGY FOX, R. I., Weissman, I. L. 1979; 123 (4): 1736-1740

  View details for Web of Science ID A1979HM71500051

  View details for PubMedID 383840

• LYMPHOID SYSTEM - ITS NORMAL ARCHITECTURE AND POTENTIAL FOR UNDERSTANDING SYSTEM THROUGH STUDY OF LYMPHOPROLIFERATIVE DISEASES HUMAN PATHOLOGY Weissman, I. L., Warnke, R., BUTCHER, E. C., Rouse, R., Levy, R. 1978; 9 (1): 25-45

  Abstract

  This article presents a view of lymphoid tissue architecture as defined by the traffic of defined lymphoid cell classes. The compartmentalization of lymphocytes is discussed in reference to specific cell-cell interactions that occur in antigen-driven immune responses. Finally, the distribution of normal and neoplastic lymphocytes in humans is defined and compared with animal model systems.

  View details for Web of Science ID A1978EK34700004

  View details for PubMedID 344190

• RETROVIRUS PURIFICATION - METHOD THAT CONSERVES ENVELOPE GLYCOPROTEIN AND MAXIMIZES INFECTIVITY JOURNAL OF VIROLOGY McGrath, M., Witte, O., Pincus, T., Weissman, I. L. 1978; 25 (3): 923-927

  Abstract

  A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques.

  View details for Web of Science ID A1978EQ58300026

  View details for PubMedID 205680

• Immunology Hood, L., Weissman, I., Wood, B. Benjamin/Cummings. 1978
• LYMPHOCYTE INTERACTION WITH HIGH ENDOTHELIAL VENULES - LACK OF SPECIES SPECIFICITY Butcher, E., Scollay, R., Weissman, I. GUSTAV FISCHER VERLAG. 1978: 304–

  View details for Web of Science ID A1978FD02400011

• Each T-cell lymphoma induced by a particular murine leukemia virus bears surface receptors specific for that virus. Birth defects original article series McGrath, M., Weissman, I. L., Baird, S., RASCHKE, W., DECLEVE, A., Lieberman, M., KAPLAN, H. S. 1978; 14 (2): 349-361

  View details for PubMedID 76488

• LYT MARKERS ON THYMUS-CELL MIGRANTS NATURE Scollay, R., Kochen, M., Butcher, E., Weissman, I. 1978; 276 (5683): 79-80

  View details for Web of Science ID A1978FU83100046

  View details for PubMedID 310964

• SPECIFICITY OF CELL-SURFACE VIRUS RECEPTORS ON RADIATION LEUKEMIA-VIRUS AND RADIATION-INDUCED THYMIC LYMPHOMAS JOURNAL OF VIROLOGY McGrath, M. S., DECLEVE, A., Lieberman, M., KAPLAN, H. S., Weissman, I. L. 1978; 28 (3): 819-827

  Abstract

  We have developed a system for analysis of murine leukemic virus (MuLV) receptors on the surface of thymic lymphoma cells utilizing the fluorescence-activated cell sorter. The binding of fluoresceinated or rhodaminated MuLV to target cells showed saturation kinetics and was blocked by homologous MuLV, and bound MuLV had a polypeptide profile identical to that of input MuLV. Thymic lymphomas bound specifically the MuLV which induced them, whereas only 0.5 to 2% of normal thymocytes showed equivalent MuLV binding. Simultaneous binding of excess fluoresceinated RadLV and rhodaminated MCF-247 AKR virus to radiation leukemia virus-induced or spontaneous AKR thymic lymphomas demonstrated that even in the presence of both viruses the cells bound preferentially the inducing MuLV. Examination of the C57BL/Ka endogenous viruses showed that radiation leukemia virus-induced thymic lymphomas bind only thymotropic-leukemogenic radiation leukemia virus and not eco- or xenofibrotropic MuLV's. Thus, virus binding in this system involves only leukemogenic isolates of these retroviruses and implies a central role of this receptor-ligand interaction in the processes of leukemic transformation.

  View details for Web of Science ID A1978FY67900017

  View details for PubMedID 215782

• CELLULAR INFILTRATE IN CARDIAC ALLOGRAFT-REJECTION IN MICE TRANSPLANTATION Billingham, M., Warnke, R., Weissman, I. L. 1977; 23 (2): 171-176

  View details for Web of Science ID A1977CW63800015

  View details for PubMedID 319582

• A RECEPTOR MEDIATED MODEL OF VIRAL LEUKEMOGENESIS HYPOTHESIS AND EXPERIMENTS Differentiation of Normal and Neoplastic Hematopoietic Cells McGrath, M., Weissman, I. 1977: 577–589
• EVIDENCE THAT MULV-INDUCED THYMIC LYMPHOMA-CELLS POSSESS SPECIFIC CELL-MEMBRANE BINDING-SITES FOR MULV INTERNATIONAL JOURNAL OF CANCER Baird, S., RASCHKE, W., Weissman, I. L. 1977; 19 (3): 403-413

  Abstract

  Cell-surface binding sites specific for thymotropic murine leukemia viruses were found in high concentrations on thymic lymphoma cell lines induced by this class of virus, but were detectable in much lower concentrations (if at all) in several non-T leukemias, plasmacytomas, and normal thymocytes or spleen cells. By specific comparison, Moloney leukemia virus (M-MuLV) binds to a lymphoma induced by M-MuLV, but not to a thymic lymphoma induced by Gross leukemia virus (G-MuLV); and G-MuLV binds to an AKR lymphoma but not to the M-MuLV-induced lymphoma. The material which binds to these T-lymphoma membrane sites is input virus, rather than a contaminant which copurifies with virus. Autoradiographic analysis demonstrates that a high proportion of T-lymphoma cells possess binding sites, whereas only a rare cell in the thymus binds murine leukemia virus to the same degree. We raise and discuss the hypothesis that each T lymphoma induced by thymotropic leukemia viruses may represent the clonal descendants of the few rate cells in the normal thymocyte population which also bind these viruses.

  View details for Web of Science ID A1977CZ75500017

  View details for PubMedID 300367

• CELLULAR MATURATION OF ONCORNAVIRUS GLYCOPROTEINS - TOPOLOGICAL ARRANGEMENT OF PRECURSOR AND PRODUCT FORMS IN CELLULAR MEMBRANES VIROLOGY Witte, O. N., TSUKAMOTOADEY, A., Weissman, I. L. 1977; 76 (2): 539-553

  View details for Web of Science ID A1977CY27900007

  View details for PubMedID 190766

• THE DEMISE OF THE FIVE-YEAR PLAN Stanford Medicine Weissman, I. 1977; 16: 6-13
• ONCORNAVIRUS LEUKEMOGENESIS AS A MODEL FOR SELECTIVE NEOPLASTIC TRANSFORMATION Life Sciences Research Report 7, Neoplastic Transformation: Mechanisms and Consequences Weissman, I., Baird, S. 1977: 135–152
• FETAL HEMATOPOIETIC ORIGINS OF THE ADULT HEMATOLYMPHOID SYSTEM Differentiation of Normal and Neoplastic Hematopoietic Cells Weissman, I., Papaioannou, V., Gardner, R. 1977: 33–47
• Normal and neoplastic maturation of T-lineage lymphocytes. Cold Spring Harbor symposia on quantitative biology Weissman, I. L., Baird, S., Gardner, R. L., Papaioannou, V. E., RASCHKE, W. 1977; 41: 9-21

  View details for PubMedID 302194

• DEVELOPMENT AND FUNCTION OF SUBPOPULATIONS OF THYMOCYTES AND T LYMPHOCYTES PROGRESS IN ALLERGY Cantor, H., Weissman, I. 1976; 20: 1-64

  View details for Web of Science ID A1976BR11800002

  View details for PubMedID 56011

• T CELL MATURATION AND THE ONTOGENY OF SPLENIC LYMPHOID ARCHITECTURE Immuno-Aspects of the Spleen Weissman, I. Elsevier/North Holland Biomedical Press. 1976: 77–87
• NORMAL AND NEOPLASTIC MATURATION OF T-LINEAGE LYMPHOCYTES COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Weissman, I. L., Baird, S., Gardner, R. L., Papaioannou, V. E., RASCHKE, W. 1976; 41: 9-21

  View details for Web of Science ID A1976DF78500004

• ONCORNAVIRUS BUDDING - KINETICS OF FORMATION AND UTILIZATION OF VIRAL MEMBRANE GLYCOPROTEIN VIROLOGY Witte, O. N., Weissman, I. L. 1976; 69 (2): 464-473

  View details for Web of Science ID A1976BJ32400009

  View details for PubMedID 176781

• Lymphoid tissue architecture. III. Germinal centers, T cells, and thymus-dependent vs thymus-independent antigens. Advances in experimental medicine and biology Weissman, I. L., GUTMAN, G. A., FRIEDBERG, S. H., Jerabek, L. 1976; 66: 229-237

  View details for PubMedID 1083633

• Evidence that uridine incorporation is not a selective marker for mouse lymphocyte subclasses. Journal of immunology GUTMAN, G. A., Weissman, I. L. 1975; 115 (3): 939-940

  Abstract

  We demonstrate here that mouse T+ and Ig+ lymphocytes do not show the differential incorporation of 3H-uridine into acid precipitable cellular components described for these lymphocyte subpopulation in the rat.

  View details for PubMedID 1097532

• THYMUS-CELL MATURATION .2. DIFFERENTIATION OF 3 MATURE SUBCLASSES INVIVO CELLULAR IMMUNOLOGY Fathman, C. G., Small, M., Herzenberg, L. A., Weissman, I. L. 1975; 15 (1): 109-128

  View details for Web of Science ID A1975V156100011

  View details for PubMedID 1109155

• LYMPHOCYTE TYPES IN LYMPHOMAS IN AN ANIMAL-MODEL OF SJOGRENS SYNDROME Greenspan, J. S., Gutman, G., Talal, N., Weissman, I. L., Sugai, S. SAGE PUBLICATIONS INC. 1975: L67–L67

  View details for Web of Science ID A1975V705200901

• FOCAL INFECTION AND TRANSFORMATION INSITU OF THYMUS-CELL SUBCLASSES BY A THYMOTROPIC MURINE LEUKEMIA-VIRUS CANCER RESEARCH DECLEVE, A., Travis, M., Weissman, I. L., Lieberman, M., KAPLAN, H. S. 1975; 35 (12): 3585-3595

  Abstract

  Studies on the maturational lineages of thymic lymphocytes have revealed several subclasses which are distinguishable on the basis of cell size, topographic distribution within the thymus, DNA synthetic and mitotic activity, migratory behavior, and other properties. Strain C57BL/Ka mice were inoculated with radiation leukemia virus at different concentrations, and tissues were removed at defined intervals. Sequential sections were analyzed for virus-specific cytoplasmic antigen expression, for morphological evidence of neoplastic transformation, and for alkaline phosphatase activity. The first detectable sign of MuLV infection was the focal appearance of cytoplasmic viral antigens in cells of the outer thymic cortex, followed by coalescence of such foci and, several weeks later, by the appearance of morphologically transformed and alkaline phosphatase-positive cells, again often focally distributed in the outer thymic cortex. These observations strongly suggest that the large, mitotically active cells of the outer thymic cortex are the principal source of target cells for both productive infection and subsequent lymphoma induction by the virus.

  View details for Web of Science ID A1975AY30900006

  View details for PubMedID 172227

• MOUSE T-CELL SURFACE GLYCOPROTEIN RECOGNIZED BY HETEROLOGOUS ANTI-THYMOCYTE SERA AND ITS RELATIONSHIP TO THY-1 ANTIGEN NATURE Trowbridge, I. S., Weissman, I. L., Bevan, M. J. 1975; 256 (5519): 652-654

  View details for Web of Science ID A1975AM56600031

  View details for PubMedID 50563

• EVIDENCE THAT URIDINE INCORPORATION IS NOT A SELECTIVE MARKER FOR MOUSE LYMPHOCYTE SUBCLASSES JOURNAL OF IMMUNOLOGY GUTMAN, G. A., Weissman, I. L. 1975; 115 (3): 739-740

  View details for Web of Science ID A1975AQ02400025

• DIFFERENTIATION AND MIGRATION OF T LYMPHOCYTES ISRAEL JOURNAL OF MEDICAL SCIENCES Weissman, I. L., Masuda, T., Olive, C., FRIEDBERG, S. H. 1975; 11 (12): 1267-1277

  View details for Web of Science ID A1975BJ21000004

  View details for PubMedID 1082869

• DEVELOPMENT AND DISTRIBUTION OF IMMUNOGLOBULIN-BEARING CELLS IN MICE TRANSPLANTATION REVIEWS Weissman, I. L. 1975; 24: 159-176

  View details for Web of Science ID A1975AH93100005

  View details for PubMedID 1096381

• INVITRO CORTISONE SENSITIVITY OF INVIVO CORTISONE-RESISTANT THYMOCYTES ISRAEL JOURNAL OF MEDICAL SCIENCES Weissman, I. L., Levy, R. 1975; 11 (9): 884-888

  Abstract

  Thymus cells from untreated or hydrocortisone-treated mice were cultured for 20 hr in the presence or absence of a water-soluble glucocorticoid, hydrocortisone sodium succinate. By two independent assays of cell viability or function, virtually all thymocytes from untreated hosts were inactivated by hydrocortisone sodium succinate, whereas most, but not all, thymocytes from hydrocortisone-treated hosts were inactivated. Thus, the thymocytes which survive after treatment of the animal with glucocorticoids are only partially resistant to the effects of glucocorticoids in vitro.

  View details for Web of Science ID A1975AQ75200003

  View details for PubMedID 1184360

• DIFFERENTIATION OF THYMUS-CELLS FEDERATION PROCEEDINGS Weissman, I. L., Small, M., Fathman, C. G., Herzenberg, L. A. 1975; 34 (2): 141-144

  View details for Web of Science ID A1975V451000006

  View details for PubMedID 1090451

• Lymphoid tissue architecture. II. Ontogeny of peripheral T and B cells in mice: evidence against Peyer's patches as the site of generation of B cells. Journal of immunology FRIEDBERG, S. H., Weissman, I. L. 1974; 113 (5): 1477-1492

  View details for PubMedID 4608249

• THYMUS-ANTIGEN AND IMMUNOGLOBULIN-POSITIVE LYMPHOCYTES IN TISSUE INFILTRATES OF NZB-NZW MICE CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY GREENSPA, J. S., GUTMAN, G. A., Weissman, I. L., Talal, N. 1974; 3 (1): 16-31

  View details for Web of Science ID A1974U158900002

  View details for PubMedID 4611670

• RADIOLABELED ANTITUMOR ANTIBODIES .3. HIGHLY IODINATED AND HIGHLY RADIOIODINATED ANTIBODIES JOURNAL OF THE NATIONAL CANCER INSTITUTE Nord, S., Weissman, I. L. 1974; 53 (4): 959-965

  View details for Web of Science ID A1974U665800009

  View details for PubMedID 4473559

• THYMUS-ANTIGEN AND IMMUNOGLOBULIN-POSITIVE CELLS IN LYMPH-NODES, THYMUS, AND MALIGNANT LYMPHOMAS OF NZB-NZW MICE CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY GREENSPA, J. S., GUTMAN, G. A., Talal, N., Weissman, I. L., Sugai, S. 1974; 3 (1): 32-51

  View details for Web of Science ID A1974U158900003

  View details for PubMedID 4611671

• HISTOPATHOLOGY AND IMMUNOPATHOLOGY OF LYMPROLIFERATIVE DISEASE OF NEW-ZEALAND BLACK-WHITE HYBRID MICE GREENSPA, J. S., GUTTMAN, G. A., Weissman, I. L., Talal, N. SAGE PUBLICATIONS INC. 1974: 1046–46

  View details for Web of Science ID A1974U131900013

• PRIMARY AND SECONDARY IMMUNE RESPONSE RELATED TO RADIATION EXPOSURE: LOCALIZED IRRADIATION Interaction of Radiation and Host Immune Defense Mechanisms in Malignancy Eltringham, J., Weissman, I. Brookhaven National Laboratory. 1974: 167–170
• RADIOLABELED ANTIBODIES: THEIR POTENTIAL AS QUANTITATIVE TOOLS FOR IN VITRO AND IN VIVO TUMOR DIAGNOSIS Interaction of Radiation and Host Immune Defense Mechanisms in Malignancy Weissman, I., Nord, S., Ellis, R. Brookhaven National Laboratory. 1974: 379–398
• MEMBRANE PROTEINS OF MSV-MLV - THEIR ROLE IN VIRION-VIRION INTERACTIONS INVITRO VIROLOGY Witte, O. N., Weissman, I. L. 1974; 61 (2): 588-593

  View details for Web of Science ID A1974U601300024

  View details for PubMedID 4371500

• RADIOLABELED ANTITUMOR ANTIBODIES .2. QUANTITATIVE-ANALYSIS OF MOLONEY TUMOR ANTIGENS ON MOLONEY LYMPHOMA-CELLS (LSTRA) JOURNAL OF THE NATIONAL CANCER INSTITUTE Nord, S., Weissman, I. L. 1974; 53 (1): 125-130

  View details for Web of Science ID A1974T743500015

  View details for PubMedID 4858065

• Tissue localization of lymphoid cells. Series haematologica (1968) Weissman, I. L., GUTMAN, G. A., FRIEDBERG, S. H. 1974; 7 (4): 482-504

  View details for PubMedID 4141797

• POLYPEPTIDES OF MOLONEY SARCOMA-LEUKEMIA VIRIONS - THEIR RESOLUTION AND INCORPORATION INTO EXTRACELLULAR VIRIONS VIROLOGY Witte, O. N., Weissman, I. L. 1974; 61 (2): 575-587

  View details for Web of Science ID A1974U601300023

  View details for PubMedID 4371461

• RADIOLABELED ANTITUMOR ANTIBODIES .1. ANTIBODY-SPECIFIC AND IMMUNOGLOBULIN-SPECIFIC BINDING-SITES ON MOLONEY LYMPHOMA-CELLS (LSTRA) JOURNAL OF THE NATIONAL CANCER INSTITUTE Nord, S., Weissman, I. L. 1974; 53 (1): 117-124

  View details for Web of Science ID A1974T743500014

  View details for PubMedID 4858154

• PRODUCTION OF ANTI-H-2 ANTIBODIES IN THYMECTOMIZED MICE EUROPEAN JOURNAL OF IMMUNOLOGY Klein, J., Livnat, S., HAUPTFEL, V., Jerabek, L., Weissman, I. 1974; 4 (1): 41-44

  View details for Web of Science ID A1974S261200010

  View details for PubMedID 4853884

• CELLULAR IMMUNITY TO HETEROLOGOUS ERYTHROCYTES IN-VITRO .1. ROLE OF SURFACE-ADHERENT CELLS AND SPECIFIC MEDIATORS IN AN EFFECTOR MECHANISM CELLULAR IMMUNOLOGY Weissman, I., Lannin, D., Jerabek, L., Barclay, T. 1973; 7 (2): 222-236

  View details for Web of Science ID A1973P622900005

  View details for PubMedID 4704917

• TUMOR IMMUNITY IN-VIVO - EVIDENCE THAT IMMUNE DESTRUCTION OF TUMOR LEAVES BYSTANDER CELLS INTACT JOURNAL OF THE NATIONAL CANCER INSTITUTE Weissman, I. L. 1973; 51 (2): 443-448

  View details for Web of Science ID A1973Q520900011

  View details for PubMedID 4765368

• TRANSFER OF TOLERANCE TRANSPLANTATION Weissman, I. L. 1973; 15 (3): 265-269

  View details for Web of Science ID A1973P092700001

  View details for PubMedID 4123075

• THYMUS CELL MATURATION - STUDIES ON ORIGIN OF CORTISONE-RESISTANT THYMIC LYMPHOCYTES JOURNAL OF EXPERIMENTAL MEDICINE Weissman, I. L. 1973; 137 (2): 504-510

  Abstract

  Outer thymic cortical large lymphocytes were labeled by transcapsular administration of tritiated thymidine. By 2-4 days after labeling, small and medium labeled lymphocyte descendants were found throughout the cortex and in the medulla. The labeled cortical lymphocytes undergo pycnosis after parenteral administration of hydrocortisone 24 h previously, but their (labeled) medullary descendants do not. In addition, parenteral administration of hydrocortisone at the time of surface labeling results in the absence of the appearance of labeled medullary descendants.

  View details for Web of Science ID A1973O791700023

  View details for PubMedID 4539848

• REGIONAL LYMPH-NODE IRRADIATION - EFFECT ON LOCAL AND DISTANT GENERATION OF ANTIBODY-FORMING CELLS JOURNAL OF IMMUNOLOGY Weissman, I. L., Peacock, M., ELTRINGH, J. R. 1973; 110 (5): 1300-1306

  View details for Web of Science ID A1973P988200013

  View details for PubMedID 4572634

• HOMING PROPERTIES OF THYMUS-INDEPENDENT FOLLICULAR LYMPHOCYTES TRANSPLANTATION GUTMAN, G. A., Weissman, I. L. 1973; 16 (6): 621-629

  View details for Web of Science ID A1973R489400013

  View details for PubMedID 4585386

• FAILURE TO DEMONSTRATE POSTNATAL TESTICULAR DEPENDENT EXPRESSION OF MALE-SPECIFIC TRANSPLANTATION ANTIGEN IN MICE TRANSPLANTATION Weissman, I. L. 1973; 16 (2): 122-125

  View details for Web of Science ID A1973Q548300007

  View details for PubMedID 4125965

• STRUCTURAL CHARACTERISTICS OF SOME MURINE RNA TUMOR-VIRUSES STUDIED BY LACTOPEROXIDASE IODINATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Witte, O. N., Weissman, I. L., KAPLAN, H. S. 1973; 70 (1): 36-40

  Abstract

  Iodination by the noninvasive enzymatic lactoperoxidase technique has been used to study the enzyme-accessible and enzyme-inaccessible proteins of three oncorna viruses (radiation leukemia virus, Moloney leukemia virus, and mouse mammary tumor virus). The number and relative molecular weight of proteins associated with virion preparations purified on sucrose gradients were characterized by scans of Coomassie blue-stained bands after dodecyl sulfate-polyacrylamide gel electrophoresis. Gel scans from the leukemia viruses are similar, each showing six distinct major protein bands on stained gels. The mammary tumor virus proteins by this analysis are not similar to those of the leukemia viruses. Enzymatic iodination of intact virion preparations led to the solitary labeling of one of the major proteins of each virus-the 80,000-dalton protein of the leukemia viruses and the 52,500-dalton protein of the mammary tumor virus. These are tentatively positioned as surface (enzyme-accessible) moieties in the virions. Disruption of each virus with a nonionic nonionic detergent before enzymatic iodination led to the labeling of the remaining stainable bands. The four lower molecular weight bands of the leukemia viruses are tentatively positioned as internal (enzyme-inaccessible) components.

  View details for Web of Science ID A1973O525600010

  View details for PubMedID 4346036

• Immunological memory in mice. 3. Memory to heterologous erythrocytes in both T cell and B cell populations and requirement for T cells in expression of B cell memory. Evidence using immunoglobulin allotype and mouse alloantigen theta markers with congenic mice. journal of experimental medicine Mitchell, G. F., Chan, E. L., Noble, M. S., Weissman, I. L., MISHELL, R. I., Herzenberg, L. A. 1972; 135 (2): 165-184

  Abstract

  Using anti-allotype sera and AKR anti thetaC3H sera, a requirement for two cell types has been demonstrated in the adoptive secondary response of mice to heterologous erythrocytes. The cell types have been designated B cells [precursors of plaque-forming cells (PFC)] and T cells (thymus-influenced cells, not providing precursors of detectable PFC). The in vivo indirect PFC response of spleen cells from primed mice is markedly reduced by in vitro treatment of the cells with a mixture of anti-theta serum and guinea pig serum (Anti theta + GPS). This B cell response is fully restored to control levels by thymus cells from normal mice which do not themselves provide precursors of indirect PFC. Thus memory is carried by the B cell lineage but the expression of this memory is dependent on the presence of a cell population which is sensitive to Anti theta + GPS and which is replaced functionally by unprimed T cells. When assayed for T cell activity, thoracic duct cells from specifically primed mice are better than cells from nonspecifically primed mice in restoring the B cell response of spleen cells from immunized mice. Moreover, the T cell activity of a reconstitutive cell population from primed mice is reduced by incubation with Anti theta + GPS. We conclude that memory to heterologous erythrocyte antigens is carried by the T cell lineage as well as the B cell lineage even though unprimed T cells are sufficient for expression of B cell memory.

  View details for PubMedID 4110524

• LYMPHOID-TISSUE ARCHITECTURE - EXPERIMENTAL ANALYSIS OF ORIGIN AND DISTRIBUTION OF T-CELLS AND B-CELLS IMMUNOLOGY GUTMAN, G. A., Weissman, I. L. 1972; 23 (4): 465-?

  View details for Web of Science ID A1972N872400001

  View details for PubMedID 4563475

• IMMUNOLOGICAL MEMORY IN MICE .3. MEMORY TO HETEROLOGOUS ERYTHROCYTES IN BOTH T-CELL AND B-CELL POPULATIONS AND REQUIREMENT FOR T-CELLS IN EXPRESSION OF B-CELL MEMORY - EVIDENCE USING IMMUNOGLOBULIN ALLOTYPE AND MOUSE ALLOANTIGEN THETA MARKERS WITH CONGENIC MIC JOURNAL OF EXPERIMENTAL MEDICINE Mitchell, G. F., MISHELL, R. I., Noble, M. S., Herzenberg, L. A., Weissman, I. L., Chan, E. L. 1972; 135 (2): 165-?

  View details for Web of Science ID A1972L529400001

• IMMUNOTHERAPY AND IMMUNODIAGNOSIS OF METASTATIC NEOPLASMS: PROSPECTS AND PROGRESS Frontiers of Radiation Therapy and Oncology, Vol. 7 Weissman, I., Nord, S., Baird, S. S. Karger A.G. and University Park Press. 1972: 161–178
• Differential effects of local lymphoid irradiation on delayed hypersensitivity and the serum antibody response in rats: antigen injection before x-rays. Journal of immunology ELTRINGHAM, J. R., Weissman, I. L. 1971; 106 (5): 1185-1190

  View details for PubMedID 5574403

• Antibody inhibition of the immune response: experimental analysis of the site of action in vivo. Journal of immunology Eisenberg, R. A., Weissman, I. L. 1971; 106 (1): 143-149

  View details for PubMedID 5547592

• DIFFERENTIAL EFFECTS OF LOCAL LYMPHOID IRRADIATION ON DELAYED HYPERSENSITIVITY AND SERUM ANTIBODY RESPONSE IN RATS - ANTIGEN INJECTION BEFORE X-RAYS JOURNAL OF IMMUNOLOGY ELTRINGH, J. R., Weissman, I. L. 1971; 106 (5): 1185-?

  View details for Web of Science ID A1971J392700008

• TUMOR IMMUNOLOGY CALIFORNIA MEDICINE Weissman, I. L. 1971; 114 (3): 76-?

  View details for Web of Science ID A1971I740300023

  View details for PubMedID 5544693

• DUAL ORIGIN OF INTIMAL CELLS IN CARDIAC-ALLOGRAFT ARTERIOSCLEROSIS NEW ENGLAND JOURNAL OF MEDICINE Kennedy, L. J., Weissman, I. L. 1971; 285 (16): 884-?

  View details for Web of Science ID A1971K520400003

  View details for PubMedID 4939537

• INHERITANCE AND STRAIN DISTRIBUTION OF A RAT IMMUNOGLOBULIN ALLOTYPE JOURNAL OF IMMUNOLOGY GUTMAN, G. A., WEISMAN, I. L. 1971; 107 (5): 1390-?

  View details for Web of Science ID A1971K827800024

  View details for PubMedID 5117672

• ANTI-THETA ANTISERA MAY CONTAIN ANTI-ALLOTYPE CONTAMINATION NATURE-NEW BIOLOGY Baird, S., Santa, J., Weissman, I. 1971; 232 (28): 56-?

  View details for Web of Science ID A1971J788900014

  View details for PubMedID 5284452

• THE BONE MARROW ORIGIN OF LYMPHOID PRIMARY FOLLICLE SMALL LYMPHOCYTES Morphological and Functional Aspects of Immunity, Vol. 12 Gutman, G., Weissman, I. Plenum Publishing Corporation. 1971: 595–602
• THE ROLE OF THE THYMUS AND EXTRATHYMIC FACTORS IN THE DEVELOPMENT OF IMMUNE COMPETENCE DEELOPMENTAL ASPECTS OF ANTIBODY FORMATION AND STRUCTURE, VOL. 1 Weissman, I. Academic Press. 1970: 55–67
• REGIONAL LYMPH NODE IRRADIATION . EFFECT ON IMMUNE RESPONSES RADIOLOGY ELTRINGH, J. R., Weissman, I. 1970; 94 (2): 438-?

  View details for Web of Science ID A1970F207000038

  View details for PubMedID 5412821

• GENETIC AND HISTOCHEMICAL STUDIES ON MOUSE SPLEEN BLACK SPOTS NATURE Weissman, I. 1967; 215 (5098): 315-?

  View details for Web of Science ID A19679613100058

  View details for PubMedID 6059528

• THYMUS CELL MIGRATION JOURNAL OF EXPERIMENTAL MEDICINE Weissman, I. L. 1967; 126 (2): 291-?

  Abstract

  THE PRECEDING STUDIES HAVE ESTABLISHED THE FOLLOWING POINTS: Intrathymic labeling of thymic lymphocytes provides an adequate marker system to detect the migration of thymus cells to peripheral lymphoid sites. In the newborn, this comprises a major portion of the total lymphocyte population in lymph nodes and spleen. In the adult, this migration is limited to specific portions of lymph nodes and spleen, i.e., those portions which serve the recirculating pool of small lymphocytes. Kinetic studies of labeling within the adult thymus indicate that large cells give rise to medium and small cells, which then migrate to the specific sites noted above. In the newborn, the kinetic pattern is similar to that of adults, with the single distinction that large cells also migrate, accelerating the tempo of migration in these hosts. The long-term fate and function of thymus cell migrants has not yet been determined.

  View details for Web of Science ID A19679726600008

  View details for PubMedID 6028489

• Studies on the mechanism of split tolerance in mice. Transplantation Weissman, I. L. 1966; 4 (5): 565-571

  View details for PubMedID 5339404

• WEAK HISTOCOMPATIBILITY LOCI Ann NY Acad Sci Eichwald, E., Weissman, I. 1966; 129 (1): 94-101
• WEAK HISTOCOMPATIBILITY LOCI ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Eichwald, E. J., Weissman, I. L. 1966; 129 (A1): 94-?

  View details for Web of Science ID A19668855100009

• THE EFFECT OF MILLIPORE FILTER PORE SIZE ON IMMUNOLOGICAL RESTORATION OF THYMECTOMIZED HOSTS The Thymus Barclay, T., Weissman, I. 1964: 117–120
• AGE EFFECT ON THYMUS GRAFT REGENERATION AND IMMUNOLOGIC RESTORATION OF THYMECTOMIZED HOSTS The Thymus Weissman, I., Barclay, T., Kaplan, H. 1964: 102–104
• PARABIOTIC SHIFTS AND THE H-2 LOCUS J Natl Cancer Inst Eichwald, E., Weissman, I., Kowalchuk, J., Lustgraaf, E. 1963; 30: 783-794
• PARABIOTIC SHIFTS AND H-2 LOCUS JOURNAL OF THE NATIONAL CANCER INSTITUTE Eichwald, E. J., KOWALCHU, J., Weissman, I. L., LUSTGRAAF, E. C. 1963; 30 (4): 783-?

  View details for Web of Science ID A19635928B00003

• TISSUE TRANSPLANTATION Advances in Biological and Medical Physics Eichwald, E., Lustgraaf, E., Weissman, I., Kowalchuk, J. Academic Press--Elsevier. 1963: 160–177
• THE "WHITE GRAFT" REACTION AND PARABIOSIS Ann NY Acad Sci Eichwald, E., Anthony, M., Weissman, I., Lustgraaf, E. 1962; 99 (5): 606-610
• WHITE GRAFT REACTION AND PARABIOSIS ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Eichwald, E. J., LUSTGRAAF, E. C., Anthony, M., Weissman, I. L. 1962; 99 (3): 606-?

  View details for Web of Science ID A19624776A00011

• Antibody formation and repressor systems. Transplantation bulletin Weissman, I. L., LUSTGRAAF, E. C. 1961; 28: 134-135

  View details for PubMedID 14005927

• PARABIOTIC ANEMIA-POLYCYTHEMIA PROCEEDINGS OF THE SOCIETY FOR EXPERIMENTAL BIOLOGY AND MEDICINE Eichwald, E. J., FUSON, R. B., Weissman, I., LUSTGRAAF, E. C. 1961; 106 (2): 441-?

  View details for Web of Science ID A19611024C00015

  View details for PubMedID 13726087

• SEX-LINKED REJECTION OF NORMAL AND NEOPLASTIC TISSUE .1. DISTRIBUTION AND SPECIFICITY JOURNAL OF THE NATIONAL CANCER INSTITUTE Eichwald, E. J., SILMSER, C. R., Weissman, I. 1958; 20 (3): 563-575

  View details for Web of Science ID A1958WD27500008

  View details for PubMedID 13539607

• ATTEMPTS TO DEMONSTRATE SEX-LINKED HISTOCOMPATIBILITY GENES TRANSPLANTATION BULLETIN Eichwald, E. J., LUSTGRAAF, E. C., Weissman, I., STRAINER, M. 1958; 5 (4): 387-388

  View details for Web of Science ID A1958WZ65800006

  View details for PubMedID 13592896

• BIBLIOGRAPHY OF TUMOR TRANSPLANTATION - ADDENDUM NO-2 TRANSPLANTATION BULLETIN Eichwald, E. J., Weissman, I. 1957; 4 (2): 88-100

  View details for Web of Science ID A1957WZ53200019

  View details for PubMedID 13422632