Hiro Nakauchi obtained a M.D. from Yokohama City University School of Medicine and a Ph.D. in immunology from University of Tokyo Graduate School of Medicine. He isolated CD8 genes during his post-doc period at the Laboratory of Prof. Leonard Herzenberg at Stanford University. After returning to Japan, he started working on hematopoietic stem cells in his laboratory at RIKEN. In 1994, he became Professor of Immunology at the University of Tsukuba where he demonstrated that a single hematopoietic stem cell could reconstitute the entire hematopoietic system, a definitive experimental proof for the “stemness”. Since April 2002, he has been a Professor of Stem Cell Therapy in the Institute of Medical Science at The University of Tokyo (IMSUT). In 2008, he was appointed Director of newly established Center for Stem Cell Biology and Regenerative Medicine at IMSUT. In 2014, he returned to Stanford University as a faculty to continue his stem cell research at the Institute of Stem Cell Biology and Regenerative Medicine. Goals of his work are to translate discoveries in basic research into practical medical applications.

Administrative Appointments

  • Assistant Professor, Department of Immunology Juntendo University, School of Medicine (1986 - 1987)
  • Associate team leader, Team leader Laboratory of Cell Growth and Differentiation,, The Institute of Physical and Chemical Research (RIKEN) (1987 - 1995)
  • Professor, Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba (1994 - 2002)
  • Professor, Laboratory of Stem Cell Therapy, Center for Exp. Medicine, Institute of Medical Science, University of Tokyo (2002 - 2007)
  • Leader, iPS Research Core Facility Program of The Project for Realization of Regenerative Medicine, University of Tokyo (2008 - 2013)
  • Research Director, Nakauchi Stem Cell and Organ Regeneration Project, Japan Science and Technology Agency, Exploratory Research for Advanced Technology (2008 - 2013)
  • Director, Center for Stem Cell Biology and Regenerative Medicine,, Institute of Medical Science, University of Tokyo (2008 - 2017)
  • Professor, Department of Genetics,, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University (2014 - Present)
  • Project Professor, Distinguished Professor Unit, Institute of Medical Science, University of Tokyo (2017 - 2022)
  • Distinguished Professor, Advanced Research Institute, Tokyo Medical and Dental University (2022 - Present)

Boards, Advisory Committees, Professional Organizations

  • Advisory board, RIKEN Center for Developmental Biology/Center for Biosystems Research (2016 - Present)
  • Guest Professor, University of Ulm, Germany (2010 - 2013)
  • President, Japanese Society of Regenerative Medicine (2007 - 2010)
  • Advisory board, RIKEN Research Center for Allergy and Immunology (2005 - 2013)
  • Board of Directors, International Society of Stem Cell Research (ISSCR) (2004 - 2008)
  • Member, International Members Committee, American Society of Hematology (2004 - 2007)
  • Advisory board, CONSERT (Concerted Safety & Efficiency Evaluation of Retroviral Transgenesis in Gene Therapy of Inherited Disease) by the European Union (2004 - 2007)

Professional Education

  • Postdoctoral Fellow, immunogenetics and molecular biology, Department of Genetics Stanford University School of Medicine
  • PhD, Department of Immunology, Graduate School of Medicine, University of Tokyo
  • MD, Yokohama City University, School of Medicine

Current Research and Scholarly Interests

Translation of discoveries in basic research into practical medical applications


  • Generation of functional cells and organs from iPS cells, Stanford University, The University of Tokyo, Meiji University, University of California Davis



  • Development of stem cell based therapy, Stanford University



  • Isolation and Clonal Characterization of Hematopoietic Stem Cells, Stanford University, The University of Tokyo



2023-24 Courses

Stanford Advisees

All Publications

  • Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors. Nature biotechnology Suchy, F. P., Karigane, D., Nakauchi, Y., Higuchi, M., Zhang, J., Pekrun, K., Hsu, I., Fan, A. C., Nishimura, T., Charlesworth, C. T., Bhadury, J., Nishimura, T., Wilkinson, A. C., Kay, M. A., Majeti, R., Nakauchi, H. 2024


    CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

    View details for DOI 10.1038/s41587-024-02171-w

    View details for PubMedID 38589662

    View details for PubMedCentralID 7846836

  • Generation of Functional Organs Using a Cell-Competitive Niche in Intra- and Inter-species Rodent Chimeras. Cell stem cell Nishimura, T., Suchy, F. P., Bhadury, J., Igarashi, K. J., Charlesworth, C. T., Nakauchi, H. 2020


    Interspecies organ generation via blastocyst complementation has succeeded in rodents, but not yet in evolutionally more distant species. Early developmental arrest hinders the formation of highly chimeric fetuses. We demonstrate that the deletion of insulin-like growth factor 1 receptor (Igf1r) in mouse embryos creates a permissive "cell-competitive niche" in several organs, significantly augmenting both mouse intraspecies and mouse/rat interspecies donor chimerism that continuously increases from embryonic day 11 onward, sometimes even taking over entire organs within intraspecies chimeras. Since Igf1r deletion allows the evasion of early developmental arrest, interspecies fetuses with high levels of organ chimerism can be generated via blastocyst complementation. This observation should facilitate donor cell contribution to host tissues, resulting in whole-organ generation via blastocyst complementation across wide evolutionary distances.

    View details for DOI 10.1016/j.stem.2020.11.019

    View details for PubMedID 33373620

  • Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature Wilkinson, A. C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, R. V., Yamamoto, R., Loh, K. M., Nakamura, Y., Watanabe, M., Nakauchi, H., Yamazaki, S. 2019


    Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

    View details for DOI 10.1038/s41586-019-1244-x

    View details for PubMedID 31142833

  • Large-Scale Clonal Analysis Resolves Aging of the Mouse Hematopoietic Stem Cell Compartment. Cell stem cell Yamamoto, R. n., Wilkinson, A. C., Ooehara, J. n., Lan, X. n., Lai, C. Y., Nakauchi, Y. n., Pritchard, J. K., Nakauchi, H. n. 2018; 22 (4): 600–607.e4


    Aging is linked to functional deterioration and hematological diseases. The hematopoietic system is maintained by hematopoietic stem cells (HSCs), and dysfunction within the HSC compartment is thought to be a key mechanism underlying age-related hematopoietic perturbations. Using single-cell transplantation assays with five blood-lineage analysis, we previously identified myeloid-restricted repopulating progenitors (MyRPs) within the phenotypic HSC compartment in young mice. Here, we determined the age-related functional changes to the HSC compartment using over 400 single-cell transplantation assays. Notably, MyRP frequency increased dramatically with age, while multipotent HSCs expanded modestly within the bone marrow. We also identified a subset of functional cells that were myeloid restricted in primary recipients but displayed multipotent (five blood-lineage) output in secondary recipients. We have termed this cell type latent-HSCs, which appear exclusive to the aged HSC compartment. These results question the traditional dogma of HSC aging and our current approaches to assay and define HSCs.

    View details for PubMedID 29625072

  • Changing concepts in hematopoietic stem cells. Science (New York, N.Y.) Yamamoto, R., Wilkinson, A. C., Nakauchi, H. 2018; 362 (6417): 895–96

    View details for PubMedID 30467158

  • Interspecies organogenesis generates autologous functional islets. Nature Yamaguchi, T., Sato, H., Kato-Itoh, M., Goto, T., Hara, H., Sanbo, M., Mizuno, N., Kobayashi, T., Yanagida, A., Umino, A., Ota, Y., Hamanaka, S., Masaki, H., Rashid, S. T., Hirabayashi, M., Nakauchi, H. 2017; 542 (7640): 191-196


    Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.

    View details for DOI 10.1038/nature21070

    View details for PubMedID 28117444

  • Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation SCIENCE Taya, Y., Ota, Y., Wilkinson, A. C., Kanazawa, A., Watarai, H., Kasai, M., Nakauchi, H., Yamazaki, S. 2016; 354 (6316): 1152-1155


    A specialized bone marrow microenvironment (niche) regulates hematopoietic stem cell (HSC) self-renewal and commitment. For successful donor-HSC engraftment, the niche must be emptied via myeloablative irradiation or chemotherapy. However, myeloablation can cause severe complications and even mortality. Here we report that the essential amino acid valine is indispensable for the proliferation and maintenance of HSCs. Both mouse and human HSCs failed to proliferate when cultured in valine-depleted conditions. In mice fed a valine-restricted diet, HSC frequency fell dramatically within 1 week. Furthermore, dietary valine restriction emptied the mouse bone marrow niche and afforded donor-HSC engraftment without chemoirradiative myeloablation. These findings indicate a critical role for valine in HSC maintenance and suggest that dietary valine restriction may reduce iatrogenic complications in HSC transplantation.

    View details for DOI 10.1126/science.aag3145

    View details for Web of Science ID 000388916400043

    View details for PubMedID 27934766

  • 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


    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

  • Clonal Analysis Unveils Self-Renewing Lineage-Restricted Progenitors Generated Directly from Hematopoietic Stem Cells CELL Yamamoto, R., Morita, Y., Ooehara, J., Hamanaka, S., Onodera, M., Rudolph, K. L., Ema, H., Nakauchi, H. 2013; 154 (5): 1112-1126


    Consensus holds that hematopoietic stem cells (HSCs) give rise to multipotent progenitors (MPPs) of reduced self-renewal potential and that MPPs eventually produce lineage-committed progenitor cells in a stepwise manner. Using a single-cell transplantation system and marker mice, we unexpectedly found myeloid-restricted progenitors with long-term repopulating activity (MyRPs), which are lineage-committed to megakaryocytes, megakaryocyte-erythroid cells, or common myeloid cells (MkRPs, MERPs, or CMRPs, respectively) in the phenotypically defined HSC compartment together with HSCs. Paired daughter cell assays combined with transplantation revealed that HSCs can give rise to HSCs via symmetric division or directly differentiate into MyRPs via asymmetric division (yielding HSC-MkRP or HSC-CMRP pairs). These myeloid bypass pathways could be essential for fast responses to ablation stress. Our results show that loss of self-renewal and stepwise progression through specific differentiation stages are not essential for lineage commitment of HSCs and suggest a revised model of hematopoietic differentiation.

    View details for DOI 10.1016/j.cell.2013.08.007

    View details for Web of Science ID 000323767300023

    View details for PubMedID 23993099

  • Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Matsunari, H., Nagashima, H., Watanabe, M., Umeyama, K., Nakano, K., Nagaya, M., Kobayashi, T., Yamaguchi, T., Sumazaki, R., Herzenberg, L. A., Nakauchi, H. 2013; 110 (12): 4557-4562


    In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.

    View details for DOI 10.1073/pnas.1222902110

    View details for Web of Science ID 000317521600039

    View details for PubMedID 23431169

  • Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation CELL STEM CELL Nishimura, T., Kaneko, S., Kawana-Tachikawa, A., Tajima, Y., Goto, H., Zhu, D., Nakayama-Hosoya, K., Iriguchi, S., Uemura, Y., Shimizu, T., Takayama, N., Yamada, D., Nishimura, K., Ohtaka, M., Watanabe, N., Takahashi, S., Iwamoto, A., Koseki, H., Nakanishi, M., Eto, K., Nakauchi, H. 2013; 12 (1): 114-126


    Adoptive immunotherapy with functional T cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. However, exhaustion of antigen-specific T cells represents a major challenge to this type of approach. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8(+) T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells were then redifferentiated into CD8(+) T cells that had a high proliferative capacity and elongated telomeres. These "rejuvenated" cells possessed antigen-specific killing activity and exhibited T cell receptor gene-rearrangement patterns identical to those of the original T cell clone from the patient. We also found that this method can be effective for generating specific T cells for other pathology-associated antigens. Thus, this type of approach may have broad applications in the field of adoptive immunotherapy.

    View details for DOI 10.1016/j.stem.2012.11.002

    View details for Web of Science ID 000313839500015

    View details for PubMedID 23290140

  • Nonmyelinating Schwann Cells Maintain Hematopoietic Stem Cell Hibernation in the Bone Marrow Niche CELL Yamazaki, S., Ema, H., Karlsson, G., Yamaguchi, T., Miyoshi, H., Shioda, S., Taketo, M. M., Karlsson, S., Iwama, A., Nakauchi, H. 2011; 147 (5): 1146-1158


    Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.

    View details for DOI 10.1016/j.cell.2011.09.053

    View details for Web of Science ID 000297376600024

    View details for PubMedID 22118468

  • Generation of Rat Pancreas in Mouse by Interspecific Blastocyst Injection of Pluripotent Stem Cells CELL Kobayashi, T., Yamaguchi, T., Hamanaka, S., Kato-Itoh, M., Yamazaki, Y., Ibata, M., Sato, H., Lee, Y., Usui, J., Knisely, A. S., Hirabayashi, M., Nakauchi, H. 2010; 142 (5): 787-799


    The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.

    View details for DOI 10.1016/j.cell.2010.07.039

    View details for Web of Science ID 000281523200021

    View details for PubMedID 20813264

  • Skin graft with dermis and appendages generated in vivo by cell competition. Nature communications Nagano, H., Mizuno, N., Sato, H., Mizutani, E., Yanagida, A., Kano, M., Kasai, M., Yamamoto, H., Watanabe, M., Suchy, F., Masaki, H., Nakauchi, H. 2024; 15 (1): 3366


    Autologous skin grafting is a standard treatment for skin defects such as burns. No artificial skin substitutes are functionally equivalent to autologous skin grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of cultured epidermal cells on a synthetic dermal substitute, can engraft deep wounds, it requires the wound bed to be well-vascularized and lacks skin appendages. In this study, we successfully generate complete skin grafts with pluripotent stem cell-derived epidermis with appendages on p63 knockout embryos' dermis. Donor pluripotent stem cell-derived keratinocytes encroach the embryos' dermis by eliminating p63 knockout keratinocytes based on cell-extracellular matrix adhesion mediated cell competition. Although the chimeric skin contains allogenic dermis, it is engraftable as long as autologous grafts. Furthermore, we could generate semi-humanized skin segments by human keratinocytes injection into the amnionic cavity of p63 knockout mice embryos. Niche encroachment opens the possibility of human skin graft production in livestock animals.

    View details for DOI 10.1038/s41467-024-47527-7

    View details for PubMedID 38684678

    View details for PubMedCentralID PMC11058811

  • Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells. Developmental cell Fowler, J. L., Zheng, S. L., Nguyen, A., Chen, A., Xiong, X., Chai, T., Chen, J. Y., Karigane, D., Banuelos, A. M., Niizuma, K., Kayamori, K., Nishimura, T., Cromer, M. K., Gonzalez-Perez, D., Mason, C., Liu, D. D., Yilmaz, L., Miquerol, L., Porteus, M. H., Luca, V. C., Majeti, R., Nakauchi, H., Red-Horse, K., Weissman, I. L., Ang, L. T., Loh, K. M. 2024


    The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

    View details for DOI 10.1016/j.devcel.2024.03.003

    View details for PubMedID 38569552

  • Rejuvenated iPSC-derived GD2-directed CART cells harbor robust cytotoxicity against small cell lung cancer. Cancer research communications Kinoshita, S., Ishii, M., Ando, J., Kimura, T., Yamaguchi, T., Harada, S., Takahashi, F., Nakashima, K., Nakazawa, Y., Yamazaki, S., Ohshima, K., Takahashi, K., Nakauchi, H., Ando, M. 2024


    Small cell lung cancer (SCLC) is exceptionally aggressive, with limited treatment options. Disialoganglioside (GD2) is highly expressed on SCLC and is considered a good target for chimeric antigen receptor (CAR) T cells (CARTs). Although GD2-directed CARTs (GD2-CARTs) exhibit cytotoxicity against various GD2-expressing tumors, they lack significant cytotoxicity against SCLC. To enhance cytotoxicity of GD2-CARTs against SCLC, we introduced GD2-CAR into iPSC-derived rejuvenated cytotoxic T lymphocytes (GD2-CARrejTs). GD2-CARrejTs acted much more strongly against SCLC cells than did GD2-CARTs both in vitro and in vivo. Single-cell RNA sequencing elucidated that levels of expression of TIGIT were significantly lower and levels of expression of genes associated with cytotoxicity were significantly higher in GD2-CARrejTs than those in GD2-CARTs. Dual blockade of TIGIT and programmed death-1 (PD-1) increased the cytotoxicity of GD2-CARTs to some extent, suggesting that low TIGIT and PD-1 expression of GD2-CARrejTs is a major factor required for robust cytotoxicity against SCLC. Not only for robust cytotoxicity but also for availability as "off-the-shelf" T cell therapy, iPSC-derived GD2-CARrejTs are a promising novel treatment for SCLC.

    View details for DOI 10.1158/2767-9764.CRC-23-0259

    View details for PubMedID 38380966

  • Publisher Correction: Hypoblast from human pluripotent stem cells regulates epiblast development. Nature Okubo, T., Rivron, N., Kabata, M., Masaki, H., Kishimoto, K., Semi, K., Nakajima-Koyama, M., Kunitomi, H., Kaswandy, B., Sato, H., Nakauchi, H., Woltjen, K., Saitou, M., Sasaki, E., Yamamoto, T., Takashima, Y. 2024

    View details for DOI 10.1038/s41586-024-07166-w

    View details for PubMedID 38347193

  • Secreted Particle Information Transfer (SPIT) - A Cellular Platform forIn VivoGenetic Engineering. bioRxiv : the preprint server for biology Charlesworth, C. T., Homma, S., Suchy, F., Wang, S., Bhadhury, J., Amaya, A. K., Camarena, J., Zhang, J., Tan, T. K., Igarishi, K., Nakauchi, H. 2024


    A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cell's nucleus, the ability of human cells to engraft into patients' long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

    View details for DOI 10.1101/2024.01.11.575257

    View details for PubMedID 38260654

  • iPSC-derived hypoimmunogenic tissue resident memory Tcells mediate robust anti-tumor activity against cervical cancer. Cell reports. Medicine Furukawa, Y., Ishii, M., Ando, J., Ikeda, K., Igarashi, K. J., Kinoshita, S., Azusawa, Y., Toyota, T., Honda, T., Nakanishi, M., Ohshima, K., Masuda, A., Yoshida, E., Kitade, M., Porteus, M., Terao, Y., Nakauchi, H., Ando, M. 2023: 101327


    Functionally rejuvenated human papilloma virus-specific cytotoxic T lymphocytes (HPV-rejTs) generated from induced pluripotent stem cells robustly suppress cervical cancer. However, autologous rejT generation is time consuming, leading to difficulty in treating patients with advanced cancer. Although use of allogeneic HPV-rejTs can obviate this, the major obstacle is rejection by the patient immune system. To overcome this, we develop HLA-A24&-E dual integrated HPV-rejTs after erasing HLA class I antigens. These rejTs effectively suppress recipient immune rejection while maintaining more robust cytotoxicity than original cytotoxic T lymphocytes. Single-cell RNA sequencing performed to gain deeper insights reveal that HPV-rejTs are highly enriched with tissue resident memory Tcells, which enhance cytotoxicity against cervical cancer through TGFbetaR signaling, with increased CD103 expression. Genes associated with the immunological synapse also are upregulated, suggesting that these features promote stronger activation of Tcell receptor (TCR) and increased TCR-mediated target cell death. We believe that our work will contribute to feasible "off-the-shelf" Tcell therapy with robust anti-cervical cancer effects.

    View details for DOI 10.1016/j.xcrm.2023.101327

    View details for PubMedID 38091985

  • Hypoblast from human pluripotent stem cells regulates epiblast development. Nature Okubo, T., Rivron, N., Kabata, M., Masaki, H., Kishimoto, K., Semi, K., Nakajima-Koyama, M., Kunitomi, H., Kaswandy, B., Sato, H., Nakauchi, H., Woltjen, K., Saitou, M., Sasaki, E., Yamamoto, T., Takashima, Y. 2023


    Recently, several studies using cultures of human embryos together with single-cell RNA-seq (scRNA-seq) analyses have revealed differences between humans and mice, necessitating the study of human embryos 1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation stage studies. Thus, recent efforts have focused on developing in vitro self-organising models using human stem cells 9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (nHyC)-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pre-gastrula stage, whose emergence can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have thus successfully modelled and revealed the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

    View details for DOI 10.1038/s41586-023-06871-2

    View details for PubMedID 38052228

  • iPSC-Derived Dual Antigen Receptor T Cells Targeting GD2 and LMP2 Antigens for Extranodal NK/T-Cell Lymphoma, Nasal Type Kinoshita, S., Ishii, M., Furukawa, Y., Sato, S., Ando, J., Nakauchi, H., Ando, M. AMER SOC HEMATOLOGY. 2023
  • iPSC-Derived CD4 T Cell Generation and Investigation of CD4/CD8 T Cell Lineage Choice Furukawa, Y., Ishii, M., Goto, A., Kinoshita, S., Ando, J., Nakauchi, H., Ando, M. AMER SOC HEMATOLOGY. 2023
  • Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells. STAR protocols Matsuzaki, T., Kawano, Y., Horikiri, M., Shimokawa, Y., Yamazaki, T., Okuma, N., Koike, H., Kimura, M., Kawamura, R., Yoneyama, Y., Furuichi, Y., Hakuno, F., Takahashi, S., Nakabayashi, S., Okamoto, S., Nakauchi, H., Taniguchi, H., Takebe, T., Yoshikawa, H. Y. 2023; 4 (3): 102471


    Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe etal. (2015) and Matsuzaki etal. (2014, 2022).1,2,3.

    View details for DOI 10.1016/j.xpro.2023.102471

    View details for PubMedID 37515762

  • Functional calcium-responsive parathyroid glands generated using single-step blastocyst complementation. Proceedings of the National Academy of Sciences of the United States of America Kano, M., Mizuno, N., Sato, H., Kimura, T., Hirochika, R., Iwasaki, Y., Inoshita, N., Nagano, H., Kasai, M., Yamamoto, H., Yamaguchi, T., Suga, H., Masaki, H., Mizutani, E., Nakauchi, H. 2023; 120 (28): e2216564120


    Patients with permanent hypoparathyroidism require lifelong replacement therapy to avoid life-threatening complications, The benefits of conventional treatment are limited, however. Transplanting a functional parathyroid gland (PTG) would yield better results. Parathyroid gland cells generated from pluripotent stem cells in vitro to date cannot mimic the physiological responses to extracellular calcium that are essential for calcium homeostasis. We thus hypothesized that blastocyst complementation (BC) could be a better strategy for generating functional PTG cells and compensating loss of parathyroid function. We here describe generation of fully functional PTGs from mouse embryonic stem cells (mESCs) with single-step BC. Using CRISPR-Cas9 knockout of Glial cells missing2 (Gcm2), we efficiently produced aparathyroid embryos for BC. In these embryos, mESCs differentiated into endocrinologically mature PTGs that rescued Gcm2-/- mice from neonatal death. The mESC-derived PTGs responded to extracellular calcium, restoring calcium homeostasis on transplantation into mice surgically rendered hypoparathyroid. We also successfully generated functional interspecies PTGs in Gcm2-/- rat neonates, an accomplishment with potential for future human PTG therapy using xenogeneic animal BC. Our results demonstrate that BC can produce functional endocrine organs and constitute a concept in treatment of hypoparathyroidism.

    View details for DOI 10.1073/pnas.2216564120

    View details for PubMedID 37379351

  • Chemically defined cytokine-free expansion of human haematopoietic stem cells. Nature Sakurai, M., Ishitsuka, K., Ito, R., Wilkinson, A. C., Kimura, T., Mizutani, E., Nishikii, H., Sudo, K., Becker, H. J., Takemoto, H., Sano, T., Kataoka, K., Takahashi, S., Nakamura, Y., Kent, D. G., Iwama, A., Chiba, S., Okamoto, S., Nakauchi, H., Yamazaki, S. 2023


    Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

    View details for DOI 10.1038/s41586-023-05739-9

    View details for PubMedID 36813966

  • Physioxia improves the selectivity of hematopoietic stem cell expansion cultures. Blood advances Igarashi, K. J., Kucinski, I., Chan, Y. Y., Tan, T., Khoo, H. M., Kealy, D., Bhadury, J., Hsu, I., Ho, P. Y., Niizuma, K., Hickey, J. W., Nolan, G., Bridge, K. S., Czechowicz, A., Gottgens, B., Nakauchi, H., Wilkinson, A. C. 2023


    Hematopoietic stem cells (HSCs) are a rare hematopoietic cell type that can entirely reconstitute the blood and immune systems following transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases, but remains a high-risk therapy due to potential side effects including poor graft function and graft-vs-host disease (GvHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution from low-cell dose grafts. Here, we demonstrate that we can improve the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures through the use of physioxic culture conditions. Single-cell transcriptomic analysis confirmed inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GvHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, as well as highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT.

    View details for DOI 10.1182/bloodadvances.2023009668

    View details for PubMedID 36809781

  • Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats JOURNAL OF REPRODUCTION AND DEVELOPMENT Torikai, K., Shimizu, K., Nagatomo, H., Kasai, M., Kato-itoh, M., Kamada, Y., Shibasaki, I., Jeon, H., Kikuchi, R., Wakayama, S., Suchy, F., Nakauchi, H., Wakayama, T., Mizutani, E. 2023; 69 (1): 48-52
  • LARGE-SCALE IN VIVO CRISPR SCREENS IDENTIFY SAGA COMPLEX MEMBERS AS KEY REGULATORS OF HAEMATOPOIESIS Wilkinson, A., Haney, M., Shankar, A., Hsu, I., Miyauchi, M., Palovics, R., Olender, L., Khoo, H., Igarashi, K., Bhadury, J., Munson, C., Mack, P., Tan, T., Nakauchi, H., Wyss-Coray, T. ELSEVIER SCIENCE INC. 2023: S43
  • Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats. The Journal of reproduction and development Torikai, K., Shimizu, K., Nagatomo, H., Kasai, M., Kato-Itoh, M., Kamada, Y., Shibasaki, I., Jeon, H., Kikuchi, R., Wakayama, S., Suchy, F., Nakauchi, H., Wakayama, T., Mizutani, E. 2022


    We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant sperm cells had a lower oocyte activation capacity; however, the use of activated oocytes resulted in the same fecundity as that of fresh, untreated sperm. Pre-activated oocytes were more resistant to physical damage, showed higher survival rates, and required less time per injection. Testing this method in rats yielded similar results, although the oocyte activation method was different. Remarkably, this method resulted in higher birth rates of rat progeny than with conventional methods of rat ICSI. Our method thereby streamlines mouse and rat ICSI, making it more accessible to laboratories across many disciplines.

    View details for DOI 10.1262/jrd.2022-065

    View details for PubMedID 36529517

  • An optimized Sendai viral vector platform for reprogramming to naive pluripotency. Cell reports methods Charlesworth, C. T., Nakauchi, H. 2022; 2 (11): 100349


    Technologies to reprogram somatic cells into iPSCs have advanced significantly, however challenges to the derivation of iPSCs remain. In this issue of Cell Reports Methods, Kunitomi et al. address some of these challenges by developing a straightforward protocol to derive naive human iPSCs using Sendai virus vectors.

    View details for DOI 10.1016/j.crmeth.2022.100349

    View details for PubMedID 36452874

    View details for PubMedCentralID PMC9701616

  • Mechanical guidance of self-condensation patterns of differentiating progeny. iScience Matsuzaki, T., Shimokawa, Y., Koike, H., Kimura, M., Kawano, Y., Okuma, N., Kawamura, R., Yoneyama, Y., Furuichi, Y., Hakuno, F., Takahashi, S., Nakabayashi, S., Okamoto, S., Nakauchi, H., Taniguchi, H., Takebe, T., Yoshikawa, H. Y. 2022; 25 (10): 105109


    Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG>0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.

    View details for DOI 10.1016/j.isci.2022.105109

    View details for PubMedID 36317160

  • 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


    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

  • Identification and characterization of invitro expanded hematopoietic stem cells. EMBO reports Che, J. L., Bode, D., Kucinski, I., Cull, A. H., Bain, F., Becker, H. J., Jassinskaja, M., Barile, M., Boyd, G., Belmonte, M., Zeng, A. G., Igarashi, K. J., Rubio-Lara, J., Shepherd, M. S., Clay, A., Dick, J. E., Wilkinson, A. C., Nakauchi, H., Yamazaki, S., Gottgens, B., Kent, D. G. 2022: e55502


    Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved >200-fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non-HSCs and single cell-initiated cultures have substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. By directly linking single-clone functional transplantation data with single-clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

    View details for DOI 10.15252/embr.202255502

    View details for PubMedID 35971894

  • Streamlined and quantitative detection of chimerism using digital PCR. Scientific reports Suchy, F. P., Nishimura, T., Seki, S., Wilkinson, A. C., Higuchi, M., Hsu, I., Zhang, J., Bhadury, J., Nakauchi, H. 2022; 12 (1): 10223


    Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues is complicated by sample preparation bias, transgenic silencing, phenotypic similarity, and low-throughput analytical pipelines. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among common albino and non-albino mouse strains. In addition, we validated that this assay is compatible with crude lysate from all solid organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Moreover, the concepts discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.

    View details for DOI 10.1038/s41598-022-14467-5

    View details for PubMedID 35715477

  • Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Cell Ang, L. T., Nguyen, A. T., Liu, K. J., Chen, A., Xiong, X., Curtis, M., Martin, R. M., Raftry, B. C., Ng, C. Y., Vogel, U., Lander, A., Lesch, B. J., Fowler, J. L., Holman, A. R., Chai, T., Vijayakumar, S., Suchy, F. P., Nishimura, T., Bhadury, J., Porteus, M. H., Nakauchi, H., Cheung, C., George, S. C., Red-Horse, K., Prescott, J. B., Loh, K. M. 2022


    Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

    View details for DOI 10.1016/j.cell.2022.05.024

    View details for PubMedID 35738284

  • Author Correction: Investigation of Cas9 antibodies in the human eye. Nature communications Toral, M. A., Charlesworth, C. T., Ng, B., Chemudupati, T., Homma, S., Nakauchi, H., Bassuk, A. G., Porteus, M. H., Mahajan, V. B. 2022; 13 (1): 2109

    View details for DOI 10.1038/s41467-022-29844-x

    View details for PubMedID 35414059

  • Functional primordial germ cell-like cells from pluripotent stem cells in rats. Science (New York, N.Y.) Oikawa, M., Kobayashi, H., Sanbo, M., Mizuno, N., Iwatsuki, K., Takashima, T., Yamauchi, K., Yoshida, F., Yamamoto, T., Shinohara, T., Nakauchi, H., Kurimoto, K., Hirabayashi, M., Kobayashi, T. 2022; 376 (6589): 176-179


    The in vitro generation of germ cells from pluripotent stem cells (PSCs) can have a substantial effect on future reproductive medicine and animal breeding. A decade ago, in vitro gametogenesis was established in the mouse. However, induction of primordial germ cell-like cells (PGCLCs) to produce gametes has not been achieved in any other species. Here, we demonstrate the induction of functional PGCLCs from rat PSCs. We show that epiblast-like cells in floating aggregates form rat PGCLCs. The gonadal somatic cells support maturation and epigenetic reprogramming of the PGCLCs. When rat PGCLCs are transplanted into the seminiferous tubules of germline-less rats, functional spermatids-that is, those capable of siring viable offspring-are generated. Insights from our rat model will elucidate conserved and divergent mechanisms essential for the broad applicability of in vitro gametogenesis.

    View details for DOI 10.1126/science.abl4412

    View details for PubMedID 35389778

  • Immunological barriers to haematopoietic stem cell gene therapy. Nature reviews. Immunology Charlesworth, C. T., Hsu, I., Wilkinson, A. C., Nakauchi, H. 2022


    Cell and gene therapies using haematopoietic stem cells (HSCs) epitomize the transformative potential of regenerative medicine. Recent clinical successes for gene therapies involving autologous HSC transplantation (HSCT) demonstrate the potential of genetic engineering in this stem cell type for curing disease. With recent advances in CRISPR gene-editing technologies, methodologies for the ex vivo expansion of HSCs and non-genotoxic conditioning protocols, the range of clinical indications for HSC-based gene therapies is expected to significantly expand. However, substantial immunological challenges need to be overcome. These include pre-existing immunity to gene-therapy reagents, immune responses to neoantigens introduced into HSCs by genetic engineering, and unique challenges associated with next-generation and off-the-shelf HSC products. By synthesizing these factors in this Review, we hope to encourage more research to address the immunological issues associated with current and next-generation HSC-based gene therapies to help realize the full potential of this field.

    View details for DOI 10.1038/s41577-022-00698-0

    View details for PubMedID 35301483

  • Investigation of Cas9 antibodies in the human eye. Nature communications Toral, M. A., Charlesworth, C. T., Ng, B., Chemudupati, T., Homma, S., Nakauchi, H., Bassuk, A. G., Porteus, M. H., Mahajan, V. B. 2022; 13 (1): 1053


    Preexisting immunity against Cas9 proteins in humans represents a safety risk for CRISPR-Cas9 technologies. However, it is unclear to what extent preexisting Cas9 immunity is relevant to the eye as it is targeted for early in vivo CRISPR-Cas9 clinical trials. While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Although precise mechanisms are unclear, intraocular inflammation remains a major cause of vision loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence of preexisting Cas9-reactive antibodies in serum but not in the eye. However, we detected intraocular antibodies reactive to S. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that serum antibody concentration may determine whether specific intraocular antibodies develop, but preexisting immunity to Cas9 may represent a lower risk in human eyes than systemically.

    View details for DOI 10.1038/s41467-022-28674-1

    View details for PubMedID 35217666

  • In vitro and in vivo functions of T cells produced in complemented thymi of chimeric mice generated by blastocyst complementation. Scientific reports Yamazaki, K., Kubara, K., Ishii, S., Li, P., Dairiki, R., Hihara, T., Ishizuka, Y., Izumi, Y., Kumai, M., Kamisako, T., Ishizaki, H., Sato, H., Masaki, H., Mizuno, N., Mitsuhashi, K., Ito, M., Hamanaka, S., Yamaguchi, T., Watanabe, M., Sugiyama, F., Nakauchi, H. 2022; 12 (1): 3242


    Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNgamma, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNgamma-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.

    View details for DOI 10.1038/s41598-022-07159-7

    View details for PubMedID 35217706

  • Advances in Allogeneic Cancer Cell Therapy and Future Perspectives on "Off-the-Shelf" T Cell Therapy Using iPSC Technology and Gene Editing. Cells Furukawa, Y., Hamano, Y., Shirane, S., Kinoshita, S., Azusawa, Y., Ando, J., Nakauchi, H., Ando, M. 1800; 11 (2)


    The concept of allogeneic cell therapy was first presented over 60 years ago with hematopoietic stem cell transplantation. However, complications such as graft versus host disease (GVHD) and regimen-related toxicities remained as major obstacles. To maximize the effect of graft versus leukemia, while minimizing the effect of GVHD, donor lymphocyte infusion was utilized. This idea, which was used against viral infections, postulated that adoptive transfer of virus-specific cytotoxic T lymphocytes could reconstitute specific immunity and eliminate virus infected cells and led to the idea of banking third party cytotoxic T cells (CTLs). T cell exhaustion sometimes became a problem and difficulty arose in creating robust CTLs. However, the introduction of induced pluripotent stem cells (iPSCs) lessens such problems, and by using iPSC technology, unlimited numbers of allogeneic rejuvenated CTLs with robust and proliferative cytotoxic activity can be created. Despite this revolutionary concept, several concerns still exist, such as immunorejection by recipient cells and safety issues of gene editing. In this review, we describe approaches to a feasible "off-the-shelf" therapy that can be distributed rapidly worldwide. We also offer perspectives on the future of allogeneic cell cancer immunotherapy.

    View details for DOI 10.3390/cells11020269

    View details for PubMedID 35053386

  • Generation of heterozygous PKD1 mutant pigs exhibiting early-onset renal cyst formation. Laboratory investigation; a journal of technical methods and pathology Watanabe, M., Umeyama, K., Nakano, K., Matsunari, H., Fukuda, T., Matsumoto, K., Tajiri, S., Yamanaka, S., Hasegawa, K., Okamoto, K., Uchikura, A., Takayanagi, S., Nagaya, M., Yokoo, T., Nakauchi, H., Nagashima, H. 1800


    Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, manifesting as the progressive development of fluid-filled renal cysts. In approximately half of all patients with ADPKD, end-stage renal disease results in decreased renal function. In this study, we used CRISPR-Cas9 and somatic cell cloning to produce pigs with the unique mutation c.152_153insG (PKD1insG/+). Pathological analysis of founder cloned animals and progeny revealed that PKD1insG/+ pigs developed many pathological conditions similar to those of patients with heterozygous mutations in PKD1. Pathological similarities included the formation of macroscopic renal cysts at the neonatal stage, number and cystogenic dynamics of the renal cysts formed, interstitial fibrosis of the renal tissue, and presence of a premature asymptomatic stage. Our findings demonstrate that PKD1insG/+ pigs recapitulate the characteristic symptoms of ADPKD.

    View details for DOI 10.1038/s41374-021-00717-z

    View details for PubMedID 34980882

  • Treatment of a genetic brain disease by CNS-wide microglia replacement. Science translational medicine Shibuya, Y., Kumar, K. K., Mader, M. M., Yoo, Y., Ayala, L. A., Zhou, M., Mohr, M. A., Neumayer, G., Kumar, I., Yamamoto, R., Marcoux, P., Liou, B., Bennett, F. C., Nakauchi, H., Sun, Y., Chen, X., Heppner, F. L., Wyss-Coray, T., Südhof, T. C., Wernig, M. 2022; 14 (636): eabl9945


    Hematopoietic cell transplantation after myeloablative conditioning has been used to treat various genetic metabolic syndromes but is largely ineffective in diseases affecting the brain presumably due to poor and variable myeloid cell incorporation into the central nervous system. Here, we developed and characterized a near-complete and homogeneous replacement of microglia with bone marrow cells in mice without the need for genetic manipulation of donor or host. The high chimerism resulted from a competitive advantage of scarce donor cells during microglia repopulation rather than enhanced recruitment from the periphery. Hematopoietic stem cells, but not immediate myeloid or monocyte progenitor cells, contained full microglia replacement potency equivalent to whole bone marrow. To explore its therapeutic potential, we applied microglia replacement to a mouse model for Prosaposin deficiency, which is characterized by a progressive neurodegeneration phenotype. We found a reduction of cerebellar neurodegeneration and gliosis in treated brains, improvement of motor and balance impairment, and life span extension even with treatment started in young adulthood. This proof-of-concept study suggests that efficient microglia replacement may have therapeutic efficacy for a variety of neurological diseases.

    View details for DOI 10.1126/scitranslmed.abl9945

    View details for PubMedID 35294256

  • Xenotransplantation and interspecies organogenesis: current status and issues. Frontiers in endocrinology Kano, M., Mizutani, E., Homma, S., Masaki, H., Nakauchi, H. 2022; 13: 963282


    Pancreas (and islet) transplantation is the only curative treatment for type 1 diabetes patients whose beta-cell functions have been abolished. However, the lack of donor organs has been the major hurdle to save a large number of patients. Therefore, transplantation of animal organs is expected to be an alternative method to solve the serious shortage of donor organs. More recently, a method to generate organs from pluripotent stem cells inside the body of other species has been developed. This interspecies organ generation using blastocyst complementation (BC) is expected to be the next-generation regenerative medicine. Here, we describe the recent advances and future prospects for these two approaches.

    View details for DOI 10.3389/fendo.2022.963282

    View details for PubMedID 35992127

  • Bioluminescent Tracking of Human Induced Pluripotent Stem Cells In Vitro and In Vivo. Methods in molecular biology (Clifton, N.J.) Nishimura, T., Niizuma, K., Nakauchi, H. 2022; 2524: 291-297


    The discovery and development of induced pluripotent stem cells (iPSCs) opened a novel venue for disease modeling, drug discovery, and personalized medicine. Additionally, iPSCs have been utilized for a wide variety of research and clinical applications without immunological and ethical concerns that arise from using embryonic stem cells. Understanding the in vivo behavior of iPSCs, as well as their derivatives, requires the monitoring of their localization, proliferation, and viability after transplantation. Bioluminescence imaging (BLI) gives investigators a non-invasive and sensitive means for spatio-temporal tracking in vivo. For scientists working within the field of iPSCs, this protocol provides a walk-through on how to conduct in vitro and in vivo experiments with an iPSCs constitutively expressing luciferase.

    View details for DOI 10.1007/978-1-0716-2453-1_22

    View details for PubMedID 35821480

  • Generation of Tfap2c-T2A-tdTomato knock-in reporter rats via adeno-associated virus-mediated efficient gene targeting. Molecular reproduction and development Oikawa, M., Nagae, M., Mizuno, N., Iwatsuki, K., Yoshida, F., Inoue, N., Uenoyama, Y., Tsukamura, H., Nakauchi, H., Hirabayashi, M., Kobayashi, T. 2022


    Gene editing in mammalian zygotes enables us to generate genetically modified animals rapidly and efficiently. In this study, we compare multiple gene targeting strategies in rat zygotes by generating a novel knock-in reporter rat line to visualize the expression pattern of transcription factor AP-2 gamma (Tfap2c). The targeting vector is designed to replace the stop codon of Tfap2c with T2A-tdTomato sequence. We show that the combination of electroporation-mediated transduction of CRISPR/Cas9 components with adeno-associated virus-mediated transduction of the targeting vector is the most efficient in generating the targeted rat line. The Tfap2c-T2A-tdTomato fluorescence reflects the endogenous expression pattern of Tfap2c in preimplantation embryo, germline, placenta, and forebrain during rat embryo development. The reporter line generated here will be a reliable resource for identifying and purifying Tfap2c expressing cells in rats, and the gene targeting strategy we used can be widely applied for generating desired animals.

    View details for DOI 10.1002/mrd.23562

    View details for PubMedID 35170139

  • Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species. Development (Cambridge, England) Kinoshita, M., Kobayashi, T., Planells, B., Klisch, D., Spindlow, D., Masaki, H., Bornelov, S., Stirparo, G. G., Matsunari, H., Uchikura, A., Lamas-Toranzo, I., Nichols, J., Nakauchi, H., Nagashima, H., Alberio, R., Smith, A. 2021; 148 (23)


    Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.

    View details for DOI 10.1242/dev.199901

    View details for PubMedID 34874452

  • Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells. Cell reports Kobayashi, T., Castillo-Venzor, A., Penfold, C. A., Morgan, M., Mizuno, N., Tang, W. W., Osada, Y., Hirao, M., Yoshida, F., Sato, H., Nakauchi, H., Hirabayashi, M., Surani, M. A. 2021; 37 (2): 109812


    Rabbit embryos develop as bilaminar discs at gastrulation as in humans and most other mammals, whereas rodents develop as egg cylinders. Primordial germ cells (PGCs) appear to originate during gastrulation according to many systematic studies on mammalian embryos. Here, we show that rabbit PGC (rbPGC) specification occurs at the posterior epiblast at the onset of gastrulation. Using newly derived rabbit pluripotent stem cells, we show robust and rapid induction of rbPGC-like cells invitro with WNT and BMP morphogens, which reveals SOX17 as the critical regulator of rbPGC fate as in several non-rodent mammals. We posit that development as a bilaminar disc is a crucial determinant of the PGC regulators, regardless of the highly diverse development of extraembryonic tissues, including the amnion. We propose that investigations on rabbits with short gestation, large litters, and where gastrulation precedes implantation can contribute significantly to advances in early mammalian development.

    View details for DOI 10.1016/j.celrep.2021.109812

    View details for PubMedID 34644585

  • Dual-antigen targeted iPSC-derived chimeric antigen receptor-T cell therapy for refractory lymphoma. Molecular therapy : the journal of the American Society of Gene Therapy Harada, S., Ando, M., Ando, J., Ishii, M., Yamaguchi, T., Yamazaki, S., Toyota, T., Ohara, K., Ohtaka, M., Nakanishi, M., Shin, C., Ota, Y., Nakashima, K., Ohshima, K., Imai, C., Nakazawa, Y., Nakauchi, H., Komatsu, N. 2021


    We generated dual-antigen receptor (DR) T cells from induced pluripotent stem cells (iPSC) to mitigate tumor antigen escape. These cells were engineered to express a chimeric antigen receptor (CAR) for the antigen cell surface latent membrane protein 1 (LMP1; LMP1-CAR) and a T cell receptor directed to cell surface latent membrane protein 2 (LMP2), in association with human leucocyte antigen A24, to treat therapy-refractory Epstein-Barr virus-associated lymphomas. We introduced LMP1-CAR into iPSC derived from LMP2-specific cytotoxic T lymphocytes (CTL) to generate rejuvenated CTL (rejT) active against LMP1 and LMP2, or DRrejT. All DRrejT-treated mice survived >100d. Furthermore, DRrejT rejected follow-up inocula of lymphoma cells, demonstrating that DRrejT persisted long-term. We also demonstrated that DRrejT targeting CD19 and LMP2 antigens exhibited a robust tumor suppressive effect and conferred a clear survival advantage. Co-operative antitumor effect and in vivo persistence, with unlimited availability of DRrejT therapy, will provide powerful and sustainable T cell immunotherapy.

    View details for DOI 10.1016/j.ymthe.2021.10.006

    View details for PubMedID 34628050

  • High glucose macrophage exosomes enhance atherosclerosis by driving cellular proliferation & hematopoiesis. iScience Bouchareychas, L., Duong, P., Phu, T. A., Alsop, E., Meechoovet, B., Reiman, R., Ng, M., Yamamoto, R., Nakauchi, H., Gasper, W. J., Van Keuren-Jensen, K., Raffai, R. L. 2021; 24 (8): 102847


    We investigated whether extracellular vesicles (EVs) produced under hyperglycemic conditions could communicate signaling to drive atherosclerosis. We did so by treating Apoe-/- mice with exosomes produced by bone marrow-derived macrophages (BMDM) exposed to high glucose (BMDM-HG-exo) or control. Infusions of BMDM-HG-exo increased hematopoiesis, circulating myeloid cell numbers, and atherosclerotic lesions with an accumulation of macrophage foam and apoptotic cells. Transcriptome-wide analysis of cultured macrophages treated with BMDM-HG-exo or plasma EVs isolated from subjects with type II diabetes revealed a reduced inflammatory state and increased metabolic activity. Furthermore, BMDM-HG-exo induced cell proliferation and reprogrammed energy metabolism by increasing glycolytic activity. Lastly, profiling microRNA in BMDM-HG-exo and plasma EVs from diabetic subjects with advanced atherosclerosis converged on miR-486-5p as commonly enriched and recognized in dysregulated hematopoiesis and Abca1 control. Together, our findings show that EVs serve to communicate detrimental properties of hyperglycemia to accelerate atherosclerosis in diabetes.

    View details for DOI 10.1016/j.isci.2021.102847

    View details for PubMedID 34381972

  • iPSC-derived neoantigen-specific cytotoxic T-lymphocyte therapy for Ewing sarcoma. Cancer immunology research Ishii, M., Ando, J., Yamazaki, S., Toyota, T., Ohara, K., Furukawa, Y., Suehara, Y., Nakanishi, M., Nakashima, K., Ohshima, K., Nakauchi, H., Ando, M. 2021


    The prognosis of Ewing sarcoma caused by EWS/FLI1 fusion is poor, especially after metastasis. Although therapy with cytotoxic T lymphocytes (CTLs) targeted against altered EWS/FLI1 sequences at the gene break/fusion site may be effective, CTLs generated from peripheral blood are often exhausted because of continuous exposure to tumor antigens. We addressed this by generating induced pluripotent stem cell (iPSC)-derived functionally rejuvenated CTLs (rejTs) directed against the neoantigen encoded by the EWS/FLI1 fusion gene. In this study, we examined the antitumor effects of EWS/FLI1-rejTs against Ewing sarcoma. The altered amino acid sequence at the break/fusion point of EWS/FLI1, when presented as a neoantigen, evokes an immune response that targets EWS/FLI1+ sarcoma. Although the frequency of generated EWS/FLI1-specific CTLs was only 0.003%, we successfully established CTL clones from a healthy donor. We established iPSCs from an EWS/FLI1-specific CTL clone and redifferentiated them into EWS/FLI1-specific rejTs. To evaluate cytotoxicity, we cocultured EWS/FLI1-rejTs with Ewing sarcoma cell lines. EWS/FLI1-rejTs rapidly and continuously suppressed the proliferation of Ewing sarcoma for >40 hours. Using an Ewing sarcoma xenograft mouse model, we verified the antitumor effect of EWS/FLI1-rejTs via imaging, and EWS/FLI1-rejTs conferred a statistically significant survival advantage. "Off-the-shelf" therapy is less destructive and disruptive than chemotherapy, and radiation is always desirable, particularly in adolescents, whom Ewing sarcoma most often affects. Thus, EWS/FLI1-rejTs targeting an Ewing sarcoma neoantigen could be a promising new therapeutic tool.

    View details for DOI 10.1158/2326-6066.CIR-21-0193

    View details for PubMedID 34385178

  • Feasibility of large experimental animal models in testing novel therapeutic strategies for diabetes. World journal of diabetes Nagaya, M., Hasegawa, K., Uchikura, A., Nakano, K., Watanabe, M., Umeyama, K., Matsunari, H., Osafune, K., Kobayashi, E., Nakauchi, H., Nagashima, H. 2021; 12 (4): 306–30


    Diabetes is among the top 10 causes of death in adults and caused approximately four million deaths worldwide in 2017. The incidence and prevalence of diabetes is predicted to increase. To alleviate this potentially severe situation, safer and more effective therapeutics are urgently required. Mice have long been the mainstay as preclinical models for basic research on diabetes, although they are not ideally suited for translating basic knowledge into clinical applications. To validate and optimize novel therapeutics for safe application in humans, an appropriate large animal model is needed. Large animals, especially pigs, are well suited for biomedical research and share many similarities with humans, including body size, anatomical features, physiology, and pathophysiology. Moreover, pigs already play an important role in translational studies, including clinical trials for xenotransplantation. Progress in genetic engineering over the past few decades has facilitated the development of transgenic animals, including porcine models of diabetes. This article discusses features that attest to the attractiveness of genetically modified porcine models of diabetes for testing novel treatment strategies using recent technical advances.

    View details for DOI 10.4239/wjd.v12.i4.306

    View details for PubMedID 33889282

  • Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice. Nature communications Wilkinson, A. C., Dever, D. P., Baik, R., Camarena, J., Hsu, I., Charlesworth, C. T., Morita, C., Nakauchi, H., Porteus, M. H. 2021; 12 (1): 686


    CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

    View details for DOI 10.1038/s41467-021-20909-x

    View details for PubMedID 33514718

  • Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats. Nature communications Kobayashi, T. n., Goto, T. n., Oikawa, M. n., Sanbo, M. n., Yoshida, F. n., Terada, R. n., Niizeki, N. n., Kajitani, N. n., Kazuki, K. n., Kazuki, Y. n., Hochi, S. n., Nakauchi, H. n., Surani, M. A., Hirabayashi, M. n. 2021; 12 (1): 1328


    Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.

    View details for DOI 10.1038/s41467-021-21557-x

    View details for PubMedID 33637711

  • ISSCR Guidelines for Stem Cell Research and Clinical Translation: The 2021 update. Stem cell reports Lovell-Badge, R., Anthony, E., Barker, R. A., Bubela, T., Brivanlou, A. H., Carpenter, M., Charo, R. A., Clark, A., Clayton, E., Cong, Y., Daley, G. Q., Fu, J., Fujita, M., Greenfield, A., Goldman, S. A., Hill, L., Hyun, I., Isasi, R., Kahn, J., Kato, K., Kim, J. S., Kimmelman, J., Knoblich, J. A., Mathews, D., Montserrat, N., Mosher, J., Munsie, M., Nakauchi, H., Naldini, L., Naughton, G., Niakan, K., Ogbogu, U., Pedersen, R., Rivron, N., Rooke, H., Rossant, J., Round, J., Saitou, M., Sipp, D., Steffann, J., Sugarman, J., Surani, A., Takahashi, J., Tang, F., Turner, L., Zettler, P. J., Zhai, X. 2021


    The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.

    View details for DOI 10.1016/j.stemcr.2021.05.012

    View details for PubMedID 34048692

  • ISSCR guidelines for the transfer of human pluripotent stem cells and their direct derivatives into animal hosts. Stem cell reports Hyun, I., Clayton, E. W., Cong, Y., Fujita, M., Goldman, S. A., Hill, L. R., Monserrat, N., Nakauchi, H., Pedersen, R. A., Rooke, H. M., Takahashi, J., Knoblich, J. A. 2021


    The newly revised 2021 ISSCR Guidelines for Stem Cell Research and Clinical Translation includes scientific and ethical guidance for the transfer of human pluripotent stem cells and their direct derivatives into animal models. In this white paper, the ISSCR subcommittee that drafted these guidelines for research involving the use of nonhuman embryos and postnatal animals explains and summarizes their recommendations.

    View details for DOI 10.1016/j.stemcr.2021.05.005

    View details for PubMedID 34048695

  • Polyvinyl alcohol hydrolysis rate and molecular weight influence human and murine HSC activity ex vivo. Stem cell research Sudo, K., Yamazaki, S., Wilkinson, A. C., Nakauchi, H., Nakamura, Y. 2021; 56: 102531


    Ex vivo expansion of hematopoietic stem cells (HSCs) is one of the most promising strategies to increase the availability of transplantable HSCs and improve bone marrow transplantation outcomes. We recently demonstrated that mouse HSCs could be efficiently expanded in polyvinyl alcohol (PVA)-containing culture medium using only recombinant stem cell factor and thrombopoietin cytokines. However, the behavior of human HSCs in these simple PVA-based media was not fully elucidated. In this study, we analyzed the compatibility of PVA of different hydrolysis rates (HR) and molecular weights (MW) to support functional human and mouse HSCs ex vivo. Human and mouse HSCs proliferated more frequently in media containing PVA with lower HR than with higher HR, but both PVA types supported HSC multilineage reconstitution potential. Importantly, human HSCs cultured in PVA-containing media engrafted not only in irradiated recipients but also in non-irradiated recipients. Our results demonstrate that human HSCs can be maintained ex vivo using PVA-based culture systems and suggest approaches for future optimization of human HSC expansion.

    View details for DOI 10.1016/j.scr.2021.102531

    View details for PubMedID 34509158

  • Genetically engineered pigs manifesting pancreatic agenesis with severe diabetes. BMJ open diabetes research & care Nagaya, M., Hasegawa, K., Watanabe, M., Nakano, K., Okamoto, K., Yamada, T., Uchikura, A., Osafune, K., Yokota, H., Nagaoka, T., Matsunari, H., Umeyama, K., Kobayashi, E., Nakauchi, H., Nagashima, H. 2020; 8 (2)


    INTRODUCTION: Pancreatic duodenum homeobox 1 (Pdx1) expression is crucial for pancreatic organogenesis and is a key regulator of insulin gene expression. Hairy and enhancer of split 1 (Hes1) controls tissue morphogenesis by maintaining undifferentiated cells. Hes1 encodes a basic helix loop helix (bHLH) transcriptional repressor and functionally antagonizes positive bHLH genes, such as the endocrine determination gene neurogenin-3. Here, we generated a new pig model for diabetes by genetic engineering Pdx1 and Hes1 genes.RESEARCH DESIGN AND METHODS: A transgenic (Tg) chimera pig with germ cells carrying a construct expressing Hes1 under the control of the Pdx1 promoter was used to mate with wild-type gilts to obtain Tg piglets.RESULTS: The Tg pigs showed perinatal death; however, this phenotype could be rescued by insulin treatment. The duodenal and splenic lobes of the Tg pigs were slender and did not fully develop, whereas the connective lobe was absent. beta cells were not detected, even in the adult pancreas, although other endocrine cells were detected, and exocrine cells functioned normally. The pigs showed no irregularities in any organs, except diabetes-associated pathological alterations, such as retinopathy and renal damage.CONCLUSION: Pdx1-Hes1 Tg pigs were an attractive model for the analysis of pancreatic development and testing of novel treatment strategies for diabetes.

    View details for DOI 10.1136/bmjdrc-2020-001792

    View details for PubMedID 33257422

  • In vivo clonal analysis of aging hematopoietic stem cells. Mechanisms of ageing and development Yamamoto, R., Nakauchi, H. 2020: 111378


    Hematopoietic stem cells (HSCs) are characterized by two key features: Self-renewal ability and multilineage differentiation potential (multipotentiality). With aging, these key features gradually change. This is thought to be related to hematological diseases. However, clonal in vivo analysis assessing the potential of HSCs to differentiate along erythroid and platelet lineages ("five-lineage tracing") has not been performed in the aged bone marrow. By contrast, in young HSCs clonal in vivo analysis combined with five-lineage tracing has provided us with novel insights into HSC biology. Understanding HSC aging at the clonal level will help us to elucidate aging mechanisms and disease progression. We review recent progress towards understanding HSC aging at the clonal cell level in the transplantation setting.

    View details for DOI 10.1016/j.mad.2020.111378

    View details for PubMedID 33022333

  • CAS9-AAV6 GENE CORRECTION OF AUTOLOGOUS HSCS IMPROVES SICKLE CELL DISEASE ERYTHROPOIESIS IN MICE Wilkinson, A., Dever, D., Baik, R., Hsu, I., Camarena, J., Charlesworth, C., Morita, C., Nakauchi, H., Porteus, M. ELSEVIER SCIENCE INC. 2020: S52
  • Sufficiency for inducible Caspase-9 safety switch in human pluripotent stem cells and disease cells. Gene therapy Nishimura, T., Xu, H., Iwasaki, M., Karigane, D., Saavedra, B., Takahashi, Y., Suchy, F. P., Monobe, S., Martin, R. M., Ohtaka, M., Nakanishi, M., Burrows, S. R., Cleary, M. L., Majeti, R., Shibuya, A., Nakauchi, H. 2020


    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promising potential for opening new avenues in regenerative medicine. However, since the tumorigenic potential of undifferentiated pluripotent stem cells (PSCs) is a major safety concern for clinical transplantation, inducible Caspase-9 (iC9) is under consideration for use as a fail-safe system. Here, we used targeted gene editing to introduce the iC9 system into human iPSCs, and then interrogated the efficiency of inducible apoptosis with normal iPSCs as well as diseased iPSCs derived from patients with acute myeloid leukemia (AML-iPSCs). The iC9 system induced quick and efficient apoptosis to iPSCs in vitro. More importantly, complete eradication of malignant cells without AML recurrence was shown in disease mouse models by using AML-iPSCs. In parallel, it shed light on several limitations of the iC9 system usage. Our results suggest that careful use of the iC9 system will serve as an important countermeasure against posttransplantation adverse events in stem cell transplantation therapies.

    View details for DOI 10.1038/s41434-020-0179-z

    View details for PubMedID 32704085

  • Sustainable Tumor-Suppressive Effect of iPSC-Derived Rejuvenated T Cells Targeting Cervical Cancers. Molecular therapy : the journal of the American Society of Gene Therapy Honda, T., Ando, M., Ando, J., Ishii, M., Sakiyama, Y., Ohara, K., Toyota, T., Ohtaka, M., Masuda, A., Terao, Y., Nakanishi, M., Nakauchi, H., Komatsu, N. 2020


    Immunotherapy utilizing induced pluripotent stem cell (iPSC) technology has great potential. Functionally rejuvenated cytotoxic T lymphocytes (CTLs) can survive long-term as young memory Tcells invivo, with continuous tumor eradication. Banking of iPSCs as an unlimited "off-the-shelf" source of therapeutic Tcells may be feasible. To generate safer iPSCs, we reprogrammed human papilloma virus type 16 (HPV16) E6-specific CTLs by Sendai virus vector without cotransduction of SV40 large T antigen. The iPSCs efficiently differentiated into HPV16-specific rejuvenated CTLs that demonstrated robust cytotoxicity against cervical cancer. The tumor-suppressive effect of rejuvenated CTLs was stronger and more persistent than that of original peripheral blood CTLs. These rejuvenated HPV16-specific CTLs provide a sustained tumor-suppressive effect even for epithelial cancers and constitute promising immunotherapy for cervical cancer.

    View details for DOI 10.1016/j.ymthe.2020.07.004

    View details for PubMedID 32710827

  • Stabilizing hematopoietic stem cells in vitro. Current opinion in genetics & development Wilkinson, A. C., Nakauchi, H. 2020; 64: 1–5


    Hematopoietic stem cells (HSCs) can regenerate all lineages of the adult blood and immune systems long-term following transplantation via a combination of self-renewal and multipotent differentiation. HSCs are therefore an important cell type in both basic research and in the clinic, where HSC transplantation is a curative therapy for a range of diseases. However, as a rare bone marrow cell population, the characterization and collection of HSCs can often be challenging. This has led to a large search for in vitro culture conditions that support the growth of functional HSCs and the in vitro stabilization of the HSC state represents a major goal in the field. Here, we review recent progress towards stabilizing HSCs in vitro.

    View details for DOI 10.1016/j.gde.2020.05.035

    View details for PubMedID 32570191

  • Stepwise strategy for generating osteoblasts from human pluripotent stem cells under fully defined xeno-free conditions with small - molecule inducers REGENERATIVE THERAPY Zujur, D., Kanke, K., Onodera, S., Tani, S., Lai, J., Azuma, T., Xin, X., Lichtler, A. C., Rowe, D. W., Saito, T., Tanaka, S., Masaki, H., Nakauchi, H., Chung, U., Hojo, H., Ohba, S. 2020; 14: 19–31
  • Hedgehog Activation Regulates Human Osteoblastogenesis. Stem cell reports Onodera, S., Saito, A., Hojo, H., Nakamura, T., Zujur, D., Watanabe, K., Morita, N., Hasegawa, D., Masaki, H., Nakauchi, H., Nomura, T., Shibahara, T., Yamaguchi, A., Chung, U., Azuma, T., Ohba, S. 2020


    Two genetic diseases, Gorlin syndrome and McCune-Albright syndrome (MAS), show completely opposite symptoms in terms of bone mineral density and hedgehog (Hh) activity. In this study, we utilized human induced pluripotent stem cell (iPSC)-based models of the two diseases to understand the roles of Hh signaling in osteogenesis. Gorlin syndrome-derived iPSCs showed increased osteoblastogenesis and mineralization with Hh signaling activation and upregulation of a set of transcription factors in an osteogenic culture, compared with the isogenic control. MAS-specific iPSCs showed poor mineralization with low Hh signaling activity in the osteogenic culture; impaired osteoblastogenesis was restored to the normal level by treatment with an Hh signaling-activating small molecule. These data suggest that Hh signaling is a key controller for differentiation of osteoblasts from precursors. This study may pave a path to new drug therapies for genetic abnormalities in calcification caused by dysregulation of Hh signaling.

    View details for DOI 10.1016/j.stemcr.2020.05.008

    View details for PubMedID 32531191

  • In vivo and ex vivo haematopoietic stem cell expansion. Current opinion in hematology Yamamoto, R., Wilkinson, A. C., Nakauchi, H. 2020


    PURPOSE OF REVIEW: Haematopoietic stem cells (HSCs) are characterized by two key features: self-renewal ability and multilineage differentiation potential. Through these cellular activities, HSCs sustain blood and immune system homeostasis throughout life and can also reconstitute the entire haematopoietic system within a bone marrow ablated recipient. This approach of HSC transplantation is used clinically as a curative treatment option for numerous haematological diseases, both malignant and nonmalignant.RECENT FINDINGS: Elucidation of the mechanism of HSC expansion represents a major focus within haematology. Here, we review the recent progress towards understanding HSC expansion in vivo and ex vivo, including a discussion of recent clonal transplantation assays and the development of novel ex vivo culture systems.SUMMARY: Recent findings provide exciting promise for improving the safety and efficacy of current HSC-based therapies as well as for the development of new therapeutic paradigms.

    View details for DOI 10.1097/MOH.0000000000000593

    View details for PubMedID 32452877

  • Vasoactive Intestinal Peptide Derived From Liver Mesenchymal Cells Mediates Tight Junction Assembly in Mouse Intrahepatic Bile Ducts. Hepatology communications Sato, A., Kakinuma, S., Miyoshi, M., Kamiya, A., Tsunoda, T., Kaneko, S., Tsuchiya, J., Shimizu, T., Takeichi, E., Nitta, S., Kawai-Kitahata, F., Murakawa, M., Itsui, Y., Nakagawa, M., Azuma, S., Koshikawa, N., Seiki, M., Nakauchi, H., Asahina, Y., Watanabe, M. 2020; 4 (2): 235-254


    Formation of intrahepatic bile ducts (IHBDs) proceeds in accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate the differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, further studies are needed to fully understand the arrangement of IHBDs into a continuous hierarchical network. This study aims to clarify the interaction between biliary and liver mesenchymal cells during IHBD formation. To identify candidate factors contributing to this cell-cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)-deficient (knockout [KO]) mice livers, in which IHBD formation is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the formation of luminal structures comprised of hepatoblast-derived cholangiocytes (cholangiocytic cysts), whereas MMP14-KO mesenchymal cells failed to promote cyst formation. Comprehensive analysis revealed that expression of vasoactive intestinal peptide (VIP) was significantly suppressed in MMP14-KO mesenchymal cells. VIP and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD development, respectively, in vivo. VIP/VIPR1 signaling significantly encouraged cholangiocytic cyst formation and up-regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly suppressed the tight junction assembly and the up-regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenous VIP administration promoted the restoration of damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP is produced by periportal mesenchymal cells during the perinatal stage. It supports bile duct development by establishing tight junctions and up-regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery from cholestatic damage through the establishment of tight junctions in the bile ducts.

    View details for DOI 10.1002/hep4.1459

    View details for PubMedID 32025608

    View details for PubMedCentralID PMC6996346

  • Long-term ex vivo expansion of mouse hematopoietic stem cells. Nature protocols Wilkinson, A. C., Ishida, R., Nakauchi, H., Yamazaki, S. 2020


    Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout life. However, the mechanism behind such remarkable abilities remains undiscovered, at least in part because of the paucity of HSCs and the modest ex vivo expansion of HSCs in media that contain poorly defined albumin supplements such as bovine serum albumin. Here, we describe a simple platform for the expansion of functional mouse HSCs ex vivo for >1 month under fully defined albumin-free conditions. The culture system affords 236- to 899-fold expansion over the course of a month and is also amenable to clonal analysis of HSC heterogeneity. The large numbers of expanded HSCs enable HSC transplantation into nonconditioned recipients, which is otherwise not routinely feasible because of the large numbers of HSCs required. This protocol therefore provides a powerful approach with which to interrogate HSC self-renewal and lineage commitment and, more broadly, to study and characterize the hematopoietic and immune systems.

    View details for DOI 10.1038/s41596-019-0263-2

    View details for PubMedID 31915389

  • Germline development in rat revealed by visualization and deletion of Prdm14. Development (Cambridge, England) Kobayashi, T. n., Kobayashi, H. n., Goto, T. n., Takashima, T. n., Oikawa, M. n., Ikeda, H. n., Terada, R. n., Yoshida, F. n., Sanbo, M. n., Nakauchi, H. n., Kurimoto, K. n., Hirabayashi, M. n. 2020


    Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and a critical PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that Prdm14's crucial role in PGC specification is conserved between rat and mice, by analyzing Prdm14 deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program: failure in maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the postimplantation epiblast and all PGC stages in rat, to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional time-line for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved vs species-specific features for germline development in mammals.

    View details for DOI 10.1242/dev.183798

    View details for PubMedID 32001439

  • Germline development in rat revealed by visualization and deletion of Prdm14. Development (Cambridge, England) Kobayashi, T., Kobayashi, H., Goto, T., Takashima, T., Oikawa, M., Ikeda, H., Terada, R., Yoshida, F., Sanbo, M., Nakauchi, H., Kurimoto, K., Hirabayashi, M. 2020


    Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and a critical PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that Prdm14's crucial role in PGC specification is conserved between rat and mice, by analyzing Prdm14 deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program: failure in maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the postimplantation epiblast and all PGC stages in rat, to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional time-line for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved vs species-specific features for germline development in mammals.

    View details for DOI 10.1242/dev.183798

    View details for PubMedID 34004822

  • Author Correction: CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. Scientific reports Vilarino, M. n., Rashid, S. T., Suchy, F. P., McNabb, B. R., van der Meulen, T. n., Fine, E. J., Ahsan, S. D., Mursaliyev, N. n., Sebastiano, V. n., Diab, S. S., Huising, M. O., Nakauchi, H. n., Ross, P. J. 2020; 10 (1): 7500


    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    View details for DOI 10.1038/s41598-020-64443-0

    View details for PubMedID 32371904

  • Macrophage Exosomes Resolve Atherosclerosis by Regulating Hematopoiesis and Inflammation via MicroRNA Cargo. Cell reports Bouchareychas, L. n., Duong, P. n., Covarrubias, S. n., Alsop, E. n., Phu, T. A., Chung, A. n., Gomes, M. n., Wong, D. n., Meechoovet, B. n., Capili, A. n., Yamamoto, R. n., Nakauchi, H. n., McManus, M. T., Carpenter, S. n., Van Keuren-Jensen, K. n., Raffai, R. L. 2020; 32 (2): 107881


    Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery.

    View details for DOI 10.1016/j.celrep.2020.107881

    View details for PubMedID 32668250

  • Stepwise strategy for generating osteoblasts from human pluripotent stem cells under fully defined xeno-free conditions with small-molecule inducers. Regenerative therapy Zujur, D. n., Kanke, K. n., Onodera, S. n., Tani, S. n., Lai, J. n., Azuma, T. n., Xin, X. n., Lichtler, A. C., Rowe, D. W., Saito, T. n., Tanaka, S. n., Masaki, H. n., Nakauchi, H. n., Chung, U. I., Hojo, H. n., Ohba, S. n. 2020; 14: 19–31


    Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

    View details for DOI 10.1016/j.reth.2019.12.010

    View details for PubMedID 31988991

    View details for PubMedCentralID PMC6965656

  • Haematopoietic stem cell self-renewal in vivo and ex vivo. Nature reviews. Genetics Wilkinson, A. C., Igarashi, K. J., Nakauchi, H. n. 2020


    The self-renewal capacity of multipotent haematopoietic stem cells (HSCs) supports blood system homeostasis throughout life and underlies the curative capacity of clinical HSC transplantation therapies. However, despite extensive characterization of the HSC state in the adult bone marrow and embryonic fetal liver, the mechanism of HSC self-renewal has remained elusive. This Review presents our current understanding of HSC self-renewal in vivo and ex vivo, and discusses important advances in ex vivo HSC expansion that are providing new biological insights and offering new therapeutic opportunities.

    View details for DOI 10.1038/s41576-020-0241-0

    View details for PubMedID 32467607

  • Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion. Experimental hematology Nishimura, T., Hsu, I., Martinez-Krams, D. C., Nakauchi, Y., Majeti, R., Yamazaki, S., Nakauchi, H., Wilkinson, A. C. 2019


    Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.

    View details for DOI 10.1016/j.exphem.2019.11.007

    View details for PubMedID 31874780

  • Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation. Stem cell reports Matsunari, H., Watanabe, M., Hasegawa, K., Uchikura, A., Nakano, K., Umeyama, K., Masaki, H., Hamanaka, S., Yamaguchi, T., Nagaya, M., Nishinakamura, R., Nakauchi, H., Nagashima, H. 2019


    We have previously established a concept of developing exogenic pancreas in a genetically modified pig fetus with an apancreatic trait, thereby proposing the possibility of invivo generation of functional human organs in xenogenic large animals. In this study, we aimed to demonstrate a further proof-of-concept of the compensation for disabled organogeneses in pig, including pancreatogenesis, nephrogenesis, hepatogenesis, and vasculogenesis. These dysorganogenetic phenotypes could be efficiently induced via genome editing of the cloned pigs. Induced dysorganogenetic traits could also be compensated by allogenic blastocyst complementation, thereby proving the extended concept of organ regeneration from exogenous pluripotent cells in empty niches during various organogeneses. These results suggest that the feasibility of blastocyst complementation using genome-edited cloned embryos permits experimentation toward the invivo organ generation in pigs from xenogenic pluripotent cells.

    View details for DOI 10.1016/j.stemcr.2019.11.008

    View details for PubMedID 31883918

  • Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds. Stem cell reports Kawata, M., Mori, D., Kanke, K., Hojo, H., Ohba, S., Chung, U., Yano, F., Masaki, H., Otsu, M., Nakauchi, H., Tanaka, S., Saito, T. 2019


    A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5-9days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues invivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and beta-catenin association with DNA showed that retinoic acid and Wnt/beta-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an invitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation.

    View details for DOI 10.1016/j.stemcr.2019.07.012

    View details for PubMedID 31402337

  • Long-term eradication of extranodal NK/T cell lymphoma, nasal type, by induced pluripotent stem cell-derived Epstein-Barr virus-specific rejuvenated T cells in vivo. Haematologica Ando, M., Ando, J., Yamazaki, S., Ishii, M., Sakiyama, Y., Harada, S., Honda, T., Yamaguchi, T., Nojima, M., Ohshima, K., Nakauchi, H., Komatsu, N. 2019


    Functionally rejuvenated induced pluripotent stem cell-derived antigen-specific cytotoxic T lymphocytes are expected to be potent immunotherapy for tumors. When L-asparaginase-containing standard chemotherapy fails in extranodal NK/T cell lymphoma, nasal type, no effective salvage therapy exists. The clinical course then is miserable. We demonstrate prolonged and robust eradication of extranodal NK/T cell lymphoma, nasal type, in vivo by Epstein-Barr virus-specific induced pluripotent stem cell-derived antigen-specific cytotoxic T lymphocytes, with induced pluripotent stem cell-derived antigen-specific cytotoxic T lymphocytes persisting as central memory T cells in mouse spleen for at least 6 months. The anti-tumor response is so strong that any concomitant effect of PD-1 blockade is unclear. These results suggest that long-term persistent Epstein-Barr virus-specific induced pluripotent stem cell-derived antigen-specific cytotoxic T lymphocytes contribute to continuous anti-tumor effect and offer an effective salvage therapy for relapsed and refractory extranodal NK/T cell lymphoma, nasal type.

    View details for DOI 10.3324/haematol.2019.223511

    View details for PubMedID 31296577

  • CRISPR/Cas9 BIO-PROTOCOL Mizuno, N., Mizutani, E., Sato, H., Kasai, M., Nakauchi, H., Yamaguchi, T. 2019; 9 (13)
  • CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice. Bio-protocol Mizuno, N., Mizutani, E., Sato, H., Kasai, M., Nakauchi, H., Yamaguchi, T. 2019; 9 (13): e3295


    Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos' genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation embryos with zona pellucida, glycoprotein membrane surrounding early embryos. We found adeno-associated virus (AAV) can infect zygotes of various mammals through intact zona pellucida. AAV-mediated donor DNA delivery following Cas9 ribonucleoprotein electroporation enables large fragment knock-in without micromanipulation.

    View details for DOI 10.21769/BioProtoc.3295

    View details for PubMedID 33654808

    View details for PubMedCentralID PMC7854279

  • Loss of fibrocystin promotes interleukin-8-dependent proliferation and CTGF production of biliary epithelium JOURNAL OF HEPATOLOGY Tsunoda, T., Kakinuma, S., Miyoshi, M., Kamiya, A., Kaneko, S., Sato, A., Tsuchiya, J., Nitta, S., Kawai-Kitahata, F., Murakawa, M., Itsui, Y., Nakagawa, M., Azuma, S., Sogo, T., Komatsu, H., Mukouchi, R., Inui, A., Fujisawa, T., Nakauchi, H., Asahina, Y., Watanabe, M. 2019; 71 (1): 143–52
  • Anephrogenic phenotype induced by SALL1 gene knockout in pigs. Scientific reports Watanabe, M., Nakano, K., Uchikura, A., Matsunari, H., Yashima, S., Umeyama, K., Takayanagi, S., Sakuma, T., Yamamoto, T., Morita, S., Horii, T., Hatada, I., Nishinakamura, R., Nakauchi, H., Nagashima, H. 2019; 9 (1): 8016


    To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1-KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1-mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.

    View details for DOI 10.1038/s41598-019-44387-w

    View details for PubMedID 31142767

  • Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination. Cell stem cell Martin, R. M., Ikeda, K., Cromer, M. K., Uchida, N., Nishimura, T., Romano, R., Tong, A. J., Lemgart, V. T., Camarena, J., Pavel-Dinu, M., Sindhu, C., Wiebking, V., Vaidyanathan, S., Dever, D. P., Bak, R. O., Laustsen, A., Lesch, B. J., Jakobsen, M. R., Sebastiano, V., Nakauchi, H., Porteus, M. H. 2019; 24 (5): 821


    Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

    View details for PubMedID 31051134

  • LIM homeobox 2 promotes interaction between human iPS-derived hepatic progenitors and iPS-derived hepatic stellate-like cells. Scientific reports Miyoshi, M., Kakinuma, S., Kamiya, A., Tsunoda, T., Tsuchiya, J., Sato, A., Kaneko, S., Nitta, S., Kawai-Kitahata, F., Murakawa, M., Itsui, Y., Nakagawa, M., Azuma, S., Nakauchi, H., Asahina, Y., Watanabe, M. 2019; 9 (1): 2072


    Human induced pluripotent stem (iPS) cells can differentiate into hepatocyte lineages, although the phenotype of the differentiated cells is immature compared to adult hepatocytes. Improvement of cell-cell interactions between epithelium and mesenchyme is a potential approach to address this phenotype issue. In this study, we developed a model system for improving interactions between human iPS-derived hepatic progenitor cells (iPS-HPCs) and human iPS-derived hepatic stellate cell-like cells (iPS-HSCs). The phenotype of iPS-HSCs, including gene and protein expression profiles and vitamin A storage, resembled that of hepatic stellate cells. Direct co-culture of iPS-HSCs with iPS-HPCs significantly improved hepatocytic maturation in iPS-HPCs, such as their capacity for albumin production. Next, we generated iPS cell lines overexpressing LIM homeobox 2 (LHX2), which suppresses myofibroblastic changes in HSCs in mice. Hepatocytic maturation in iPS-HPCs was significantly increased in direct co-culture with iPS-HSCs overexpressing LHX2, but not in co-culture with a human hepatic stellate cell line (LX-2) overexpressing LHX2. LHX2 regulated the expression of extracellular matrices, such as laminin and collagen, in iPS-HSCs. In conclusion, this study provides an evidence that LHX2 upregulation in iPS-HSCs promotes hepatocytic maturation of iPS-HPCs, and indicates that genetically modified iPS-HSCs will be of value for research into cell-cell interactions.

    View details for PubMedID 30765795

  • Generation of pluripotent stem cell-derived mouse kidneys in Sall1-targeted anephric rats. Nature communications Goto, T., Hara, H., Sanbo, M., Masaki, H., Sato, H., Yamaguchi, T., Hochi, S., Kobayashi, T., Nakauchi, H., Hirabayashi, M. 2019; 10 (1): 451


    Regeneration of human kidneys in animal models would help combat the severe shortage of donors in transplantation therapy. Previously, we demonstrated by interspecific blastocyst complementation between mouse and rats, generation of pluripotent stem cell (PSC)-derived functional pancreas, in apancreatic Pdx1 mutant mice. We, however, were unable to obtain rat PSC-derived kidneys in anephric Sall1 mutant mice, likely due to the poor contribution of rat PSCs to the mouse metanephric mesenchyme, a nephron progenitor. Here, conversely, we show that mouse PSCs can efficiently differentiate into the metanephric mesenchyme in rat, allowing the generation of mouse PSC-derived kidney in anephric Sall1 mutant rat. Glomerular epithelium and renal tubules in the kidneys are entirely composed of mouse PSC-derived cells expressing key functional markers. Importantly, the ureter-bladder junction is normally formed. These data provide proof-of-principle for interspecific blastocyst complementation as a viable approach for kidney generation.

    View details for PubMedID 30723213

  • Author Correction: Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature Wilkinson, A. C., Ishida, R. n., Kikuchi, M. n., Sudo, K. n., Morita, M. n., Crisostomo, R. V., Yamamoto, R. n., Loh, K. M., Nakamura, Y. n., Watanabe, M. n., Nakauchi, H. n., Yamazaki, S. n. 2019


    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

    View details for DOI 10.1038/s41586-019-1395-9

    View details for PubMedID 31289376

  • Human iPSC Generation from Antigen-Specific T Cells. Methods in molecular biology (Clifton, N.J.) Nishimura, T., Murmann, Y., Nakauchi, H. 2019; 2048: 53–57


    The discovery and development of induced pluripotent stem cells (iPSCs) opened a novel venue for disease modeling, drug discovery, and personalized medicine. Additionally, iPSCs have been utilized for a wide variety of research and clinical applications without immunological and ethical concerns that encounter embryonic stem cells. Adoptive T cell immunotherapy is a form of cellular immunotherapy that involves transfusion of functional T cells. However, this approach requires T cell expansion and the process causes T cell exhaustion. As a result, highly expanded T cells have not proven to be particularly effective for treatments. This exhaustion issue could be overcome due to rejuvenation of T cells by reprogramming to pluripotency and redifferentiation to T cells. This is a potential therapeutic strategy for combating various types of cancer.

    View details for DOI 10.1007/978-1-4939-9728-2_5

    View details for PubMedID 31396928

  • Using the Inducible Caspase-9 Suicide-Safeguard System with iPSC and Bioluminescent Tracking. Methods in molecular biology (Clifton, N.J.) Villanueva, J., Nishimura, T., Nakauchi, H. 2019; 2048: 259–64


    For scientists working within the field of induced pluripotent stem cells (iPSCs), this protocol will provide a thorough walk-through on how to conduct in vitro and in vivo experiments that validate the function of a particular safeguard system technology. In short, we provide instructions on how to generate inducible Caspase-9 (iC9) safeguard system with human iPSCs that act as normal or abnormal models of the cells for therapeutics to be tried after differentiation. These iC9-iPSCs should be modified prior to beginning this protocol by constitutively expressing luciferase, an enzyme capable of generating bioluminescent signals through the oxidation of the substrate luciferin. Monitoring the bioluminescent signal over time provides the information on whether a safeguard system is working or not.

    View details for DOI 10.1007/978-1-4939-9728-2_20

    View details for PubMedID 31396943

  • Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells. Nature medicine Mori, M. n., Furuhashi, K. n., Danielsson, J. A., Hirata, Y. n., Kakiuchi, M. n., Lin, C. S., Ohta, M. n., Riccio, P. n., Takahashi, Y. n., Xu, X. n., Emala, C. W., Lu, C. n., Nakauchi, H. n., Cardoso, W. V. 2019


    Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation1. Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange2,3. Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1cnull mutation) or expand (due to Fgfr2cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions4-6.

    View details for DOI 10.1038/s41591-019-0635-8

    View details for PubMedID 31700187

  • Generation of Antigen-Specific T Cells from Human Induced Pluripotent Stem Cells. Methods in molecular biology (Clifton, N.J.) Nishimura, T., Nakauchi, H. 2019; 1899: 25–40


    Human induced pluripotent stem cells (iPSCs) are a potential source of blood cells for transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. The development of widely varying reprogramming methods has enabled us nowadays to obtain iPSCs even from a small number of antigen-specific T cells from patients. As these T-cell-derived iPSCs (T-iPSCs) carry TCR gene rearrangements in their genomic DNA, they are likely useful for producing antigen-specific T cells and for studying T-cell development. T-cell immunotherapy is potentially an effective therapeutic strategy against many types of cancers and viral infections. If antigen-specific T cells tailored against diseases and for patients can be easily obtained, T-cell immunotherapy should become a popular choice of therapy. Here, we show the in vitro way to guide T-iPSCs sequentially to yield hematopoietic stem/progenitor cells (HSPCs), T-lineage cells, and mature CD8 single-positive T cells. These in vitro-generated CD8+ T cells display antigen-specific cytotoxity and perform general T-cell functions. This novel protocol thus provides means to generate antigen-specific T cells as well as chances to study normal human lymphopoiesis. It may help identify, and then clear away, barriers to T-cell immunotherapy such as immunological tolerance and cell exhaustion. T-iPSCs can confer their juvenile status upon their descendant T cells during pluripotency reprogramming and redifferentiation. This phenomenon should help to eliminate T-cell exhaustion.

    View details for PubMedID 30649763

  • 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)


    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

  • Single cell analysis of human foetal liver captures the transcriptional profile of hepatobiliary hybrid progenitors. Nature communications Segal, J. M., Kent, D. n., Wesche, D. J., Ng, S. S., Serra, M. n., Oulès, B. n., Kar, G. n., Emerton, G. n., Blackford, S. J., Darmanis, S. n., Miquel, R. n., Luong, T. V., Yamamoto, R. n., Bonham, A. n., Jassem, W. n., Heaton, N. n., Vigilante, A. n., King, A. n., Sancho, R. n., Teichmann, S. n., Quake, S. R., Nakauchi, H. n., Rashid, S. T. 2019; 10 (1): 3350


    The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM+ population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.

    View details for DOI 10.1038/s41467-019-11266-x

    View details for PubMedID 31350390

  • Efficient scarless genome editing in human pluripotent stem cells. Nature methods Ikeda, K., Uchida, N., Nishimura, T., White, J., Martin, R. M., Nakauchi, H., Sebastiano, V., Weinberg, K. I., Porteus, M. H. 2018; 15 (12): 1045–47


    Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.

    View details for PubMedID 30504872

  • Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice Kon, A., Yamazaki, S., Nannya, Y., Kataoka, K., Ota, Y., Nakagawa, M., Yoshida, K., Yoshizato, T., Sanada, M., Nakayama, M., Koseki, H., Nakauchi, H., Ogawa, S. WILEY. 2018: 364
  • Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector. iScience Mizuno, N., Mizutani, E., Sato, H., Kasai, M., Ogawa, A., Suchy, F., Yamaguchi, T., Nakauchi, H. 2018; 9: 286–97


    Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome editing have been established in only a small set of animals. To overcome these issues, we developed a method of large-fragment DNA knockin without micromanipulation. In this study, we successfully delivered the knockin donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large-DNA fragment knockin and whole exon exchange with electroporation of CRISPR/Cas9 ribonucleoprotein. By this method, we can exchange large DNA fragments conveniently in various animal species without micromanipulation.

    View details for PubMedID 30447647

  • Chimeric liver transplantation reveals interspecific graft remodelling JOURNAL OF HEPATOLOGY Oldani, G., Peloso, A., Vijgen, S., Wilson, E. M., Slits, F., Gex, Q., Morel, P., Delaune, V., Orci, L. A., Yamaguchi, T., Kobayashi, T., Rubbia-Brandt, L., Nakauchi, H., Lacotte, S., Toso, C. 2018; 69 (5): 1025–36


    A major limitation in the field of liver transplantation is the shortage of transplantable organs. Chimeric animals carrying human tissue have the potential to solve this problem. However, currently available chimeric organs retain a high level of xenogeneic cells, and the transplantation of impure organs needs to be tested.We created chimeric livers by injecting Lewis rat hepatocytes into C57Bl/6Fah-/-Rag2-/-Il2rg-/- mice, and further transplanted them into newly weaned Lewis rats (45 ± 3 g) with or without suboptimal immunosuppression (tacrolimus 0.6 mg/kg/day for 56 or 112 days). Control donors included wild-type C57Bl/6 mice (xenogeneic) and Lewis rats (syngeneic).Without immunosuppression, recipients of chimeric livers experienced acute rejection, and died within 8 to 11 days. With immunosuppression, they all survived for >112 days with normal weight gain compared to syngeneic controls, while all xenogeneic controls died within 98 days due to rejection with Banff scores >6 (p = 0.0014). The chimeric grafts underwent post-transplant remodelling, growing by 670% on average. Rat hepatocytes fully replaced mouse hepatocytes starting from day 56 (absence of detectable mouse serum albumin, histological clearance of mouse hepatocytes). In addition, rat albumin levels reached those of syngeneic recipients. Four months after transplantation of chimeric livers, we observed the development of diffuse mature rat bile ducts through transdifferentiation of hepatocytes (up to 72% of cholangiocytes), and patchy areas of portal endothelium originating from the host (seen in one out of five recipients).Taken together, these data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant recipient-oriented graft remodelling. Validation in a large animal model is still needed.Chimeric animals are composed of cells from different species. Chimeric animals carrying human tissue have the potential to increase the availability of transplantable organs. We transplanted rat-to-mouse liver grafts into newly weaned rats. The chimeric grafts underwent post-transplant remodelling with rat hepatocytes replacing all mouse hepatocytes within 56 days. In addition, we observed the post-transplant development of diffuse mature rat bile ducts through the transformation of hepatocytes, and patchy areas of portal endothelium originating from the host. These data demonstrate the efficacy of transplanting rat-to-mouse chimeric livers into rats, with a high potential for post-transplant graft remodelling.

    View details for PubMedID 30031887

  • Integrated Stress Response Activity Marks Stem Cells in Normal Hematopoiesis and Leukemia. Cell reports van Galen, P., Mbong, N., Kreso, A., Schoof, E. M., Wagenblast, E., Ng, S. W., Krivdova, G., Jin, L., Nakauchi, H., Dick, J. E. 2018; 25 (5): 1109


    Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Severe perturbations of cellular homeostasis result in rapid HSC loss to maintain clonal purity. However, normal homeostatic processes can also generate lower-level stress; how HSCs survive these conditions remains unknown. Here we show that the integrated stress response (ISR) is uniquely active in HSCs and facilitates their persistence. Activating transcription factor 4 (ATF4) mediates the ISR and is highly expressed in HSCs due to scarcity of the eIF2 translation initiation complex. Amino acid deprivation results in eIF2alpha phosphorylation-dependent upregulation of ATF4, promoting HSC survival. Primitive acute myeloid leukemia (AML) cells also display eIF2 scarcity and ISR activity marks leukemia stem cells (LSCs) in primary AML samples. These findings identify a link between the ISR and stem cell survival in the normal and leukemic contexts.

    View details for PubMedID 30380403

  • An interspecies barrier to tetraploid complementation and chimera formation SCIENTIFIC REPORTS Yamaguchi, T., Sato, H., Kobayashi, T., Kato-itoh, M., Goto, T., Hara, H., Mizuno, N., Yanagida, A., Umino, A., Hamanaka, S., Suchy, F., Masaki, H., Ota, Y., Hirabayashi, M., Nakauchi, H. 2018; 8: 15289


    To study development of the conceptus in xenogeneic environments, we assessed interspecies chimera formation as well as tetraploid complementation between mouse and rat. Overall contribution of donor PSC-derived cells was lower in interspecies chimeras than in intraspecies chimeras, and high donor chimerism was associated with anomalies or embryonic death. Organ to organ variation in donor chimerism was greater in interspecies chimeras than in intraspecies chimeras, suggesting species-specific affinity differences among interacting molecules necessary for organogenesis. In interspecies tetraploid complementation, embryo development was near normal until the stage of placental formation, after which no embryos survived.

    View details for PubMedID 30327488

  • Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation STEM CELL REPORTS Hamanaka, S., Umino, A., Sato, H., Hayama, T., Yanagida, A., Mizuno, N., Kobayashi, T., Kasai, M., Suchy, F., Yamazaki, S., Masaki, H., Yamaguchi, T., Nakauchi, H. 2018; 11 (4): 988–97


    In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived.

    View details for PubMedID 30245211

  • Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep. Transgenic research Vilarino, M., Suchy, F. P., Rashid, S. T., Lindsay, H., Reyes, J., McNabb, B. R., van der Meulen, T., Huising, M. O., Nakauchi, H., Ross, P. J. 2018


    The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR(ddPCR). Next, we determined the presence of mosaicism in~50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

    View details for PubMedID 30284144

  • Interspecies chimeras CURRENT OPINION IN GENETICS & DEVELOPMENT Suchy, F., Nakauchi, H. 2018; 52: 36–41
  • Generation of HIV-Resistant Macrophages from IPSCs by Using Transcriptional Gene Silencing and Promoter-Targeted RNA MOLECULAR THERAPY-NUCLEIC ACIDS Higaki, K., Hirao, M., Kawana-Tachikawa, A., Iriguchi, S., Kumagai, A., Ueda, N., Bo, W., Kamibayashi, S., Watanabe, A., Nakauchi, H., Suzuki, K., Kaneko, S. 2018; 12: 793–804


    Highly active antiretroviral therapy (HAART) has markedly prolonged the prognosis of HIV-1 patients. However, lifelong dependency on HAART is a continuing challenge, and an effective therapeutic is much desired. Recently, introduction of short hairpin RNA (shRNA) targeting the HIV-1 promoter was found to suppress HIV-1 replication via transcriptional gene silencing (TGS). The technology is expected to be applied with hemato-lymphopoietic cell transplantation of HIV patients to suppress HIV transcription in transplanted hemato-lymphopoietic cells. Combination of the TGS technology with new cell transplantation strategy with induced pluripotent stem cell (iPSC)-derived hemato-lymphopoietic cells might contribute to new gene therapy in the HIV field. In this study, we evaluated iPSC-derived macrophage functions and feasibility of TGS technology in macrophages. Human iPSCs were transduced with shRNAs targeting the HIV-1 promoter region (shPromA) by using a lentiviral vector. The shPromA-transfected iPSCs were successfully differentiated into functional macrophages, and they exhibited strong protection against HIV-1 replication with alteration in the histone structure of the HIV-1 promoter region to induce heterochromatin formation. These results indicated that iPS-derived macrophage is a useful tool to investigate HIV infection and protection, and that the TGS technology targeting the HIV promoter is a potential candidate of new gene therapy.

    View details for PubMedID 30141412

  • In vitro platform of allogeneic stem cell-derived cardiomyocyte transplantation for cardiac conduction defects EUROPACE Yoshida, A., Lee, J., Tomoyama, S., Miwa, K., Shirakawa, K., Hamanaka, S., Yamaguchi, T., Nakauchi, H., Miyagawa, S., Sawa, Y., Komuro, I., Sakata, Y. 2018; 20 (9): 1553–60


    The aim of the present study is to develop in vitro experimental analytical method for the electrophysiological properties of allogeneic induced pluripotent stem cell-derived cardiomyocytes (CMs) in cardiac conduction defect model.Cardiomyocytes were derived from rat induced pluripotent stem cells CMs (riPSC-CMs) using an embryoid body-based differentiation method with the serial application of growth factors including activin-A, bone morphogenetic protein 4 (BMP-4), and inhibitor of wnt production 2 (IWP-2). Flow cytometry analysis showed that 74.0 ± 2.7% of riPSC-CMs expressed cardiac troponin-T (n = 3). Immunostaining analysis revealed organized sarcomeric structure in riPSC-CMs and the expression of connexin 43 between riPSC-CMs and neonatal rat ventricular CMs (NRVMs). Ca2+ transient recordings revealed the simultaneous excitement of riPSC-CMs and NRVMs, and prolonged Ca2+ transient duration of riPSC-CMs as compared with NRVMs (731 ± 15.9 vs. 610 ± 7.72 ms, P < 0.01, n = 3). Isolated NRVMs were cultured in two discrete regions to mimic cardiac conduction defects on multi-electrode array dish, and riPSC-CMs were seeded in the channel between the two discrete regions. Membrane potential imaging with di-8-ANEPPS discerned the propagation of the electrical impulse from one NRVM region to the other through a riPSC-CM pathway. This pathway had significantly longer action potential duration as compared with NRVMs. Electrophysiological studies using a multi-electrode array platform demonstrated the longer conduction time and functional refractory period of the riPSC-CM pathway compared with the NRVM pathway.Using an in vitro experimental system to mimic cardiac conduction defect, transplanted allogeneic riPSC-CMs showed electrical coupling between two discrete regions of NRVMs. Electrophysiological testing using our platform will enable electrophysiological screening prior to transplantation of stem cell-derived CMs.

    View details for PubMedID 29554331

  • [iPSC-derived rejuvenated T-cell therapy for Epstein-Barr virus-associated lymphomas]. [Rinsho ketsueki] The Japanese journal of clinical hematology Ando, M., Nakauchi, H., Komatsu, N. 2018; 59 (7): 932-938


    Extranodal natural killer (NK) /T-cell lymphoma (ENKL), nasal-type, is an aggressive lymphoma that is relatively common in Asia and is resistant to anthracycline-based chemotherapy. Antigen-specific cytotoxic T-lymphocyte (CTL) therapy can induce durable remission in selected tumors, such as melanomas and virus-related tumors. ENKLs are commonly infected with the Epstein-Barr virus (EBV) ; therefore, CTL therapy can be effective for these lymphomas. However, CTLs that are continuously exposed to viral or tumor antigens usually get exhausted. Antigen-specific CTLs generated from iPSCs have a higher proliferative capacity and longer telomeres than original CTLs and are functionally rejuvenated (rejuvenated T cell, rejT). For clinical translation, the tumorigenic potential of iPSCs and the malignant transformation of differentiated iPSCs are major safety concerns. To address these issues, we introduced inducible caspase-9 (iC9) -based safeguard system into iPSCs. iC9-rejTs exert strong anti-tumor effects against EBV-infected tumors in vivo, and the iC9 suicide gene system provides a reliable safeguard for rejT therapy. We initiated a preclinical study using this novel immunotherapy for EBV-associated lymphoma. We believe that rejT therapy can be a potential safe approach as an "off-the-shelf" therapy for refractory ENKL in the future.

    View details for DOI 10.11406/rinketsu.59.932

    View details for PubMedID 30078805

  • Human iPS derived progenitors bioengineered into liver organoids using an inverted colloidal crystal poly (ethylene glycol) scaffold. Biomaterials Ng, S. S., Saeb-Parsy, K., Blackford, S. J., Segal, J. M., Serra, M. P., Horcas-Lopez, M., No, D. Y., Mastoridis, S., Jassem, W., Frank, C. W., Cho, N. J., Nakauchi, H., Glenn, J. S., Rashid, S. T. 2018; 182: 299–311


    Generation of human organoids from induced pluripotent stem cells (iPSCs) offers exciting possibilities for developmental biology, disease modelling and cell therapy. Significant advances towards those goals have been hampered by dependence on animal derived matrices (e.g. Matrigel), immortalized cell lines and resultant structures that are difficult to control or scale. To address these challenges, we aimed to develop a fully defined liver organoid platform using inverted colloid crystal (ICC) whose 3-dimensional mechanical properties could be engineered to recapitulate the extracellular niche sensed by hepatic progenitors during human development. iPSC derived hepatic progenitors (IH) formed organoids most optimally in ICC scaffolds constructed with 140 mum diameter pores coated with type I collagen in a two-step process mimicking liver bud formation. The resultant organoids were closer to adult tissue, compared to 2D and 3D controls, with respect to morphology, gene expression, protein secretion, drug metabolism and viral infection and could integrate, vascularise and function following implantation into livers of immune-deficient mice. Preliminary interrogation of the underpinning mechanisms highlighted the importance of TGFbeta and hedgehog signalling pathways. The combination of functional relevance with tuneable mechanical properties leads us to propose this bioengineered platform to be ideally suited for a range of future mechanistic and clinical organoid related applications.

    View details for PubMedID 30149262

  • Designing Motif-Engineered Receptors To Elucidate Signaling Molecules Important for Proliferation of Hematopoietic Stem Cells ACS SYNTHETIC BIOLOGY Ishizuka, S., Lai, C., Otsu, M., Nakauchi, H., Nagamune, T., Kawahara, M. 2018; 7 (7): 1709–14


    The understanding of signaling events is critical for attaining long-term expansion of hematopoietic stem cells ex vivo. In this study, we aim to analyze the contribution of multiple signaling molecules in proliferation of hematopoietic stem cells. To this end, we design a bottom-up engineered receptor with multiple tyrosine motifs, which can recruit multiple signaling molecules of interest. This is followed by a top-down approach, where one of the multiple tyrosine motifs in the bottom-up engineered receptor is functionally knocked out by tyrosine-to-phenylalanine mutation. The combination of these two approaches demonstrates the importance of Shc in cooperation with STAT3 or STAT5 in the proliferation of hematopoietic stem cells. The platform developed herein may be applied for analyzing other cells and/or other cell fate regulation systems.

    View details for PubMedID 29920201

  • Interspecies chimeras. Current opinion in genetics & development Suchy, F., Nakauchi, H. 2018; 52: 36–41


    By probing early embryogenesis and regeneration, interspecies chimeras provide a unique platform for discovery and clinical use. Although efficient generation of human:animal chimeric embryos remains elusive, recent advancements attempt to overcome incompatibilities in xenogeneic development and transplantation.

    View details for PubMedID 29859382

  • Spred1 Safeguards Hematopoietic Homeostasis against Diet-Induced Systemic Stress CELL STEM CELL Tadokoro, Y., Hoshii, T., Yamazaki, S., Eto, K., Ema, H., Kobayashi, M., Ueno, M., Ohta, K., Arai, Y., Hara, E., Harada, K., Oshima, M., Oshima, H., Arai, F., Yoshimura, A., Nakauchi, H., Hirao, A. 2018; 22 (5): 713-+


    Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis.

    View details for PubMedID 29706577

  • Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications STEM CELL REPORTS Ong, J., Serra, M., Segal, J., Cujba, A., Ng, S., Butler, R., Millar, V., Hatch, S., Zimri, S., Koike, H., Chan, K., Bonham, A., Walk, M., Voss, T., Heaton, N., Mitry, R., Dhawan, A., Ebner, D., Danovi, D., Nakauchi, H., Rashid, S. 2018; 10 (3): 693–702


    Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC) lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications.

    View details for PubMedID 29478892

  • Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice BLOOD Kon, A., Yamazaki, S., Nannya, Y., Kataoka, K., Ota, Y., Nakagawa, M., Yoshida, K., Shiozawa, Y., Morita, M., Yoshizato, T., Sanada, M., Nakayama, M., Koseki, H., Nakauchi, H., Ogawa, S. 2018; 131 (6): 621–35


    Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre-mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2-mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2-mutated mice. RNA sequencing of the Srsf2-mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r, Fyn, Gnas, Nsd1, Hnrnpa2b1, and Trp53bp1 Among the mutant Srsf2-associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults.

    View details for PubMedID 29146882

    View details for PubMedCentralID PMC5888285

  • Dissection of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic Stem Cells Via Motif-Engineered Chimeric Receptors STEM CELL REVIEWS AND REPORTS Saka, K., Lai, C., Nojima, M., Kawahara, M., Otsu, M., Nakauchi, H., Nagamune, T. 2018; 14 (1): 101–9


    Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.

    View details for PubMedID 28948469

    View details for PubMedCentralID PMC5801400

  • Modeling lethal X-linked genetic disorders in pigs with ensured fertility PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Matsunari, H., Watanabe, M., Nakano, K., Enosawa, S., Umeyama, K., Uchikura, A., Yashima, S., Fukuda, T., Klymiuk, N., Kurome, M., Kessler, B., Wuenschd, A., Zakhartchenko, V., Wolf, E., Hanazono, Y., Nagaya, M., Umezawa, A., Nakauchi, H., Nagashima, H. 2018; 115 (4): 708–13


    Genetically engineered pigs play an indispensable role in the study of rare monogenic diseases. Pigs harboring a gene responsible for a specific disease can be efficiently generated via somatic cell cloning. The generation of somatic cell-cloned pigs from male cells with mutation(s) in an X chromosomal gene is a reliable and straightforward method for reproducing X-linked genetic diseases (XLGDs) in pigs. However, the severe symptoms of XLGDs are often accompanied by impaired growth and reproductive disorders, which hinder the reproduction of these valuable model animals. Here, we generated unique chimeric boars composed of mutant cells harboring a lethal XLGD and normal cells. The chimeric boars exhibited the cured phenotype with fertility while carrying and transmitting the genotype of the XLGD. This unique reproduction system permits routine production of XLGD model pigs through the male-based breeding, thereby opening an avenue for translational research using disease model pigs.

    View details for PubMedID 29311328

  • iPSC-Derived Organs In Vivo: Challenges and Promise CELL STEM CELL Suchy, F., Yamaguchi, T., Nakauchi, H. 2018; 22 (1): 21–24


    Transplanting iPSCs into the embryos of another species can generate functional organs for basic research and translational applications. We discuss forward-looking approaches and address key remaining challenges of generating iPSC-derived human organs in vivo.

    View details for PubMedID 29304339

  • Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity. Experimental hematology Wilkinson, A. C., Morita, M. n., Nakauchia, H. n., Yamazaki, S. n. 2018


    Hematopoietic stem cells (HSCs) are used clinically in bone marrow (BM) transplantation due to their unique ability to reform the entire hematopoietic system. Recently, we reported that HSCs are highly sensitive to valine, one of the three branched-chain amino acids (BCAAs) in addition to isoleucine and leucine. Dietary depletion of valine could even be used as a conditioning regimen for HSC transplantation. Here, we report that HSCs are highly sensitive to the balance of BCAAs, with both proliferation and survival reduced by BCAA imbalance. However, low but balanced BCAA levels failed to rescue HSC maintenance. Importantly, in vivo depletion of all three BCAAs was significantly less toxic than depletion of valine only. We demonstrate that BCAA depletion can replace valine depletion as a safer alternative to BM conditioning. In summary, by determining HSC metabolic requirements, we can improve metabolic approaches to BM conditioning.

    View details for PubMedID 29705267

  • CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep SCIENTIFIC REPORTS Vilarino, M., Rashid, S., Suchy, F., McNabb, B., van der Meulen, T., Fine, E. J., Ahsan, S., Mursaliyev, N., Sebastiano, V., Diab, S., Huising, M. O., Nakauchi, H., Ross, P. J. 2017; 7: 17472


    One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

    View details for PubMedID 29234093

  • Haploid embryonic stem cell lines derived from androgenetic and parthenogenetic rat blastocysts JOURNAL OF REPRODUCTION AND DEVELOPMENT Hirabayashi, M., Hara, H., Goto, T., Takizawa, A., Dwinell, M. R., Yamanaka, T., Hochi, S., Nakauchi, H. 2017; 63 (6): 611–16


    The present study was conducted to establish haploid embryonic stem (ES) cell lines using fluorescent marker-carrying rats. In the first series, 7 ES cell lines were established from 26 androgenetic haploid blastocysts. However, only 1 ES cell line (ahES-2) was found to contain haploid cells (1n = 20 + X) by fluorescence-activated cell sorting (FACS) and karyotypic analyses. No chimeras were detected among the 10 fetuses and 41 offspring derived from blastocyst injection with the FACS-purified haploid cells. In the second series, 2 ES cell lines containing haploid cells (13% in phES-1 and 1% in phES-2) were established from 2 parthenogenetic haploid blastocysts. Only the phES-2 cell population was purified by repeated FACS to obtain 33% haploid cells. Following blastocyst injection with the FACS-purified haploid cells, no chimera was observed among the 11 fetuses; however, 1 chimeric male was found among the 47 offspring. Although haploid rat ES cell lines can be established from both blastocyst sources, FACS purification may be necessary for maintenance and chimera production.

    View details for PubMedID 28824040

    View details for PubMedCentralID PMC5735273

  • Mammalian Transcription Factor Networks: Recent Advances in Interrogating Biological Complexity. Cell systems Wilkinson, A. C., Nakauchi, H., Göttgens, B. 2017; 5 (4): 319-331


    Transcription factor (TF) networks are a key determinant of cell fate decisions in mammalian development and adult tissue homeostasis and are frequently corrupted in disease. However, our inability to experimentally resolve and interrogate the complexity of mammalian TF networks has hampered the progress in this field. Recent technological advances, in particular large-scale genome-wide approaches, single-cell methodologies, live-cell imaging, and genome editing, are emerging as important technologies in TF network biology. Several recent studies even suggest a need to re-evaluate established models of mammalian TF networks. Here, we provide a brief overview of current and emerging methods to define mammalian TF networks. We also discuss how these emerging technologies facilitate new ways to interrogate complex TF networks, consider the current open questions in the field, and comment on potential future directions and biomedical applications.

    View details for DOI 10.1016/j.cels.2017.07.004

    View details for PubMedID 29073372

  • In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells by Gfi1b, c-Fos, and Gata2 Overexpression within Teratoma. Stem cell reports Tsukada, M., Ota, Y., Wilkinson, A. C., Becker, H. J., Osato, M., Nakauchi, H., Yamazaki, S. 2017; 9 (4): 1024-1033


    Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) could potentially provide unlimited HSCs for clinical transplantation, a curative treatment for numerous blood diseases. However, to date, bona fide HSC generation has been largely unsuccessful in vitro. We have previously described proof of concept for in vivo HSC generation from PSCs via teratoma formation. However, our first-generation system was complex and the output low. Here, we further optimize this technology and demonstrate the following: (1) simplified HSC generation using transcription factor overexpression; (2) improved HSC output using c-Kit-deficient host mice, and (3) that teratomas can be transplanted and cryopreserved. We demonstrate that overexpression of Gfi1b, c-Fos, and Gata2, previously reported to transdifferentiate fibroblasts into hematopoietic progenitors in vitro, can induce long-term HSC formation in vivo. Our in vivo system provides a useful platform to investigate new strategies and re-evaluate existing strategies to generate HSCs and study HSC development.

    View details for DOI 10.1016/j.stemcr.2017.08.010

    View details for PubMedID 28943250

    View details for PubMedCentralID PMC5639260

  • A Novel Mouse Model of iNKT Cell-deficiency Generated by CRISPR/Cas9 Reveals a Pathogenic Role of iNKT Cells in Metabolic Disease. Scientific reports Ren, Y., Sekine-Kondo, E., Shibata, R., Kato-Itoh, M., Umino, A., Yanagida, A., Satoh, M., Inoue, K., Yamaguchi, T., Mochida, K., Nakae, S., Van Kaer, L., Iwabuchi, K., Nakauchi, H., Watarai, H. 2017; 7 (1): 12765


    iNKT cells play important roles in immune regulation by bridging the innate and acquired immune systems. The functions of iNKT cells have been investigated in mice lacking the Traj18 gene segment that were generated by traditional embryonic stem cell technology, but these animals contain a biased T cell receptor (TCR) repertoire that might affect immune responses. To circumvent this confounding factor, we have generated a new strain of iNKT cell-deficient mice by deleting the Traj18 locus using CRISPR/Cas9 technology, and these animals contain an unbiased TCR repertoire. We employed these mice to investigate the contribution of iNKT cells to metabolic disease and found a pathogenic role of these cells in obesity-associated insulin-resistance. The new Traj18-deficient mouse strain will assist in studies of iNKT cell biology.

    View details for DOI 10.1038/s41598-017-12475-4

    View details for PubMedID 28986544

    View details for PubMedCentralID PMC5630609

  • Interspecies chimeras for human stem cell research DEVELOPMENT Masaki, H., Nakauchi, H. 2017; 144 (14): 2544–47


    Interspecies chimeric assays are a valuable tool for investigating the potential of human stem and progenitor cells, as well as their differentiated progeny. This Spotlight article discusses the different factors that affect interspecies chimera generation, such as evolutionary distance, developmental timing, and apoptosis of the transplanted cells, and suggests some possible strategies to address them. A refined approach to generating interspecies chimeras could contribute not only to a better understanding of cellular potential, but also to understanding the nature of xenogeneic barriers and mechanisms of heterochronicity, to modeling human development, and to the creation of human transplantable organs.

    View details for PubMedID 28720651

  • Pre-Transplantation Blockade of TNF-alpha-Mediated Oxygen Species Accumulation Protects Hematopoietic Stem Cells STEM CELLS Ishida, T., Suzuki, S., Lai, C., Yamazaki, S., Kakuta, S., Iwakura, Y., Nojima, M., Takeuchi, Y., Higashihara, M., Nakauchi, H., Otsu, M. 2017; 35 (4): 989-1002


    Hematopoietic stem cell (HSC) transplantation (HSCT) for malignancy requires toxic pre-conditioning to maximize anti-tumor effects and donor-HSC engraftment. While this induces bone marrow (BM)-localized inflammation, how this BM environmental change affects transplanted HSCs in vivo remains largely unknown. We here report that, depending on interval between irradiation and HSCT, residence within lethally irradiated recipient BM compromises donor-HSC reconstitution ability. Both in vivo and in vitro we demonstrate that, among inflammatory cytokines, TNF-α plays a role in HSC damage: TNF-α stimulation leads to accumulation of reactive oxygen species (ROS) in highly purified hematopoietic stem/progenitor cells (HSCs/HSPCs). Transplantation of flow-cytometry-sorted murine HSCs reveals damaging effects of accumulated ROS on HSCs. Short-term incubation either with an specific inhibitor of tumor necrosis factor receptor 1 signaling or an antioxidant N-acetyl-L-cysteine (NAC) prevents TNF-α-mediated ROS accumulation in HSCs. Importantly, pre-transplantation exposure to NAC successfully demonstrats protective effects in inflammatory BM on graft-HSCs, exhibiting better reconstitution capability than that of nonprotected control grafts. We thus suggest that in vivo protection of graft-HSCs from BM inflammation is a feasible and attractive approach, which may lead to improved hematopoietic reconstitution kinetics in transplantation with myeloablative conditioning that inevitably causes inflammation in recipient BM. Stem Cells 2016.

    View details for DOI 10.1002/stem.2524

    View details for Web of Science ID 000398102000014

  • Pharmacological targeting of plasmin prevents lethality in a murine model of macrophage activation syndrome. Blood Shimazu, H., Munakata, S., Tashiro, Y., Salama, Y., Dhahri, D., Eiamboonsert, S., Ota, Y., Onoda, H., Tsuda, Y., Okada, Y., Nakauchi, H., Heissig, B., Hattori, K. 2017


    Macrophage activation syndrome (MAS) is a life-threatening disorder characterized by a cytokine storm and multiorgan dysfunction due to excessive immune activation. Although abnormalities of coagulation and fibrinolysis are major components of MAS, the role of the fibrinolytic system and its key player, plasmin, in the development of MAS remains to be solved. We established a murine model of fulminant MAS by repeated injections of Toll-like receptor-9 (TLR-9) agonist and D-galactosamine (DG) in immunocompetent mice. We found plasmin was excessively activated during the progression of fulminant MAS in mice. Genetic and pharmacological inhibition of plasmin counteracted MAS-associated lethality and other related symptoms. We show that plasmin regulates the influx of inflammatory cells and the production of inflammatory cytokines/chemokines. Collectively, our findings identify plasmin as a decisive checkpoint in the inflammatory response during MAS and a potential novel therapeutic target for MAS.

    View details for DOI 10.1182/blood-2016-09-738096

    View details for PubMedID 28325863

  • 'Off-the-shelf' immunotherapy with iPSC-derived rejuvenated cytotoxic T lymphocytes. Experimental hematology Ando, M., Nakauchi, H. 2017; 47: 2-12


    Adoptive T-cell therapy to target and kill tumor cells shows promise and induces durable remissions in selected malignancies. However, for most cancers, clinical utility is limited. Cytotoxic T lymphocytes continuously exposed to viral or tumor antigens, with long-term expansion, may become unable to proliferate ("exhausted"). To exploit fully rejuvenated induced pluripotent stem cell (iPSC)-derived antigen-specific cytotoxic T lymphocytes is a potentially powerful approach. We review recent progress in engineering iPSC-derived T cells and prospects for clinical translation. We also describe the importance of introducing a suicide gene safeguard system into adoptive T-cell therapy, including iPSC-derived T-cell therapy, to protect from unexpected events in first-in-humans clinical trials.

    View details for DOI 10.1016/j.exphem.2016.10.009

    View details for PubMedID 27826124

  • Fail-Safe System against Potential Tumorigenicity after Transplantation of iPSC Derivatives. Stem cell reports Itakura, G., Kawabata, S., Ando, M., Nishiyama, Y., Sugai, K., Ozaki, M., Iida, T., Ookubo, T., Kojima, K., Kashiwagi, R., Yasutake, K., Nakauchi, H., Miyoshi, H., Nagoshi, N., Kohyama, J., Iwanami, A., Matsumoto, M., Nakamura, M., Okano, H. 2017


    Human induced pluripotent stem cells (iPSCs) are promising in regenerative medicine. However, the risks of teratoma formation and the overgrowth of the transplanted cells continue to be major hurdles that must be overcome. Here, we examined the efficacy of the inducible caspase-9 (iCaspase9) gene as a fail-safe against undesired tumorigenic transformation of iPSC-derived somatic cells. We used a lentiviral vector to transduce iCaspase9 into two iPSC lines and assessed its efficacy in vitro and in vivo. In vitro, the iCaspase9 system induced apoptosis in approximately 95% of both iPSCs and iPSC-derived neural stem/progenitor cells (iPSC-NS/PCs). To determine in vivo function, we transplanted iPSC-NS/PCs into the injured spinal cord of NOD/SCID mice. All transplanted cells whose mass effect was hindering motor function recovery were ablated upon transduction of iCaspase9. Our results suggest that the iCaspase9 system may serve as an important countermeasure against post-transplantation adverse events in stem cell transplant therapies.

    View details for DOI 10.1016/j.stemcr.2017.02.003

    View details for PubMedID 28262544

    View details for PubMedCentralID PMC5355810

  • An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors. Stem cell reports Ieyasu, A., Ishida, R., Kimura, T., Morita, M., Wilkinson, A. C., Sudo, K., Nishimura, T., Ohehara, J., Tajima, Y., Lai, C., Otsu, M., Nakamura, Y., Ema, H., Nakauchi, H., Yamazaki, S. 2017


    Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

    View details for DOI 10.1016/j.stemcr.2017.01.015

    View details for PubMedID 28238792

  • Continuous cell supply from Krt7-expressing hematopoietic stem cells during native hematopoiesis revealed by targeted in vivo gene transfer method SCIENTIFIC REPORTS Tajima, Y., Ito, K., Umino, A., Wilkinson, A. C., Nakauchi, H., Yamazaki, S. 2017; 7


    The nature of hematopoietic stem cells under normal hematopoiesis remained largely unknown due to the limited assays available to monitor their behavior in situ. Here, we develop a new mouse model to transfer genes specifically into the primitive hematopoietic stem cell compartment through the utilization of a modified Rcas/TVA system. We succeeded in transferring a GFP reporter gene into adult hematopoietic stem cells in vivo, which are predominantly quiescent, by generating pseudotyped-lentivirus. Furthermore, we demonstrate the utility of this system to study neonatal hematopoiesis, a developmental stage that has been difficult to analyze to date. Using the system developed in this study, we observed continuous multi-lineage hematopoietic cell supply in peripheral blood from Krt7-positive hematopoietic stem cells during unperturbed homeostatic condition. This powerful experimental system could provide a new standard tool to analyze hematopoiesis under physiological condition without transplantation.

    View details for DOI 10.1038/srep40684

    View details for Web of Science ID 000392272500001

    View details for PubMedID 28098173

    View details for PubMedCentralID PMC5241640

  • Thalidomide induces apoptosis in undifferentiated human induced pluripotent stem cells. In vitro cellular & developmental biology. Animal Tachikawa, S. n., Nishimura, T. n., Nakauchi, H. n., Ohnuma, K. n. 2017


    Thalidomide, which was formerly available commercially to control the symptoms of morning sickness, is a strong teratogen that causes fetal abnormalities. However, the mechanism of thalidomide teratogenicity is not fully understood; thalidomide toxicity is not apparent in rodents, and the use of human embryos is ethically and technically untenable. In this study, we designed an experimental system featuring human-induced pluripotent stem cells (hiPSCs) to investigate the effects of thalidomide. These cells exhibit the same characteristics as those of epiblasts originating from implanted fertilized ova, which give rise to the fetus. Therefore, theoretically, thalidomide exposure during hiPSC differentiation is equivalent to that in the human fetus. We examined the effects of thalidomide on undifferentiated hiPSCs and early-differentiated hiPSCs cultured in media containing bone morphogenetic protein-4, which correspond, respectively, to epiblast (future fetus) and trophoblast (future extra-embryonic tissue). We found that only the number of undifferentiated cells was reduced. In undifferentiated cells, application of thalidomide increased the number of apoptotic and dead cells at day 2 but not day 4. Application of thalidomide did not affect the cell cycle. Furthermore, immunostaining and flow cytometric analysis revealed that thalidomide exposure had no effect on the expression of specific markers of undifferentiated and early trophectodermal differentiated cells. These results suggest that the effect of thalidomide was successfully detected in our experimental system and that thalidomide eliminated a subpopulation of undifferentiated hiPSCs. This study may help to elucidate the mechanisms underlying thalidomide teratogenicity and reveal potential strategies for safely prescribing this drug to pregnant women.

    View details for PubMedID 28849348

  • Lessons from Interspecies Mammalian Chimeras ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 33 Suchy, F., Nakauchi, H., Schekman, R. 2017; 33: 203–17


    As chimeras transform from beasts of Greek mythology into tools of contemporary bioscience, secrets of developmental biology and evolutionary divergence are being revealed. Recent advances in stem cell biology and interspecies chimerism have generated new models with extensive basic and translational applications, including generation of transplantable, patient-specific organs.

    View details for PubMedID 28806099

  • Establishment of mouse expanded potential stem cells. Nature Yang, J. n., Ryan, D. J., Wang, W. n., Tsang, J. C., Lan, G. n., Masaki, H. n., Gao, X. n., Antunes, L. n., Yu, Y. n., Zhu, Z. n., Wang, J. n., Kolodziejczyk, A. A., Campos, L. S., Wang, C. n., Yang, F. n., Zhong, Z. n., Fu, B. n., Eckersley-Maslin, M. A., Woods, M. n., Tanaka, Y. n., Chen, X. n., Wilkinson, A. C., Bussell, J. n., White, J. n., Ramirez-Solis, R. n., Reik, W. n., Göttgens, B. n., Teichmann, S. A., Tam, P. P., Nakauchi, H. n., Zou, X. n., Lu, L. n., Liu, P. n. 2017; 550 (7676): 393–97


    Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

    View details for PubMedID 29019987

  • Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins BMC BIOLOGY Tarusawa, E., Sanbo, M., Okayama, A., Miyashita, T., Kitsukawa, T., Hirayama, T., Hirabayashi, T., Hasegawa, S., Kaneko, R., Toyoda, S., Kobayashi, T., Kato-Itoh, M., Nakauchi, H., Hirabayashi, M., Yagi, T., Yoshimura, Y. 2016; 14


    The specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections.We show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity.Our results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.

    View details for DOI 10.1186/s12915-016-0326-6

    View details for Web of Science ID 000389550400001

    View details for PubMedID 27912755

    View details for PubMedCentralID PMC5133762

  • Stem cells and interspecies chimaeras NATURE Wu, J., Greely, H. T., Jaenisch, R., Nakauchi, H., Rossant, J., Belmonte, J. C. 2016; 540 (7631): 51-59


    Chimaeras are both monsters of the ancient imagination and a long-established research tool. Recent advances, particularly those dealing with the identification and generation of various kinds of stem cells, have broadened the repertoire and utility of mammalian interspecies chimaeras and carved out new paths towards understanding fundamental biology as well as potential clinical applications.

    View details for DOI 10.1038/nature20573

    View details for Web of Science ID 000388916600047

    View details for PubMedID 27905428

  • Non-myeloablative preconditioning with ACK2 (anti-c-kit antibody) is efficient in bone marrow transplantation for murine models of mucopolysaccharidosis type II. Molecular genetics and metabolism Yokoi, T., Yokoi, K., Akiyama, K., Higuchi, T., Shimada, Y., Kobayashi, H., Sato, T., Ohteki, T., Otsu, M., Nakauchi, H., Ida, H., Ohashi, T. 2016; 119 (3): 232-238


    Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by the deficient activity of iduronate 2-sulfatase (IDS), which is involved in the lysosomal catabolism of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. Such a deficiency leads to the accumulation of undegraded GAGs in some organs. Although enzyme replacement therapy is available as a treatment of MPS II, there are some limitations, such as the requirement of weekly administration for whole life. To avoid such limitations, hematopoietic cell transplantation (HSCT) is a possible alternative. In fact, some report suggested positive effects of HSCT for MPS II. However, HSCT has also some limitations. Strong conditioning regimens can cause severe side effects. For overcome this obstacle, we studied the efficacy of ACK2, an antibody that blocks KIT, followed by low-dose irradiation as a preconditioning regimen for HSCT using a murine model of MPS II. This protocol achieves 58.7±4.92% donor chimerism at 16weeks after transplantation in the peripheral blood of recipient mice. GAG levels were significantly reduced in liver, spleen, heart and intestine. These results indicated that ACK2-based preconditioning might be one of the choices for MPS II patients who receive HSCT.

    View details for DOI 10.1016/j.ymgme.2016.08.003

    View details for PubMedID 27590924

  • Pre-transplantation blockade of TNF-a-mediated oxygen species accumulation protects hematopoietic stem cells. Stem cells Ishida, T., Suzuki, S., Lai, C., Yamazaki, S., Kakuta, S., Iwakura, Y., Nojima, M., Takeuchi, Y., Higashihara, M., Nakauchi, H., Otsu, M. 2016


    Hematopoietic stem cell (HSC) transplantation (HSCT) for malignancy requires toxic pre-conditioning to maximize anti-tumor effects and donor-HSC engraftment. While this induces bone marrow (BM)-localized inflammation, how this BM environmental change affects transplanted HSCs in vivo remains largely unknown. We here report that, depending on interval between irradiation and HSCT, residence within lethally irradiated recipient BM compromises donor-HSC reconstitution ability. Both in vivo and in vitro we demonstrate that, among inflammatory cytokines, TNF-α plays a role in HSC damage: TNF-α stimulation leads to accumulation of reactive oxygen species (ROS) in highly purified hematopoietic stem/progenitor cells (HSCs/HSPCs). Transplantation of flow-cytometry-sorted murine HSCs reveals damaging effects of accumulated ROS on HSCs. Short-term incubation either with an specific inhibitor of tumor necrosis factor receptor 1 signaling or an antioxidant N-acetyl-L-cysteine (NAC) prevents TNF-α-mediated ROS accumulation in HSCs. Importantly, pre-transplantation exposure to NAC successfully demonstrats protective effects in inflammatory BM on graft-HSCs, exhibiting better reconstitution capability than that of nonprotected control grafts. We thus suggest that in vivo protection of graft-HSCs from BM inflammation is a feasible and attractive approach, which may lead to improved hematopoietic reconstitution kinetics in transplantation with myeloablative conditioning that inevitably causes inflammation in recipient BM. Stem Cells 2016.

    View details for DOI 10.1002/stem.2524

    View details for PubMedID 27753160

  • Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy. Human gene therapy methods Lin, H., Okumura, T., Yatsuda, Y., Ito, S., Nakauchi, H., Otsu, M. 2016; 27 (5): 197-208


    Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer-probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.

    View details for PubMedID 27763786

  • Fibrinolytic crosstalk with endothelial cells expands murine mesenchymal stromal cells. Blood Dhahri, D., Sato-Kusubata, K., Ohki-Koizumi, M., Nishida, C., Tashiro, Y., Munakata, S., Shimazu, H., Salama, Y., Eiamboonsert, S., Nakauchi, H., Hattori, K., Heissig, B. 2016; 128 (8): 1063-1075


    Tissue plasminogen activator (tPA), aside from its vascular fibrinolytic action, exerts various effects within the body, ranging from synaptic plasticity to control of cell fate. Here, we observed that by activating plasminogen and matrix metalloproteinase-9, tPA expands murine bone marrow-derived CD45(-)TER119(-)Sca-1(+)PDGFRα(+) mesenchymal stromal cells (PαS-MSCs) in vivo through a crosstalk between PαS-MSCs and endothelial cells. Mechanistically, tPA induces the release of Kit ligand from PαS-MSCs, which activates c-Kit(+) endothelial cells to secrete MSC growth factors: platelet-derived growth factor-BB (PDGF-BB) and fibroblast growth factor 2 (FGF2). In synergy, FGF2 and PDGF-BB upregulate PDGFRα expression in PαS-MSCs, which ultimately leads to PαS-MSC expansion. These data show a novel mechanism by which the fibrinolytic system expands PαS-MSCs through a cytokine crosstalk between niche cells.

    View details for DOI 10.1182/blood-2015-10-673103

    View details for PubMedID 27283026

  • Multiple allogeneic progenitors in combination function as a unit to support early transient hematopoiesis in transplantation. journal of experimental medicine Ishida, T., Takahashi, S., Lai, C., Nojima, M., Yamamoto, R., Takeuchi, E., Takeuchi, Y., Higashihara, M., Nakauchi, H., Otsu, M. 2016; 213 (9): 1865-1880


    Cord blood (CB) is a valuable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in CB transplantation (CBT) is often delayed because of low graft cell numbers. This limits the use of CB. To overcome this cell dose barrier, we modeled an insufficient dose CBT setting in lethally irradiated mice and then added hematopoietic stem/progenitor cells (HSCs/HPCs; HSPCs) derived from four mouse allogeneic strains. The mixture of HSPCs rescued recipients and significantly accelerated hematopoietic recovery. Including T cells from one strain favored single-donor chimerism through graft versus graft reactions, with early hematopoietic recovery unaffected. Furthermore, using clinically relevant procedures, we successfully isolated a mixture of CD34(+) cells from multiple frozen CB units at one time regardless of HLA-type disparities. These CD34(+) cells in combination proved transplantable into immunodeficient mice. This work provides proof of concept that when circumstances require support of hematopoiesis, combined multiple units of allogeneic HSPCs are capable of early hematopoietic reconstitution while allowing single-donor hematopoiesis by a principal graft.

    View details for DOI 10.1084/jem.20151493

    View details for PubMedID 27503070

    View details for PubMedCentralID PMC4995077

  • Setdb1 maintains hematopoietic stem and progenitor cells by restricting the ectopic activation of nonhematopoietic genes. Blood Koide, S., Oshima, M., Takubo, K., Yamazaki, S., Nitta, E., Saraya, A., Aoyama, K., Kato, Y., Miyagi, S., Nakajima-Takagi, Y., Chiba, T., Matsui, H., Arai, F., Suzuki, Y., Kimura, H., Nakauchi, H., Suda, T., Shinkai, Y., Iwama, A. 2016; 128 (5): 638-649


    Setdb1, also known as Eset, is a methyltransferase that catalyzes trimethylation of H3K9 (H3K9me3) and plays an essential role in the silencing of endogenous retroviral elements (ERVs) in the developing embryo and embryonic stem cells (ESCs). Its role in somatic stem cells, however, remains unclear because of the early death of Setdb1-deficient embryos. We demonstrate here that Setdb1 is the first H3K9 methyltransferase shown to be essential for the maintenance of hematopoietic stem and progenitor cells (HSPCs) in mice. The deletion of Setdb1 caused the rapid depletion of hematopoietic stem and progenitor cells (HSPCs), as well as leukemic stem cells. In contrast to ESCs, ERVs were largely repressed in Setdb1-deficient HSPCs. A list of nonhematopoietic genes was instead ectopically activated in HSPCs after reductions in H3K9me3 levels, including key gluconeogenic enzyme genes fructose-1,6-bisphosphatase 1 (Fbp1) and Fbp2 The ectopic activation of gluconeogenic enzymes antagonized glycolysis and impaired ATP production, resulting in a compromised repopulating capacity of HSPCs. Our results demonstrate that Setdb1 maintains HSPCs by restricting the ectopic activation of nonhematopoietic genes detrimental to their function and uncover that the gluconeogenic pathway is one of the critical targets of Setdb1 in HSPCs.

    View details for DOI 10.1182/blood-2016-01-694810

    View details for PubMedID 27301860

  • Spatiotemporal Reconstruction of the Human Blastocyst by Single-Cell Gene-Expression Analysis Informs Induction of Naive Pluripotency DEVELOPMENTAL CELL Durruthy-Durruthy, J., Wossidlo, M., Pai, S., Takahashi, Y., Kang, G., Omberg, L., Chen, B., Nakauchi, H., Pera, R. R., Sebastiano, V. 2016; 38 (1): 100-115


    Human preimplantation embryo development involves complex cellular and molecular events that lead to the establishment of three cell lineages in the blastocyst: trophectoderm, primitive endoderm, and epiblast. Owing to limited resources of biological specimens, our understanding of how the earliest lineage commitments are regulated remains narrow. Here, we examined gene expression in 241 individual cells from early and late human blastocysts to delineate dynamic gene-expression changes. We distinguished all three lineages and further developed a 3D model of the inner cell mass and trophectoderm in which individual cells were mapped into distinct expression domains. We identified in silico precursors of the epiblast and primitive endoderm lineages and revealed a role for MCRS1, TET1, and THAP11 in epiblast formation and their ability to induce naive pluripotency in vitro. Our results highlight the potential of single-cell gene-expression analysis in human preimplantation development to instruct human stem cell biology.

    View details for DOI 10.1016/j.devcel.2016.06.014

    View details for Web of Science ID 000380073400013

    View details for PubMedID 27404362

  • Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus SCIENTIFIC REPORTS Kaneko, S., Kakinuma, S., Asahina, Y., Kamiya, A., Miyoshi, M., Tsunoda, T., Nitta, S., Asano, Y., Nagata, H., Otani, S., Kawai-Kitahata, F., Murakawa, M., Itsui, Y., Nakagawa, M., Azuma, S., Nakauchi, H., Nishitsuji, H., Ujino, S., Shimotohno, K., Iwamoto, M., Watashi, K., Wakita, T., Watanabe, M. 2016; 6


    Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies.

    View details for DOI 10.1038/srep29358

    View details for Web of Science ID 000379242500001

    View details for PubMedID 27386799

  • Transition of differential histone H3 methylation in photoreceptors and other retinal cells during retinal differentiation SCIENTIFIC REPORTS Ueno, K., Iwagawa, T., Kuribayashi, H., Baba, Y., Nakauchi, H., Murakami, A., Nagasaki, M., Suzuki, Y., Watanabe, S. 2016; 6


    To analyze cell lineage-specific transitions in global transcriptional and epigenetic changes during retinogenesis, we purified retinal cells from normal mice during postnatal development into two fractions, namely, photoreceptors and other retinal cells, based on Cd73 expression, and performed RNA sequencing and ChIP sequencing of H3K27me3 and H3K4me3. Genes expressed in the photoreceptor lineage were marked with H3K4me3 in the Cd73-positive cell fraction; however, the level of H3K27me3 was very low in both Cd73-positive and -negative populations. H3K27me3 may be involved in spatio-temporal onset of a subset of bipolar-related genes. Subsets of genes expressed in amacrine and retinal ganglion cells, which are early-born retinal cell types, were suggested to be maintained in a silent state by H3K27me3 during late-stage retinogenesis. In the outer nuclear layer, upregulation of Rho and rod-related genes were observed in Ezh2-ablated retina, suggesting a role for H3K27me3 in the maintenance of proper expression levels. Taken together, our data on the transition of lineage-specific molecular signatures during development suggest that histone methylation is involved in retinal differentiation and maintenance through cell lineage-specific mechanisms.

    View details for DOI 10.1038/srep29264

    View details for Web of Science ID 000379266700001

    View details for PubMedID 27377164

  • Investigation of bipotent differentiation of hepatoblasts using inducible diphtheria toxin receptor-transgenic mice HEPATOLOGY RESEARCH Yanagida, A., Mizuno, N., Yamazaki, Y., Kato-Itoh, M., Umino, A., Sato, H., Ito, K., Yamaguchi, T., Nakauchi, H., Kamiya, A. 2016; 46 (8): 816-828


    Hepatic progenitor cells, called hepatoblasts, are highly proliferative and exhibit bi-potential differentiation into hepatocytes and cholangiocytes in the fetal liver. Thus, they are the ideal source for transplantation therapy. Although several studies have been performed in vitro, the molecular mechanisms regulating hepatoblast differentiation in vivo following transplantation remain poorly understood. The aim of this study was to investigate an in vivo model to analyze hepatoblast bi-potency and proliferative ability.Hepatic transplantation model using Cre-inducible diphtheria toxin receptor transgenic mice (iDTR), and albafpCre mice expressing Cre under the control of albumin and α-fetoprotein regulatory elements were established. Fresh hepatoblasts were transplanted into diphtheria toxin (DT)-injected iDTRalbafpCre mice and analyzed their differentiation and proliferation abilities by immunostaining and genes expression profiles.Fresh hepatoblasts transplanted into DT-injected iDTRalbafpCre mice engrafted and differentiated into both hepatocytes and cholangiocytes. Additionally, the number of engrafted hepatoblast-derived hepatocytes increased following partial hepatectomy and serial DT injections. Expression levels of hepatic functional genes in transplanted hepatoblast-derived hepatocytes were similar to that of normal hepatocytes.In our iDTRalbafpCre transplantation model, fresh hepatoblasts could differentiate into hepatocytes and cholangiocytes. In addition, these donor cells were induced to proliferate by the following liver injury stimulation. This result suggests that this model is valuable for investigating hepatoblast differentiation pathways in vivo. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1111/hepr.12622

    View details for Web of Science ID 000380920000012

    View details for PubMedID 26584962

  • Practical Selection Methods for Rat and Mouse Round Spermatids Without DNA Staining by Flow Cytometric Cell Sorting MOLECULAR REPRODUCTION AND DEVELOPMENT Hayama, T., Yamaguchi, T., Kato-Itoh, M., Ishii, Y., Mizuno, N., Umino, A., Sato, H., Sanbo, M., Hamanaka, S., Masaki, H., Hirabayashi, M., Nakauchi, H. 2016; 83 (6): 488-496


    Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc.

    View details for DOI 10.1002/mrd.22644

    View details for Web of Science ID 000385291900002

    View details for PubMedID 27031189

  • Rat Blastocysts from Nuclear Injection and Time-Lagged Enucleation and Their Commitment to Embryonic Stem Cells. Cellular reprogramming Hara, H., Goto, T., Takizawa, A., Sanbo, M., Jacob, H. J., Kobayashi, T., Nakauchi, H., Hochi, S., Hirabayashi, M. 2016; 18 (2): 108-115


    Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B-tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes (n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs.

    View details for DOI 10.1089/cell.2015.0084

    View details for PubMedID 26990947

  • Hypomorphic phenotype of Foxn1 gene-modified rats by CRISPR/Cas9 system. Transgenic research Goto, T., Hara, H., Nakauchi, H., Hochi, S., Hirabayashi, M. 2016


    The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.

    View details for DOI 10.1007/s11248-016-9941-9

    View details for PubMedID 26931321

  • 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-?


    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

  • Matrix metalloproteinase-14 mediates formation of bile ducts and hepatic maturation of fetal hepatic progenitor cells. Biochemical and biophysical research communications Otani, S., Kakinuma, S., Kamiya, A., Goto, F., Kaneko, S., Miyoshi, M., Tsunoda, T., Asano, Y., Kawai-Kitahata, F., Nitta, S., Nakata, T., Okamoto, R., Itsui, Y., Nakagawa, M., Azuma, S., Asahina, Y., Yamaguchi, T., Koshikawa, N., Seiki, M., Nakauchi, H., Watanabe, M. 2016; 469 (4): 1062-1068


    Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14-deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14-deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14-deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14-deficient livers. We demonstrate that MMP-14-mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes.

    View details for DOI 10.1016/j.bbrc.2015.12.105

    View details for PubMedID 26724533

  • Fetal Hematopoietic Stem Cell Transplantation Fails to Fully Regenerate the B-Lymphocyte Compartment. Stem cell reports Ghosn, E. E., Waters, J., Phillips, M., Yamamoto, R., Long, B. R., Yang, Y., Gerstein, R., Stoddart, C. A., Nakauchi, H., Herzenberg, L. A. 2016; 6 (1): 137-149


    B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.

    View details for DOI 10.1016/j.stemcr.2015.11.011

    View details for PubMedID 26724903

  • Fetal Hematopoietic Stem Cell Transplantation Fails to Fully Regenerate the B-Lymphocyte Compartment STEM CELL REPORTS Ghosn, E. E., Waters, J., Phillips, M., Yamamoto, R., Long, B. R., Yang, Y., Gerstein, R., Stoddart, C. A., Nakauchi, H., Herzenberg, L. A. 2016; 6 (1): 137-149


    B cells are key components of cellular and humoral immunity and, like all lymphocytes, are thought to originate and renew from hematopoietic stem cells (HSCs). However, our recent single-HSC transfer studies demonstrate that adult bone marrow HSCs do not regenerate B-1a, a subset of tissue B cells required for protection against pneumonia, influenza, and other infections. Since B-1a are regenerated by transfers of fetal liver, the question arises as to whether B-1a derive from fetal, but not adult, HSCs. Here we show that, similar to adult HSCs, fetal HSCs selectively fail to regenerate B-1a. We also show that, in humanized mice, human fetal liver regenerates tissue B cells that are phenotypically similar to murine B-1a, raising the question of whether human HSC transplantation, the mainstay of such models, is sufficient to regenerate human B-1a. Thus, our studies overtly challenge the current paradigm that HSCs give rise to all components of the immune system.

    View details for DOI 10.1016/j.stemcr.2015.11.011

    View details for Web of Science ID 000368099500014

  • Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells. Methods in molecular biology (Clifton, N.J.) Yanagida, A., Nakauchi, H., Kamiya, A. 2016; 1357: 295-310


    Stem cells have the unique properties of self-renewal and multipotency (producing progeny belonging to two or more lineages). Induced pluripotent stem (iPS) cells can be generated from somatic cells by simultaneous expression of pluripotent factors (Oct3/4, Klf4, Sox2, and c-Myc). They share the same properties as embryonic stem (ES) cells and can differentiate into several tissue cells, i.e., neurons, hematopoietic cells, and liver cells. Therefore, iPS cells are suitable candidate cells for regenerative medicine and analyses of disease mechanisms.The liver is the major organ that regulates a multitude of metabolic functions. Hepatocytes are the major cell type populating the liver parenchyma and express several metabolic enzymes that are necessary for liver functions. Although hepatocytes are essential for maintaining homeostasis, it is difficult to alter artificial and transplanted cells because of their multifunctionality, donor shortage, and immunorejection risk. During liver development, hepatic progenitor cells in the fetal liver differentiate into both mature hepatocytes and cholangiocytes. As hepatic progenitor cells have bipotency and high proliferation ability, they could present a potential source for generating transplantable cells or as a liver study model. Here we describe the induction and purification of hepatic progenitor cells derived from human iPS cells. These cells can proliferate for a long term under suitable culture conditions.

    View details for DOI 10.1007/7651_2015_199

    View details for PubMedID 25697415

  • Liver maturation deficiency in p57(Kip2)-/- mice occurs in a hepatocytic p57(Kip2) expression-independent manner. Developmental biology Yanagida, A., Chikada, H., Ito, K., Umino, A., Kato-Itoh, M., Yamazaki, Y., Sato, H., Kobayashi, T., Yamaguchi, T., Nakayama, K. I., Nakauchi, H., Kamiya, A. 2015; 407 (2): 331-343

    View details for DOI 10.1016/j.ydbio.2015.07.004

    View details for PubMedID 26165599

  • MEK-ERK Activity Regulates the Proliferative Activity of Fetal Hepatoblasts Through Accumulation of p16/19(cdkn2a) STEM CELLS AND DEVELOPMENT Kamiya, A., Ito, K., Yanagida, A., Chikada, H., Iwama, A., Nakauchi, H. 2015; 24 (21): 2525-2535

    View details for DOI 10.1089/scd.2015.0015

    View details for PubMedID 26181762

  • A Safeguard System for Induced Pluripotent Stem Cell-Derived Rejuvenated T Cell Therapy STEM CELL REPORTS Ando, M., Nishimura, T., Yamazaki, S., Yamaguchi, T., Kawana-Tachikawa, A., Hayama, T., Nakauchi, Y., Ando, J., Ota, Y., Takahashi, S., Nishimura, K., Ohtaka, M., Nakanishi, M., Miles, J. J., Burrows, S. R., Brenner, M. K., Nakauchi, H. 2015; 5 (4): 597-608


    The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

    View details for DOI 10.1016/j.stemcr.2015.07.011

    View details for Web of Science ID 000364990900013

    View details for PubMedID 26321144

  • The basic helix-loop-helix transcription factor, Mist1, induces maturation of mouse fetal hepatoblasts SCIENTIFIC REPORTS Chikada, H., Ito, K., Yanagida, A., Nakauchi, H., Kamiya, A. 2015; 5

    View details for DOI 10.1038/srep14989

    View details for PubMedID 26456005

  • Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells TISSUE ENGINEERING PART C-METHODS Okeyo, K. O., Kurosawa, O., Yamazaki, S., Oana, H., Kotera, H., Nakauchi, H., Washizu, M. 2015; 21 (10): 1105-1115

    View details for DOI 10.1089/ten.tec.2015.0038

    View details for Web of Science ID 000362087300012

    View details for PubMedID 25914965

  • Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells. Development Masaki, H., Kato-Itoh, M., Umino, A., Sato, H., Hamanaka, S., Kobayashi, T., Yamaguchi, T., Nishimura, K., Ohtaka, M., Nakanishi, M., Nakauchi, H. 2015; 142 (18): 3222-3230


    Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs.

    View details for DOI 10.1242/dev.124016

    View details for PubMedID 26023098

  • Roles of histone H3K27 trimethylase Ezh2 in retinal proliferation and differentiation DEVELOPMENTAL NEUROBIOLOGY Iida, A., Iwagawa, T., Baba, Y., Satoh, S., Mochizuki, Y., Nakauchi, H., Furukawa, T., Koseki, H., Murakami, A., Watanabe, S. 2015; 75 (9): 947-960

    View details for DOI 10.1002/dneu.22261

    View details for Web of Science ID 000359705000005

  • Dipeptide species regulate p38MAPK-Smad3 signalling to maintain chronic myelogenous leukaemia stem cells NATURE COMMUNICATIONS Naka, K., Jomen, Y., Ishihara, K., Kim, J., Ishimoto, T., Bae, E., Mohney, R. P., Stirdivant, S. M., Oshima, H., Oshima, M., Kim, D., Nakauchi, H., Takihara, Y., Kato, Y., Ooshima, A., Kim, S. 2015; 6


    Understanding the specific survival of the rare chronic myelogenous leukaemia (CML) stem cell population could provide a target for therapeutics aimed at eradicating these cells. However, little is known about how survival signalling is regulated in CML stem cells. In this study, we survey global metabolic differences between murine normal haematopoietic stem cells (HSCs) and CML stem cells using metabolomics techniques. Strikingly, we show that CML stem cells accumulate significantly higher levels of certain dipeptide species than normal HSCs. Once internalized, these dipeptide species activate amino-acid signalling via a pathway involving p38MAPK and the stemness transcription factor Smad3, which promotes CML stem cell maintenance. Importantly, pharmacological inhibition of dipeptide uptake inhibits CML stem cell activity in vivo. Our results demonstrate that dipeptide species support CML stem cell maintenance by activating p38MAPK-Smad3 signalling in vivo, and thus point towards a potential therapeutic target for CML treatment.

    View details for DOI 10.1038/ncomms9039

    View details for Web of Science ID 000360352300001

    View details for PubMedID 26289811

    View details for PubMedCentralID PMC4560789

  • Curative haploidentical BMT in a murine model of X-linked chronic granulomatous disease INTERNATIONAL JOURNAL OF HEMATOLOGY Takeuchi, Y., Takeuchi, E., Ishida, T., Onodera, M., Nakauchi, H., Otsu, M. 2015; 102 (1): 111-120


    Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder characterized by defective microbial killing in phagocytes. Long-term prognosis for CGD patients is generally poor, highlighting the need to develop minimally toxic, curative therapeutic approaches. We here describe the establishment of a mouse model in which X-linked CGD can be cured by allogeneic bone marrow transplantation. Using a combination of non-myeloablative-dose total body irradiation and a single injection of anti-CD40 ligand monoclonal antibody, transplantation of whole bone marrow cells achieved long-lasting mixed chimerism in X-linked CGD mice in a haploidentical transplantation setting. Stable mixed chimerism was maintained for up to 1 year even at a low range (<20 % donor cells), indicating induction of donor-specific tolerance. The regimen induced mild myelosuppression without severe acute complications. Stable chimerism was therapeutic, as it suppressed cutaneous granuloma formation in an in vivo test suited for evaluation of treatment efficacy in murine CGD models. These results warrant future development of a simplified allogeneic hematopoietic cell transplantation regimen that would benefit CGD patients by allowing the use of haploidentical donor grafts without serious concerns of severe treatment-related toxicity.

    View details for DOI 10.1007/s12185-015-1799-8

    View details for Web of Science ID 000358146600015

    View details for PubMedID 25921405

  • Hyaline cartilage formation and tumorigenesis of implanted tissues derived from human induced pluripotent stem cells BIOMEDICAL RESEARCH-TOKYO Saito, T., Yano, F., Mori, D., Kawata, M., Hoshi, K., Takato, T., Masaki, H., Otsu, M., Eto, K., Nakauchi, H., Chung, U., Tanaka, S. 2015; 36 (3): 179-186


    Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

    View details for Web of Science ID 000357237100003

    View details for PubMedID 26106047

  • Vascularized and Complex Organ Buds from Diverse Tissues via Mesenchymal Cell-Driven Condensation CELL STEM CELL Takebe, T., Enomura, M., Yoshizawa, E., Kimura, M., Koike, H., Ueno, Y., Matsuzaki, T., Yamazaki, T., Toyohara, T., Osafune, K., Nakauchi, H., Yoshikawa, H. Y., Taniguchi, H. 2015; 16 (5): 556-565


    Transplantation of in-vitro-generated organ buds is a promising approach toward regenerating functional and vascularized organs. Though it has been recently shown in the context of liver models, demonstrating the applicability of this approach to other systems by delineating the molecular mechanisms guiding organ bud formation is critical. Here, we demonstrate a generalized method for organ bud formation from diverse tissues by combining pluripotent stem cell-derived tissue-specific progenitors or relevant tissue samples with endothelial cells and mesenchymal stem cells (MSCs). The MSCs initiated condensation within these heterotypic cell mixtures, which was dependent upon substrate matrix stiffness. Defining optimal mechanical properties promoted formation of 3D, transplantable organ buds from tissues including kidney, pancreas, intestine, heart, lung, and brain. Transplanted pancreatic and renal buds were rapidly vascularized and self-organized into functional, tissue-specific structures. These findings provide a general platform for harnessing mechanical properties to generate vascularized, complex organ buds with broad applications for regenerative medicine.

    View details for DOI 10.1016/j.stem.2015.03.004

    View details for Web of Science ID 000354188600015

    View details for PubMedID 25891906

  • Inhibition of Plasmin Protects Against Colitis in Mice by Suppressing Matrix Metalloproteinase 9-Mediated Cytokine Release From Myeloid Cells. Gastroenterology Munakata, S., Tashiro, Y., Nishida, C., Sato, A., Komiyama, H., Shimazu, H., Dhahri, D., Salama, Y., Eiamboonsert, S., Takeda, K., Yagita, H., Tsuda, Y., Okada, Y., Nakauchi, H., Sakamoto, K., Heissig, B., Hattori, K. 2015; 148 (3): 565-578 e4


    Activated proteases such as plasmin and matrix metalloproteinases (MMPs) are activated in intestinal tissues of patients with active inflammatory bowel diseases. We investigated the effect of plasmin on progression of acute colitis.Colitis was induced in Mmp9-/-, Plg-/-, and C57BL/6 (control) mice by administration of dextran sulfate sodium, trinitrobenzene sulfonic acid, or CD40 antibody. Plasmin was inhibited in control mice by intraperitoneal injection of YO-2, which blocks its active site. Mucosal and blood samples were collected and analyzed by reverse transcription polymerase chain reaction and immunohistochemical analyses, as well as for mucosal inflammation and levels of cytokines and chemokines.Circulating levels of plasmin were increased in mice with colitis, compared with controls. Colitis did not develop in control mice injected with YO-2 or in Plg-/- mice. Colons from these mice had reduced infiltration of Gr1+ neutrophils and F4/80+ macrophages, and reduced levels of inflammatory cytokines and chemokines. Colonic inflammation and colitis induction required activation of endogenous MMP9. Following colitis induction, mice given YO-2, Plg-/- mice, and Mmp9-/- mice had reduced serum levels of tumor necrosis factor and CXCL5, compared to control mice.In mice, plasmin induces a feedback mechanism in which activation of the fibrinolytic system promotes development of colitis, via activation of MMP9 or proteolytic enzymes. The proteolytic environment stimulates influx of myeloid cells into the colonic epithelium and production of tumor necrosis factor and CXCL5. In turn, myeloid CD11b+ cells release the urokinase plasminogen activator, which accelerates plasmin production. Disruption of the plasmin-induced chronic inflammatory circuit might therefore be a strategy for treatment of colitis.

    View details for DOI 10.1053/j.gastro.2014.12.001

    View details for PubMedID 25490065

  • Effect of donor chimerism to reduce the level of glycosaminoglycans following bone marrow transplantation in a murine model of mucopolysaccharidosis type II JOURNAL OF INHERITED METABOLIC DISEASE Yokoi, K., Akiyama, K., Kaneshiro, E., Higuchi, T., Shimada, Y., Kobayashi, H., Akiyama, M., Otsu, M., Nakauchi, H., Ohashi, T., Ida, H. 2015; 38 (2): 333-340


    Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficient activity of the iduronate-2-sulfatase. This leads to accumulation of glycosaminoglycans (GAGs) in the lysosomes of various cells. Although it has been proposed that bone marrow transplantation (BMT) may have a beneficial effect for patients with MPS II, the requirement for donor-cell chimerism to reduce GAG levels is unknown. To address this issue, we transplanted various ratios of normal and MPS II bone marrow cells in a mouse model of MPS II and analyzed GAG accumulation in various tissues. Chimerism of whole leukocytes and each lineage of BMT recipients' peripheral blood was similar to infusion ratios. GAGs were significantly reduced in the liver, spleen, and heart of recipients. The level of GAG reduction in these tissues depends on the percentage of normal-cell chimerism. In contrast to these tissues, a reduction in GAGs was not observed in the kidney and brain, even if 100 % donor chimerism was achieved. These observations suggest that a high degree of chimerism is necessary to achieve the maximum effect of BMT, and donor lymphocyte infusion or enzyme replacement therapy might be considered options in cases of low-level chimerism in MPS II patients.

    View details for DOI 10.1007/s10545-014-9800-x

    View details for Web of Science ID 000350360200015

    View details for PubMedID 25503568

  • Effective treatment against severe graft-versus-host disease with allele-specific anti-HLA monoclonal antibody in a humanized mouse model. Experimental hematology Nakauchi, Y., Yamazaki, S., Napier, S. C., Usui, J., Ota, Y., Takahashi, S., Watanabe, N., Nakauchi, H. 2015; 43 (2): 79-88 e1 4


    Graft-versus-host disease (GVHD), mediated by donor-derived alloreactive T cells, is a major cause of non-relapse mortality in allogeneic hematopoietic stem-cell transplantation (allo-HSCT). Its therapy is not well-defined. We established allele-specific anti-HLA monoclonal antibodies (ASHmAbs) that specifically target HLA molecules, with steady death of target-expressing cells. One such ASHmAb, against HLA-A*02:01 (A2-kASHmAb), was examined in a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγ(null) (NOG) mice were injected with healthy-donor human peripheral blood mononuclear cells (PBMCs), some expressing HLA-A*02:01, some not. Administration of A2-kASHmAb promoted the survival of mice injected with HLA-A*02:01-expressing PBMCs (p<0.0001) and, in humanized NOG mice, immediately cleared HLA-A*02:01-expressing human blood cells from mouse peripheral blood. Human PBMCs were again detectable in mouse blood 2-4 weeks after A2-kASHmAb administration, suggesting that kASHmAb may be safely administered to GVHD patients without permanently ablating the graft. This approach, different from those of existing GVHD pharmacotherapy, may open a new door for treatment of GVHD in HLA-mismatched allo-HSCT.

    View details for DOI 10.1016/j.exphem.2014.10.008

    View details for PubMedID 25448490

  • Effective treatment against severe graft-versus-host disease with allele-specific anti-HLA monoclonal antibody in a humanized mouse model. Experimental hematology Nakauchi, Y., Yamazaki, S., Napier, S. C., Usui, J., Ota, Y., Takahashi, S., Watanabe, N., Nakauchi, H. 2015; 43 (2): 79-88 e4

    View details for DOI 10.1016/j.exphem.2014.10.008

    View details for PubMedID 25448490

  • Characterization of mesenchymal progenitor cells in the crown and root pulp of primary teeth BIOMEDICAL RESEARCH-TOKYO Toriumi, T., Takayama, N., Murakami, M., Sato, M., Yuguchi, M., Yamazaki, Y., Eto, K., Otsu, M., Nakauchi, H., Shirakawa, T., Isokawa, K., Honda, M. J. 2015; 36 (1): 31-45


    The existence of progenitor/mesenchymal stem cells (MSCs) was demonstrated previously in human primary/deciduous teeth. In this study, we examined dental pulp cells from root portion (root cells) of primary teeth without discernible root resorption and compared them with pulp cells from the crown portion (crown cells). Root cells and crown cells were characterized and compared to each other based on progenitor/MSC characteristics and on their generation efficiency of induced pluripotent stem (iPS) cells. Root cells and crown cells included cells manifesting typical progenitor/MSC properties such as osteogenic and adipogenic differentiation potential and clonogenicity. Interestingly, root cells showed a higher expression level of embryonic stem cell marker, KLF4, than crown cells. Moreover, the number of colony-forming unit-fibroblast and cell proliferation rate were higher for root cells than crown cells, and the efficiency of generating iPS cells from root cells was approximately four times higher than that from crown cells. Taken together, these results suggest that root cells from primary teeth show the MSC-like properties and thus could be a potent alternative source for iPS cell generation and the subsequent transplantation therapy.

    View details for Web of Science ID 000350009900004

    View details for PubMedID 25749149

  • Targeted organ generation using mixl1-inducible mouse pluripotent stem cells in blastocyst complementation. Stem cells and development Kobayashi, T., Kato-Itoh, M., Nakauchi, H. 2015; 24 (2): 182-189


    Generation of functional organs from patients' own cells is one of the ultimate goals of regenerative medicine. As a novel approach to creation of organs from pluripotent stem cells (PSCs), we employed blastocyst complementation in organogenesis-disabled animals and successfully generated PSC-derived pancreas and kidneys. Blastocyst complementation, which exploits the capacity of PSCs to participate in forming chimeras, does not, however, exclude contribution of PSCs to the development of tissues-including neural cells or germ cells-other than those specifically targeted by disabling of organogenesis. This fact provokes ethical controversy if human PSCs are to be used. In this study, we demonstrated that forced expression of Mix-like protein 1 (encoded by Mixl1) can be used to guide contribution of mouse embryonic stem cells to endodermal organs after blastocyst injection. We then succeeded in applying this method to generate functional pancreas in pancreatogenesis-disabled Pdx1 knockout mice using a newly developed tetraploid-based organ-complementation method. These findings hold promise for targeted organ generation from patients' own PSCs in livestock animals.

    View details for DOI 10.1089/scd.2014.0270

    View details for PubMedID 25192056

  • Successful Reprogramming of Epiblast Stem Cells by Blocking Nuclear Localization of beta-Catenin STEM CELL REPORTS Murayama, H., Masaki, H., Sato, H., Hayama, T., Yamaguchi, T., Nakauchi, H. 2015; 4 (1): 103-113


    Epiblast stem cells (EpiSCs) in mice and rats are primed pluripotent stem cells (PSCs). They barely contribute to chimeric embryos when injected into blastocysts. Reprogramming of EpiSCs to embryonic stem cell (ESC)-like cells (rESCs) may occur in response to LIF-STAT3 signaling; however, low reprogramming efficiency hampers potential use of rESCs in generating chimeras. Here, we describe dramatic improvement of conversion efficiency from primed to naive-like PSCs through upregulation of E-cadherin in the presence of the cytokine LIF. Analysis revealed that blocking nuclear localization of β-CATENIN with small-molecule inhibitors significantly enhances reprogramming efficiency of mouse EpiSCs. Although activation of Wnt/β-catenin signals has been thought desirable for maintenance of naive PSCs, this study provides the evidence that inhibition of nuclear translocation of β-CATENIN enhances conversion of mouse EpiSCs to naive-like PSCs (rESCs). This affords better understanding of gene regulatory circuits underlying pluripotency and reprogramming of PSCs.

    View details for DOI 10.1016/j.stemcr.2014.12.003

    View details for Web of Science ID 000348036800010

    View details for PubMedID 25556568

    View details for PubMedCentralID PMC4297867

  • T-cell restricted T-bet overexpression induces aberrant hematopoiesis of myeloid cells and impairs function of macrophages in the lung BLOOD Iriguchi, S., Kikuchi, N., Kaneko, S., Noguchi, E., Morishima, Y., Matsuyama, M., Yoh, K., Takahashi, S., Nakauchi, H., Ishii, Y. 2015; 125 (2): 370-382


    Although overexpression of T-bet, a master transcription factor in type 1 helper T lymphocytes, has been reported in several hematological and immune diseases, its role in their pathogenesis is not fully understood. In the present study, we used transgenic model mice (T-bet(tg/wt) and T-bet(tg/tg)) to investigate the effects of T-bet overexpression selectively in T lymphocytes on the development of hematological and immune diseases. The results showed that T-bet overexpression in T cells spontaneously induces maturation arrest in the mononuclear phagocyte lineage, as well as spontaneous dermatitis and pulmonary alveolar proteinosis (PAP)-like disease in T-bet(tg/wt) and T-bet(tg/tg) mice, respectively. T-bet(tg/tg) alveoli with the PAP phenotype showed remarkable reorganization of alveolar mononuclear phagocyte subpopulations and impaired function in addition to augmented T cell infiltration. In addition, PAP development in T-bet(tg/tg) mice was found to associate with increased migration of myeloid cells from the bone marrow into the peripheral blood. These findings reveal an unexpected link between T-bet overexpression in T lymphocytes and the development of PAP caused by reorganization of mononuclear phagocytes in the lung, and provide new insight into the molecular pathogenesis of secondary PAP accompanied by hematological disorders.

    View details for DOI 10.1182/blood-2014-05-575225

    View details for Web of Science ID 000350810200025

    View details for PubMedID 25349175

  • Inhibition of plasmin attenuates murine acute graft-versus-host disease mortality by suppressing the matrix metalloproteinase-9-dependent inflammatory cytokine storm and effector cell trafficking. Leukemia Sato, A., Nishida, C., Sato-Kusubata, K., Ishihara, M., Tashiro, Y., Gritli, I., Shimazu, H., Munakata, S., Yagita, H., Okumura, K., Tsuda, Y., Okada, Y., Tojo, A., Nakauchi, H., Takahashi, S., Heissig, B., Hattori, K. 2015; 29 (1): 145-156


    The systemic inflammatory response observed during acute graft-versus-host disease (aGVHD) is driven by proinflammatory cytokines, a 'cytokine storm'. The function of plasmin in regulating the inflammatory response is not fully understood, and its role in the development of aGVHD remains unresolved. Here we show that plasmin is activated during the early phase of aGVHD in mice, and its activation correlated with aGVHD severity in humans. Pharmacological plasmin inhibition protected against aGVHD-associated lethality in mice. Mechanistically, plasmin inhibition impaired the infiltration of inflammatory cells, the release of membrane-associated proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and Fas-ligand directly, or indirectly via matrix metalloproteinases (MMPs) and alters monocyte chemoattractant protein-1 (MCP-1) signaling. We propose that plasmin and potentially MMP-9 inhibition offers a novel therapeutic strategy to control the deadly cytokine storm in patients with aGVHD, thereby preventing tissue destruction.Leukemia advance online publication, 3 June 2014; doi:10.1038/leu.2014.151.

    View details for DOI 10.1038/leu.2014.151

    View details for PubMedID 24791857

  • An assessment of the effects of ectopic gp91phox expression in XCGD iPSC-derived neutrophils MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT Lin, H., Masaki, H., Yamaguchi, T., Wada, T., Yachie, A., Nishimura, K., Ohtaka, M., Nakanishi, M., Nakauchi, H., Otsu, M. 2015; 2
  • An assessment of the effects of ectopic gp91phox expression in XCGD iPSC-derived neutrophils. Molecular therapy. Methods & clinical development Lin, H., Masaki, H., Yamaguchi, T., Wada, T., Yachie, A., Nishimura, K., Ohtaka, M., Nakanishi, M., Nakauchi, H., Otsu, M. 2015; 2: 15046-?


    For the treatment of monogenetic hematological disorders, restoration of transgene expression in affected cell populations is generally considered to have beneficial effects. However, X-linked chronic granulomatous disease (XCGD) is unique since the appearance of functional neutrophils in the peripheral blood following hematopoietic stem cell gene therapy is transient only. One contributing factor could be the occurrence of detrimental effects secondary to ectopic gp91phox expression in neutrophils, which has not been formally demonstrated previously. This study uses iPSCs to model XCGD, which allows the process of differentiation to be studied intensely in vitro. Alpharetroviral vectors carrying a ubiquitous promoter were used to drive the "ectopic" expression of codon optimized gp91phox cDNA. In the mature fraction of neutrophils differentiated from transduced XCGD-iPSCs, cellular recovery in terms of gp91phox expression and reactive oxygen species production was abruptly lost before cells had fully differentiated. Most critically, ectopic gp91phox expression could be identified clearly in the developing fraction of the transduced groups, which appeared to correspond with reduced cell viability. It is possible that this impedes further differentiation of developing neutrophils. Therefore, affording cellular protection from the detrimental effects of ectopic gp91phox expression may improve XCGD clinical outcomes.

    View details for DOI 10.1038/mtm.2015.46

    View details for PubMedID 26682238

  • 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

  • Screening of Drugs to Treat 8p11 Myeloproliferative Syndrome Using Patient-Derived Induced Pluripotent Stem Cells with Fusion Gene CEP110-FGFR1. PloS one Yamamoto, S., Otsu, M., Matsuzaka, E., Konishi, C., Takagi, H., Hanada, S., Mochizuki, S., Nakauchi, H., Imai, K., Tsuji, K., Ebihara, Y. 2015; 10 (3)


    Induced pluripotent stem (iPS) cells provide powerful tools for studying disease mechanisms and developing therapies for diseases. The 8p11 myeloproliferative syndrome (EMS) is an aggressive chronic myeloproliferative disorder (MPD) that is caused by constitutive activation of fibroblast growth factor receptor 1. EMS is rare and, consequently, effective treatment for this disease has not been established. Here, iPS cells were generated from an EMS patient (EMS-iPS cells) to assist the development of effective therapies for EMS. When iPS cells were co-cultured with murine embryonic stromal cells, EMS-iPS cells produced more hematopoietic progenitor and hematopoietic cells, and CD34+ cells derived from EMS-iPS cells exhibited 3.2-7.2-fold more macrophage and erythroid colony forming units (CFUs) than those derived from control iPS cells. These data indicate that EMS-iPS cells have an increased hematopoietic differentiation capacity, which is characteristic of MPDs. To determine whether a tyrosine kinase inhibitor (TKI) could suppress the increased number of CFUs formed by EMS-iPS-induced CD34+ cells, cells were treated with one of four TKIs (CHIR258, PKC 412, ponatinib, and imatinib). CHIR258, PKC 412, and ponatinib reduced the number of CFUs formed by EMS-iPS-induced CD34+ cells in a dose-dependent manner, whereas imatinib did not. Similar effects were observed on primary peripheral blood cells (more than 90% of which were blasts) isolated from the patient. This study provides evidence that the EMS-iPS cell line is a useful tool for the screening of drugs to treat EMS and to investigate the mechanism underlying this disease.

    View details for DOI 10.1371/journal.pone.0120841

    View details for PubMedID 25803811

  • Novel strategies for liver therapy using stem cells GUT Rashid, T., Takebe, T., Nakauchi, H. 2015; 64 (1): 1-4

    View details for DOI 10.1136/gutjnl-2014-307480

    View details for Web of Science ID 000346167700001

    View details for PubMedID 25183202

  • Quantification of adult T-cell leukemia/lymphoma cells using simple four-color flow cytometry CLINICAL CHEMISTRY AND LABORATORY MEDICINE Ishigaki, T., Zaike, Y., Nojima, M., Kobayashi, S., Ohno, N., Uchimaru, K., Tojo, A., Nakauchi, H., Watanabe, N. 2015; 53 (1): 85-93


    Abstract Background: The absolute number of adult T-cell leukemia/lymphoma (ATL) cells in peripheral blood is an essential indicator to evaluate disease status. However, microscopically counting ATL cells based on morphology requires experience and tends to be inaccurate due to the rarity of ATL. Methods: Based on our research showing that acute-type ATL cells are specifically enriched in the CD4+/CD7- (CD7N) fraction, a new analytical method to accurately quantify ATL cells was established using an internal bead standard and simple four-color flow cytometry. This method was verified by comparison with microscopic examination of 49 peripheral blood samples and used to follow up patients. Results: A strong correlation was observed between the number of CD7N cells measured by flow cytometry and the number of abnormal lymphocytes measured microscopically by experienced technicians [Pearson's R, 0.963; Spearman's rho, 0.921; intercorrelation coefficient, 0.962]. The linear regression coefficient was close to 1 (β=1.013). Our method could detect 1 cell/μL, and the limit of quantitation was between 2.9 and 9.8 cells/μL. The frequency of CD7N cells among CD4+ cells changed during chemotherapy, which reflected differences between chemosensitive and chemoresistant cases. Kaplan-Meier analysis with a log-rank test showed that patients with decreased CD7N proportion after chemotherapy had significantly longer disease-specific survival (p=0.003). Conclusions: Our newly established method quantified tumor cells in patients with acute-type ATL. Furthermore, this method was useful for assessing the efficacy of chemotherapy, and the change of the CD7N proportion could be more important to predict prognosis.

    View details for DOI 10.1515/cclm-2014-0183

    View details for Web of Science ID 000346021000017

    View details for PubMedID 25060346

  • Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients LEUKEMIA Sakurai, M., Kunimoto, H., Watanabe, N., Fukuchi, Y., Yuasa, S., Yamazaki, S., Nishimura, T., Sadahira, K., Fukuda, K., Okano, H., Nakauchi, H., Morita, Y., Matsumura, I., Kudo, K., Ito, E., Ebihara, Y., Tsuji, K., Harada, Y., Harada, H., Okamoto, S., Nakajima, H. 2014; 28 (12): 2344-2354


    Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.Leukemia advance online publication, 13 May 2014; doi:10.1038/leu.2014.136.

    View details for DOI 10.1038/leu.2014.136

    View details for Web of Science ID 000346177500011

    View details for PubMedID 24732596

  • Generation of Recombination Activating Gene-1-Deficient Neonatal Piglets: A Model of T and B Cell Deficient Severe Combined Immune Deficiency PLOS ONE Ito, T., Sendai, Y., Yamazaki, S., Seki-Soma, M., Hirose, K., Watanabe, M., Fukawa, K., Nakauchi, H. 2014; 9 (12)


    Although severe combined immune deficiency (SCID) is a very important research model for mice and SCID mice are widely used, there are only few reports describing the SCID pig models. Therefore, additional research in this area is needed. In this study, we describe the generation of Recombination activating gene-1 (Rag-1)-deficient neonatal piglets in Duroc breed using somatic cell nuclear transfer (SCNT) with gene targeting and analysis using fluorescence-activated cell sorting (FACS) and histology. We constructed porcine Rag-1 gene targeting vectors for the Exon 2 region and obtained heterozygous/homozygous Rag-1 knockout cell colonies using SCNT. We generated two Rag-1-deficient neonatal piglets and compared them with wild-type neonatal piglets. FACS analysis showed that Rag-1 disruption causes a lack of Immunoglobulin M-positive B cells and CD3-positive T cells in peripheral blood mononuclear cells. Consistent with FACS analysis, histological analysis revealed structural defects and an absence of mature lymphocytes in the spleen, mesenteric lymph node (MLNs), and thymus in Rag-1-deficient piglets. These results confirm that Rag-1 is necessary for the generation of lymphocytes in pigs, and Rag-1-deficient piglets exhibit a T and B cell deficient SCID (T-B-SCID) phenotype similar to that of rodents and humans. The T-B-SCID pigs with Rag-1 deficiency generated in this study could be a suitably versatile model for laboratory, translational, and biomedical research, including the development of a humanized model and assessment of pluripotent stem cells.

    View details for DOI 10.1371/journal.pone.0113833

    View details for Web of Science ID 000347114900083

    View details for PubMedID 25437445

  • 73 application of the hollow fiber vitrification method to the cryopreservation of highly cryosensitive embryos. Reproduction, fertility, and development Uchikura, A., Matsunari, H., Nakano, K., Hatae, S., Matsumura, Y., Asano, Y., Takeishi, T., Nakauchi, H., Nagashima, H. 2014; 27 (1): 129-30


    We recently demonstrated that the hollow fibre vitrification (HFV) method (Matsunari et al. 2012) could effectively be applied to the cryopreservation of embryos from diverse species. In this study, we applied the HFV method to the cryopreservation of highly cryosensitive specimens, such as in vitro matured (IVM)/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, as well as IVM/IVF-derived cattle embryos at early cleavage stages. Porcine parthenogenetic morulae (d-4) derived from IVM oocytes were treated with 0.25% pronase to remove zona pellucidae. The resulting blastomeres were isolated from the zona-free morulae by a decompaction treatment followed by gentle pipetting. Bovine IVM-IVF embryos at the 2 to 4 cell (d-1), 8 to 16 cell (d-3), and morula stages (d-5) were then subjected to vitrification. The HFV procedure was performed as described previously using 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5M trehalose as cryoprotectants. Four to twenty embryos, or all of the blastomeres isolated from a single morula, were individually loaded into a cellulose acetate hollow fibre (25mm long, 185μm φ, 15μm membrane thickness) and vitrified. Survival of the vitrified embryos was assessed by in vitro development to blastocysts. Blastomeres recovered after vitrification were aggregated in micro-wells to examine their ability to form blastocysts. The HFV method was demonstrated to be effective for cryopreserving zona-free in vitro-produced porcine morulae and the blastomeres isolated from them (Table 1), as well as bovine IVM-IVF embryos at early cleavage stages. These data demonstrate that the HFV method is effective for highly cryosensitive specimens, such as IVM/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, and IVM/IVF-derived cattle embryos at early cleavage stages. These achievements may expand the technological options in the production of cloned and genetically modified pigs that are useful for biomedical research.

    View details for DOI 10.1071/RDv27n1Ab73

    View details for PubMedID 25472122

  • 31 production efficiency of gene knockout pigs using genome editing and somatic cell cloning. Reproduction, fertility, and development Matsunari, H., Watanabe, M., Nakano, K., Uchikura, A., Asano, Y., Hatae, S., Takeishi, T., Umeyama, K., Nagaya, M., Miyagawa, S., Hanazono, Y., Nakauchi, H., Nagashima, H. 2014; 27 (1): 108


    Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5-83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN).

    View details for DOI 10.1071/RDv27n1Ab31

    View details for PubMedID 25472080

  • Circulating Cells Contribute to Cardiomyocyte Regeneration After Injury. Circulation research Wu, J. M., Hsueh, Y. C., Ch'ang, H. J., Luo, C. Y., Wu, L. W., Nakauchi, H., Hsieh, P. C. 2014


    Rationale: The contribution of bone marrow-borne hematopoietic cells to the ischemic myocardium has been documented. However, a pivotal study reported no evidence of myocardial regeneration from hematopoietic-derived cells. The study did not take into account the possible effect of early injury-induced signaling as the test mice were parabiotically paired to partners immediately after surgery-induced myocardial injury when cross circulation has not yet developed. Objective: To re-evaluate the role of circulating cells in the injured myocardium. Methods and Results: By combining pulse-chase labeling and parabiosis model, we show that circulating cells derived from the parabiont expressed cardiac-specific markers in the injured myocardium. Genetic fate-mapping also revealed that circulating hematopoietic cells acquired cardiac cell fate by means of cell fusion and transdifferentiation. Conclusions: These results suggest that circulating cells participate in cardiomyocyte regeneration in a mouse model of parabiosis when the circulatory system is fully developed before surgery-induced heart injury.

    View details for DOI 10.1161/CIRCRESAHA.116.304564

    View details for PubMedID 25398235

  • A melanocyte-melanoma precursor niche in sweat glands of volar skin PIGMENT CELL & MELANOMA RESEARCH Okamoto, N., Aoto, T., Uhara, H., Yamazaki, S., Akutsu, H., Umezawa, A., Nakauchi, H., Miyachi, Y., Saida, T., Nishimura, E. K. 2014; 27 (6): 1039-1050


    Determination of the niche for early-stage cancer remains a challenging issue. Melanoma is an aggressive cancer of the melanocyte lineage. Early melanoma cells are often found in the epidermis around sweat ducts of human volar skin, and the skin pigmentation pattern is an early diagnostic sign of acral melanoma. However, the niche for melanoma precursors has not been determined yet. Here, we report that the secretory portion (SP) of eccrine sweat glands provide an anatomical niche for melanocyte-melanoma precursor cells. Using lineage-tagged H2B-GFP reporter mice, we found that melanoblasts that colonize sweat glands during development are maintained in an immature, slow-cycling state but renew themselves in response to genomic stress and provide their differentiating progeny to the epidermis. FISH analysis of human acral melanoma expanding in the epidermis revealed that unpigmented melanoblasts with significant cyclin D1 gene amplification reside deep in the SP of particular sweat gland(s). These findings indicate that sweat glands maintain melanocyte-melanoma precursors in an immature state in the niche and explain the preferential distribution of early melanoma cells around sweat glands in human volar skin.

    View details for DOI 10.1111/pcmr.12298

    View details for Web of Science ID 000344349300010

    View details for PubMedID 25065272

  • Gene targeting study reveals unexpected expression of brain-expressed X-linked 2 in endocrine and tissue stem/progenitor cells in mice. The Journal of biological chemistry Ito, K., Yamazaki, S., Yamamoto, R., Tajima, Y., Yanagida, A., Kobayashi, T., Kato-Itoh, M., Kakuta, S., Iwakura, Y., Nakauchi, H., Kamiya, A. 2014; 289 (43): 29892-911


    Identification of genes specifically expressed in stem/progenitor cells is an important issue in developmental and stem cell biology. Genome-wide gene expression analyses in liver cells performed in this study have revealed a strong expression of X-linked genes that include members of the brain-expressed X-linked (Bex) gene family in stem/progenitor cells. Bex family genes are expressed abundantly in the neural cells and have been suggested to play important roles in the development of nervous tissues. However, the physiological role of its individual members and the precise expression pattern outside the nervous system remain largely unknown. Here, we focused on Bex2 and examined its role and expression pattern by generating knock-in mice; the enhanced green fluorescence protein (EGFP) was inserted into the Bex2 locus. Bex2-deficient mice were viable and fertile under laboratory growth conditions showing no obvious phenotypic abnormalities. Through an immunohistochemical analysis and flow cytometry-based approach, we observed unique EGFP reporter expression patterns in endocrine and stem/progenitor cells of the liver, pyloric stomach, and hematopoietic system. Although Bex2 seems to play redundant roles in vivo, these results suggest the significance and potential applications of Bex2 in studies of endocrine and stem/progenitor cells.

    View details for DOI 10.1074/jbc.M114.580084

    View details for PubMedID 25143383

    View details for PubMedCentralID PMC4208000

  • Gene Targeting Study Reveals Unexpected Expression of Brain-expressed X-linked 2 in Endocrine and Tissue Stem/Progenitor Cells in Mice JOURNAL OF BIOLOGICAL CHEMISTRY Ito, K., Yamazaki, S., Yamamoto, R., Tajima, Y., Yanagida, A., Kobayashi, T., Kato-Itoh, M., Kakuta, S., Iwakura, Y., Nakauchi, H., Kamiya, A. 2014; 289 (43): 29892-29911
  • Revisiting the flight of Icarus: making human organs from PSCs with large animal chimeras. Cell stem cell Rashid, T., Kobayashi, T., Nakauchi, H. 2014; 15 (4): 406-9


    While cell therapies hold great potential for treating many conditions, their utility for treating patients that require whole organ replacement is unclear. To address this challenge, we propose using genetically engineered "organ niches" in large animals to generate human organs from pluripotent stem cells and discuss the hurdles facing such strategies.

    View details for DOI 10.1016/j.stem.2014.09.013

    View details for PubMedID 25280216

  • Generation of Mouse Functional Oocytes in Rat by Xeno-Ectopic Transplantation of Primordial Germ Cells BIOLOGY OF REPRODUCTION Hayama, T., Yamaguchi, T., Kato-Itoh, M., Hamanaka, S., Kawarai, M., Sanbo, M., Tamura, C., Lee, Y., Yanagida, A., Murayama, H., Mizuno, N., Umino, A., Sato, H., Yamazaki, S., Masaki, H., Kobayashi, T., Hirabayashi, M., Nakauchi, H. 2014; 91 (4)


    Primordial germ cells (PGCs) are germ cell progenitors in the fetal genital ridge; female PGCs give rise to definitive oocytes that contribute to the next generation. Artificial PGCs have been induced in vitro from pluripotent stem cells and gonad-like tissue has been induced in vivo by cotransplantation of PGCs with PGC-free gonadal cells. To apply these technologies to human infertility treatment or conservation of rare species, PGC transplantation must be established in xenogenic animals. Here, we established a xenogeneic transplantation model by inducing ovary-like tissue from PGCs in xenogenic animals. We transplanted enzymatically dispersed PGCs with PGC-free gonadal cells under the kidney capsule of xenogenic immunodeficient animals. The transplanted cells formed ovary-like tissues under the kidney capsule. These tissues were histologically similar to the normal gonad and expressed the oocyte markers Vasa and Stella. In addition, mouse germinal vesicle-stage oocyte-like cells collected from ovary-like tissue in rats matured to metaphase II via in vitro maturation and gave rise to offspring by intracytoplasmic sperm injection. Our studies show that rat/mouse female PGCs and PGC-free gonadal cells can develop and reconstruct ovary-like tissue containing functional oocytes in an ectopic xenogenic microenvironment.

    View details for DOI 10.1095/biolreprod.114.121640

    View details for Web of Science ID 000343288000013

  • Generation of mouse functional oocytes in rat by xeno-ectopic transplantation of primordial germ cells. Biology of reproduction Hayama, T., Yamaguchi, T., Kato-Itoh, M., Hamanaka, S., Kawarai, M., Sanbo, M., Tamura, C., Lee, Y. S., Yanagida, A., Murayama, H., Mizuno, N., Umino, A., Sato, H., Yamazaki, S., Masaki, H., Kobayashi, T., Hirabayashi, M., Nakauchi, H. 2014; 91 (4): 89


    Primordial germ cells (PGCs) are germ cell progenitors in the fetal genital ridge; female PGCs give rise to definitive oocytes that contribute to the next generation. Artificial PGCs have been induced in vitro from pluripotent stem cells and gonad-like tissue has been induced in vivo by cotransplantation of PGCs with PGC-free gonadal cells. To apply these technologies to human infertility treatment or conservation of rare species, PGC transplantation must be established in xenogenic animals. Here, we established a xenogeneic transplantation model by inducing ovary-like tissue from PGCs in xenogenic animals. We transplanted enzymatically dispersed PGCs with PGC-free gonadal cells under the kidney capsule of xenogenic immunodeficient animals. The transplanted cells formed ovary-like tissues under the kidney capsule. These tissues were histologically similar to the normal gonad and expressed the oocyte markers Vasa and Stella. In addition, mouse germinal vesicle-stage oocyte-like cells collected from ovary-like tissue in rats matured to metaphase II via in vitro maturation and gave rise to offspring by intracytoplasmic sperm injection. Our studies show that rat/mouse female PGCs and PGC-free gonadal cells can develop and reconstruct ovary-like tissue containing functional oocytes in an ectopic xenogenic microenvironment.

    View details for DOI 10.1095/biolreprod.114.121640

    View details for PubMedID 25165118

  • Mesenchymal progenitor cells in mouse foetal liver regulate differentiation and proliferation of hepatoblasts LIVER INTERNATIONAL Ito, K., Yanagida, A., Okada, K., Yamazaki, Y., Nakauchi, H., Kamiya, A. 2014; 34 (9): 1378-1390

    View details for DOI 10.1111/liv.12387

    View details for Web of Science ID 000342579400011

  • Haploinsufficiency of Sf3b1 leads to compromised stem cell function but not to myelodysplasia LEUKEMIA Matsunawa, M., Yamamoto, R., Sanada, M., Sato-Otsubo, A., Shiozawa, Y., Yoshida, K., Otsu, M., Shiraishi, Y., Miyano, S., Isono, K., Koseki, H., Nakauchi, H., Ogawa, S. 2014; 28 (9): 1844-1850


    SF3B1 is a core component of the mRNA splicing machinery and frequently mutated in myeloid neoplasms with myelodysplasia, particularly in those characterized by the presence of increased ring sideroblasts. Deregulated RNA splicing is implicated in the pathogenesis of SF3B1-mutated neoplasms, but the exact mechanism by which the SF3B1 mutation is associated with myelodysplasia and the increased ring sideroblasts formation is still unknown. We investigated the functional role of SF3B1 in normal hematopoiesis utilizing Sf3b1 heterozygous-deficient mice. Sf3b1(+/-) mice had a significantly reduced number of hematopoietic stem cells (CD34(-)cKit(+)ScaI(+)Lin(-) cells or CD34(-)KSL cells) compared with Sf3b1(+/+) mice, but hematopoiesis was grossly normal in Sf3b1(+/-) mice. When transplanted competitively with Sf3b1(+/+) bone marrow cells, Sf3b1(+/-) stem cells showed compromised reconstitution capacity in lethally irradiated mice. There was no increase in the number of ring sideroblasts or evidence of myeloid dysplasia in Sf3b1(+/-) mice. These data suggest that SF3B1 plays an important role in the regulation of hematopoietic stem cells, whereas SF3B1 haploinsufficiency itself is not associated with the myelodysplastic syndrome phenotype with ring sideroblasts.

    View details for DOI 10.1038/leu.2014.73

    View details for Web of Science ID 000341783000011

    View details for PubMedID 24535406

  • Generation of induced pluripotent stem cells derived from primary and secondary myelofibrosis patient samples EXPERIMENTAL HEMATOLOGY Hosoi, M., Kumano, K., Taoka, K., Arai, S., Kataoka, K., Ueda, K., Kamikubo, Y., Takayama, N., Otsu, M., Eto, K., Nakauchi, H., Kurokawa, M. 2014; 42 (9): 816-825
  • Down syndrome-associated haematopoiesis abnormalities created by chromosome transfer and genome editing technologies SCIENTIFIC REPORTS Kazuki, Y., Yakura, Y., Abe, S., Osaki, M., Kajitani, N., Kazuki, K., Takehara, S., Honma, K., Suemori, H., Yamazaki, S., Sakuma, T., Toki, T., Shimizu, R., Nakauchi, H., Yamamoto, T., Oshimura, M. 2014; 4


    Infants with Down syndrome (DS) are at a high risk of developing transient abnormal myelopoiesis (TAM). A GATA1 mutation leading to the production of N-terminally truncated GATA1 (GATA1s) in early megakaryocyte/erythroid progenitors is linked to the onset of TAM and cooperated with the effect of trisomy 21 (Ts21). To gain insights into the underlying mechanisms of the progression to TAM in DS patients, we generated human pluripotent stem cells harbouring Ts21 and/or GATA1s by combining microcell-mediated chromosome transfer and genome editing technologies. In vitro haematopoietic differentiation assays showed that the GATA1s mutation blocked erythropoiesis irrespective of an extra chromosome 21, while Ts21 and the GATA1s mutation independently perturbed megakaryopoiesis and the combination of Ts21 and the GATA1s mutation synergistically contributed to an aberrant accumulation of skewed megakaryocytes. Thus, the DS model cells generated by these two technologies are useful in assessing how GATA1s mutation is involved in the onset of TAM in patients with DS.

    View details for DOI 10.1038/srep06136

    View details for Web of Science ID 000340932200001

    View details for PubMedID 25159877

  • Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer's disease. journal of experimental medicine Lee, J. K., Jin, H. K., Park, M. H., Kim, B., Lee, P. H., Nakauchi, H., Carter, J. E., He, X., Schuchman, E. H., Bae, J. 2014; 211 (8): 1551-1570


    In Alzheimer's disease (AD), abnormal sphingolipid metabolism has been reported, although the pathogenic consequences of these changes have not been fully characterized. We show that acid sphingomyelinase (ASM) is increased in fibroblasts, brain, and/or plasma from patients with AD and in AD mice, leading to defective autophagic degradation due to lysosomal depletion. Partial genetic inhibition of ASM (ASM(+/-)) in a mouse model of familial AD (FAD; amyloid precursor protein [APP]/presenilin 1 [PS1]) ameliorated the autophagocytic defect by restoring lysosomal biogenesis, resulting in improved AD clinical and pathological findings, including reduction of amyloid-β (Aβ) deposition and improvement of memory impairment. Similar effects were noted after pharmacologic restoration of ASM to the normal range in APP/PS1 mice. Autophagic dysfunction in neurons derived from FAD patient induced pluripotent stem cells (iPSCs) was restored by partial ASM inhibition. Overall, these results reveal a novel mechanism of ASM pathogenesis in AD that leads to defective autophagy due to impaired lysosomal biogenesis and suggests that partial ASM inhibition is a potential new therapeutic intervention for the disease.

    View details for DOI 10.1084/jem.20132451

    View details for PubMedID 25049335

  • Stage-Specific Roles for Cxcr4 Signaling in Murine Hematopoietic Stem/Progenitor Cells in the Process of Bone Marrow Repopulation STEM CELLS Lai, C., Yamazaki, S., Okabe, M., Suzuki, S., Maeyama, Y., Iimura, Y., Onodera, M., Kakuta, S., Iwakura, Y., Nojima, M., Otsu, M., Nakauchi, H. 2014; 32 (7): 1929-1942


    Hematopoietic cell transplantation has proven beneficial for various intractable diseases, but it remains unclear how hematopoietic stem/progenitor cells (HSPCs) home to the bone marrow (BM) microenvironment, initiate hematopoietic reconstitution, and maintain life-long hematopoiesis. The use of newly elucidated molecular determinants for overall HSPC engraftment should benefit patients. Here, we report that modification of C-X-C chemokine receptor type 4 (Cxcr4) signaling in murine HSPCs does not significantly affect initial homing/lodging events, but leads to alteration in subsequent BM repopulation kinetics, with observations confirmed by both gain- and loss-of-function approaches. By using C-terminal truncated Cxcr4 as a gain-of-function effector, we demonstrated that signal augmentation likely led to favorable in vivo repopulation of primitive cell populations in BM. These improved features were correlated with enhanced seeding efficiencies in stromal cell cocultures and altered ligand-mediated phosphorylation kinetics of extracellular signal-regulated kinases observed in Cxcr4 signal-augmented HSPCs in vitro. Unexpectedly, however, sustained signal enhancement even with wild-type Cxcr4 overexpression resulted in impaired peripheral blood (PB) reconstitution, most likely by preventing release of donor hematopoietic cells from the marrow environment. We thus conclude that timely regulation of Cxcr4/CXCR4 signaling is key in providing donor HSPCs with enhanced repopulation potential following transplantation, whilst preserving the ability to release HSPC progeny into PB for improved transplantation outcomes.

    View details for DOI 10.1002/stem.1670

    View details for Web of Science ID 000337785200020

    View details for PubMedID 24510783

  • Multicolor staining of globin subtypes reveals impaired globin switching during erythropoiesis in human pluripotent stem cells. Stem cells translational medicine Ochi, K., Takayama, N., Hirose, S., Nakahata, T., Nakauchi, H., Eto, K. 2014; 3 (7): 792-800


    Adult hemoglobin composed of α- and β-globin reflects a change from expression of embryonic ε- and fetal γ-globin to adult β-globin in human erythroid cells, so-called globin switching. Human pluripotent stem cells (hPSCs) are a potential source for in vitro erythrocyte production, but they show prominent expression of γ-globin with little β-globin expression, which indicates incomplete globin switching. To examine the mechanism of this impaired globin switching, we optimized multicolor flow cytometry to simultaneously follow expression of different globin subtypes using different immunofluorescent probes. This enabled us to detect upregulation of β-globin and the corresponding silencing of γ-globin at the single-cell level during cord blood CD34(+) cell-derived erythropoiesis, examined as an endogenous control. Using this approach, we initially characterized the heterogeneous β-globin expression in erythroblasts from several hPSC clones and confirmed the predominant expression of γ-globin. These hPSC-derived erythroid cells also displayed reduced expression of BCL11A-L. However, doxycycline-induced overexpression of BCL11A-L in selected hPSCs promoted γ-globin silencing. These results strongly suggest that impaired γ-globin silencing is associated with downregulated BCL11A-L in hPSC-derived erythroblasts and that multicolor staining of globin subtypes is an effective approach to studying globin switching in vitro.

    View details for DOI 10.5966/sctm.2013-0216

    View details for PubMedID 24873860

  • Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions STEM CELL REPORTS Kanke, K., Masaki, H., Saito, T., Komiyama, Y., Hojo, H., Nakauchi, H., Lichtler, A. C., Takato, T., Chung, U., Ohba, S. 2014; 2 (6): 751-760


    Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4',3':4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

    View details for DOI 10.1016/j.stemcr.2014.04.016

    View details for Web of Science ID 000336653200002

    View details for PubMedID 24936463

  • Bone marrow Schwann cells induce hematopoietic stem cell hibernation INTERNATIONAL JOURNAL OF HEMATOLOGY Yamazaki, S., Nakauchi, H. 2014; 99 (6): 695-698


    Hematopoietic stem cells (HSCs) are clonogenic cells capable of both self-renewal and multilineage differentiation. In adult mouse bone marrow (BM), most HSCs remain in the non-dividing G0-phase of cell cycle, in close contact with supporting cells known as the HSC "niche". In the present study, we focused on signaling mechanisms that regulate stem cell dormancy in the BM niche. We show that TGF-β type II receptor deficiency causes reduced phosphorylation of Smad2/3 and impairs long-term repopulating activity in HSCs, suggesting a significant role for TGF-β/Smad signaling in hematopoiesis. Furthermore, we aimed at defining the candidate BM niche responsible for homeostasis of hematopoiesis, and revealed that non-myelinating Schwann cells sustain HSC hibernation by converting TGF-β from its latent to its active form.

    View details for DOI 10.1007/s12185-014-1588-9

    View details for Web of Science ID 000337603400004

    View details for PubMedID 24817152

  • Destruction of polychlorinated naphthalenes by a high-temperature melting treatment (GeoMelt process) ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH Yamamoto, T., Kai, Y., Nakauchi, H., Abuku, T., Noma, Y. 2014; 21 (12): 7557-7566


    A series of treatment experiments were carried out to evaluate the applicability of a high-temperature melting treatment (GeoMelt process) to the destruction of polychlorinated naphthalene (PCN) formulation. We started with 10-kg-scale experiments in which a small melting furnace was used and then scaled up to a 1-t-scale experiment in which a melting furnace that resembled an actual treatment system was used. These runs were evaluated whether destruction efficiency (DE) of total PCNs was more than 99.999% and whether concentrations of PCNs and polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/DFs) in vitrified materials, emission gas, and scrubber water were below the target levels. Because DE values and the target levels of PCNs and PCDDs/DFs in these runs were satisfactory, then we carried out a demonstrative experiment using the actual treatment system and confirmed destruction of PCNs. Based on good results of the demonstrative experiment, stock of PCN formulation was successfully treated continuously.

    View details for DOI 10.1007/s11356-014-2643-z

    View details for Web of Science ID 000337086300031

    View details for PubMedID 24595750

  • Transgenic Pigs with Pancreas-specific Expression of Green Fluorescent Protein JOURNAL OF REPRODUCTION AND DEVELOPMENT Matsunari, H., Kobayashi, T., Watanabe, M., Umeyama, K., Nakano, K., Kanai, T., Matsuda, T., Nagaya, M., Hara, M., Nakauchi, H., Nagashima, H. 2014; 60 (3): 230-237


    The development and regeneration of the pancreas is of considerable interest because of the role of these processes in pancreatic diseases, such as diabetes. Here, we sought to develop a large animal model in which the pancreatic cell lineage could be tracked. The pancreatic and duodenal homeobox-1 (Pdx1) gene promoter was conjugated to Venus, a green fluorescent protein, and introduced into 370 in vitro-matured porcine oocytes by intracytoplasmic sperm injection-mediated gene transfer. These oocytes were transferred into four recipient gilts, all of which became pregnant. Three gilts were sacrificed at 47-65 days of gestation, and the fourth was allowed to farrow. Seven of 16 fetuses obtained were transgenic (Tg) and exhibited pancreas-specific green fluorescence. The fourth recipient gilt produced a litter of six piglets, two of which were Tg. The founder Tg offspring matured normally and produced healthy first-generation (G1) progeny. A postweaning autopsy of four 27-day-old G1 Tg piglets confirmed the pancreas-specific Venus expression. Immunostaining of the pancreatic tissue indicated the transgene was expressed in β-cells. Pancreatic islets from Tg pigs were transplanted under the renal capsules of NOD/SCID mice and expressed fluorescence up to one month after transplantation. Tg G1 pigs developed normally and had blood glucose levels within the normal range. Insulin levels before and after sexual maturity were within normal ranges, as were other blood biochemistry parameters, indicating that pancreatic function was normal. We conclude that Pdx1-Venus Tg pigs represent a large animal model suitable for research on pancreatic development/regeneration and diabetes.

    View details for Web of Science ID 000338271700009

    View details for PubMedID 24748398

  • Generation of induced pluripotent stem cells derived from primary and secondary myelofibrosis patient samples. Experimental hematology Hosoi, M., Kumano, K., Taoka, K., Arai, S., Kataoka, K., Ueda, K., Kamikubo, Y., Takayama, N., Otsu, M., Eto, K., Nakauchi, H., Kurokawa, M. 2014


    Induced pluripotent stem cells (iPS) derived from disease cells are expected to provide a new experimental material, especially for diseases from which samples are difficult to obtain. In this study, we generated iPS from samples from patients with primary and secondary myelofibrosis. The primary myelofibrosis cells had chromosome 13q deletions, and the secondary myelofibrosis (SMF) cells had JAK2V617F mutations. The myelofibrosis patient cell-derived iPS (MF-iPS) were confirmed as possessing these parental disease-specific genomic markers. The capacity to form three germ layers was confirmed by teratoma assay. By co-culture with specific feeder cells and cytokines, MF-iPS can re-differentiate into blood progenitor cells and finally into megakaryocytes. We found that mRNA levels of interleukin-8, one of the candidate cytokines related to the pathogenesis of myelofibrosis, was elevated predominantly in megakaryocytes derived from MF-iPS. Because megakaryocytes from myelofibrosis clones are considered to produce critical mediators to proliferate fibroblasts in the bone marrow and iPS can provide differentiated cells abundantly, the disease-specific iPS we established should be a good research tool for this intractable disease.

    View details for DOI 10.1016/j.exphem.2014.03.010

    View details for PubMedID 24859480

  • The generation of induced pluripotent stem cells (iPSCs) from patients with infantile and late-onset types of Pompe disease and the effects of treatment with acid-alpha-glucosidase in Pompe's iPSCs MOLECULAR GENETICS AND METABOLISM Higuchi, T., Kawagoe, S., Otsu, M., Shimada, Y., Kobayashi, H., Hirayama, R., Eto, K., Ida, H., Ohashi, T., Nakauchi, H., Eto, Y. 2014; 112 (1): 44-48


    Pompe disease (PD), which is also called glycogen storage disease type II (GSDII), is one of the lysosomal storage diseases (LSDs) caused by a deficiency in acid-α-glucosidase (GAA) in the lysosome and is characterized by the accumulation of glycogen in various cells. PD has been treated by enzyme replacement therapy (ERT). We generated induced pluripotent stem cells (iPSCs) from the cells of patients with infantile-type and late-onset-type PD using a retrovirus vector to deliver transgenes encoding four reprogramming factors, namely, OCT4, SOX2, c-MYC, and KLF4. We confirmed that the two types of PD-iPSCs exhibited an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. The PD-iPSCs exhibited strong positive staining with Periodic acid-Schiff (PAS). Moreover, ultrastructural features of these iPSCs exhibited massive glycogen granules in the cytoplasm, particularly in the infantile-type but to a lesser degree in the late-onset type. Glycogen granules of the infantile-type iPSCs treated with rhGAA were markedly decreased in a dose-dependent manner. Human induced pluripotent stem cell provides an opportunity to build up glycogen storage of Pompe disease in vitro. It represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies for Pompe disease.

    View details for DOI 10.1016/j.ymgme.2014.02.012

    View details for Web of Science ID 000335539700007

    View details for PubMedID 24642446

  • A Comparison of the Rest Complex Binding Patterns in Embryonic Stem Cells and Epiblast Stem Cells PLOS ONE Seki, M., Masaki, H., Arauchi, T., Nakauchi, H., Sugano, S., Suzuki, Y. 2014; 9 (4)


    We detected and characterized the binding sites of the representative Rest complex components Rest, Sin3A, and Lsd1. We compared their binding patterns in mouse embryonic stem (ES) cells and epiblast stem (EpiS) cells. We found few Rest sites unique to the EpiS cells. The ES-unique site features were distinct from those of the common sites, namely, the signal intensities were weaker, and the characteristic gene function categories differed. Our analyses showed that the Rest binding sites do not always overlap with the Sin3A and Lsd1 binding sites. The Sin3A binding pattern differed remarkably between the ES and EpiS cells and was accompanied by significant changes in acetylated-histone patterns in the surrounding regions. A series of transcriptome analyses in the same cell types unexpectedly showed that the putative target gene transcript levels were not dramatically different despite dynamic changes in the Rest complex binding patterns and chromatin statuses, which suggests that Rest is not the sole determinant of repression at its targets. Nevertheless, we identified putative Rest targets with explicitly enhanced transcription upon Rest knock-down in 143 and 60 common and ES-unique Rest target genes, respectively. Among such sites, several genes are involved in ES cell proliferation. In addition, we also found that long, intergenic non-coding RNAs were apparent Rest targets and shared similar features with the protein-coding target genes. Interestingly, such non-coding target genes showed less conservation through evolution than protein-coding targets. As a result of differences in the components and targets of the Rest complex, its functional roles may differ in ES and EpiS cells.

    View details for DOI 10.1371/journal.pone.0095374

    View details for Web of Science ID 000335227400057

    View details for PubMedID 24752154

  • Expandable Megakaryocyte Cell Lines Enable Clinically Applicable Generation of Platelets from Human Induced Pluripotent Stem Cells CELL STEM CELL Nakamura, S., Takayama, N., Hirata, S., Seo, H., Endo, H., Ochi, K., Fujita, K., Koike, T., Harimoto, K., Dohda, T., Watanabe, A., Okita, K., Takahashi, N., Sawaguchi, A., Yamanaka, S., Nakauchi, H., Nishimura, S., Eto, K. 2014; 14 (4): 535-548


    The donor-dependent supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs). This approach involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) from PSC-derived hematopoietic progenitors through the overexpression of BMI1 and BCL-XL to respectively suppress senescence and apoptosis and the constrained overexpression of c-MYC to promote proliferation. The resulting imMKCLs can be expanded in culture over extended periods (4-5 months), even after cryopreservation. Halting the overexpression of c-MYC, BMI1, and BCL-XL in growing imMKCLs led to the production of CD42b(+) platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo. The combination of robust expansion capacity and efficient platelet production means that appropriately selected imMKCL clones represent a potentially inexhaustible source of hPSC-derived platelets for clinical application.

    View details for DOI 10.1016/j.stem.2014.01.011

    View details for Web of Science ID 000334766400016

    View details for PubMedID 24529595

  • Developmental Epigenetic Modification Regulates Stochastic Expression of Clustered Protocadherin Genes, Generating Single Neuron Diversity NEURON Toyoda, S., Kawaguchi, M., Kobayashi, T., Tarusawa, E., Toyama, T., Okano, M., Oda, M., Nakauchi, H., Yoshimura, Y., Sanbo, M., Hirabayashi, M., Hirayama, T., Hirabayashi, T., Yagi, T. 2014; 82 (1): 94-108


    In the brain, enormous numbers of neurons have functional individuality and distinct circuit specificities. Clustered Protocadherins (Pcdhs), diversified cell-surface proteins, are stochastically expressed by alternative promoter choice and affect dendritic arborization in individual neurons. Here we found that the Pcdh promoters are differentially methylated by the de novo DNA methyltransferase Dnmt3b during early embryogenesis. To determine this methylation's role in neurons, we produced chimeric mice from Dnmt3b-deficient induced pluripotent stem cells (iPSCs). Single-cell expression analysis revealed that individual Dnmt3b-deficient Purkinje cells expressed increased numbers of Pcdh isoforms; in vivo, they exhibited abnormal dendritic arborization. These results indicate that DNA methylation by Dnmt3b at early embryonic stages regulates the probability of expression for the stochastically expressed Pcdh isoforms. They also suggest a mechanism for a rare human recessive disease, the ICF (Immunodeficiency, Centromere instability, and Facial anomalies) syndrome, which is caused by Dnmt3b mutations.

    View details for DOI 10.1016/j.neuron.2014.02.005

    View details for Web of Science ID 000333804800011

    View details for PubMedID 24698270

  • Nov/CCN3 regulates long-term repopulating activity of murine hematopoietic stem cells via integrin alpha v beta 3 INTERNATIONAL JOURNAL OF HEMATOLOGY Ishihara, J., Umemoto, T., Yamato, M., Shiratsuchi, Y., Takaki, S., Petrich, B. G., Nakauchi, H., Eto, K., Kitamura, T., Okano, T. 2014; 99 (4): 393-406


    Throughout life, hematopoietic stem cells (HSCs) sustain the blood cell supply through their capacities for self-renewal and multilineage differentiation. These processes are regulated within a specialized microenvironment termed the 'niche'. Here, we show a novel mechanism for regulating HSC function that is mediated by nephroblastoma overexpressed (Nov/CCN3), a matricellular protein member of the CCN family. We found that Nov contributes to the maintenance of long-term repopulating (LTR) activity through association with integrin αvβ3 on HSCs. The resultant β3 integrin outside-in signaling is dependent on thrombopoietin (TPO), a crucial cytokine involved in HSC maintenance. TPO was required for Nov binding to integrin αvβ3, and stimulated Nov expression in HSCs. However, in the presence of IFNγ, a cytokine known to impair HSC function, not only was TPO-induced expression of Nov suppressed, but the LTR activity was conversely impaired by TPO-mediated ligation of integrin αvβ3 with exogenous ligands, including Nov, as well. Thus, Nov/integrin αvβ3-mediated maintenance of HSCs appears to be modulated by simultaneous stimulation by other cytokines. Our finding suggests that this system contributes to the regulation of HSCs within the bone marrow niche.

    View details for DOI 10.1007/s12185-014-1534-x

    View details for Web of Science ID 000334446400007

    View details for PubMedID 24563081

  • Use of cell type-specific transcriptome to identify genes specifically involved in Muller glia differentiation during retinal development DEVELOPMENTAL NEUROBIOLOGY Mochizuki, Y., Iida, A., Lyons, E., Kageyama, R., Nakauchi, H., Murakami, A., Watanabe, S. 2014; 74 (4): 426-437


    Retinal progenitor cells alter their properties over the course of development, and sequentially produce different sub-populations of retinal cells. We had previously found that early and late retinal progenitor cell populations can be distinguished by their surface antigens, SSEA-1 and c-kit, respectively. Using DNA microarray analysis, we examined the transcriptomes of SSEA-1 positive cells at E14, and c-kit positive, and c-kit negative cells at P1. By comparing data, we identified genes specifically expressed in c-kit positive late retinal progenitor cells. The previous literature suggests that most of the c-kit positive cell-specific genes are related to glia differentiation in brain or are expressed in Müller glia. Since Notch signaling promotes Müller glia differentiation in retina, we examined the effects of gain- and loss-of-Notch signaling on expression of these genes and found that all the genes were positively affected by Notch signaling. Finally, we screened the genes for their function in retinal development by shRNA-based suppression in retinal explants. In about half the genes, Müller glia differentiation was perturbed when their expression was suppressed. Taken together, these results show that at P1, c-kit positive retinal progenitor cells, which include Müller glia precursor cells, are enriched for genes related to glial differentiation. We propose analysis of purified subsets of retinal cells as a powerful tool to elucidate the molecular basis of retinal development.

    View details for DOI 10.1002/dneu.22131

    View details for Web of Science ID 000332185800002

    View details for PubMedID 24124169

  • A Comprehensive System for Generation and Evaluation of Induced Pluripotent Stem Cells Using piggyBac Transposition PLOS ONE Tsukiyama, T., Kato-Itoh, M., Nakauchi, H., Ohinata, Y. 2014; 9 (3)


    The most stringent criterion for evaluating pluripotency is generation of chimeric animals with germline transmission ability. Because the quality of induced pluripotent stem cell (iPSC) lines is heterogeneous, an easy and accurate system to evaluate these abilities would be useful. In this study, we describe a simple but comprehensive system for generating and evaluating iPSCs by single transfection of multiple piggyBac (PB) plasmid vectors encoding Tet-inducible polycistronic reprogramming factors, a pluripotent-cell-specific reporter, a constitutively active reporter, and a sperm-specific reporter. Using this system, we reprogrammed 129 and NOD mouse embryonic fibroblasts into iPSCs, and then evaluated the molecular and functional properties of the resultant iPSCs by quantitative RT-PCR analysis and chimera formation assays. The iPSCs contributed extensively to chimeras, as indicated by the constitutively active TagRFP reporter, and also differentiated into sperm, as indicated by the late-spermatogenesis-specific Acr (acrosin)-EGFP reporter. Next, we established secondary MEFs from E13.5 chimeric embryos and efficiently generated secondary iPSCs by simple addition of doxycycline. Finally, we applied this system to establishment and evaluation of rat iPSCs and production of rat sperm in mouse-rat interspecific chimeras. By monitoring the fluorescence of Acr-EGFP reporter, we could easily detect seminiferous tubules containing rat iPSC-derived spermatids and sperm. And, we succeeded to obtain viable offspring by intracytoplasmic sperm injection (ICSI) using these haploid male germ cells. We propose that this system will enable robust strategies for induction and evaluation of iPSCs, not only in rodents but also in other mammals. Such strategies will be especially valuable in non-rodent species, in which verification of germline transmission by mating is inefficient and time-consuming.

    View details for DOI 10.1371/journal.pone.0092973

    View details for Web of Science ID 000333675600114

    View details for PubMedID 24667806

  • Histone demethylase Jmjd3 is required for the development of subsets of retinal bipolar cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Iida, A., Iwagawa, T., Kuribayashi, H., Satoh, S., Mochizuki, Y., Baba, Y., Nakauchi, H., Furukawa, T., Koseki, H., Murakami, A., Watanabe, S. 2014; 111 (10): 3751-3756


    Di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) is an important gene repression mechanism. H3K27me2/3-specific demethylase, Jmjd3, was expressed in the inner nuclear layer during late retinal development. In contrast, H3K27 methyltransferase, Ezh2, was highly expressed in the embryonic retina but its expression decreased rapidly after birth. Jmjd3 loss of function in the developing retina resulted in failed differentiation of PKC-positive bipolar cell subsets (rod-ON-BP) and reduced transcription factor Bhlhb4 expression, which is critical for the differentiation of rod-ON-BP cells. Overexpression of Bhlhb4, but not of other BP cell-related genes, such as transcription factors Neurod and Chx10, in Jmjd3-knockdown retina rescued loss of PKC-positive BP cells. Populations of other retinal cell subsets were not significantly affected. In addition, proliferation activity and apoptotic cell number during retinal development were not affected by the loss of Jmjd3. Levels of histone H3 trimethyl Lys27 (H3K27me3) in the Bhlhb4 locus were lower in Islet-1-positive BP cells and amacrine cells than in the Islet-1-negative cell fraction. The Islet-1-negative cell fraction consisted mainly of photoreceptors, suggestive of lineage-specific demethylation of H3K27me3 in the Bhlhb4 locus. We propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal cells.

    View details for DOI 10.1073/pnas.1311480111

    View details for Web of Science ID 000332564800035

    View details for PubMedID 24572572

  • A Chemical Probe that Labels Human Pluripotent Stem Cells CELL REPORTS Hirata, N., Nakagawa, M., Fujibayashi, Y., Yamauchi, K., Murata, A., Minami, I., Tomioka, M., Kondo, T., Kuo, T., Endo, H., Inoue, H., Sato, S., Ando, S., Kawazoe, Y., Aiba, K., Nagata, K., Kawase, E., Chang, Y., Suemori, H., Eto, K., Nakauchi, H., Yamanaka, S., Nakatsuji, N., Ueda, K., Uesugi, M. 2014; 6 (6): 1165-1174


    A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

    View details for DOI 10.1016/j.celrep.2014.02.006

    View details for Web of Science ID 000333465000020

    View details for PubMedID 24613351

  • The TIF1 beta-HP1 System Maintains Transcriptional Integrity of Hematopoietic Stem Cells STEM CELL REPORTS Miyagi, S., Koide, S., Saraya, A., Wendt, G. R., Oshima, M., Konuma, T., Yamazaki, S., Mochizuki-Kashio, M., Nakajima-Takagi, Y., Wang, C., Chiba, T., Kitabayashi, I., Nakauchi, H., Iwama, A. 2014; 2 (2): 145-152


    TIF1β is a transcriptional corepressor that recruits repressive chromatin modifiers to target genes. Its biological function and physiological targets in somatic stem cells remain largely unknown. Here, we show that TIF1β is essential for the maintenance of hematopoietic stem cells (HSCs). Deletion of Tif1b in mice induced active cycling and apoptosis of HSCs and promoted egression of HSCs from the bone marrow, leading to rapid depletion of HSCs. Strikingly, Tif1b-deficient HSCs showed a strong trend of ectopic expression of nonhematopoietic genes. Levels of heterochromatin protein 1 (HP1α, β and γ) proteins, which form a complex with TIF1β, were significantly reduced in the absence of TIF1β and depletion of HP1 recapitulated a part of the phenotypes of Tif1b-deficient HSCs. These results demonstrate that the TIF1β-HP1 system functions as a critical repressive machinery that targets genes not normally activated in the hematopoietic compartment, thereby maintaining the transcriptional signature specific to HSCs.

    View details for DOI 10.1016/j.stemcr.2013.12.008

    View details for Web of Science ID 000336647600004

    View details for PubMedID 24527388

  • Homeodomain Transcription Factor Meis1 Is a Critical Regulator of Adult Bone Marrow Hematopoiesis PLOS ONE Ariki, R., Morikawa, S., Mabuchi, Y., Suzuki, S., Nakatake, M., Yoshioka, K., Hidano, S., Nakauchi, H., Matsuzaki, Y., Nakamura, T., Goitsuka, R. 2014; 9 (2)


    Hematopoietic stem cells in the bone marrow have the capacity to both self-renew and to generate all cells of the hematopoietic system. The balance of these two activities is controlled by hematopoietic stem cell-intrinsic regulatory mechanisms as well as extrinsic signals from the microenvironment. Here we demonstrate that Meis1, a TALE family homeodomain transcription factor involved in numerous embryonic developmental processes, is selectively expressed in hematopoietic stem/progenitor cells. Conditional Meis1 knockout in adult hematopoietic cells resulted in a significant reduction in the hematopoietic stem/progenitor cells. Suppression of hematopoiesis by Meis1 deletion appears to be caused by impaired self-renewal activity and reduced cellular quiescence of hematopoietic stem/progenitor cells in a cell autonomous manner, resulting in stem cell exhaustion and defective long-term hematopoiesis. Meis1 deficiency down-regulated a subset of Pbx1-dependent hematopoietic stem cell signature genes, suggesting a functional link between them in the maintenance of hematopoietic stem/progenitor cells. These results show the importance of Meis1 in adult hematopoiesis.

    View details for DOI 10.1371/journal.pone.0087646

    View details for Web of Science ID 000330626900092

    View details for PubMedID 24498346

  • Enzyme augmentation therapy enhances the therapeutic efficacy of bone marrow transplantation in mucopolysaccharidosis type II mice MOLECULAR GENETICS AND METABOLISM Akiyama, K., Shimada, Y., Higuchi, T., Ohtsu, M., Nakauchi, H., Kobayashi, H., Fukuda, T., Ida, H., Eto, Y., Crawford, B. E., Brown, J. R., Ohashi, T. 2014; 111 (2): 139-146


    Before the availability of an enzyme replacement therapy (ERT) for mucopolysaccharidosis type II (MPS II), patients were treated by bone marrow transplantation (BMT). However, the effectiveness of BMT for MPS II was equivocal, particularly at addressing the CNS manifestations. To study this further, we subjected a murine model of MPS II to BMT and evaluated the effect at correcting the biochemical and pathological aberrations in the viscera and CNS. Our results indicated that BMT reduced the accumulation of glycosaminoglycans (GAGs) in a variety of visceral organs, but not in the CNS. With the availability of an approved ERT for MPS II, we investigated and compared the relative merits of the two strategies either as a mono or combination therapy. We showed that the combination of BMT and ERT was additive at reducing tissue levels of GAGs in the heart, kidney and lung. Moreover, ERT conferred greater efficacy if the immunological response against the infused recombinant enzyme was low. Finally, we showed that pathologic GAGs might potentially represent a sensitive biomarker to monitor the therapeutic efficacy of therapies for MPS II.

    View details for DOI 10.1016/j.ymgme.2013.09.013

    View details for Web of Science ID 000330746000294

    View details for PubMedID 24100247

  • Derivation of Embryonic Stem Cell Lines from Parthenogenetically Developing Rat Blastocysts STEM CELLS AND DEVELOPMENT Hirabayashi, M., Goto, T., Tamura, C., Sanbo, M., Hara, H., Kato-Itoh, M., Sato, H., Kobayashi, T., Nakauchi, H., Hochi, S. 2014; 23 (2): 107-114


    This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.

    View details for DOI 10.1089/scd.2013.0200

    View details for Web of Science ID 000329356800003

    View details for PubMedID 24010570

  • Clock gene Bmal1 is dispensable for intrinsic properties of murine hematopoietic stem cells. Journal of negative results in biomedicine Ieyasu, A., Tajima, Y., Shimba, S., Nakauchi, H., Yamazaki, S. 2014; 13: 4-?


    Circadian rhythms are known to influence a variety of biological phenomena such as cell cycle, sleep-wake rhythm, hormone release and other important physiological functions. Given that cell cycle entry of hibernating hematopoietic stem cells (HSCs) plays a critical role in controlling hematopoiesis, we asked functional significance of the clock gene Bmal1, which plays a central role in regulating circadian rhythms as a transcription factor. Here we investigated the necessity of Bmal1 for HSC functions using Bmal1 deficient (Bmal1⁻/⁻) mice.Using colony-forming assays in vitro, we found that the frequency of mixed colony formation between Bmal1⁺/⁺ and Bmal1⁻/⁻ CD34-KSL cells does not differ significantly. Competitive bone marrow assays also revealed that Bmal1⁻/⁻ bone marrow cells competed normally with wild-type cells and displayed long-term multi-hematopoietic lineage reconstitution. In addition, there were no significant differences in the frequencies and hibernation state of bone marrow HSCs between Bmal1⁺/⁺ and Bmal1⁻/⁻ mice, suggesting that they are independent of circadian rhythms.This paper discusses the necessity of circadian rhythms for HSC functions. Our data clearly shows that a key circadian clock gene Bmal1 is dispensable for intrinsic functions of HSCs, such as differentiation, proliferation and repopulating ability.

    View details for DOI 10.1186/1477-5751-13-4

    View details for PubMedID 24606809

  • DNA Methylation Is Involved in the Expression of miR-142-3p in Fibroblasts and Induced Pluripotent Stem Cells. Stem cells international Abdul Razak, S. R., Baba, Y., Nakauchi, H., Otsu, M., Watanabe, S. 2014; 2014: 101349-?


    MicroRNAs are differentially expressed in cells and regulate multiple biological processes. We have been analyzing comprehensive expression patterns of microRNA in human and mouse embryonic stem and induced pluripotent stem cells. We determined microRNAs specifically expressed in these pluripotent stem cells, and miR-142-3p is one of such microRNAs. miR-142-3p is expressed at higher levels in induced pluripotent stem cells relative to fibroblasts in mice. Level of expression of miR142-3p decreased during embryoid body formation from induced pluripotent stem cells. Loss-of-function analyses of miR-142-3p suggested that miR-142-3p plays roles in the proliferation and differentiation of induced pluripotent stem cells. CpG motifs were found in the 5' genomic region of the miR-142-3p; they were highly methylated in fibroblasts, but not in undifferentiated induced pluripotent stem cells. Treating fibroblasts with 5-aza-2'-deoxycytidine increased the expression of miR-142-3p significantly and reduced methylation at the CpG sites, suggesting that the expression of miR-142-3p is suppressed by DNA methylation in fibroblasts. Luciferase analysis using various lengths of the 5' genomic region of miR142-3p indicated that CpGs in the proximal enhancer region may play roles in suppressing the expression of miR-142-3p in fibroblasts.

    View details for DOI 10.1155/2014/101349

    View details for PubMedID 25544846

  • DNA Methylation Is Involved in the Expression of miR-142-3p in Fibroblasts and Induced Pluripotent Stem Cells STEM CELLS INTERNATIONAL Razak, S. R., Baba, Y., Nakauchi, H., Otsu, M., Watanabe, S. 2014
  • The generation and maintenance of rat induced pluripotent stem cells. Methods in molecular biology (Clifton, N.J.) Yamaguchi, T., Hamanaka, S., Nakauchi, H. 2014; 1210: 143-150


    This chapter describes a newly developed method for generating and maintaining rat induced pluripotent stem cells (riPSCs). We first provide a detailed protocol for the generation of lentiviral vector carrying three reprogramming factors to produce high-quality riPSCs. This technique allows reprogramming of rat somatic cells to ground state with germ-line competence. Subsequently, we elaborate a detailed protocol for the generation of riPSCs from rat embryonic fibroblast (REF). Finally, the protocols for the optimal culture conditions of riPSCs and preparation of frozen stock are described. We also outline the advantages of generating riPSCs.

    View details for DOI 10.1007/978-1-4939-1435-7_11

    View details for PubMedID 25173166

  • Five-lineage clonal analysis of hematopoietic stem/progenitor cells. Methods in molecular biology (Clifton, N.J.) Yamamoto, R., Morita, Y., Nakauchi, H. 2014; 1185: 237-245


    Hematopoietic stem cells (HSCs) have self-renewal activity and multipotency. Clonal analysis and determination of HSC differentiation potential into platelets and erythrocytes as well as leukocytes are essential for the study of self-renewal and lineage commitment in HSC. However, due to technical limitations, platelet and erythrocyte differentiation potentials have not been assessed. This chapter describes principles and methods for single-cell sorting, single-cell transplantation, and identification and quantitative analysis of cell contribution to platelets and erythrocytes in addition to leukocytes in mouse chimeras.

    View details for DOI 10.1007/978-1-4939-1133-2_16

    View details for PubMedID 25062633

  • Immortalization of Erythroblasts by c-MYC and BCL-XL Enables Large-Scale Erythrocyte Production from Human Pluripotent Stem Cells STEM CELL REPORTS Hirose, S., Takayama, N., Nakamura, S., Nagasawa, K., Ochi, K., Hirata, S., Yamazaki, S., Yamaguchi, T., Otsu, M., Sano, S., Takahashi, N., Sawaguchi, A., Ito, M., Kato, T., Nakauchi, H., Eto, K. 2013; 1 (6): 499-508


    The lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A(+) erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.

    View details for DOI 10.1016/j.stemcr.2013.10.010

    View details for Web of Science ID 000336647100004

    View details for PubMedID 24371805

  • Top-down motif engineering of a cytokine receptor for directing ex vivo expansion of hematopoietic stem cells JOURNAL OF BIOTECHNOLOGY Saka, K., Kawahara, M., Teng, J., Otsu, M., Nakauchi, H., Nagamune, T. 2013; 168 (4): 659-665


    The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.

    View details for DOI 10.1016/j.jbiotec.2013.09.012

    View details for Web of Science ID 000327843200049

    View details for PubMedID 24070902

  • Mesenchymal progenitor cells in mouse foetal liver regulate differentiation and proliferation of hepatoblasts. Liver international : official journal of the International Association for the Study of the Liver Ito, K., Yanagida, A., Okada, K., Yamazaki, Y., Nakauchi, H., Kamiya, A. 2013


    Hepatoblasts are somatic progenitor cells of the foetal liver that possess high proliferative capacity and bi-potency for differentiation into both hepatocytes and cholangiocytes. Although mesenchymal cells are known to be important for liver ontogeny, current understanding of their interaction with hepatoblasts remains obscure. Mesenchymal cell populations in the developing liver were purified and their potential to support proliferation and differentiation of hepatoblasts was examined.Foetal liver cells were fractionated with a flow cytometer using antibodies against cell surface markers. Gene expression of mesenchymal-specific transcripts and morphological characteristics were analysed. The ability of the mesenchymal cells to support hepatoblast function was analysed using a transwell and direct coculture system.CD45(-) Ter119(-) CD71(-) Dlk1(mid) PDGFRα(+) cells from the mid-foetal stage liver expressed the mesenchymal cell-specific transcription factors H2.0-like homeobox 1 and LIM homeobox 2 at high levels. Foetal mesenchymal cells make contact with hepatoblasts in vivo and possess the potential to differentiate into chondrocytes, osteocytes and adipocytes under appropriate cell culture conditions, indicating that these cells are possible candidates for mesenchymal stem/progenitor cells. Foetal mesenchymal cells expressed pleiotrophin, hepatocyte growth factor and midkine 1, which are involved in the growth of hepatoblasts. Using the coculture system with hepatoblasts and foetal mesenchymal cells, these cells were shown to support proliferation and maturation of hepatoblasts through indirect and direct interactions respectively.Dlk1(mid) PDGFRα(+) cells in non-haematopoetic fraction derived from the foetal liver exhibit mesenchymal stem/progenitor cell characteristics and have abilities to support proliferation and differentiation of hepatoblasts.

    View details for DOI 10.1111/liv.12387

    View details for PubMedID 24238062

  • Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms NATURE GENETICS Kon, A., Shih, L., Minamino, M., Sanada, M., Shiraishi, Y., Nagata, Y., Yoshida, K., Okuno, Y., Bando, M., Nakato, R., Ishikawa, S., Sato-Otsubo, A., Nagae, G., Nishimoto, A., Haferlach, C., Nowak, D., Sato, Y., Alpermann, T., Nagasaki, M., Shimamura, T., Tanaka, H., Chiba, K., Yamamoto, R., Yamaguchi, T., Otsu, M., Obara, N., Sakata-Yanagimoto, M., Nakamaki, T., Ishiyama, K., Nolte, F., Hofmann, W., Miyawaki, S., Chiba, S., Mori, H., Nakauchi, H., Koeffler, H. P., Aburatani, H., Haferlach, T., Shirahige, K., Miyano, S., Ogawa, S. 2013; 45 (10): 1232-U187


    Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.

    View details for DOI 10.1038/ng.2731

    View details for Web of Science ID 000324989600021

    View details for PubMedID 23955599

  • Profiling of MicroRNA in Human and Mouse ES and iPS Cells Reveals Overlapping but Distinct MicroRNA Expression Patterns PLOS ONE Razak, S. R., Ueno, K., Takayama, N., Nariai, N., Nagasaki, M., Saito, R., Koso, H., Lai, C., Murakami, M., Tsuji, K., Michiue, T., Nakauchi, H., Otsu, M., Watanabe, S. 2013; 8 (9)


    Using quantitative PCR-based miRNA arrays, we comprehensively analyzed the expression profiles of miRNAs in human and mouse embryonic stem (ES), induced pluripotent stem (iPS), and somatic cells. Immature pluripotent cells were purified using SSEA-1 or SSEA-4 and were used for miRNA profiling. Hierarchical clustering and consensus clustering by nonnegative matrix factorization showed two major clusters, human ES/iPS cells and other cell groups, as previously reported. Principal components analysis (PCA) to identify miRNAs that segregate in these two groups identified miR-187, 299-3p, 499-5p, 628-5p, and 888 as new miRNAs that specifically characterize human ES/iPS cells. Detailed direct comparisons of miRNA expression levels in human ES and iPS cells showed that several miRNAs included in the chromosome 19 miRNA cluster were more strongly expressed in iPS cells than in ES cells. Similar analysis was conducted with mouse ES/iPS cells and somatic cells, and several miRNAs that had not been reported to be expressed in mouse ES/iPS cells were suggested to be ES/iPS cell-specific miRNAs by PCA. Comparison of the average expression levels of miRNAs in ES/iPS cells in humans and mice showed quite similar expression patterns of human/mouse miRNAs. However, several mouse- or human-specific miRNAs are ranked as high expressers. Time course tracing of miRNA levels during embryoid body formation revealed drastic and different patterns of changes in their levels. In summary, our miRNA expression profiling encompassing human and mouse ES and iPS cells gave various perspectives in understanding the miRNA core regulatory networks regulating pluripotent cells characteristics.

    View details for DOI 10.1371/journal.pone.0073532

    View details for Web of Science ID 000326520200010

    View details for PubMedID 24086284

  • Generation of Col2a1-EGFP iPS Cells for Monitoring Chondrogenic Differentiation PLOS ONE Saito, T., Yano, F., Mori, D., Ohba, S., Hojo, H., Otsu, M., Eto, K., Nakauchi, H., Tanaka, S., Chung, U., Kawaguchi, H. 2013; 8 (9)


    Induced pluripotent stem cells (iPSC) are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC) marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

    View details for DOI 10.1371/journal.pone.0074137

    View details for Web of Science ID 000324494000088

    View details for PubMedID 24066106

  • Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling JOURNAL OF CLINICAL INVESTIGATION Hirata, S., Takayama, N., Jono-Ohnishi, R., Endo, H., Nakamura, S., Dohda, T., Nishi, M., Hamazaki, Y., Ishii, E., Kaneko, S., Otsu, M., Nakauchi, H., Kunishima, S., Eto, K. 2013; 123 (9): 3802-3814


    Congenital amegakaryocytic thrombocytopenia (CAMT) is caused by the loss of thrombopoietin receptor-mediated (MPL-mediated) signaling, which causes severe pancytopenia leading to bone marrow failure with onset of thrombocytopenia and anemia prior to leukopenia. Because Mpl(-/-) mice do not exhibit the human disease phenotype, we used an in vitro disease tracing system with induced pluripotent stem cells (iPSCs) derived from a CAMT patient (CAMT iPSCs) and normal iPSCs to investigate the role of MPL signaling in hematopoiesis. We found that MPL signaling is essential for maintenance of the CD34+ multipotent hematopoietic progenitor (MPP) population and development of the CD41+GPA+ megakaryocyte-erythrocyte progenitor (MEP) population, and its role in the fate decision leading differentiation toward megakaryopoiesis or erythropoiesis differs considerably between normal and CAMT cells. Surprisingly, complimentary transduction of MPL into normal or CAMT iPSCs using a retroviral vector showed that MPL overexpression promoted erythropoiesis in normal CD34+ hematopoietic progenitor cells (HPCs), but impaired erythropoiesis and increased aberrant megakaryocyte production in CAMT iPSC-derived CD34+ HPCs, reflecting a difference in the expression of the transcription factor FLI1. These results demonstrate that impaired transcriptional regulation of the MPL signaling that normally governs megakaryopoiesis and erythropoiesis underlies CAMT.

    View details for DOI 10.1172/JCI64721

    View details for Web of Science ID 000324562600026

    View details for PubMedID 23908116

  • Two differential flows in a bioreactor promoted platelet generation from human pluripotent stem cell-derived megakaryocytes EXPERIMENTAL HEMATOLOGY Nakagawa, Y., Nakamura, S., Nakajima, M., Endo, H., Dohda, T., Takayama, N., Nakauchi, H., Arai, F., Fukuda, T., Eto, K. 2013; 41 (8): 742-748


    Induced pluripotent stem cell (iPSC) technology enables us to investigate various potential iPSC-based therapies. Although the safety of iPSC derivation has not been completely validated, anucleate cells, such as platelets or erythrocytes, derived from iPSCs are promising targets. However, the efficiency of in vitro platelet generation from megakaryocytes (MKs) under static culture conditions is lower than is seen in vivo. In this study, we demonstrate the proof of concept by a two-dimensional flow culture system that enabled us to increase platelet yield from human embryonic stem cell or iPSC-derived MKs using a biomimetic artificial blood vessel system. The bioreactor was composed of biodegradable scaffolds with ordered arrays of pores made to mimic in vivo bone marrow through salt leaching. Within the system, two flows in different directions in which the angle between the directions of flow is 60 degrees but not 90 degrees contributed to suitable pressure and shear stress applied to MKs to promote platelet generation. Generated platelets derived from human embryonic stem cells or human induced pluripotent stem cells through the bioreactor with a 60-degree angle revealed intact integrin αIIbβ3 activation after agonist stimulation. Collectively, our findings indicate that two flows in different directions of two-dimensional flow culture may be a feasible system for in vitro generation of platelets from pluripotent stem cells (i.e., iPSC-derived MKs) in numbers sufficient for transfusion therapy.

    View details for DOI 10.1016/j.exphem.2013.04.007

    View details for Web of Science ID 000323024700008

    View details for PubMedID 23618622

  • An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential PLOS ONE Yanagida, A., Ito, K., Chikada, H., Nakauchi, H., Kamiya, A. 2013; 8 (7)


    Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

    View details for DOI 10.1371/journal.pone.0067541

    View details for Web of Science ID 000322433300003

    View details for PubMedID 23935837

  • Generation of Engraftable Hematopoietic Stem Cells From Induced Pluripotent Stem Cells by Way of Teratoma Formation MOLECULAR THERAPY Suzuki, N., Yamazaki, S., Yamaguchi, T., Okabe, M., Masaki, H., Takaki, S., Otsu, M., Nakauchi, H. 2013; 21 (7): 1424-1431


    In vitro generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic approaches for replacing bone marrow (BM) transplantation without rejection or graft versus host disease. Hitherto, however, it has proved difficult to generate truly functional HSCs transplantable to adult host mice. Here, we demonstrate a unique in vivo differentiation system yielding engraftable HSCs from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis. In mice, we found that iPSC-derived HSCs migrate from teratomas into the BM and their intravenous injection into irradiated recipients resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Using this in vivo generation system, we could demonstrate that X-linked severe combined immunodeficiency (X-SCID) mice can be treated by HSCs derived from gene-corrected clonal iPSCs. It should also be noted that neither leukemia nor tumors were observed in recipients after transplantation of iPSC-derived HSCs. Taken our findings together, our system presented in this report should provide a useful tool not only for the study of HSCs, but also for practical application of iPSCs in the treatment of hematologic and immunologic diseases.

    View details for DOI 10.1038/mt.2013.71

    View details for Web of Science ID 000321112500016

    View details for PubMedID 23670574

  • Generation of transgenic mouse line expressing Kusabira Orange throughout body, including erythrocytes, by random segregation of provirus method BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hamanaka, S., Ooehara, J., Morita, Y., Ema, H., Takahashi, S., Miyawaki, A., Otsu, M., Yamaguchi, T., Onodera, M., Nakauchi, H. 2013; 435 (4): 586-591


    Fluorescent-protein transgenic mice are useful for obtaining marked somatic cells to study kinetics of development or differentiation. Fluorescence-marked hematopoietic stem cells in particular are commonly used for studying hematopoiesis. However, as far as we know, no transgenic mouse line is described in which a fluorescent protein is stably and constitutively expressed in all hematopoietic cells, including erythrocytes and platelets. Using the random segregation of provirus (RSP) method, we generated from retrovirally transduced mouse embryonic stem cells a transgenic mouse line expressing a red/orange fluorescent protein, Kusabira Orange (KuO). KuO transgenic mouse line cells carry only one proviral integration site and stably express KuO in all hematopoietic-lineage elements, including erythrocytes and platelets. Moreover, bone-marrow transplantation in KuO transgenic mice demonstrated normal hematopoieisis. KuO transgenic mice likely will prove useful for study of hematopoiesis that includes erythropoiesis and megakaryopoiesis.

    View details for DOI 10.1016/j.bbrc.2013.05.017

    View details for Web of Science ID 000321025800014

    View details for PubMedID 23685154

  • Wnt5a signaling mediates biliary differentiation of fetal hepatic stem/progenitor cells in mice HEPATOLOGY Kiyohashi, K., Kakinuma, S., Kamiya, A., Sakamoto, N., Nitta, S., Yamanaka, H., Yoshino, K., Fujiki, J., Murakawa, M., Kusano-Kitazume, A., Shimizu, H., Okamoto, R., Azuma, S., Nakagawa, M., Asahina, Y., Tanimizu, N., Kikuchi, A., Nakauchi, H., Watanabe, M. 2013; 57 (6): 2502-2513


    The molecular mechanisms regulating differentiation of fetal hepatic stem/progenitor cells, called hepatoblasts, which play pivotal roles in liver development, remain obscure. Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. Although a β-catenin-independent noncanonical Wnt pathway is essential for cell adhesion and polarity, the physiological functions of noncanonical Wnt pathways in liver development are unknown. Here we describe a functional role for Wnt5a, a noncanonical Wnt ligand, in the differentiation of mouse hepatoblasts. Wnt5a was expressed in mesenchymal cells and other cells of wild-type (WT) midgestational fetal liver. We analyzed fetal liver phenotypes in Wnt5a-deficient mice using a combination of histological and molecular techniques. Expression levels of Sox9 and the number of hepatocyte nuclear factor (HNF)1β(+) HNF4α(-) biliary precursor cells were significantly higher in Wnt5a-deficient liver relative to WT liver. In Wnt5a-deficient fetal liver, in vivo formation of primitive bile ductal structures was significantly enhanced relative to WT littermates. We also investigated the function of Wnt5a protein and downstream signaling molecules using a three-dimensional culture system that included primary hepatoblasts or a hepatic progenitor cell line. In vitro differentiation assays showed that Wnt5a retarded the formation of bile duct-like structures in hepatoblasts, leading instead to hepatic maturation of such cells. Whereas Wnt5a signaling increased steady-state levels of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in fetal liver, inhibition of CaMKII activity resulted in the formation of significantly more and larger-sized bile duct-like structures in vitro compared with those in vehicle-supplemented controls.Wnt5a-mediated signaling in fetal hepatic stem/progenitor cells suppresses biliary differentiation. These findings also suggest that activation of CaMKII by Wnt5a signaling suppresses biliary differentiation. (HEPATOLOGY 2013;).

    View details for DOI 10.1002/hep.26293

    View details for Web of Science ID 000320276400043

    View details for PubMedID 23386589

  • Sal-like protein 4 (SALL4), a stem cell biomarker in liver cancers HEPATOLOGY Oikawa, T., Kamiya, A., Zeniya, M., Chikada, H., Hyuck, A. D., Yamazaki, Y., Wauthier, E., Tajiri, H., Miller, L. D., Wang, X. W., Reid, L. M., Nakauchi, H. 2013; 57 (4): 1469-1483


    Liver cancers, including hepatocellular carcinomas (HCCs), cholangiocarcinomas (CCs), and fibrolamellar HCCs (FL-HCCs) are among the most common cancers worldwide and are associated with a poor prognosis. Investigations of genes important in liver cancers have focused on Sal-like protein 4 (SALL4), a member of a family of zinc finger transcription factors. It is a regulator of embryogenesis, organogenesis, pluripotency, can elicit reprogramming of somatic cells, and is a marker of stem cells. We found it expressed in normal murine hepatoblasts, normal human hepatic stem cells, hepatoblasts and biliary tree stem cells, but not in mature parenchymal cells of liver or biliary tree. It was strongly expressed in surgical specimens of human HCCs, CCs, a combined hepatocellular and cholangiocarcinoma, a FL-HCC, and in derivative, transplantable tumor lines in immune-compromised hosts. Bioinformatics analyses indicated that elevated expression of SALL4 in tumors is associated with poor survival of HCC patients. Experimental manipulation of SALL4's expression results in changes in proliferation versus differentiation in human HCC cell lines in vitro and in vivo in immune-compromised hosts. Virus-mediated gene transfer of SALL4 was used for gain- and loss-of-function analyses in the cell lines. Significant growth inhibition in vitro and in vivo, accompanied by an increase in differentiation occurred with down-regulation of SALL4. Overexpression of SALL4 resulted in increased cell proliferation in vitro, correlating with an increase in expression of cytokeratin19 (CK19), epithelial cell adhesion molecules (EpCAM), and adenosine triphosphate (ATP)-binding cassette-G2 (ABCG2).SALL4's expression is an indicator of stem cells, a prognostic marker in liver cancers, correlates with cell and tumor growth, with resistance to 5-FU, and its suppression results in differentiation and slowed tumor growth. SALL4 is a novel therapeutic target for liver cancers.

    View details for DOI 10.1002/hep.26159

    View details for Web of Science ID 000317363600021

    View details for PubMedID 23175232

  • A retrospective analysis of germline competence in rat embryonic stem cell lines TRANSGENIC RESEARCH Hirabayashi, M., Tamura, C., Sanbo, M., Kato-Itoh, M., Kobayashi, T., Nakauchi, H., Hochi, S. 2013; 22 (2): 411-416


    The factors responsible for conferring germline competence in embryonic stem (ES) cell lines remain unidentified. In the present study, rat ES cell lines (n = 17) were established with 3i medium (SU5402, PD0325901, CHIR99021), 2i medium (PD0325901, CHIR99021) or 2iF medium (PD0325901, CHIR99021, forskolin), and their potential for germline transmission to the G1 generation was examined. Rat strains were divided into an albino group (F344, Wistar or CAG/Venus transgenic rats with the Wistar background) or a colored coat group (Brown-Norway, Dark-Agouti, or BLK rats selected from >F3 generations of Wistar × Dark-Agouti rats based on their black coat color). Successful germline transmission was observed in 57 % (4/7), 40 % (2/5) and 100 % (5/5) of the ES cells established with 3i, 2i and 2iF media, respectively. ES cell lines from the homozygous CAG/Venus transgenic rats were established in all three media, but only the lines established with the 2iF medium were germline-competent. Neither coat-color (albino: 64 %, 7/11; colored: 67 %, 4/6) nor gender of the ES cell lines (XX: 67 %, 2/3; XY: 64 %, 9/14) were likely to affect germline transmission.

    View details for DOI 10.1007/s11248-012-9638-7

    View details for Web of Science ID 000316065900013

    View details for PubMedID 22875289

  • Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hiramoto, T., Ebihara, Y., Mizoguchi, Y., Nakamura, K., Yamaguchi, K., Ueno, K., Nariai, N., Mochizuki, S., Yamamoto, S., Nagasaki, M., Furukawa, Y., Tani, K., Nakauchi, H., Kobayashi, M., Tsuji, K. 2013; 110 (8): 3023-3028


    The derivation of induced pluripotent stem (iPS) cells from individuals of genetic disorders offers new opportunities for basic research into these diseases and the development of therapeutic compounds. Severe congenital neutropenia (SCN) is a serious disorder characterized by severe neutropenia at birth. SCN is associated with heterozygous mutations in the neutrophil elastase [elastase, neutrophil-expressed (ELANE)] gene, but the mechanisms that disrupt neutrophil development have not yet been clarified because of the current lack of an appropriate disease model. Here, we generated iPS cells from an individual with SCN (SCN-iPS cells). Granulopoiesis from SCN-iPS cells revealed neutrophil maturation arrest and little sensitivity to granulocyte-colony stimulating factor, reflecting a disease status of SCN. Molecular analysis of the granulopoiesis from the SCN-iPS cells vs. control iPS cells showed reduced expression of genes related to the wingless-type mmtv integration site family, member 3a (Wnt3a)/β-catenin pathway [e.g., lymphoid enhancer-binding factor 1], whereas Wnt3a administration induced elevation lymphoid enhancer-binding factor 1-expression and the maturation of SCN-iPS cell-derived neutrophils. These results indicate that SCN-iPS cells provide a useful disease model for SCN, and the activation of the Wnt3a/β-catenin pathway may offer a novel therapy for SCN with ELANE mutation.

    View details for DOI 10.1073/pnas.1217039110

    View details for Web of Science ID 000315954400084

    View details for PubMedID 23382209

  • The early retinal progenitor-expressed gene Sox11 regulates the timing of the differentiation of retinal cells DEVELOPMENT Usui, A., Mochizuki, Y., Iida, A., Miyauchi, E., Satoh, S., Sock, E., Nakauchi, H., Aburatani, H., Murakami, A., Wegner, M., Watanabe, S. 2013; 140 (4): 740-750


    Sry-related HMG box (Sox) proteins, Sox11 and Sox4 are members of the SoxC subtype. We found that Sox11 was strongly expressed in early retinal progenitor cells and that Sox4 expression began around birth, when expression of Sox11 subsided. To analyze the roles of Sox11 and Sox4 in retinal development, we perturbed their expression patterns in retinal explant cultures. Overexpression of Sox11 and Sox4 in retinal progenitors resulted in similar phenotypes: an increased number of cone cells and dramatically decreased numbers of rod cells and Müller glia. Birth-date analysis showed that cone cells were produced at a later developmental stage than that in which cone genesis normally occurs. Sox11-knockout retinas showed delayed onset and progress of differentiation of subsets of retinal cells during the embryonic period. After birth, retinal differentiation took place relatively normally, probably because of the redundant activity of Sox4, which starts to be expressed around birth. Overexpression and loss-of-function analysis failed to provide any evidence that Sox11 and Sox4 directly regulate the transcription of genes crucial to the differentiation of subsets of retinal cells. However, histone H3 acetylation of some early proneural genes was reduced in knockout retina. Thus, Sox11 may create an epigenetic state that helps to establish the competency to differentiate. Taking our findings together, we propose that the sequential expression of Sox11 and Sox4 during retinogenesis leads to the fine adjustment of retinal differentiation by helping to establish the competency of retinal progenitors.

    View details for DOI 10.1242/dev.090274

    View details for Web of Science ID 000314405000005

    View details for PubMedID 23318640

  • Role of SOX17 in hematopoietic development from human embryonic stem cells BLOOD Nakajima-Takagi, Y., Osawa, M., Oshima, M., Takagi, H., Miyagi, S., Endoh, M., Endo, T. A., Takayama, N., Eto, K., Toyoda, T., Koseki, H., Nakauchi, H., Iwama, A. 2013; 121 (3): 447-458


    To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34(+)CD43(-) endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested, only Sox17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34(+)CD43(+)CD45(-/low) cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34(+)CD43(-) ECs compared with low levels in CD34(+)CD43(+)CD45(-) pre-hematopoietic progenitor cells (pre-HPCs) and CD34(+)CD43(+)CD45(+) HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34(+)CD43(+)CD45(-/low) cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation directly. Depletion of SOX17 in CD34(+)CD43(-) ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs, thereby regulating hematopoietic development from hESCs/iPSCs.

    View details for DOI 10.1182/blood-2012-05-431403

    View details for Web of Science ID 000313727500008

    View details for PubMedID 23169777

  • Stem cell therapy: an exercise in patience and prudence PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Lin, H., Otsu, M., Nakauchi, H. 2013; 368 (1609)


    In recent times, the epigenetic study of pluripotency based on cellular reprogramming techniques led to the creation of induced pluripotent stem cells. It has come to represent the forefront of a new wave of alternative therapeutic approaches in the field of stem cell therapy. Progress in drug development has saved countless lives, but there are numerous intractable diseases where curative treatment cannot be achieved through pharmacological intervention alone. Consequently, there has been an unfortunate rise in incidences of organ failures, degenerative disorders and cancers, hence novel therapeutic interventions are required. Stem cells have unique self-renewal and multilineage differentiation capabilities that could be harnessed for therapeutic purposes. Although a number of mature differentiated cells have been characterized in vitro, few have been demonstrated to function in a physiologically relevant context. Despite fervent levels of enthusiasm in the field, the reality is that other than the employment of haematopoietic stem cells, many other therapies have yet to be thoroughly proven for their therapeutic benefit and safety in application. This review shall focus on a discussion regarding the current status of stem cell therapy, the issues surrounding it and its future prospects with a general background on the regulatory networks underlying pluripotency.

    View details for DOI 10.1098/rstb.2011.0334

    View details for Web of Science ID 000311945800007

    View details for PubMedID 23166396

  • 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


    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

  • Enrichment and clonal culture of hepatic stem/progenitor cells during mouse liver development. Methods in molecular biology (Clifton, N.J.) Kamiya, A., Nakauchi, H. 2013; 945: 273-286


    Liver regenerates after hepatectomy or chemical-induced injury. In contrast to cells in other tissues that can regenerate, mature cells (hepatocytes), but not undifferentiated stem cells, are mainly responsible for acute liver regeneration. Liver stem cells take part in liver regeneration in some forms of chronic liver injury, when the proliferative ability of differentiated hepatocytes is impaired. During liver development, both hepatocytes and cholangiocytes are differentiated from common precursor cells, called hepatoblasts. By combining fluorescence-activated cell sorting (FACS) and an in vitro clonal culture system for stem/progenitor cells, we established a method to isolate stem/progenitor cells prospectively from mouse fetal and adult livers. FACS clone-sorted single CD45(-)Ter119(-)c-kit(-)CD13(+)CD133(+) cells (from fetal mid-gestational livers) or CD45(-)Ter119(-)c-kit(-)Sca1(-)CD13(+)CD49f(+)CD133(+) cells (from adult livers) can form a colony containing both albumin-positive hepatocytes and cytokeratin 19-positive bile ductal cells, indicating that these cells have the characters of liver stem/progenitor cells (proliferative capability and bipotency for hepatic and for biliary epithelial differentiation). These cells can maintain these capabilities for several months in culture.

    View details for DOI 10.1007/978-1-62703-125-7_16

    View details for PubMedID 23097112

  • Rapid T-cell chimerism switch and memory T-cell expansion are associated with pre-engraftment immune reaction early after cord blood transplantation BRITISH JOURNAL OF HAEMATOLOGY Matsuno, N., Yamamoto, H., Watanabe, N., Uchida, N., Ota, H., Nishida, A., Ikebe, T., Ishiwata, K., Nakano, N., Tsuji, M., Asano-Mori, Y., Izutsu, K., Masuoka, K., Wake, A., Yoneyama, A., Nakauchi, H., Taniguchi, S. 2013; 160 (2): 255-258

    View details for DOI 10.1111/bjh.12097

    View details for Web of Science ID 000313520300018

    View details for PubMedID 23116322

  • Identification of Rat Rosa26 Locus Enables Generation of Knock-In Rat Lines Ubiquitously Expressing tdTomato STEM CELLS AND DEVELOPMENT Kobayashi, T., Kato-Itoh, M., Yamaguchi, T., Tamura, C., Sanbo, M., Hirabayashi, M., Nakauchi, H. 2012; 21 (16): 2981-2986


    Recent discovery of a method for derivation and culture of germline-competent rat pluripotent stem cells (PSCs) enables generation of transgenic rats or knock-out rats via genetic modification of such PSCs. This opens the way to use rats, as is routine in mice, for analyses of gene functions or physiological features. In mouse or human, one widely used technique to express a gene of interest stably and ubiquitously is to insert that gene into the Rosa26 locus via gene targeting of PSCs. Rosa26 knock-in mice conditionally expressing a reporter or a toxin gene have contributed to tracing or ablation of specific cell lineages. We successfully identified a rat orthologue of the mouse Rosa26 locus. Insertion of tdTomato, a variant of red fluorescent protein, into the Rosa26 locus of PSCs of various rat strains allows ubiquitous expression of tdTomato. Through germline transmission of one Rosa26-tdTomato knock-in embryonic stem cell line, we also obtained tdTomato knock-in rats. These expressed tdTomato ubiquitously throughout their bodies, which indicates that the rat Rosa26 locus conserves functions of its orthologues in mouse and human. The new tools described here (targeting vectors, knock-in PSCs, and rats) should be useful for a variety of research using rats.

    View details for DOI 10.1089/scd.2012.0065

    View details for Web of Science ID 000310060900008

    View details for PubMedID 22564063

  • Development of an All-in-One Inducible Lentiviral Vector for Gene Specific Analysis of Reprogramming PLOS ONE Yamaguchi, T., Hamanaka, S., Kamiya, A., Okabe, M., Kawarai, M., Wakiyama, Y., Umino, A., Hayama, T., Sato, H., Lee, Y., Kato-Itoh, M., Masaki, H., Kobayashi, T., Yamazaki, S., Nakauchi, H. 2012; 7 (7)


    Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.

    View details for DOI 10.1371/journal.pone.0041007

    View details for Web of Science ID 000306548900067

    View details for PubMedID 22815895

  • Inhibition of PAI-1 induces neutrophil-driven neoangiogenesis and promotes tissue regeneration via production of angiocrine factors in mice BLOOD Tashiro, Y., Nishida, C., Sato-Kusubata, K., Ohki-Koizumi, M., Ishihara, M., Sato, A., Gritli, I., Komiyama, H., Sato, Y., Dan, T., Miyata, T., Okumura, K., Tomiki, Y., Sakamoto, K., Nakauchi, H., Heissig, B., Hattori, K. 2012; 119 (26): 6382-6393


    Plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of a major fibrinolytic factor, tissue-type plasminogen activator, can both promote and inhibit angiogenesis. However, the physiologic role and the precise mechanisms underlying the angiogenic effects of PAI-1 remain unclear. In the present study, we report that pharmacologic inhibition of PAI-1 promoted angiogenesis and prevented tissue necrosis in a mouse model of hind-limb ischemia. Improved tissue regeneration was due to an expansion of circulating and tissue-resident granulocyte-1 marker (Gr-1(+)) neutrophils and to increased release of the angiogenic factor VEGF-A, the hematopoietic growth factor kit ligand, and G-CSF. Immunohistochemical analysis indicated increased amounts of fibroblast growth factor-2 (FGF-2) in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated animals. Ab neutralization and genetic knockout studies indicated that both the improved tissue regeneration and the increase in circulating and ischemic tissue-resident Gr-1(+) neutrophils depended on the activation of tissue-type plasminogen activator and matrix metalloproteinase-9 and on VEGF-A and FGF-2. These results suggest that pharmacologic PAI-1 inhibition activates the proangiogenic FGF-2 and VEGF-A pathways, which orchestrates neutrophil-driven angiogenesis and induces cell-driven revascularization and is therefore a potential therapy for ischemic diseases.

    View details for DOI 10.1182/blood-2011-12-399659

    View details for Web of Science ID 000307400700031

    View details for PubMedID 22573404

  • Generation of induced pluripotent stem cells from primary chronic myelogenous leukemia patient samples BLOOD Kumano, K., Arai, S., Hosoi, M., Taoka, K., Takayama, N., Otsu, M., Nagae, G., Ueda, K., Nakazaki, K., Kamikubo, Y., Eto, K., Aburatani, H., Nakauchi, H., Kurokawa, M. 2012; 119 (26): 6234-6242


    Induced pluripotent stem cells (iPSCs) can be generated by the expression of defined transcription factors not only from normal tissue, but also from malignant cells. Cancer-derived iPSCs are expected to provide a novel experimental opportunity to establish the disease model. We generated iPSCs from imatinib-sensitive chronic myelogenous leukemia (CML) patient samples. Remarkably, the CML-iPSCs were resistant to imatinib although they consistently expressed BCR-ABL oncoprotein. In CML-iPSCs, the phosphorylation of ERK1/2, AKT, and JNK, which are essential for the maintenance of both BCR-ABL (+) leukemia cells and iPSCs, were unchanged after imatinib treatment, whereas the phosphorylation of signal transducer and activator of transcription (STAT)5 and CRKL was significantly decreased. These results suggest that the signaling for iPSCs maintenance compensates for the inhibition of BCR-ABL. CML-iPSC-derived hematopoietic cells recovered the sensitivity to imatinib although CD34(+)38(-)90(+)45(+) immature cells were resistant to imatinib, which recapitulated the pathophysiologic feature of the initial CML. CML-iPSCs provide us with a novel platform to investigate CML pathogenesis on the basis of patient-derived samples.

    View details for DOI 10.1182/blood-2011-07-367441

    View details for Web of Science ID 000307400700014

    View details for PubMedID 22592606

  • MT1-MMP plays a critical role in hematopoiesis by regulating HIF-mediated chemokine/cytokine gene transcription within niche cells BLOOD Nishida, C., Kusubata, K., Tashiro, Y., Gritli, I., Sato, A., Ohki-Koizumi, M., Morita, Y., Nagano, M., Sakamoto, T., Koshikawa, N., Kuchimaru, T., Kizaka-Kondoh, S., Seiki, M., Nakauchi, H., Heissig, B., Hattori, K. 2012; 119 (23): 5405-5416


    HSC fate decisions are regulated by cell-intrinsic and cell-extrinsic cues. The latter cues are derived from the BM niche. Membrane-type 1 matrix metalloproteinase (MT1-MMP), which is best known for its proteolytic role in pericellular matrix remodeling, is highly expressed in HSCs and stromal/niche cells. We found that, in MT1-MMP(-/-) mice, in addition to a stem cell defect, the transcription and release of kit ligand (KitL), stromal cell-derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hematopoietic differentiation block, while addition of exogenous KitL and SDF-1 restored hematopoiesis. Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis, by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration.

    View details for DOI 10.1182/blood-2011-11-390849

    View details for Web of Science ID 000307391400015

    View details for PubMedID 22544701

  • The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer TRANSGENIC RESEARCH Watanabe, M., Kurome, M., Matsunari, H., Nakano, K., Umeyema, K., Shiota, A., Nakauchi, H., Nagashima, H. 2012; 21 (3): 605-618


    In most transgenic (Tg) animals created to date, a transgene consisting of the minimum promoter region linked to a cDNA has been used. However, transgenes on small plasmids are susceptible to the position effect, increasing the difficulty of controlling transgene expression. In this study, we attempted to create Tg pigs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) using two large genomic transgenes derived from a bacterial artificial chromosome (BAC) containing the full genomic region encoding two human proteins, type I collagen and albumin. The production efficiencies (Tg piglets/live offspring) of type I collagen and albumin Tg pigs were 11.8% (6/51) and 18.2% (2/11), respectively. In all of the Tg pigs examined by real-time PCR analysis, tissue-specific expression of the transgene was confirmed (type I collagen: skin, tendon, vessels, genitalia; albumin: liver). The production of human proteins derived from BAC transgenes was also confirmed. Fluorescence in situ hybridization analysis indicated that the BAC transgenes transferred into porcine oocytes by ICSI-MGT were integrated into single or multiple sites on the host chromosomes. These data demonstrate that Tg pigs expressing human proteins in a tissue-specific manner can be created using a BAC transgenic construct and the ICSI-MGT method.

    View details for DOI 10.1007/s11248-011-9561-3

    View details for Web of Science ID 000304097400013

    View details for PubMedID 22038447

  • Ability of tetraploid rat blastocysts to support fetal development after complementation with embryonic stem cells MOLECULAR REPRODUCTION AND DEVELOPMENT Hirabayashi, M., Tamura, C., Sanbo, M., Goto, T., Kato-Itoh, M., Kobayashi, T., Nakauchi, H., Hochi, S. 2012; 79 (6): 402-412


    This study was undertaken to generate rat offspring via tetraploid blastocyst complementation with embryonic stem (ES) cells. Tetraploid blastocysts were prepared by electrofusion of blastomeres from two-cell stage embryos, and subsequent in vivo culture for 4 days. Microinjection into the tetraploid blastocoel of an inner cell mass isolated by immunosurgery resulted in the generation of rat offspring, suggesting the successful contribution of tetraploid blastocysts to their placenta. Tetraploid blastocyst complementation was attempted with a total of 4 ES cell lines (2 lines of female karyotype and 2 lines of male karyotype). In the rESWIv-3i-5 (XX) cell line, normal-sized fetuses with heartbeats were harvested on E11.5 (12.1%), E12.5 (9.5%), and E13.5 (9.1%), but no viable fetuses were detected on E14.5. Similarly, use of the rESWIv-3i-1 (XX) cell line resulted in no viable fetus production on E14.5. Using the rESBLK2i-1 (XY) cell line, viable fetuses were harvested not only on E11.5-E13.5 (2.6-5.5%), but also on E14.5 (3.0%). The transfer of a total of 487 tetraploid blastocysts complemented with rESBLK2i-1 cells resulted in 256 implantation sites (52.6%) on E21.5, but no viable offspring was detected. Use of the rESBLK2i-1/huKO (XY) cell line also resulted in no viable offspring production on E21.5. Analyses of the methylation pattern in differentially methylated regions and transcript level of genes that are imprinted in mice (H19, Meg3, Igf2r, Peg5, and Peg10) in the E14.5 conceptuses indicated a marked difference between the ES cell-derived and control normal fetuses, but not between the tetraploid and control diploid placenta.

    View details for DOI 10.1002/mrd.22043

    View details for Web of Science ID 000304037600005

    View details for PubMedID 22499253

  • Generation of Kidney from Pluripotent Stem Cells via Blastocyst Complementation AMERICAN JOURNAL OF PATHOLOGY Usui, J., Kobayashi, T., Yamaguchi, T., Knisely, A. S., Nishinakamura, R., Nakauchi, H. 2012; 180 (6): 2417-2426


    Because a shortage of donor organs has been a major obstacle to the expansion of organ transplantation programs, the generation of transplantable organs is among the ultimate goals of regenerative medicine. However, the complex cellular interactions among and within tissues that are required for organogenesis are difficult to recapitulate in vitro. As an alternative, we used blastocyst complementation to generate pluripotent stem cell (PSC)-derived donor organs in vivo. We hypothesized that if we injected PSCs into blastocysts obtained from mutant mice in which the development of a certain organ was precluded by genetic manipulation, thereby leaving a niche for organ development, the PSC-derived cells would developmentally compensate for the defect and form the missing organ. In our previous work, we showed proof-of-principle findings of pancreas generation by injection of PSCs into pancreas-deficient Pdx1(-/-) mouse blastocysts. In this study, we have extended this technique to kidney generation using Sall1(-/-) mouse blastocysts. As a result, the defective cells were totally replaced, and the kidneys were entirely formed by the injected mouse PSC-derived cells, except for structures not under the influence of Sall1 expression (ie, collecting ducts and microvasculature). These findings indicate that blastocyst complementation can be extended to generate PSC-derived kidneys. This system may therefore provide novel insights into renal organogenesis.

    View details for DOI 10.1016/j.ajpath.2012.03.007

    View details for Web of Science ID 000305101300025

    View details for PubMedID 22507837

  • Bmi1 Confers Resistance to Oxidative Stress on Hematopoietic Stem Cells PLOS ONE Nakamura, S., Oshima, M., Yuan, J., Saraya, A., Miyagi, S., Konuma, T., Yamazaki, S., Osawa, M., Nakauchi, H., Koseki, H., Iwama, A. 2012; 7 (5)


    The polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed.In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26Stop(FL)Bmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26Stop(FL)Bmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS).Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it.

    View details for DOI 10.1371/journal.pone.0036209

    View details for Web of Science ID 000305338200017

    View details for PubMedID 22606246

  • Highly Efficient Generation of Definitive Endoderm Lineage from Human Induced Pluripotent Stem Cells 12th Congress of the Asian-Society-of-Transplantation (AST) Sekine, K., Takebe, T., Suzuki, Y., Kamiya, A., Nakauchi, H., Taniguchi, H. ELSEVIER SCIENCE INC. 2012: 1127–29


    Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology.Human iPS cells are maintained on Mitomycin C-treated mouse embryonic fibroblast layers in DMEM-Ham F12-based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling.We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation.

    View details for DOI 10.1016/j.transproceed.2012.03.001

    View details for Web of Science ID 000304240400086

    View details for PubMedID 22564643

  • Prospective Isolation and Characterization of Bipotent Progenitor Cells in Early Mouse Liver Development STEM CELLS AND DEVELOPMENT Okada, K., Kamiya, A., Ito, K., Yanagida, A., Ito, H., Kondou, H., Nishina, H., Nakauchi, H. 2012; 21 (7): 1124-1133


    Outgrowth of the foregut endoderm to form the liver bud is considered the initial event of liver development. Hepatic stem/progenitor cells (HSPCs) in the liver bud are postulated to migrate into septum transversum mesenchyme at around embryonic day (E) 9 in mice. The studies of liver development focused on the mid-fetal stage (E11.5-14.5) have identified HSPCs at this stage. However, the in vitro characteristics of HSPCs before E11.5 have not been elucidated. This is probably partly because purification and characterization of HSPCs in early fetal livers have not been fully established. To permit detailed phenotypic analyses of early fetal HSPC candidates, we developed a new coculture system, using mouse embryonic fibroblast cells. In this coculture system, CD13(+)Dlk(+) cells purified from mouse early fetal livers (E9.5 and E10.5) formed colonies composed of both albumin-positive hepatocytic cells and cytokeratin (CK) 19-positive cholangiocytic cells, indicating that early fetal CD13(+)Dlk(+) cells have properties of bipotent progenitor cells. Inhibition of signaling by Rho-associated coiled-coil containing protein kinase (Rock) or by nonmuscle myosin II (downstream from Rock) was necessary for effective expansion of early fetal CD13(+)Dlk(+) cells in vitro. In sorted CD13(+)Dlk(+) cells, expression of the hepatocyte marker genes albumin and α-fetoprotein increased with fetal liver age, whereas expression of CK19 and Sox17, endodermal progenitor cell markers, was highest at E9.5 but decreased dramatically thereafter. These first prospective studies of early fetal HSPC candidates demonstrate that bipotent stem/progenitor cells exist before E11.5 and implicate Rock-myosin II signaling in their development.

    View details for DOI 10.1089/scd.2011.0229

    View details for Web of Science ID 000303268000010

    View details for PubMedID 21861758

  • Distinct B-cell lineage commitment distinguishes adult bone marrow hematopoietic stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ghosn, E. E., Yamamoto, R., Hamanaka, S., Yang, Y., Herzenberg, L. A., Nakauchi, H., Herzenberg, L. A. 2012; 109 (14): 5394-5398


    The question of whether a single hematopoietic stem cell (HSC) gives rise to all of the B-cell subsets [B-1a, B-1b, B-2, and marginal zone (MZ) B cells] in the mouse has been discussed for many years without resolution. Studies here finally demonstrate that individual HSCs sorted from adult bone marrow and transferred to lethally irradiated recipients clearly give rise to B-2, MZ B, and B-1b, but does not detectably reconstitute B-1a cells. These findings place B-2, MZ, and B-1b in a single adult developmental lineage and place B-1a in a separate lineage derived from HSCs that are rare or missing in adults. We discuss these findings with respect to known developmental heterogeneity in other HSC-derived lymphoid, myeloid, and erythroid lineages, and how HSC developmental heterogeneity conforms to the layered model of the evolution of the immune system that we proposed some years ago. In addition, of importance to contemporary medicine, we consider the implications that HSC developmental heterogeneity may have for selecting HSC sources for human transplantation.

    View details for DOI 10.1073/pnas.1121632109

    View details for Web of Science ID 000302294700059

    View details for PubMedID 22431624

    View details for PubMedCentralID PMC3325648

  • In vitro expansion and functional recovery of mature hepatocytes from mouse adult liver LIVER INTERNATIONAL Ito, H., Kamiya, A., Ito, K., Yanagida, A., Okada, K., Nakauchi, H. 2012; 32 (4): 592-601


    Mature hepatocytes retain the ability to regenerate the liver lobule fully in vivo following injury. Several cytokines and soluble factors (hepatocyte growth factors, epidermal growth factors, insulin and nicotinamide) are known to be important for proliferation of mature hepatocytes in vitro. However, hepatocytes monolayer-cultured on extracellular matrices have gradually lost their specific functions, particularly those in drug metabolism.We have explored and established a new culture system for expansion of functional hepatocytes.We evaluated two approaches for efficient expansion of mature hepatocytes: (i) Co-culture with mouse embryonic fibroblasts (MEF); (ii) Addition to culture of inhibitors of cell signals involved in liver regeneration. After expansion steps, 3-dimensional spheroid-forming culture was used to re-induce mature hepatocellular function.The addition of inhibitors for tumour growth factor (TGF) β and glycogen synthase kinase (GSK) 3β efficiently induced in vitro expansion of mature hepatocytes. Although expression of hepatocellular functional genes decreased after expansion in monolayer culture, their expression and the activity of cytochrome P450 enzymes significantly increased with spheroid formation. Furthermore, when hepatocytes were co-cultured with MEF, addition of a MAPK/ERK kinase (MEK) inhibitor at the spheroid formation step enhanced drug-metabolism-related gene expression.Combination of the MEF co-culture system with the addition of inhibitors of TGFβ and GSK3β induced in vitro expansion of hepatocytes. Moreover, expression of mature hepatocellular genes and the activity of drug-metabolism enzymes in expanded hepatocytes were re-induced after spheroid culture.

    View details for DOI 10.1111/j.1478-3231.2011.02741.x

    View details for Web of Science ID 000301233800010

    View details for PubMedID 22222094

  • [Utilization of the ES/iPS cell technology for hematopoietic stem cell transplantation]. Nihon rinsho. Japanese journal of clinical medicine Otsu, M., Nakauchi, H. 2012; 70: 146-150

    View details for PubMedID 23133943

  • Lethal myelofibrosis induced by Bmi1-deficient hematopoietic cells unveils a tumor suppressor function of the polycomb group genes JOURNAL OF EXPERIMENTAL MEDICINE Oguro, H., Yuan, J., Tanaka, S., Miyagi, S., Mochizuki-Kashio, M., Ichikawa, H., Yamazaki, S., Koseki, H., Nakauchi, H., Iwama, A. 2012; 209 (3): 445-454


    Polycomb-group (PcG) proteins form the multiprotein polycomb repressive complexes (PRC) 1 and 2, and function as transcriptional repressors through histone modifications. They maintain the proliferative capacity of hematopoietic stem and progenitor cells by repressing the transcription of tumor suppressor genes, namely Ink4a and Arf, and thus have been characterized as oncogenes. However, the identification of inactivating mutations in the PcG gene, EZH2, unveiled a tumor suppressor function in myeloid malignancies, including primary myelofibrosis (PMF). Here, we show that loss of another PcG gene, Bmi1, causes pathological hematopoiesis similar to PMF. In a mouse model, loss of Bmi1 in Ink4a-Arf(-/-) hematopoietic cells induced abnormal megakaryocytopoiesis accompanied by marked extramedullary hematopoiesis, which eventually resulted in lethal myelofibrosis. Absence of Bmi1 caused derepression of a cohort of genes, including Hmga2, which is an oncogene overexpressed in PMF. Chromatin immunoprecipitation assays revealed that Bmi1 directly represses the transcription of Hmga2. Overexpression of Hmga2 in hematopoietic stem cells induced a myeloproliferative state with enhanced megakaryocytopoiesis in mice, implicating Hmga2 in the development of pathological hematopoiesis in the absence of Bmi1. Our findings provide the first genetic evidence of a tumor suppressor function of Bmi1 and uncover the role of PcG proteins in restricting growth by silencing oncogenes.

    View details for DOI 10.1084/jem.20111709

    View details for Web of Science ID 000302635900005

    View details for PubMedID 22351929

  • A Differentiation Checkpoint Limits Hematopoietic Stem Cell Self-Renewal in Response to DNA Damage CELL Wang, J., Sun, Q., Morita, Y., Jiang, H., Gross, A., Lechel, A., Hildner, K., Guachalla, L. M., Gompf, A., Hartmann, D., Schambach, A., Wuestefeld, T., Dauch, D., Schrezenmeier, H., Hofmann, W., Nakauchi, H., Ju, Z., Kestler, H. A., Zender, L., Rudolph, K. L. 2012; 148 (5): 1001-1014


    Checkpoints that limit stem cell self-renewal in response to DNA damage can contribute to cancer protection but may also promote tissue aging. Molecular components that control stem cell responses to DNA damage remain to be delineated. Using in vivo RNAi screens, we identified basic leucine zipper transcription factor, ATF-like (BATF) as a major component limiting self-renewal of hematopoietic stem cells (HSCs) in response to telomere dysfunction and γ-irradiation. DNA damage induces BATF in a G-CSF/STAT3-dependent manner resulting in lymphoid differentiation of HSCs. BATF deletion improves HSC self-renewal and function in response to γ-irradiation or telomere shortening but results in accumulation of DNA damage in HSCs. Analysis of bone marrow from patients with myelodysplastic syndrome supports the conclusion that DNA damage-dependent induction of BATF is conserved in human HSCs. Together, these results provide experimental evidence that a BATF-dependent differentiation checkpoint limits self-renewal of HSCs in response to DNA damage.

    View details for DOI 10.1016/j.cell.2012.01.040

    View details for Web of Science ID 000300985000019

    View details for PubMedID 22385964

  • Characterization of the ICSI-mediated gene transfer method in the production of transgenic pigs MOLECULAR REPRODUCTION AND DEVELOPMENT Umeyama, K., Saito, H., Kurome, M., Matsunari, H., Watanabe, M., Nakauchi, H., Nagashima, H. 2012; 79 (3): 218-228


    Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI-MGT yielded significantly more embryos with non-mosaic transgene expression (P < 0.01). In the ICSI-MGT group, 61.5% (24/39) of the embryos were EGFP-positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP-positive in the PN-injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI-MGT was transgenic, suggesting that ICSI-MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI-MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple-copy, single-site integration of a transgene is the primary outcome of ICSI-MGT in the pig and that ICSI-MGT is less likely than PN injection to cause transgene integration in a mosaic manner.

    View details for DOI 10.1002/mrd.22015

    View details for Web of Science ID 000300681900008

    View details for PubMedID 22213433

  • In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling BLOOD Nishimura, S., Manabe, I., Nagasaki, M., Kakuta, S., Iwakura, Y., Takayama, N., Ooehara, J., Otsu, M., Kamiya, A., Petrich, B. G., Urano, T., Kadono, T., Sato, S., Aiba, A., Yamashita, H., Sugiura, S., Kadowaki, T., Nakauchi, H., Eto, K., Nagai, R. 2012; 119 (8): E45-E56


    The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

    View details for DOI 10.1182/blood-2011-09-381400

    View details for Web of Science ID 000300713800001

    View details for PubMedID 22096246

  • Plasminogen deficiency attenuates postnatal erythropoiesis in male C57BL/6 mice through decreased activity of the LH-testosterone axis EXPERIMENTAL HEMATOLOGY Okaji, Y., Tashiro, Y., Gritli, I., Nishida, C., Sato, A., Ueno, Y., Gonzalez, S. D., Ohki-Koizumi, M., Akiyama, H., Nakauchi, H., Hattori, K., Heissig, B. 2012; 40 (2): 143-154


    Novel roles for the serine protease plasmin have been implicated recently in physiological and pathological processes. However, whether plasmin is involved in erythropoiesis is not known. In the present study, we studied the consequences of plasminogen deficiency on erythropoiesis in plasminogen-deficient (Plg knockout [KO]) mice. Erythroid differentiation was attenuated in male Plg KO mice and resulted in erythroblastic accumulation within the spleen and bone marrow, with increased apoptosis in the former, erythrocytosis, and splenomegaly, whereas similar erythropoietic defect was less prominent in female Plg KO mice. In addition, erythrocyte lifespan was shorter in both male and female Plg KO mice. Erythropoietin levels were compensatory increased in both male and female Plg KO mice, and resulted in a higher frequency of burst-forming units-erythroid within the spleen and bone marrow. Surprisingly, we found that male Plg KO mice, but not their female counterparts, exhibited normochromic normocytic anemia. The observed sex-linked erythropoietic defect was attributed to decreased serum testosterone levels in Plg KO mice as a consequence of impaired secretion of the pituitary luteinizing hormone (LH) under steady-state condition. Surgical castration causing testosterone deficiency and stimulating LH release attenuated erythroid differentiation and induced anemia in wild-type animals, but did not further decrease the hematocrit levels in Plg KO mice. In addition, complementation of LH using human choriogonadotropin, which increases testosterone production, improved the erythropoietic defect and anemia in Plg KO mice. The present results identify a novel role for plasmin in the hormonal regulation of postnatal erythropoiesis by the LH-testosterone axis.

    View details for DOI 10.1016/j.exphem.2011.10.008

    View details for Web of Science ID 000299586200006

    View details for PubMedID 22056679

  • Plasmin inhibitor reduces T-cell lymphoid tumor growth by suppressing matrix metalloproteinase-9-dependent CD11b(+)/F4/80(+) myeloid cell recruitment LEUKEMIA Ishihara, M., Nishida, C., Tashiro, Y., Gritli, I., Rosenkvist, J., Koizumi, M., Okaji, Y., Yamamoto, R., Yagita, H., Okumura, K., Nishikori, M., Wanaka, K., Tsuda, Y., Okada, Y., Nakauchi, H., Heissig, B., Hattori, K. 2012; 26 (2): 332-339


    Activation of the fibrinolytic system during lymphoma progression is a well-documented clinical phenomenon. But the mechanism by which the fibrinolytic system can modulate lymphoma progression has been elusive. The main fibrinolytic enzyme, plasminogen (Plg)/plasmin (Plm), can activate matrix metalloproteinases (MMPs), such as MMP-9, which has been linked to various malignancies. Here we provide the evidence that blockade of Plg reduces T-cell lymphoma growth by inhibiting MMP-9-dependent recruitment of CD11b(+)F4/80(+) myeloid cells locally within the lymphoma tissue. Genetic Plg deficiency and drug-mediated Plm blockade delayed T-cell lymphoma growth and diminished MMP-9-dependent CD11b(+)F4/80(+) myeloid cell infiltration into lymphoma tissues. A neutralizing antibody against CD11b inhibited T-cell lymphoma growth in vivo, which indicates that CD11b(+) myeloid cells have a role in T-cell lymphoma growth. Plg deficiency in T-cell lymphoma-bearing mice resulted in reduced plasma levels of the growth factors vascular endothelial growth-A and Kit ligand, both of which are known to enhance myeloid cell proliferation. Collectively, the data presented in this study demonstrate a previously undescribed role of Plm in lymphoproliferative disorders and provide strong evidence that specific blockade of Plg represents a promising approach for the regulation of T-cell lymphoma growth.

    View details for DOI 10.1038/leu.2011.203

    View details for Web of Science ID 000300419100016

    View details for PubMedID 21931322

  • Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL EXPERIMENTAL HEMATOLOGY Suzuki, N., Yamazaki, S., Ema, H., Yamaguchi, T., Nakauchi, H., Takaki, S. 2012; 40 (2): 166-174


    Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

    View details for DOI 10.1016/j.exphem.2011.11.003

    View details for Web of Science ID 000299586200008

    View details for PubMedID 22101255

  • Integrin-alpha v beta 3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells BLOOD Umemoto, T., Yamato, M., Ishihara, J., Shiratsuchi, Y., Utsumi, M., Morita, Y., Tsukui, H., Terasawa, M., Shibata, T., Nishida, K., Kobayashi, Y., Petrich, B. G., Nakauchi, H., Eto, K., Okano, T. 2012; 119 (1): 83-94


    Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin "inside-out" signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent "outside-in" signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.

    View details for DOI 10.1182/blood-2011-02-335430

    View details for Web of Science ID 000299012400014

    View details for PubMedID 22096247

  • Serine/threonine kinase, Melk, regulates proliferation and glial differentiation of retinal progenitor cells CANCER SCIENCE Saito, R., Nakauchi, H., Watanabe, S. 2012; 103 (1): 42-49


    Serine/threonine kinase, Melk, was initially cloned in oocytes, but it is expressed in normal tissues and especially in cancer cells. We had previously identified Melk as a gene that is highly expressed in immature mouse retinal progenitors. To analyze the function of Melk in embryogenesis, we cloned zebrafish Melk and reported that morpholino-based downregulation of Melk in zebrafish resulted in severe anemia. Melk-morpholino-treated zebrafish also showed microphthalmia, suggesting the participation of Melk in retinal development. In Melk-depleted retinas, differentiation of retinal neurons took place but was delayed, and the proliferative period of retinal progenitor cells was prolonged, suggesting that Melk might regulate the timing of the transition from proliferation to differentiation. For more detailed examination, we performed gain- and loss-of-function analyses of Melk in mouse retinas. Knockdown of Melk by shRNA in mouse embryonic retinal explant culture resulted in decreased proliferative activity of retinal progenitors, and accordingly, overexpression of Melk slightly enhanced proliferation. Differentiation of retinal progenitor into subtypes of retinal neurons was not significantly affected, but Müller glia differentiation was perturbed by the level of Melk. Furthermore, process extension of glial cells was enhanced in the absence of Melk, suggesting that Melk is involved in the morphological differentiation of retinal cells. Taken together, our results suggest that Melk is primarily required for proper proliferation, and might play multiple roles in retinal development in vertebrates.

    View details for DOI 10.1111/j.1349-7006.2011.02104.x

    View details for Web of Science ID 000298877600006

    View details for PubMedID 21923749

  • [Potential usefulness of human iPS cells on the generation of platelets]. Nihon rinsho. Japanese journal of clinical medicine Takayama, N., Eto, K., Nakauchi, H. 2011; 69 (12): 2161-2165


    Repetitive transfusion of platelets expressing HLA not corresponded to recipient matched often induces anti-HLA antibody-based undesired events including unresponsiveness in platelet transfusion therapy. It is desirable to use platelets derived from HLA-matched iPS cells. In this context, we have recently established an in vitro culture system whereby human pluripotent stem cells can be differentiated into'unique sac-like structures' (ES-/iPS-sacs) containing hematopoietic progenitors generating platelets. Among various iPS clones, we also found critical role of c-MYC in human megakaryopoiesis, leading to efficient platelet production with an intact in vivo functionality of hemostasis and thrombosis within the vessel. We propose that use of HLA-matched hiPS cells may be one of useful strategies for the treatment of thrombocytopenia in patients requiring repeated transfusion.

    View details for PubMedID 22242314

  • The role of Zic family zinc finger transcription factors in the proliferation and differentiation of retinal progenitor cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Watabe, Y., Baba, Y., Nakauchi, H., Mizota, A., Watanabe, S. 2011; 415 (1): 42-47


    Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. Using DNA microarray analysis, we found that Zics are strongly expressed in SSEA-1-positive early retinal progenitors in the peripheral region of the mouse retina. Reverse-transcription polymerase chain reaction using mRNA from the retina at various developmental stages showed that Zic1 and Zic2 are expressed in the embryonic retina and then gradually disappear during retinal development. Zic3 is also expressed in the embryonic retina; its expression level slightly decreases but it is expressed until adulthood. We overexpressed Zic1, Zic2, or Zic3 in retinal progenitors at embryonic day 17.5 and cultured the retina as explants for 2 weeks. The number of rod photoreceptors was fewer than in the control, but no other cell types showed significant differences between control and Zic overexpressing cells. The proliferation activity of normal retinal progenitors decreased after 5 days in culture, as observed in normal in vivo developmental processes. However, Zic expressing retinal cells continued to proliferate at days 5 and 7, suggesting that Zics sustain the proliferation activities of retinal progenitor cells. Since the effects of Zic1, 2, and 3 are indistinguishable in terms of differentiation and proliferation of retinal progenitors, the redundant function of Zics in retinal development is suggested.

    View details for DOI 10.1016/j.bbrc.2011.10.007

    View details for Web of Science ID 000297385000008

    View details for PubMedID 22024047

  • Frequent pathway mutations of splicing machinery in myelodysplasia NATURE Yoshida, K., Sanada, M., Shiraishi, Y., Nowak, D., Nagata, Y., Yamamoto, R., Sato, Y., Sato-Otsubo, A., Kon, A., Nagasaki, M., Chalkidis, G., Suzuki, Y., Shiosaka, M., Kawahata, R., Yamaguchi, T., Otsu, M., Obara, N., Sakata-Yanagimoto, M., Ishiyama, K., Mori, H., Nolte, F., Hofmann, W., Miyawaki, S., Sugano, S., Haferlach, C., Koeffler, H. P., Shih, L., Haferlach, T., Chiba, S., Nakauchi, H., Miyano, S., Ogawa, S. 2011; 478 (7367): 64-69


    Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent (∼45 to ∼85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 3'-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.

    View details for DOI 10.1038/nature10496

    View details for Web of Science ID 000295575400035

    View details for PubMedID 21909114

  • Ex Vivo Expansion of Human Hematopoietic Stem Cells by Garcinol, a Potent Inhibitor of Histone Acetyltransferase PLOS ONE Nishino, T., Wang, C., Mochizuki-Kashio, M., Osawa, M., Nakauchi, H., Iwama, A. 2011; 6 (9)


    Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established.In this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34(+)CD38(-) HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34(+)CD38(-) HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34(+) HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not.Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs.

    View details for DOI 10.1371/journal.pone.0024298

    View details for Web of Science ID 000294803200012

    View details for PubMedID 21931675

  • Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera CYTOKINE Kawahara, M., Chen, J., Sogo, T., Teng, J., Otsu, M., Onodera, M., Nakauchi, H., Ueda, H., Nagamune, T. 2011; 55 (3): 402-408


    Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling.

    View details for DOI 10.1016/j.cyto.2011.05.024

    View details for Web of Science ID 000294034100013

    View details for PubMedID 21700475

  • Generation of Germline-Competent Rat Induced Pluripotent Stem Cells PLOS ONE Hamanaka, S., Yamaguchi, T., Kobayashi, T., Kato-Itoh, M., Yamazaki, S., Sato, H., Umino, A., Wakiyama, Y., Arai, M., Sanbo, M., Hirabayashi, M., Nakauchi, H. 2011; 6 (7)


    Recent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.We generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.Our data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.

    View details for DOI 10.1371/journal.pone.0022008

    View details for Web of Science ID 000292811700013

    View details for PubMedID 21789202

  • Minimum requirement of donor cells to reduce the glycolipid storage following bone marrow transplantation in a murine model of Fabry disease JOURNAL OF GENE MEDICINE Yokoi, T., Kobayashi, H., Shimada, Y., Eto, Y., Ishige, N., Kitagawa, T., Otsu, M., Nakauchi, H., Ida, H., Ohashi, T. 2011; 13 (5): 262-268


    Fabry disease (FD) is a lysosomal storage disorders characterized by a deficiency of the lysosomal enzyme, α-galactosidase A. This results in the accumulation of glycolipids, mainly globotriaosylceramide (GL-3), in the lysosomes of various organs. Although bone marrow transplantation and hematopoietic stem cell-based gene therapy can offer the potential of a curative therapeutic outcome for FD, the minimum requirement of donor cells or gene-corrected cells to reduce GL-3 levels is not known.Lethally-irradiated FD mice were transplanted intravenously with normal bone marrow cells (Ly5.1 positive) mixed with those of FD mice (Ly5.2 positive) at various ratios to investigate the level of engraftment and enzyme activity necessary to effect a reduction in GL-3 storage.Chimerism of whole white blood cells of recipients' peripheral blood remained stable at 8 weeks after transplantation, and chimerism of granulocytes, monocytes, B cells and T cells was equal to that of white blood cells. GL-3 levels were significantly reduced in the lung and heart of animals with a 30% and 50% chimera, respectively. The extent of reduction in these mice was almost identical to that with 100% chimera.In FD mice, reconstitution with 100% donor cells is not required to obtain a therapeutic effect following bone marrow transplantation. These results suggest that a 30% gene correction might be sufficient to reverse disease manifestations in FD.

    View details for DOI 10.1002/jgm.1566

    View details for Web of Science ID 000291447000002

    View details for PubMedID 21520359

  • Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Onozuka, I., Kakinuma, S., Kamiya, A., Miyoshi, M., Sakamoto, N., Kiyohashi, K., Watanabe, T., Funaoka, Y., Ueyama, M., Nakagawa, M., Koshikawa, N., Seiki, M., Nakauchi, H., Watanabe, M. 2011; 406 (1): 134-140


    Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.

    View details for DOI 10.1016/j.bbrc.2011.02.012

    View details for Web of Science ID 000288302100025

    View details for PubMedID 21300023

  • Functional characterization of hematopoietic stem cells in the spleen EXPERIMENTAL HEMATOLOGY Morita, Y., Iseki, A., Okamura, S., Suzuki, S., Nakauchi, H., Ema, H. 2011; 39 (3): 351-359


    Hematopoietic stem cells (HSCs) reside in both bone marrow (BM) and spleen in adult mice. However, whether BM and spleen HSCs are functionally similar is not known. Spleen HSCs were compared with BM HSCs by various assays.Whole BM and spleen cells were quantitatively analyzed by competitive repopulation. Single-cell transplantation was performed with HSCs purified from BM and spleen. A parabiosis model was used to distinguish organ-specific HSCs from circulating HSCs. The cell cycle was analyzed with pyronin Y staining and bromodeoxyuridine uptake.Repopulating and self-renewal potentials were similar on a clonal basis between BM and spleen HSCs, whereas the HSC frequency in the spleen was significantly lower than that in the BM. Analysis of parabiotic mice suggested that most HSCs are long-term residents in each organ. Cell-cycle analysis revealed that spleen HSCs cycle twice as frequently as do BM HSCs, suggesting that G(0) phase length is longer in BM HSCs than in spleen HSCs. The cycling difference between BM and spleen HSCs was also observed in mice that had been reconstituted with BM or spleen cells, suggesting that HSC quiescence is regulated in an organ-specific manner.Spleen HSCs and BM HSCs are functionally similar, but their cycling behaviors differ.

    View details for DOI 10.1016/j.exphem.2010.12.008

    View details for Web of Science ID 000288288200009

    View details for PubMedID 21185906

  • Hair Follicle Stem Cells Provide a Functional Niche for Melanocyte Stem Cells CELL STEM CELL Tanimura, S., Tadokoro, Y., Inomata, K., Nguyen Thanh Binh, T. B., Nishie, W., Yamazaki, S., Nakauchi, H., Tanaka, Y., McMillan, J. R., Sawamura, D., Yancey, K., Shimizu, H., Nishimura, E. K. 2011; 8 (2): 177-187


    In most stem cell systems, the organization of the stem cell niche and the anchoring matrix required for stem cell maintenance are largely unknown. We report here that collagen XVII (COL17A1/BP180/BPAG2), a hemidesmosomal transmembrane collagen, is highly expressed in hair follicle stem cells (HFSCs) and is required for the maintenance not only of HFSCs but also of melanocyte stem cells (MSCs), which do not express Col17a1 but directly adhere to HFSCs. Mice lacking Col17a1 show premature hair graying and hair loss. Analysis of Col17a1-null mice revealed that COL17A1 is critical for the self-renewal of HFSCs through maintaining their quiescence and immaturity, potentially explaining the mechanism underlying hair loss in human COL17A1 deficiency. Moreover, forced expression of COL17A1 in basal keratinocytes, including HFSCs, in Col17a1-null mice rescues MSCs from premature differentiation and restores TGF-β signaling, demonstrating that HFSCs function as a critical regulatory component of the MSC niche.

    View details for DOI 10.1016/j.stem.2010.11.029

    View details for Web of Science ID 000287633400011

    View details for PubMedID 21295274

  • Development of Defective and Persistent Sendai Virus Vector A UNIQUE GENE DELIVERY/EXPRESSION SYSTEM IDEAL FOR CELL REPROGRAMMING JOURNAL OF BIOLOGICAL CHEMISTRY Nishimura, K., Sano, M., Ohtaka, M., Furuta, B., Umemura, Y., Nakajima, Y., Ikehara, Y., Kobayashi, T., Segawa, H., Takayasu, S., Sato, H., Motomura, K., Uchida, E., Kanayasu-Toyoda, T., Asashima, M., Nakauchi, H., Yamaguchi, T., Nakanishia, M. 2011; 286 (6): 4760-4771


    The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research.

    View details for DOI 10.1074/jbc.M110.183780

    View details for Web of Science ID 000286975700074

    View details for PubMedID 21138846

  • Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells JOURNAL OF EXPERIMENTAL MEDICINE Takayama, N., Nishimura, S., Nakamura, S., Shimizu, T., Ohnishi, R., Endo, H., Yamaguchi, T., Otsu, M., Nishimura, K., Nakanishi, M., Sawaguchi, A., Nagai, R., Takahashi, K., Yamanaka, S., Nakauchi, H., Eto, K. 2010; 207 (13): 2817-2830


    Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a(+)CD42b(+) platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b(+) platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones.

    View details for DOI 10.1084/jem.20100844

    View details for Web of Science ID 000285503000006

    View details for PubMedID 21098095

  • Reduction of N-Glycolylneuraminic Acid in Human Induced Pluripotent Stem Cells Generated or Cultured under Feeder- and Serum-Free Defined Conditions PLOS ONE Hayashi, Y., Chan, T., Warashina, M., Fukuda, M., Ariizumi, T., Okabayashi, K., Takayama, N., Otsu, M., Eto, K., Furue, M. K., Michiue, T., Ohnuma, K., Nakauchi, H., Asashima, M. 2010; 5 (11)


    The successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine. In particular, hiPSCs are a promising source of cells for regenerative medicine and pharmacology. However, one of the major obstacles to such uses for hiPSCs is the risk of contamination from undefined pathogens in conventional culture conditions that use serum replacement and mouse embryonic fibroblasts as feeder cells.Here we report a simple method for generating or culturing hiPSCs under feeder- and serum-free defined culture conditions that we developed previously for human embryonic stem cells. The defined culture condition comprises a basal medium with a minimal number of defined components including five highly purified proteins and fibronectin as a substrate. First, hiPSCs, which were generated using Yamanaka's four factors and conventional undefined culture conditions, adapted to the defined culture conditions. These adapted cells retained the property of self renewal as evaluated morphologically, the expression of self-renewal marker proteins, standard growth rates, and pluripotency as evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo (teratoma formation in immunodeficient mice). Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions. Second, we successfully generated hiPSCs using adult dermal fibroblast under the defined culture conditions from the reprogramming step. For a long therm culture, the generated cells also had the property of self renewal and pluripotency, they carried a normal karyotype, and they were Neu5Gc negative.This study suggested that generation or adaption culturing under defined culture conditions can eliminate the risk posed by undefined pathogens. This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

    View details for DOI 10.1371/journal.pone.0014099

    View details for Web of Science ID 000284527900011

    View details for PubMedID 21124894

  • Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Watanabe, M., Umeyama, K., Matsunari, H., Takayanagi, S., Haruyama, E., Nakano, K., Fujiwara, T., Ikezawa, Y., Nakauchi, H., Nagashima, H. 2010; 402 (1): 14-18


    Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

    View details for DOI 10.1016/j.bbrc.2010.09.092

    View details for Web of Science ID 000284182600003

    View details for PubMedID 20875794

  • Mice lacking Dok-1, Dok-2, and Dok-3 succumb to aggressive histiocytic sarcoma LABORATORY INVESTIGATION Mashima, R., Honda, K., Yang, Y., Morita, Y., Inoue, A., Arimura, S., Nishina, H., Ema, H., Nakauchi, H., Seed, B., Oda, H., Yamanashi, Y. 2010; 90 (9): 1357-1364


    Histiocytic sarcoma (HS), a rare hematological malignancy, is an aggressive neoplasm that responds poorly to therapy. The molecular etiology and pathology of this disease remain unclear, hampering the development of an effective therapy, and there remains a need for more, and more realistic, animal models. HS cells typically show a histiocytic (ie, tissue macrophage-like) morphology and express histiocyte/macrophage markers in the absence of lymphocyte markers. In this study, we report that Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice develop HS, but do not exhibit elevated incidence of other types of tumors. These mutant mice showed earlier mortality than wild-type (WT) or the other mutant mice, and this mortality was associated with HS. In total, 17 of 21 tumor-bearing Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice necropsied at 25-66 weeks of age showed multiple organ spread, with osteolytic lesions and orthotopic invasion from the bone marrow to skeletal muscle. Tumors from the mice were transplantable. In addition, all Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice, but only a small proportion of Dok-3(-/-) mice and no Dok-1(-/-)Dok-2(-/-) mice, exhibited abnormal accumulation of macrophages in the lung on necropsy at 8-12 weeks of age. Macrophages derived from Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice displayed an exaggerated proliferative response to macrophage colony-stimulating factor (M-CSF) or granulocyte- macrophage colony-stimulating factor (GM-CSF) compared with WT and mutant controls. Together, these findings indicate that Dok-1, Dok-2, and Dok-3 cooperatively suppress aggressive HS, and commend Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice as a useful model for the study of this neoplasia.

    View details for DOI 10.1038/labinvest.2010.121

    View details for Web of Science ID 000281467800008

    View details for PubMedID 20548287

  • Bmi1 Promotes Hepatic Stem Cell Expansion and Tumorigenicity in Both Ink4a/Arf-Dependent and -Independent Manners in Mice HEPATOLOGY Chiba, T., Seki, A., Aoki, R., Ichikawa, H., Negishi, M., Miyagi, S., Oguro, H., Saraya, A., Kamiya, A., Nakauchi, H., Yokosuka, O., Iwama, A. 2010; 52 (3): 1111-1123


    We previously reported that forced expression of Bmi1 (B lymphoma Moloney murine leukemia virus insertion region 1 homolog) in murine hepatic stem/progenitor cells purified from fetal liver enhances their self-renewal and drives cancer initiation. In the present study, we examined the contribution of the Ink4a/Arf tumor suppressor gene locus, one of the major targets of Bmi1, to stem cell expansion and cancer initiation. Bmi1(-/-) Delta-like protein (Dlk)(+) hepatic stem/progenitor cells showed de-repression of the Ink4a/Arf locus and displayed impaired growth activity. In contrast, Ink4a/Arf(-/-) Dlk(+) cells gave rise to considerably larger colonies containing a greater number of bipotent cells than wild-type Dlk(+) cells. Although Ink4a/Arf(-/-) Dlk(+) cells did not initiate tumors in recipient nonobese diabetic/severe combined immunodeficiency mice, enforced expression of Bmi1 in Ink4a/Arf(-/-) Dlk(+) cells further augmented their self-renewal capacity and resulted in tumor formation in vivo. Microarray analyses successfully identified five down-regulated genes as candidate downstream targets for Bmi1 in hepatic stem/progenitor cells. Of these genes, enforced expression of sex determining region Y-box 17 (Sox17) in Dlk(+) cells strongly suppressed colony propagation and tumor growth.These results indicate that repression of targets of Bmi1 other than the Ink4a/Arf locus plays a crucial role in the oncogenic transformation of hepatic stem/progenitor cells. Functional analyses of Bmi1 target genes would be of importance to elucidate the molecular machinery underlying hepatic stem cell system and explore therapeutic approaches for the eradication of liver cancer stem cells.

    View details for DOI 10.1002/hep.23793

    View details for Web of Science ID 000281423000033

    View details for PubMedID 20648475

  • FET family proto-oncogene Fus contributes to self-renewal of hematopoietic stem cells EXPERIMENTAL HEMATOLOGY Sugawara, T., Oguro, H., Negishi, M., Morita, Y., Ichikawa, H., Iseki, T., Yokosuka, O., Nakauchi, H., Iwama, A. 2010; 38 (8): 696-706


    Fus is the gene for a member of the FET family of RNA-binding proteins often involved in chromosomal translocations to generate oncogenic fusion genes in human cancers. Fus participates in multiple cellular functions, including RNA processing and transport, transcriptional regulation, and genome integrity. However, its role in hematopoiesis remains obscure. In this study, we examined its role in the self-renewal of hematopoietic stem cells (HSCs).HSCs in Fus(-/-) fetal livers were analyzed for proliferative capacity in vitro and long-term repopulating capacity in recipient mice. Radiation sensitivity of Fus(-/-) HSCs was evaluated in recipient mice repopulated by Fus(-/-) fetal liver cells.Fus(-/-) fetal livers developed normally, except for a mild reduction in numbers of hematopoietic stem and progenitor cells compared to wild-type. The proliferation and differentiation of Fus(-/-) hematopoietic progenitors were normal in vitro. However, the number of colony-forming cells present in long-term cocultures of Fus(-/-) hematopoietic progenitors and stromal cells was significantly reduced. Fus(-/-) HSCs had an impaired long-term repopulating capacity and failed to repopulate in tertiary recipient mice. Fus(-/-) HSCs were highly susceptible to radiation both in vitro and in vivo and showed retardation of radiation-induced DNA damage repair.Our findings define Fus as a novel regulator of self-renewal and radioprotection of HSCs and also implicate it in stress-resistance and maintenance of the genomic integrity of HSCs.

    View details for DOI 10.1016/j.exphem.2010.0.4.006

    View details for Web of Science ID 000280330400010

    View details for PubMedID 20412831

  • Deregulated Intracellular Signaling by Mutated c-CBL in Myeloid Neoplasms CLINICAL CANCER RESEARCH Ogawa, S., Shih, L., Suzuki, T., Otsu, M., Nakauchi, H., Koeffler, H. P., Sanada, M. 2010; 16 (15): 3825-3831


    c-CBL encodes a 120-kDa protein involved in intracellular signal transduction in a wide variety of cell types. Recently, frequent mutations of c-CBL have been reported in myeloid neoplasms showing both myelodysplastic and myeloproliferative features, in which most mutations are present in a homozygous state, as a result of allelic conversion in 11q. c-CBL has ubiquitin E3 ligase activity for a wide variety of tyrosine kinases, and thereby, negatively regulates tyrosine kinase signaling. Accordingly, c-CBL seems to have tumor suppressor functions, loss of which promotes tumorigenesis. On the other hand, once mutated, it is converted to an oncogenic protein and commits to myeloid leukemogenesis through a kind of gain of function causing aberrant signal transduction. The inhibition of mutant CBL protein or signaling pathways that it activates would have a role in therapeutics of myeloid neoplasms with CBL mutations.

    View details for DOI 10.1158/1078-0432.CCR-09-2341

    View details for Web of Science ID 000280530300004

    View details for PubMedID 20547695

  • Downregulation of ZEB1 and overexpression of Smad7 contribute to resistance to TGF-beta 1-mediated growth suppression in adult T-cell leukemia/lymphoma ONCOGENE Nakahata, S., Yamazaki, S., Nakauchi, H., Morishita, K. 2010; 29 (29): 4157-4169


    Zinc-finger E-box binding homeobox 1 (ZEB1) is a candidate tumor-suppressor gene in adult T-cell leukemia/lymphoma (ATLL). ZEB1 binds phosphorylated Smad2/3 to enhance transforming growth factor-beta1 (TGF-beta1) signaling. In addition to downregulation of ZEB1 mRNA, we found overexpression of inhibitory Smad, Smad7, in resistance of ATLL cells to growth suppression by TGF-beta1. A protein complex of Smad7 and histone deacetylase constantly bound to the promoter region of TGF-beta1 responsive genes with the Smad-responsive element (SRE) to inhibit TGF-beta1 signaling; however, ectopic expression of ZEB1 reactivated TGF-beta1 signaling by binding to Smad7 and recruiting the Smad3/p300 histone acetyltransferase complex to the promoter after TGF-beta1 stimulation in ATLL. Conversely, because ZEB1 mRNA was detected in the late stages of T-cell development, we used CTLL2 cells with ZEB1 expression, a murine peripheral T-cell lymphoma, and found that a complex of Smad3, Smad7 and ZEB1 was bound to the SRE of the p21(CDKN1A) promoter after the induction of Smad7 by TGF-beta1 treatment. Because the duration of TGF-beta1-induced transcriptional activation of PAI-1 and p21 was shortened in shZEB1-expressing CTLL2 cells, ZEB1 may have a role in enhancing TGF-beta1 signaling by binding not only to Smad3 but also to Smad7 in the nucleus. Altogether, these results suggest that both ZEB1 downregulation and Smad7 overexpression contribute to resistance to TGF-beta1-mediated growth suppression in ATLL.

    View details for DOI 10.1038/onc.2010.172

    View details for Web of Science ID 000280151500004

    View details for PubMedID 20514018

  • Reprogramming adult hematopoietic cells CURRENT OPINION IN HEMATOLOGY Kaneko, S., Otsu, M., Nakauchi, H. 2010; 17 (4): 271-275


    Recent advances in molecular biology research have culminated in development of technologies to generate pluripotent stem cells from somatic cells. In addition to skin fibroblasts, hematopoietic cells also have been shown to be amenable to reprogramming to pluripotency. The present review discusses the relevance of these findings to basic researches and regenerative medicine, and how researchers can take advantage of hematopoietic cell reprogramming technologies.In 2006, Yamanaka and his colleagues published their amazing observation that murine somatic cells can be reprogrammed to the embryonic stem cell-like state simply by retroviral-mediated introduction of three or four defined factors. Soon after, human cells also were shown to be amenable to similar reprogramming. Generation of induced pluripotent cells from several types of hematopoietic cells of both murine and human origins now has been reported.Reprogramming adult hematopoietic cells will provide opportunities to obtain valuable materials with minimum risk and burden to patients. Reprogrammed cells can be used in research to elucidate disease mechanisms and in drug or toxicity screening. In clinical settings, patient-derived induced pluripotent stem cells may be used to generate mature functional cells for various therapies.

    View details for DOI 10.1097/MOH.0b013e32833a25ee

    View details for Web of Science ID 000279023700001

    View details for PubMedID 20571391

  • Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment JOURNAL OF EXPERIMENTAL MEDICINE Morita, Y., Ema, H., Nakauchi, H. 2010; 207 (6): 1173-1182


    Hematopoietic stem cells (HSCs) have been extensively characterized based on functional definitions determined by experimental transplantation into lethally irradiated mice. In mice, HSCs are heterogeneous with regard to self-renewal potential, in vitro colony-forming activity, and in vivo behavior. We attempted prospective isolation of HSC subsets with distinct properties among CD34(-/low) c-Kit+Sca-1+Lin- (CD34-KSL) cells. CD34-KSL cells were divided, based on CD150 expression, into three fractions: CD150high, CD150med, and CD150neg cells. Compared with the other two fractions, CD150high cells were significantly enriched in HSCs, with great self-renewal potential. In vitro colony assays revealed that decreased expression of CD150 was associated with reduced erythroblast/megakaryocyte differentiation potential. All three fractions were regenerated only from CD150high cells in recipient mice. Using single-cell transplantation studies, we found that a fraction of CD150high cells displayed latent and barely detectable myeloid engraftment in primary-recipient mice but progressive and multilineage reconstitution in secondary-recipient mice. These findings highlight the complexity and hierarchy of reconstitution capability, even among HSCs in the most primitive compartment.

    View details for DOI 10.1084/jem.20091318

    View details for Web of Science ID 000278554200007

    View details for PubMedID 20421392

  • Rat Transgenesis Via Embryonic Stem Cells Electroporated With the Kusabira-Orange Gene MOLECULAR REPRODUCTION AND DEVELOPMENT Hirabayashi, M., Kato, M., Sanbo, M., Kobayashi, T., Hochi, S., Nakauchi, H. 2010; 77 (6): 474-474

    View details for DOI 10.1002/mrd.21181

    View details for Web of Science ID 000277525500002

    View details for PubMedID 20422708

  • Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration BLOOD Ohki, M., Ohki, Y., Ishihara, M., Nishida, C., Tashiro, Y., Akiyama, H., Komiyama, H., Lund, L. R., Nitta, A., Yamada, K., Zhu, Z., Ogawa, H., Yagita, H., Okumura, K., Nakauchi, H., Werb, Z., Heissig, B., Hattori, K. 2010; 115 (21): 4302-4312


    Ischemia of the heart, brain, and limbs is a leading cause of morbidity and mortality worldwide. Treatment with tissue type plasminogen activator (tPA) can dissolve blood clots and can ameliorate the clinical outcome in ischemic diseases. But the underlying mechanism by which tPA improves ischemic tissue regeneration is not well understood. Bone marrow (BM)-derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool and mobilizes CD45(+)CD11b(+) proangiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signaling. tPA improves the incorporation of CD11b(+) cells into ischemic tissues and increases expression of neoangiogenesis-related genes, including VEGF-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b(+) cells and VEGFR-1(+) cells, but not carrier-mobilized cells or CD11b(-) cells, accelerates neovascularization and ischemic tissue regeneration. Inhibition of VEGF signaling suppresses tPA-induced neovascularization in a model of hind limb ischemia. Thus, tPA mobilizes CD11b(+) cells from the BM and increases systemic and local (cellular) VEGF-A, which can locally promote angiogenesis during ischemic recovery. tPA might be useful to induce therapeutic revascularization in the growing field of regenerative medicine.

    View details for DOI 10.1182/blood-2009-08-236851

    View details for Web of Science ID 000278117900022

    View details for PubMedID 20110420

  • Gain-of-function c-CBL mutations associated with uniparental disomy of 11q in myeloid neoplasms CELL CYCLE Ogawa, S., Sanada, M., Shih, L., Suzuki, T., Otsu, M., Nakauchi, H., Koeffler, H. P. 2010; 9 (6): 1051-1056


    c-CBL (CBL) encodes a multifunctional protein engaged in the regulation of intracellular signaling pathways. It was first identified as a cellular counterpart of the viral oncogene, v-CBL, that causes murine lymphoma. Although no genetic evidence existed suggesting its role in human carcinogenesis, the recent discovery of c-CBL mutations in myeloid cancers has unveiled a unique oncogenic mechanism mediated by gain-of-function of a mutated tumor suppressor, closely associated with allelic conversion of 11q arms. In this review, we summarize our current knowledge about c-CBL mutations and discuss the molecular mechanisms of their gain-of-function.

    View details for Web of Science ID 000275636900010

    View details for PubMedID 20237427

  • Poised Lineage Specification in Multipotential Hematopoietic Stem and Progenitor Cells by the Polycomb Protein Bmi1 CELL STEM CELL Oguro, H., Yuan, J., Ichikawa, H., Ikawa, T., Yamazaki, S., Kawamoto, H., Nakauchi, H., Iwama, A. 2010; 6 (3): 279-286


    Polycomb group (PcG) proteins are essential regulators of stem cells. PcG and trithorax group proteins mark developmental regulator gene promoters with bivalent domains consisting of overlapping repressive and activating histone modifications to keep them poised for activation in embryonic stem cells. Bmi1, a component of PcG complexes, maintains the self-renewal capacity of adult stem cells, but its role in multipotency remains obscure. Here we show that Bmi1 is critical for multipotency of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). B cell lineage developmental regulator genes, Ebf1 and Pax5, appeared to be transcriptionally repressed by bivalent domains before lineage commitment. Loss of Bmi1 resulted in a resolution of bivalent domains at the Ebf1 and Pax5 loci, leading to their premature expression in HSC/MPPs accompanied by accelerated lymphoid specification and a marked reduction in HSC/MPPs. Thus, Bmi1 is required to reinforce bivalent domains at key developmental regulator gene loci to maintain lineage specification poised for activation in adult stem cells.

    View details for DOI 10.1016/j.stem.2010.01.005

    View details for Web of Science ID 000276254600014

    View details for PubMedID 20207230

  • Establishment of Rat Embryonic Stem Cell Lines That Can Participate in Germline Chimerae at High Efficiency MOLECULAR REPRODUCTION AND DEVELOPMENT Hirabayashi, M., Kato, M., Kobayashi, T., Sanbo, M., Yagi, T., Hochi, S., Nakauchi, H. 2010; 77 (2): 94-94

    View details for DOI 10.1002/mrd.21123

    View details for Web of Science ID 000273518200002

    View details for PubMedID 19899136

  • Cardiac mast cells cause atrial fibrillation through PDGF-A-mediated fibrosis in pressure-overloaded mouse hearts JOURNAL OF CLINICAL INVESTIGATION Liao, C., Akazawa, H., Tamagawa, M., Ito, K., Yasuda, N., Kudo, Y., Yamamoto, R., Ozasa, Y., Fujimoto, M., Wang, P., Nakauchi, H., Nakaya, H., Komuro, I. 2010; 120 (1): 242-253


    Atrial fibrillation (AF) is a common arrhythmia that increases the risk of stroke and heart failure. Here, we have shown that mast cells, key mediators of allergic and immune responses, are critically involved in AF pathogenesis in stressed mouse hearts. Pressure overload induced mast cell infiltration and fibrosis in the atrium and enhanced AF susceptibility following atrial burst stimulation. Both atrial fibrosis and AF inducibility were attenuated by stabilization of mast cells with cromolyn and by BM reconstitution from mast cell-deficient WBB6F1-KitW/W-v mice. When cocultured with cardiac myocytes or fibroblasts, BM-derived mouse mast cells increased platelet-derived growth factor A (PDGF-A) synthesis and promoted cell proliferation and collagen expression in cardiac fibroblasts. These changes were abolished by treatment with a neutralizing antibody specific for PDGF alpha-receptor (PDGFR-alpha). Consistent with these data, upregulation of atrial Pdgfa expression in pressure-overloaded hearts was suppressed by BM reconstitution from WBB6F1-KitW/W-v mice. Furthermore, injection of the neutralizing PDGFR-alpha-specific antibody attenuated atrial fibrosis and AF inducibility in pressure-overloaded hearts, whereas administration of homodimer of PDGF-A (PDGF-AA) promoted atrial fibrosis and enhanced AF susceptibility in normal hearts. Our results suggest a crucial role for mast cells in AF and highlight a potential application of controlling the mast cell/PDGF-A axis to achieve upstream prevention of AF in stressed hearts.

    View details for DOI 10.1172/JCI39942

    View details for Web of Science ID 000273495700028

    View details for PubMedID 20038802

  • Lnk regulates integrin alpha IIb beta 3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo JOURNAL OF CLINICAL INVESTIGATION Takizawa, H., Nishimura, S., Takayama, N., Oda, A., Nishikii, H., Morita, Y., Kakinuma, S., Yamazaki, S., Okamura, S., Tamura, N., Goto, S., Sawaguchi, A., Manabe, I., Takatsu, K., Nakauchi, H., Takaki, S., Eto, K. 2010; 120 (1): 179-190


    The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk-/- mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin alphaIIbbeta3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk-/- mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk-/- platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the beta3 integrin subunit, and reduced binding of Fyn to integrin alphaIIbbeta3. These results provide new insight into the mechanism of alphaIIbbeta3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.

    View details for DOI 10.1172/JCI39503

    View details for Web of Science ID 000273495700023

    View details for PubMedID 20038804

  • Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL EXPERIMENTAL HEMATOLOGY Nishino, T., Miyaji, K., Ishiwata, N., Arai, K., Yui, M., Asai, Y., Nakauchi, H., Iwama, A. 2009; 37 (11): 1364-1377


    The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs.hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays.During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes.This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.

    View details for DOI 10.1016/j.exphem.2009.09.001

    View details for Web of Science ID 000271495000011

    View details for PubMedID 19744539

  • Enrichment and Clonal Culture of Progenitor Cells During Mouse Postnatal Liver Development in Mice GASTROENTEROLOGY Kamiya, A., Kakinuma, S., Yamazaki, Y., Nakauchi, H. 2009; 137 (3): 1114-1126


    Stem and progenitor cells exist in normal postnatal livers. However, it has not been possible to clonally isolate or analyze postnatal liver stem/progenitor-like cells (PLSCs) derived from noninjured livers because of a lack of specific surface markers. This study aimed to establish a primary culture system for clone-sorted PLSCs.To investigate proliferation and differentiation of PLSCs, subpopulations of nonparenchymal cells derived from noninjured livers were purified and cultured using a single-cell culture system. Cells were grown in fetal liver cell-derived conditioned medium in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632.We identified CD13 and CD133 as markers expressed on the PLSC-containing population in noninjured livers and established an efficient single-cell culture system to clonally analyze PLSCs. Culture of PLSCs is difficult, even using conditioned medium, but the addition of Y-27632 increased PLSC cell proliferation. The proportion of progenitor cells among nonparenchymal cells decreased during postnatal liver development; however, a PLSC population was still preserved in 3-month-old mice. Long-term cultivated cells derived from clone-sorted cells in normal livers were established and were called normal-liver-derived stem-like cells (NLS cells). NLS cells could differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions and underwent self-renewal-like activity in serial reclone-sorted culture. CD13 and CD133 were expressed on progenitor cells derived from fetal and postnatal liver, whereas CD49f (integrin alpha6 subunit) was strongly expressed only on PLSCs.These results demonstrate the presence of progenitor cells in the CD13(+)CD49f(+)CD133(+) subpopulation of nonhematopoietic cells derived from noninjured postnatal livers.

    View details for DOI 10.1053/j.gastro.2009.06.001

    View details for Web of Science ID 000269432200049

    View details for PubMedID 19524574

  • Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton's jelly explants of human umbilical cord INTERNATIONAL JOURNAL OF HEMATOLOGY Ishige, I., Nagamura-Inoue, T., Honda, M. J., Harnprasopwat, R., Kido, M., Sugimoto, M., Nakauchi, H., Tojo, A. 2009; 90 (2): 261-269


    We isolated mesenchymal stem cells (MSC) from arteries (UCA), veins (UCV), and Wharton's jelly (UCWJ) of human umbilical cords (UC) and determined their relative capacities for sustained proliferation and multilineage differentiation. Individual UC components were dissected, diced into 1-2 mm(3) fragments, and aligned in explant cultures from which migrating cells were isolated using trypsinization. Preparations from 13 UCs produced 13 UCWJ, 11 UCV, and 10 UCA cultures of fibroblast-like, spindle-shaped cells negative for CD31, CD34, CD45, CD271, and HLA-class II, but positive for CD13, CD29, CD44, CD73, CD90, CD105, and HLA-class I. UCV cells exhibited a significantly higher frequency of colony-forming units fibroblasts than did UCWJ and UCA cells. Individual MSCs could be selectively differentiated into osteoblasts, chondrocytes, and adipocytes. When compared for osteogenic potential, UCWJ cells were the least effective precursors, whereas UCA-derived cells developed alkaline phosphatase activity with or without an osteogenic stimulus. UC components, especially blood vessels, could provide a promising source of MSCs with important clinical applications.

    View details for DOI 10.1007/s12185-009-0377-3

    View details for Web of Science ID 000269208000024

    View details for PubMedID 19657615

  • Definitive proof for direct reprogramming of hematopoietic cells to pluripotency BLOOD Okabe, M., Otsu, M., Ahn, D. H., Kobayashi, T., Morita, Y., Wakiyama, Y., Onodera, M., Eto, K., Ema, H., Nakauchi, H. 2009; 114 (9): 1764-1767


    Generation of induced pluripotent stem cells (iPSCs) generally uses fibroblastic cells, but other cell sources may prove useful in both research and clinical settings. Although proof of cellular origin requires genetic-marker identification in both target cells and established iPSCs, somatic cells other than mature lymphocytes mostly lack such markers. Here we show definitive proof of direct reprogramming of murine hematopoietic cells with no rearranged genes. Using iPSC factor transduction, we successfully derived iPSCs from bone marrow progenitor cells obtained from a mouse whose hematopoiesis was reconstituted from a single congenic hematopoietic stem cell. Established clones were demonstrated to be genetically identical to the transplanted single hematopoietic stem cell, thus proving their cellular origin. These hematopoietic cell-derived iPSCs showed typical characteristics of iPSCs, including the ability to contribute to chimerism in mice. These results will prompt further use of hematopoietic cells for iPSC generation while enabling definitive studies to test how cellular sources influence characteristics of descendant iPSCs.

    View details for DOI 10.1182/blood-2009-02-203695

    View details for Web of Science ID 000269380600011

    View details for PubMedID 19564635

  • Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms NATURE Sanada, M., Suzuki, T., Shih, L., Otsu, M., Kato, M., Yamazaki, S., Tamura, A., Honda, H., Sakata-Yanagimoto, M., Kumano, K., Oda, H., Yamagata, T., Takita, J., Gotoh, N., Nakazaki, K., Kawamata, N., Onodera, M., Nobuyoshi, M., Hayashi, Y., Harada, H., Kurokawa, M., Chiba, S., Mori, H., Ozawa, K., Omine, M., Hirai, H., Nakauchi, H., Koeffler, H. P., Ogawa, S. 2009; 460 (7257): 904-U145


    Acquired uniparental disomy (aUPD) is a common feature of cancer genomes, leading to loss of heterozygosity. aUPD is associated not only with loss-of-function mutations of tumour suppressor genes, but also with gain-of-function mutations of proto-oncogenes. Here we show unique gain-of-function mutations of the C-CBL (also known as CBL) tumour suppressor that are tightly associated with aUPD of the 11q arm in myeloid neoplasms showing myeloproliferative features. The C-CBL proto-oncogene, a cellular homologue of v-Cbl, encodes an E3 ubiquitin ligase and negatively regulates signal transduction of tyrosine kinases. Homozygous C-CBL mutations were found in most 11q-aUPD-positive myeloid malignancies. Although the C-CBL mutations were oncogenic in NIH3T3 cells, c-Cbl was shown to functionally and genetically act as a tumour suppressor. C-CBL mutants did not have E3 ubiquitin ligase activity, but inhibited that of wild-type C-CBL and CBL-B (also known as CBLB), leading to prolonged activation of tyrosine kinases after cytokine stimulation. c-Cbl(-/-) haematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines compared to c-Cbl(+/+) HSPCs, and transduction of C-CBL mutants into c-Cbl(-/-) HSPCs further augmented their sensitivities to a broader spectrum of cytokines, including stem-cell factor (SCF, also known as KITLG), thrombopoietin (TPO, also known as THPO), IL3 and FLT3 ligand (FLT3LG), indicating the presence of a gain-of-function that could not be attributed to a simple loss-of-function. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in a c-Cbl(+/+) background or by co-transduction of wild-type C-CBL, which suggests the pathogenic importance of loss of wild-type C-CBL alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a new insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some myeloid cancer subsets.

    View details for DOI 10.1038/nature08240

    View details for Web of Science ID 000268938300041

    View details for PubMedID 19620960

  • Tumor Suppression by Phospholipase C-beta 3 via SHP-1-Mediated Dephosphorylation of Stat5 CANCER CELL Xiao, W., Hong, H., Kawakami, Y., Kato, Y., Wu, D., Yasudo, H., Kimura, A., Kubagawa, H., Bertoli, L. F., Davis, R. S., Chau, L. A., Madrenas, J., Hsia, C. C., Xenocostas, A., Kipps, T. J., Hennighausen, L., Iwama, A., Nakauchi, H., Kawakami, T. 2009; 16 (2): 161-171


    Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-beta3-deficient mice develop myeloproliferative disease, lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-beta3 deficiency was suggested in mouse lymphomas in PLC-beta3(-/-) and in Emicro-myc;PLC-beta3(+/-) mice and human Burkitt's lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-beta3 is likely a tumor suppressor.

    View details for DOI 10.1016/j.ccr.2009.05.018

    View details for Web of Science ID 000268762400012

    View details for PubMedID 19647226

  • Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver JOURNAL OF HEPATOLOGY Kakinuma, S., Ohta, H., Kamiya, A., Yamazaki, Y., Oikawa, T., Okada, K., Nakauchi, H. 2009; 51 (1): 127-138


    Hepatic stem/progenitor cells possess active proliferative ability and the capacity for differentiation into hepatic and cholangiocytic lineages. Our group and others have shown that a prospectively defined population in mid-gestational fetal liver contains hepatic stem/progenitor cells. However, the phenotypes of such cells are incompletely elucidated. We analyzed the profile of cell-surface molecules on primary hepatic stem/progenitor cells.Expression of cell surface molecules on primary hepatic stem/progenitor cells in mouse mid-gestational fetal liver was analyzed using flow cytometric multicolor analyses and colony-formation assays. The potential of the cells for liver repopulation was examined by transplantation assay.We found that CD13 (aminopeptidase N) was detected on the cells of the previously reported (Dlk/Pref-1(+)) hepatic stem/progenitor fraction. Colony-formation assays revealed that the CD13(+) fraction, compared with the Dlk(+) fraction, of non-hematopoietic cells in fetal liver was enriched in hepatic stem/progenitor cells. Transplantation assay showed the former fraction exhibited repopulating potential in regenerating liver. Moreover, flow cytometric analysis for over 90 antigens demonstrated enrichment of hepatic stem/progenitor cells using several positive selection markers, including (hitherto unknown) CD13, CD73, CD106, and CD133.Our data indicated that CD13 is a positive selection marker for hepatic stem/progenitor cells in mid-gestational fetal liver.

    View details for DOI 10.1016/j.jhep.2009.02.033

    View details for Web of Science ID 000267713400015

    View details for PubMedID 19439389

  • Insights into signaling and function of hematopoietic stem cells at the single-cell level CURRENT OPINION IN HEMATOLOGY Yamazaki, S., Nakauchi, H. 2009; 16 (4): 255-258


    Development of a technique prospectively to isolate hematopoietic stem cells (HSCs) to near homogeneity has enabled clonal analysis and thus converted our understanding of HSCs from conceptual and qualitative to realistic and quantitative. Recent studies have revealed that despite their high proliferation potential, most HSCs are in G0 and enter cell cycle only after a long interval. This dormancy of HSCs, which seems to be important for long-term maintenance of 'stemness', appears to be regulated by the exchange of signals between HSCs and the bone marrow niche. Analysis of intersignaling and intrasignaling events in HSCs in and out of the bone marrow niche has begun.With the help of advances in confocal microscopy, laser scanning microscopy, and personal computer computational power over the last decade, it has become evident that thrombopoietin/c-Mpl signaling plays a role in HSC self-renewal and AKT-forkhead box O signaling in HSC dormancy. Furthermore, transforming growth factor-beta has been indicated as a candidate niche signal to induce hibernation in HSCs.Understanding of the signaling events between HSCs and niche is critical not only for stem cell biology in general and for transplantation medicine but also for the development of novel cancer therapy.

    View details for DOI 10.1097/MOH.0b013e32832c6705

    View details for Web of Science ID 000267641700004

    View details for PubMedID 19465850

  • T cell growth control using hapten-specific antibody/interleukin-2 receptor chimera CYTOKINE Sogo, T., Kawahara, M., Ueda, H., Otsu, M., Onodera, M., Nakauchi, H., Nagamune, T. 2009; 46 (1): 127-136


    IL-2 is a cytokine that is essential for the expansion and survival of activated T cells. Although adoptive transfer of tumor-specific T cells with IL-2 is one of strategies for cancer immunotherapy, it is essential to replace IL-2 that exerts severe side effects in vivo. To solve this problem, we propose to use an antibody/IL-2R chimera, which can transduce a growth signal in response to a cognate antigen. We constructed two chimeras, in which ScFv of anti-fluorescein antibody was tethered to extracellular D2 domain of erythropoietin receptor and transmembrane/cytoplasmic domains of IL-2Rbeta or gamma chain. When the chimeras were co-expressed in IL-3-dependent pro-B cell line Ba/F3 and IL-2-dependent T cell line CTLL-2, gene-modified cells were selectively expanded in the absence of IL-3 and IL-2, respectively, by adding fluorescein-conjugated BSA (BSA-FL) as a cognate antigen. Growth assay revealed that the cells with the chimeras transduced a growth signal in a BSA-FL dose-dependent manner. Furthermore, STAT3, STAT5, ERK1/2 and Akt, which are hallmarks for IL-2R signaling, were all activated by the chimeras in CTLL-2 transfectant. We also demonstrated that the chimeras were functional in murine primary T cells. These results demonstrate that the antibody/IL-2R chimeras could substantially mimic the wild-type IL-2R and could specifically expand gene-modified T cells in the presence of the cognate antigen.

    View details for DOI 10.1016/j.cyto.2008.12.020

    View details for Web of Science ID 000265446600019

    View details for PubMedID 19223197

  • A rapid and efficient strategy to generate allele-specific anti-HLA monoclonal antibodies JOURNAL OF IMMUNOLOGICAL METHODS Yamazaki, S., Suzuki, N., Saito, T., Ishii, Y., Takiguchi, M., Nakauchi, H., Watanabe, N. 2009; 343 (1): 56-60


    That generation of allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAb) is very difficult is well known. This is thought to be due to the unique epitope structure, an assemblage of amino acid residues that lie separately in the amino acid sequence of human HLA, and to its low antigenicity compared with that of common epitopes recognized as xenogeneic determinants by mice. Here we report a rapid and efficient strategy to generate ASHmAb. Different from usual immunization methods is that we suppressed the production of non-allele-specific anti-HLA antibodies against xenogeneic determinants of HLA molecules by immunizing human HLA-B51 transgenic mice against non-HLA-B51 HLA tetramers. In addition, HLA-coated beads enabled rapid and efficient screening for ASHmAb. ASHmAb generated by this strategy will be useful for HLA typing and for clinical diagnosis, such as flow cytometry-based chimerism analysis for early detection of graft failure and relapse of leukemia after HLA-mismatched hematopoietic stem cell transplantation.

    View details for DOI 10.1016/j.jim.2009.01.007

    View details for Web of Science ID 000264734000007

    View details for PubMedID 19187783

  • Stepwise Development of Hematopoietic Stem Cells from Embryonic Stem Cells PLOS ONE Matsumoto, K., Isagawa, T., Nishimura, T., Ogaeri, T., Eto, K., Miyazaki, S., Miyazaki, J., Aburatani, H., Nakauchi, H., Ema, H. 2009; 4 (3)


    The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+)CD41(+)CD45(-) phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

    View details for DOI 10.1371/journal.pone.0004820

    View details for Web of Science ID 000265496400002

    View details for PubMedID 19287487

  • Hepatic stem/progenitor cells and stem-cell transplantation for the treatment of liver disease JOURNAL OF GASTROENTEROLOGY Kakinuma, S., Nakauchi, H., Watanabe, M. 2009; 44 (3): 167-172


    Allogeneic liver transplantation is still the only effective treatment available to patients with liver failure. However, because there is a serious shortage of liver donors, an alternative therapeutic approach is needed. Transplantation of mature hepatocytes has been evaluated in clinical trials, but the long-term efficacy remains unclear and the paucity of donor cells limits this strategy. Stem-cell transplantation is a more promising alternative approach. Several studies have provided information about the mechanism underlying the proliferation and differentiation of hepatic stem/progenitor cells. Moreover, in experimental models of liver disease, transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells led to donor cell-mediated repopulation of the liver and improved survival rates. However, before stem-cell transplantation can be applied in the clinic to treat liver failure in humans, it will be necessary to overcome several difficulties associated with the technique.

    View details for DOI 10.1007/s00535-008-2297-z

    View details for Web of Science ID 000263379200001

    View details for PubMedID 19214659

  • Sall4 Regulates Cell Fate Decision in Fetal Hepatic Stem/Progenitor Cells GASTROENTEROLOGY Oikawa, T., Kamiya, A., Kakinuma, S., Zeniya, M., Nishinakamura, R., Tajiri, H., Nakauchi, H. 2009; 136 (3): 1000-1011


    Fetal hepatic stem/progenitor cells, called hepatoblasts, differentiate into both hepatocytes and cholangiocytes. The molecular mechanisms regulating this lineage segmentation process remain unknown. Sall4 has been shown to be among the regulators of organogenesis, embryogenesis, maintenance of pluripotency, and early embryonic cell fate decisions in embryonic stem cells. The expression and functional roles of Sall4 during liver development have not been elucidated. We here provide their first description in hepatoblasts.To investigate functions of Sall4 in fetal liver development, Dlk(+)CD45(-)Ter119(-) hepatoblasts derived from embryonic day 14 mouse livers were purified, and in vitro gain and loss of function analyses and in vivo transplantation analyses were performed using retrovirus- or lentivirus-mediated gene transfer.We demonstrated that Sall4 was expressed in fetal hepatoblasts but not adult hepatocytes. The expression level of Sall4 gradually fell during liver development. Overexpression of Sall4 in hepatoblasts significantly inhibited maturation induced by oncostatin M and extracellular matrix in vitro, as evidenced by morphologic changes and suppression of hepatic maturation marker gene expression. When bile duct-like structures were induced by collagen gel-embedded culture, overexpression of Sall4 markedly augmented size and number of cytokeratin19(+)-branching structures. Knockdown of Sall4 inhibited formation of these branching structures. With in vivo transplantation, Sall4 enhanced differentiation of cytokeratin19(+)-bile ducts derived from transplanted hepatoblasts.These results suggest that Sall4 plays a crucial role in controlling the lineage commitment of hepatoblasts not only inhibiting their differentiation into hepatocytes but also driving their differentiation toward cholangiocytes.

    View details for DOI 10.1053/j.gastro.2008.11.018

    View details for Web of Science ID 000263751400038

    View details for PubMedID 19185577

  • TGF-beta as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation BLOOD Yamazaki, S., Iwama, A., Takayanagi, S., Eto, K., Ema, H., Nakauchi, H. 2009; 113 (6): 1250-1256


    Hematopoietic stem cells (HSCs) reside in a bone marrow niche in a nondividing state from which they occasionally are aroused to undergo cell division. Yet, the mechanism underlying this unique feature remains largely unknown. We have recently shown that freshly isolated CD34-KSL hematopoietic stem cells (HSCs) in a hibernation state exhibit inhibited lipid raft clustering. Lipid raft clustering induced by cytokines is essential for HSCs to augment cytokine signals to the level enough to re-enter the cell cycle. Here we screened candidate niche signals that inhibit lipid raft clustering, and identified that transforming growth factor-beta (TGF-beta) efficiently inhibits cytokine-mediated lipid raft clustering and induces HSC hibernation ex vivo. Smad2 and Smad3, the signaling molecules directly downstream from and activated by TGF-beta receptors were specifically activated in CD34-KSL HSCs in a hibernation state, but not in cycling CD34+KSL progenitors. These data uncover a critical role for TGF-beta as a candidate niche signal in the control of HSC hibernation and provide TGF-beta as a novel tool for ex vivo modeling of the HSC niche.

    View details for DOI 10.1182/blood-2008-04-146480

    View details for Web of Science ID 000263119300009

    View details for PubMedID 18945958

  • Transcriptional profiling of hematopoietic stem cells by high-throughput sequencing INTERNATIONAL JOURNAL OF HEMATOLOGY Yashiro, Y., Bannai, H., Minowa, T., Yabiku, T., Miyano, S., Osawa, M., Iwama, A., Nakauchi, H. 2009; 89 (1): 24-33


    Microarray analysis has made it feasible to carry out extensive gene expression profiling in a single assay. Various hematopoietic stem cell (HSC) populations have been subjected to microarray analyses and their profiles of gene expression have been reported. However, this approach is not suitable to identify novel transcripts or for profiling of genes with low expression levels. To obtain a detailed gene expression profile of CD34(-)c-Kit(+)Sca-1(+)lineage marker-negative (Lin(-)) (CD34(-)KSL) HSCs, we constructed a CD34(-)KSL cDNA library, performed high-throughput sequencing, and compared the generated profile with that of another HSC fraction, side population (SP) Lin(-) (SP Lin(-)) cells. Sequencing of the 5'-termini of about 9,500 cDNAs from each HSC library identified 1,424 and 2,078 different genes from the CD34(-)KSL and SP Lin(-) libraries, respectively. To exclude ubiquitously expressed genes including housekeeping genes, digital subtraction was successfully performed against EST databases of other organs, leaving 25 HSC-specific genes including five novel genes. Among 4,450 transcripts from the CD34(-)KSL cDNA library that showed no homology to the presumable protein-coding genes, 29 were identified as strong candidates for mRNA-like non-coding RNAs by in silico analyses. Our cyclopedic approaches may contribute to understanding of novel molecular aspects of HSC function.

    View details for DOI 10.1007/s12185-008-0212-2

    View details for Web of Science ID 000262490600004

    View details for PubMedID 19050837

  • The Actin Polymerization Regulator WAVE2 Is Required for Early Bone Marrow Repopulation by Hematopoietic Stem Cells STEM CELLS Ogaeri, T., Eto, K., Otsu, M., Ema, H., Nakauchi, H. 2009; 27 (5): 1120-1129


    The Rho GTPase family members play essential roles in hematopoiesis. Of these, Rac1 is thought to be required for the appropriate spatial localization of hematopoietic stem and/or progenitor cells (HSPCs) within the bone marrow (BM), whereas Rac2 likely plays a role in BM retention of HSPCs. To elucidate the molecular mechanisms underlying Rac-mediated functions in hematopoietic stem cells (HSCs), we studied Wiskott-Aldrich syndrome protein family verprolin-homologous proteins (WAVEs), the specific effectors downstream of the Rac GTPases in actin polymerization. We here showed that CD34(-/low)c-Kit(+)Sca-1(+)lineage(-) HSCs (CD34(-)KSL HSCs) express WAVE2 but neither WAVE1 nor WAVE3. Because WAVE2 knockout mice are embryonic-lethal, we utilized HSCs in which the expression of WAVE2 was reduced by small interfering RNA. We found that knockdown (KD) of WAVE2 in HSCs affected neither in vitro colony formation nor cell proliferation but did impair in vivo long-term reconstitution. Interestingly, WAVE2 KD HSCs exhibited unaltered homing but showed poor BM repopulation detected as early as day 5 after transplantation. The mechanistic studies on WAVE2 KD HSCs revealed modest but significant impairment in both cobblestone-like area-forming on stromal layers and actin polymerization upon integrin ligation by fibronectin. These results suggested that WAVE2-mediated actin polymerization, potentially downstream of Rac1, plays an important role in intramarrow mobilization and proliferation of HSCs, which are believed to be crucial steps for long-term marrow reconstitution after transplantation.

    View details for DOI 10.1002/stem.42

    View details for Web of Science ID 000266179500015

    View details for PubMedID 19415782

  • New ISSCR Guidelines Underscore Major Principles for Responsible Translational Stem Cell Research CELL STEM CELL Hyun, I., Lindvall, O., Ahrlund-Richter, L., Cattaneo, E., Cavazzana-Calvo, M., Cossu, G., De Luca, M., Fox, I. J., Gerstle, C., Goldstein, R. A., Hermeren, G., High, K. A., Kim, H. O., Lee, H. P., Levy-Lahad, E., Li, L., Lo, B., Marshak, D. R., McNab, A., Munsie, M., Nakauchi, H., Rao, M., Rooke, H. M., Valles, C. S., Srivastava, A., Sugarman, J., Taylor, P. L., Veiga, A., Wong, A. L., Zoloth, L., Daley, G. Q. 2008; 3 (6): 607-609


    The International Society for Stem Cell Research (ISSCR) task force that developed new Guidelines for the Clinical Translation of Stem Cells discusses core principles that should guide the responsible transition of basic stem cell research into appropriate clinical applications.

    View details for DOI 10.1016/j.stem.2008.11.009

    View details for Web of Science ID 000261670900009

    View details for PubMedID 19041777

  • Flow Cytometric Isolation and Clonal Identification of Self-Renewing Bipotent Hepatic Progenitor Cells in Adult Mouse Liver HEPATOLOGY Suzuki, A., Sekiya, S., Onishi, M., Oshima, N., Kiyonari, H., Nakauchi, H., Taniguchi, H. 2008; 48 (6): 1964-1978


    The adult liver progenitor cells appear in response to several types of pathological liver injury, especially when hepatocyte replication is blocked. These cells are histologically identified as cells that express cholangiocyte markers and proliferate in the portal area of the hepatic lobule. Although these cells play an important role in liver regeneration, the precise characterization that determines these cells as self-renewing bipotent primitive hepatic cells remains to be shown. Here we attempted to isolate cells that express a cholangiocyte marker from the adult mouse liver and perform single cell-based analysis to examine precisely bilineage differentiation potential and self-renewing capability of these cells. Based on the results of microarray analysis and immunohistochemistry, we used an antibody against CD133 and isolate CD133(+) cells via flow cytometry. We then cultured and propagated isolated cells in a single cell culture condition and examined their potential for proliferation and differentiation in vitro and in vivo. Isolated cells that could form large colonies (LCs) in culture gave rise to both hepatocytes and cholangiocytes as descendants, while maintaining undifferentiated cells by self-renewing cell divisions. The clonogenic progeny of an LC-forming cell is capable of reconstituting hepatic tissues in vivo by differentiating into fully functional hepatocytes. Moreover, the deletion of p53 in isolated LC-forming cells resulted in the formation of tumors with some characteristics of hepatocellular carcinoma and cholangiocarcinoma upon subcutaneous injection into immunodeficient mutant mice. These data provide evidence for the stem cell-like capacity of isolated and clonally cultured CD133(+) LC-forming cells.Our method for prospectively isolating hepatic progenitor cells from the adult mouse liver will facilitate study of their roles in liver regeneration and carcinogenesis.

    View details for DOI 10.1002/hep.22558

    View details for Web of Science ID 000261219200026

    View details for PubMedID 18837044

  • The polycomb gene product BMI1 contributes to the maintenance of tumor-initiating side population cells in hepatocellular carcinoma CANCER RESEARCH Chiba, T., Miyagi, S., Saraya, A., Aoki, R., Seki, A., Morita, Y., Yonemitsu, Y., Yokosuka, O., Taniguchi, H., Nakauchi, H., Iwama, A. 2008; 68 (19): 7742-7749


    Side population (SP) cell analysis and sorting have been successfully applied to hepatocellular carcinoma (HCC) cell lines to identify a minor cell population with cancer stem cell properties. However, the molecular mechanisms operating in SP cells remain unclear. The polycomb gene product BMI1 plays a central role in the self-renewal of somatic stem cells in a variety of tissues and organs and seems to be implicated in tumor development. In this study, we determined the critical role of BMI1 in the maintenance of cancer stem cells with the SP phenotype in HCC cell lines. BMI1 was preferentially expressed in SP cells in Huh7 and PLC/PRF/5 HCC cells compared with the corresponding non-SP cells. Lentiviral knockdown of BMI1 considerably decreased the number of SP cells in both Huh7 and PLC/PRF/5 cells. Long-term culture of purified SP cells resulted in a drastic reduction in the SP subpopulation upon the BMI1 knockdown, indicating that BMI1 is required for the self-renewal of SP cells in culture. More importantly, the BMI1 knockdown abolished the tumor-initiating ability of SP cells in nonobese diabetic/severe combined immunodeficiency mice. Derepression of the INK4A and ARF genes that are major targets for BMI1 was not necessarily associated with impaired self-renewal of SP cells caused by BMI1 knockdown. In conclusion, our findings define an important role for BMI1 in the maintenance of tumor-initiating SP cells in HCC. BMI1 might be a novel therapeutic target for the eradication of cancer stem cells in HCC.

    View details for DOI 10.1158/0008-5472.CAN-07-5882

    View details for Web of Science ID 000260029900008

    View details for PubMedID 18829528

  • Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ma, F., Ebihara, Y., Umeda, K., Sakai, H., Hanada, S., Zhang, H., Zaike, Y., Tsuchida, E., Nakahata, T., Nakauchi, H., Tsuji, K. 2008; 105 (35): 13087-13092


    A critical issue for clinical utilization of human ES cells (hESCs) is whether they can generate terminally mature progenies with normal function. We recently developed a method for efficient production of hematopoietic progenitors from hESCs by coculture with murine fetal liver-derived stromal cells. Large numbers of hESCs-derived erythroid progenitors generated by the coculture enabled us to analyze the development of erythropoiesis at a clone level and investigate their function. The results showed that the globin expression in the erythroid cells in individual clones changed in a time-dependent manner. In particular, embryonic epsilon-globin-expressing erythroid cells from individual clones decreased, whereas adult-type beta-globin-expressing cells increased to approximately 100% in all clones we examined, indicating that the cells undergo definitive hematopoiesis. Enucleated erythrocytes also appeared among the clonal progeny. A comparison analysis showed that hESC-derived erythroid cells took a similar differentiation pathway to human cord blood CD34(+) progenitor-derived cells when examined for the expression of glycophorin A, CD71 and CD81. Furthermore, these hESC-derived erythroid cells could function as oxygen carriers and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study should provide an experimental model for exploring early development of human erythropoiesis and hemoglobin switching and may help in the discovery of drugs for hereditary diseases in erythrocyte development.

    View details for DOI 10.1073/pnas.0802220105

    View details for Web of Science ID 000259343000083

    View details for PubMedID 18755895

  • A new red fluorescent protein that allows efficient marking of murine hematopoietic stem cells JOURNAL OF GENE MEDICINE Sanuki, S., Hamanaka, S., Kaneko, S., Otsu, M., Karasawa, S., Miyawaki, A., Nakauchi, H., Nagasawa, T., Onodera, M. 2008; 10 (9): 965-971


    Genetic marking of hematopoietic stem cells (HSCs) with multiple fluorescent proteins (FPs) would allow analysis of their features, including interaction with adjacent cells. However, there are few red FPs that are comparable to green FPs in terms of low toxicity and high fluorescent intensity. This study has evaluated the usefulness of Kusabira Orange (KO) originated from the coral stone Fungia concinna as a red FP for marking of HSCsA vector used was the MSCV-type retroviral vector, D Delta Nsap that has the PCC4 cell-passaged myeloproliferative sarcoma virus derived long terminal repeat devoid of a binding site for YY1 and the primer-binding site derived from the dl587rev, respectively. The vector was cloned with the codon-optimized KO cDNA for higher expression in mammalian cells (huKO) and converted to the corresponding retroviruses pseudotyped with the vesicular stomatitis virus G envelope protein, then transduced into c-KIT(+)Sca-1(+)Lineage(-) cells obtained from C57BL/6 (Ly5.1) mice followed by transplantation into lethally irradiated Ly5.2 mice.Approximately 70% of donor-derived cells highly expressed huKO at 16 weeks post-transplantation. Furthermore, the high expression of huKO was also detected in serially transplanted mice, suggesting that expression of huKO per se had little deleterious effect on murine hematopoiesis. In double marking experiments, huKO-expressing hematopoietic cells were easily distinguished from those expressing EGFP by flow cytometry and fluorescent microscope analysis.Overall, the results obtained from the present study suggest that huKO can be used as a valuable and versatile red fluorescent marker for HSCs.

    View details for DOI 10.1002/jgm.1232

    View details for Web of Science ID 000260361600002

    View details for PubMedID 18613301

  • Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells JOURNAL OF EXPERIMENTAL MEDICINE Nishikii, H., Eto, K., Tamura, N., Hattori, K., Heissig, B., Kanaji, T., Sawaguchi, A., Goto, S., Ware, J., Nakauchi, H. 2008; 205 (8): 1917-1927


    Embryonic stem cells (ESCs) could potentially compensate for the lack of blood platelets available for use in transfusions. Here, we describe a new method for generating mouse ESC-derived platelets (ESPs) that can contribute to hemostasis in vivo. Flow cytometric sorting of cells from embryoid bodies on day 6 demonstrated that c-Kit(+) integrin alpha IIb (alpha IIb)(+) cells, but not CD31(+) cells or vascular endothelial cadherin(+) cells, are capable of megakaryopoiesis and the release of platelet-like structures by day 12. alpha IIb beta 3-expressing ESPs exhibited ectodomain shedding of glycoprotein (GP)Ibalpha, GPV, and GPVI, but not alpha IIb beta 3 or GPIb beta. ESPs showed impaired alpha IIb beta 3 activation and integrin-mediated actin reorganization, critical events for normal platelet function. However, the administration of metalloproteinase inhibitors GM6001 or TAPI-1 during differentiation increased the expression of GPIb alpha, improving both thrombogenesis in vitro and posttransfusion recovery in vivo. Thus, the regulation of metalloproteinases in culture could be useful for obtaining high-quality, efficacious ESPs as an alternative platelet source for transfusions.

    View details for DOI 10.1084/jem.20071482

    View details for Web of Science ID 000258528500020

    View details for PubMedID 18663123

  • Prospero-related homeobox 1 and liver receptor homolog 1 coordinately regulate long-term proliferation of murine fetal hepatoblasts HEPATOLOGY Kamiya, A., Kakinuma, S., Onodera, M., Miyajima, A., Nakauchi, H. 2008; 48 (1): 252-264


    During early to late-fetal liver development, bipotential hepatoblasts proliferate and differentiate into hepatocytes and cholangiocytes. The prospero-related homeobox 1 gene (Prox1) is expressed in hepatoblasts, and the inactivation of Prox1 causes defective early liver development, in particular, faulty migration of fetal hepatoblasts. Prox1 binds to another hepatocyte-enriched transcription factor, liver receptor homolog 1 (Lrh1), and suppresses its transcriptional activity. However, the molecular mechanism by which Prox1 and Lrh1 regulate the characteristics of fetal hepatic cells remains unknown. We investigated the contribution of Prox1 and Lrh1 in early liver development. Embryonic day 13 liver-derived CD45-Ter119-Dlk+ cells were purified as fetal hepatic stem/progenitor cells, and formation of colonies derived from single cells was detected under low-density culture conditions. We found that overexpression of Prox1 using retrovirus infection induced migration and proliferation of fetal hepatic stem/progenitor cells. In contrast, overexpression of Lrh1 suppressed colony formation. Prox1 induced the long-term proliferation of fetal hepatic stem/progenitor cells, which exhibited both high proliferative activity and bipotency for differentiation. Prox1 up-regulated expression of cyclins D2, E1, and E2, whereas it suppressed expression of p16(ink4a), the cdk inhibitor. In addition, overexpression of Prox1 significantly inhibited the proximal promoter activity of p16(ink4a).These results suggested that Prox1 and Lrh1 coordinately regulate development of hepatic stem/progenitor cells and that Prox1 induces fetal hepatocytic proliferation through the suppression of the promoter activity of p16(ink4a).

    View details for DOI 10.1002/hep.22303

    View details for Web of Science ID 000257301100030

    View details for PubMedID 18571787

  • Transporter-mediated protection against thiopurine-induced hematopoietic toxicity CANCER RESEARCH Krishnamurthy, P., Schwab, M., Takenaka, K., Nachagari, D., Morgan, J., Leslie, M., Du, W., Boyd, K., Cheok, M., Nakauchi, H., Marzolini, C., Kim, R. B., Poonkuzhali, B., Schuetz, E., Evans, W., Relling, M., Schuetz, J. D. 2008; 68 (13): 4983-4989


    Thiopurines are effective immunosuppressants and anticancer agents, but intracellular accumulation of their active metabolites (6-thioguanine nucleotides, 6-TGN) causes dose-limiting hematopoietic toxicity. Thiopurine S-methyltransferase deficiency is known to exacerbate thiopurine toxicity. However, many patients are highly sensitive to thiopurines for unknown reasons. We show that multidrug-resistance protein 4 (Mrp4) is abundant in myeloid progenitors and tested the role of the Mrp4, an ATP transporter of monophosphorylated nucleosides, in this unexplained thiopurine sensitivity. Mrp4-deficient mice experienced Mrp4 gene dosage-dependent toxicity caused by accumulation of 6-TGNs in their myelopoietic cells. Therefore, Mrp4 protects against thiopurine-induced hematopoietic toxicity by actively exporting thiopurine nucleotides. We then identified a single-nucleotide polymorphism (SNP) in human MRP4 (rs3765534) that dramatically reduces MRP4 function by impairing its cell membrane localization. This SNP is common (>18%) in the Japanese population and indicates that the increased sensitivity of some Japanese patients to thiopurines may reflect the greater frequency of this MRP4 SNP.

    View details for DOI 10.1158/0008-5472.CAN-07-6790

    View details for Web of Science ID 000257415300006

    View details for PubMedID 18593894

  • Growth and maturation of megakaryocytes is regulated by Lnk/Sh2b3 adaptor protein through crosstalk between cytokine- and integrin-mediated signals EXPERIMENTAL HEMATOLOGY Takizawa, H., Eto, K., Yoshikawa, A., Nakauchi, H., Takatsu, K., Takaki, S. 2008; 36 (7): 897-906


    Various cytokines and growth factors control the differentiation and maturation of megakaryocytes (MKs). However, the mechanism regulating platelet release from MKs is not well understood. Here, we investigated a role of Lnk/Sh2b3, an intracellular adaptor protein, in megakaryopoiesis.Number of MK progenitor in bone marrow (BM) of wild-type or Lnk(-/-) mice and their sensitivity to thrombopoietin (TPO) were determined in colony-forming unit assay. Using BM-derived wild-type or Lnk(-/-) MKs stimulated with TPO, activation of the signaling molecules was biochemically analyzed and effect of integrin stimulation on TPO signals was studied by addition of vascular cell adhesion molecule (VCAM-1). Platelet production from MKs in the presence of VCAM-1 was counted by flow cytometry and their morphological change was observed by time-lapse microscopy.Lnk(-/-) mice showed elevated platelets and mature MKs due to enhanced sensitivity of progenitors to TPO. Erk1/2 phosphorylation induced by TPO was augmented and prolonged in Lnk(-/-) MKs while activation of signal transducers and activators of transcription (Stat)3, Stat5, and Akt was normal. Wild-type MKs, but not in Lnk(-/-) MKs on VCAM-1 showed reduced Stat5 phosphorylation and mitogen-activated protein kinases activation upon stimulation with TPO. Additionally, the presence of VCAM in culture accelerated spontaneous platelet release from mature wild-type MKs, but not from Lnk(-/-) MKs.Results suggest that contact of MKs with adhesion molecules via integrins might contribute to platelet release, which is under Lnk-mediated regulation of Stat-5 activation and show that Lnk functions in responses controlled by cell adhesion and in crosstalk between integrin- and cytokine-mediated signaling.

    View details for DOI 10.1016/j.exphem.2008.02.004

    View details for Web of Science ID 000257349400015

    View details for PubMedID 18456388

  • Bloodlines of haematopoietic stem cell research in Japan PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Ema, H., Nakauchi, H. 2008; 363 (1500): 2089-2097


    Haematopoietic stem cells (HSCs) can supply all blood cells throughout the adult life of individuals. Based on this property, HSCs have been used for bone marrow and cord blood transplantation. Among various stem cells, HSCs were recognized earliest and were studied most extensively, providing a model for other stem cells. Knowledge of HSC regulation has rapidly accumulated of late. Contributions of scientists in Japan to progress HSC biology are here briefly overviewed. Focusing on the original work accomplished in Japan in the last two decades, people who have led such activities are introduced and their relationships with one another are sketched.

    View details for DOI 10.1098/rstb.2008.2263

    View details for Web of Science ID 000256140700005

    View details for PubMedID 18375375

  • Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors BLOOD Takayama, N., Nishikii, H., Usui, J., Tsukui, H., Sawaguchi, A., Hiroyama, T., Eto, K., Nakauchi, H. 2008; 111 (11): 5298-5306


    Human embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell-derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin alpha IIb beta 3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

    View details for DOI 10.1182/blood-2007-10-117622

    View details for Web of Science ID 000256336500011

    View details for PubMedID 18388179

  • Recipient-derived cells after cord blood transplantation: Dynamics elucidated by multicolor FACS, reflecting graft failure and relapse BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Watanabe, N., Takahashi, S., Ishige, M., Ishii, Y., Ooi, J., Tomonari, A., Tsukada, N., Konuma, T., Kato, S., Sato, A., Tojo, A., Nakauchi, H. 2008; 14 (6): 693-701


    Although umbilical cord blood has been increasingly used as an alternative donor source to treat hematologic malignancies, cord blood transplantation (CBT) is frequently complicated by graft failure and relapse of primary diseases. Because persistence or increase of recipient-derived hematopoietic or malignant cells has pathogenic import under these conditions, analysis of recipient-derived cells should be useful to understand the pathogenesis of graft failure and relapse of primary disease. Because most CBT involves human leukocyte antigen (HLA)-mismatched transplantation, we developed a 9-color fluorescence activated cell sorter (FACS)-based method of mixed chimerism (MC) analysis using anti-HLA antibodies to detect mismatched antigens (HLA-Flow method). Among CD4(+) T cells, CD8(+) T cells, B cells, NK cells, monocytes, and granulocytes, donor- and recipient-derived cells alike could be individually analyzed simultaneously in a rapid, quantitative and highly sensitive manner, making the HLA-Flow method very valuable in monitoring the engraftment process. In addition, this method was also useful in monitoring recipient-derived cells with leukemia-specific phenotypes, both as minimal residual disease (MRD) and as early harbingers of relapse. Leukemia relapse can be definitively diagnosed by cytogenetic or PCR studies using recipient-derived cells sorted for leukemia markers. Multicolor HLA-fFlow analysis and cell sorting in early diagnosis of graft failure and relapse was confirmed as valuable in 14 patients who had received HLA-mismatched CBT.

    View details for DOI 10.1016/j.bbmt.2008.04.001

    View details for Web of Science ID 000256279900010

    View details for PubMedID 18489995

  • An immunotherapy approach with dendritic cells genetically modified to express the tumor-associated antigen, HER2 CANCER IMMUNOLOGY IMMUNOTHERAPY Nabekura, T., Nagasawa, T., Nakauchi, H., Onodera, M. 2008; 57 (5): 611-622


    Dendritic cells (DC), genetically modified to express ovalbumin by the retroviral vector GCDNsap, can elicit stronger anti-tumor immunity than those loaded with the peptides. To assess the clinical feasibility of the strategy, such DC were prepared by differentiation of hematopoietic progenitor cells transduced with the human epidermal growth factor receptor 2 (HER2). When inoculated in mice, the DC primed both HER2-specific cytotoxic T lymphocytes and type 1 T helper lymphocytes, resulting in production of HER2-specific antibody. Of importance is that the antibody mediated antibody-dependent cellular cytotoxicity and opsonization. The potent anti-tumor effects were also confirmed by results of experiments using HER2-transgenic mice. Inoculation of HER2-transduced DC resulted in longer disease-free survival of treated mice that showed significant reduction of primary and metastatic tumors. Interestingly, footpad inoculation resulted in stronger anti-tumor effects compared to subcutaneous administration and induced higher levels of the HER2-specific antibody, suggesting that an important role of humoral immunity in anti-tumor effects for malignancies with membrane-type tumor-associated antigens (TAA). Taken together, vaccination of the TAA-transduced DC may represent a promising form of therapy for breast cancers expressing HER2.

    View details for DOI 10.1007/s00262-007-0399-8

    View details for Web of Science ID 000253524600002

    View details for PubMedID 17786440

  • Interleukin-27 directly induces differentiation in hematopoietic stem cells BLOOD Seita, J., Asakawa, M., Ooehara, J., Takayanagi, S., Morita, Y., Watanabe, N., Fujita, K., Kudo, M., Mizuguchi, J., Ema, H., Nakauchi, H., Yoshimoto, T. 2008; 111 (4): 1903-1912


    Interleukin (IL)-27, one of the most recently discovered IL-6 family cytokines, activates both the signal transducer and activator of transcription (STAT)1 and STAT3, and plays multiple roles in pro- and anti-inflammatory immune responses. IL-27 acts on various types of cells including T, B, and macrophage through the common signal-transducing receptor gp130 and its specific receptor WSX-1, but the effect of IL-27 on hematopoietic stem cells (HSCs) remains unknown. Here, we show that IL-27 together with stem cell factor (SCF) directly acts on HSCs and supports their early differentiation in vitro and in vivo. CD34(-/low)c-Kit(+)Sca-1(+)lineage marker(-) (CD34(-)KSL) cells, a population highly enriched in mouse HSCs, were found to express both IL-27 receptor subunits. In vitro cultures of CD34(-)KSL cells with IL-27 and SCF resulted in an expansion of progenitors including short-term repopulating cells, while some of their long-term repopulating activity also was maintained. To examine its in vivo effect, transgenic mice expressing IL-27 were generated. These mice exhibited enhanced myelopoiesis and impaired B lymphopoiesis in the bone marrow with extramedullary hematopoiesis in the spleen. Moreover, IL-27 similarly acted on human CD34(+) cells. These results suggest that IL-27 is one of the limited cytokines that play a role in HSC regulation.

    View details for DOI 10.1182/blood-2007-06-093328

    View details for Web of Science ID 000253251100035

    View details for PubMedID 18042804

  • The plasminogen fibrinolytic pathway is required for hematopoietic regeneration CELL STEM CELL Heissig, B., Lund, L. R., Akiyama, H., Ohki, M., Morita, Y., Romer, J., Nakauchi, H., Okumura, K., Ogawa, H., Werb, Z., Dano, K., Hattori, K. 2007; 1 (6): 658-670


    Hematopoietic stem cells within the bone marrow exist in a quiescent state. They can differentiate and proliferate in response to hematopoietic stress (e.g., myelosuppression), thereby ensuring a well-regulated supply of mature and immature hematopoietic cells within the circulation. However, little is known about how this stress response is coordinated. Here, we show that plasminogen (Plg), a classical fibrinolytic factor, is a key player in controlling this stress response. Deletion of Plg in mice prevented hematopoietic stem cells from entering the cell cycle and undergoing multilineage differentiation after myelosuppression, leading to the death of the mice. Activation of Plg by administration of tissue-type plasminogen activator promoted matrix metalloproteinase-mediated release of Kit ligand from stromal cells, thereby promoting hematopoietic progenitor cell proliferation and differentiation. Thus, activation of the fibrinolytic cascade is a critical step in regulating the hematopoietic stress response.

    View details for DOI 10.1016/j.stem.2007.10.012

    View details for Web of Science ID 000251784300012

    View details for PubMedID 18371407

  • The WAVE2/Abi 1 complex differentially regulates megakaryocyte development and spreading: implications for platelet biogenesis and spreading machinery BLOOD Eto, K., Nishikii, H., Ogaeri, T., Suetsugu, S., Kamiya, A., Kobayashi, T., Yamazaki, D., Oda, A., Takenawa, T., Nakauchi, H. 2007; 110 (10): 3637-3647


    Actin polymerization is crucial in throm-bopoiesis, platelet adhesion, and mega-karyocyte (MK) and platelet spreading. The Wiskott-Aldrich syndrome protein (WASp) homolog WAVE functions downstream of Rac and plays a pivotal role in lamellipodia formation. While MKs and platelets principally express WAVE1 and WAVE2, which are associated with Abi1, the physiologic significance of WAVE isoforms remains undefined. We generated WAVE2(-/-) embryonic stem (ES) cells because WAVE2-null mice die by embryonic day (E) 12.5. We found that while WAVE2(-/-) ES cells differentiated into immature MKs on OP9 stroma, they were severely impaired in terminal differentiation and in platelet production. WAVE2(-/-) MKs exhibited a defect in peripheral lamellipodia on fibrinogen even with phorbol 12-myristate 13-acetate (PMA) costimulation, indicating a requirement of WAVE2 for integrin alpha(IIb)beta(3)-mediated full spreading. MKs in which expression of Abi1 was reduced by small interfering RNA (siRNA) exhibited striking similarity to WAVE2(-/-) MKs in maturation and spreading. Interestingly, the knockdown of IRSp53, a Rac effector that preferentially binds to WAVE2, impaired the development of lamellipodia without affecting proplatelet production. In contrast, thrombopoiesis in vivo and platelet spreading on fibrinogen in vitro were intact in WAVE1-null mice. These observations clarify indispensable roles for the WAVE2/Abi1 complex in alpha(IIb)beta(3)-mediated lamellipodia by MKs and platelets through Rac and IRSp53, and additionally in thrombopoiesis independent of Rac and IRSp53.

    View details for DOI 10.1182/blood-2007-04-085860

    View details for Web of Science ID 000250946300030

    View details for PubMedID 17664349

  • Enhanced self-renewal capability in hepatic stem/progenitor cells drives cancer initiation GASTROENTEROLOGY Chiba, T., Zheng, Y., Kita, K., Yokosuka, O., Saisho, H., Onoidera, M., Miyoshi, H., Nakano, M., Zen, Y., Nakanuma, Y., Nakauchi, H., Iwama, A., Taniguchi, H. 2007; 133 (3): 937-950


    Transformed hematopoietic stem/progenitor cells with an enhanced or acquired self-renewal capability function as leukemic stem cells. In a variety of solid cancers, stem/progenitor cells could be also targets of carcinogenesis. However, it remains unclear whether disruption of stem cell function directly contributes to cancer initiation. We sought to elucidate the mechanisms of self-renewal in hepatic stem/progenitor cells and the relation between stem cell function and hepatocarcinogenesis.Functional analyses of polycomb-group protein Bmi1 and Wnt/beta-catenin, the molecules that are responsible for the self-renewal capability of many types of stem cells, were conducted in c-Kit(-)CD29(+)CD49f(+/low)CD45(-)Ter-119(-) hepatic stem/progenitor cells using retrovirus- or lentivirus-mediated gene transfer. The tumorigenicity of these cells transduced with the indicated retroviruses was also assessed by transplantation into nonobese diabetic/severe combined immunodeficient mice.Forced expression of Bmi1 and constitutively active beta-catenin mutant similarly promoted the self-renewal of hepatic stem/progenitor cells. The transplantation of Bmi1- or beta-catenin-transduced cells clonally expanded from single hepatic stem/progenitor cells produced tumors, which exhibited the histologic features of combined hepatocellular and cholangiocarcinoma.These observations imply that the dysregulated self-renewal of hepatic stem/progenitor cells serves as an early event in hepatocarcinogenesis, and they highlight the important roles of Bmi1 and the Wnt/beta-catenin pathway in regulating the self-renewal of normal or cancer stem cells in liver.

    View details for DOI 10.1053/j.gasrtro.2007.06.016

    View details for Web of Science ID 000249326900027

    View details for PubMedID 17673212

  • Foxo3a is essential for maintenance of the hematopoietic stem cell pool CELL STEM CELL Miyamoto, K., Araki, K. Y., Naka, K., Arai, F., Takubo, K., Yamazaki, S., Matsuoka, S., Miyamoto, T., Ito, K., Ohmura, M., Chen, C., Hosokawa, K., Nakauchi, H., Nakayama, K., Nakayama, K. I., Harada, M., Motoyama, N., Suda, T., Hirao, A. 2007; 1 (1): 101-112


    Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a(-/-) mice and showed that, although the proliferation and differentiation of Foxo3a(-/-) hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a(-/-) bone marrow cells and stromal cells was reduced. The ability of Foxo3a(-/-) HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a(-/-) HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a(-/-) mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool.

    View details for DOI 10.1016/j.stem.2007.02.001

    View details for Web of Science ID 000250872600005

    View details for PubMedID 18371339

  • Bone marrow (BM) transplantation promotes beta-cell regeneration after acute injury through BM cell mobilization ENDOCRINOLOGY Hasegawa, Y., Ogihara, T., Yamada, T., Ishigaki, Y., Imai, J., Uno, K., Gao, J., Kaneko, K., Ishihara, H., Sasano, H., Nakauchi, H., Oka, Y., Katagiri, H. 2007; 148 (5): 2006-2015


    There is controversy regarding the roles of bone marrow (BM)-derived cells in pancreatic beta-cell regeneration. To examine these roles in vivo, mice were treated with streptozotocin (STZ), followed by bone marrow transplantation (BMT; lethal irradiation and subsequent BM cell infusion) from green fluorescence protein transgenic mice. BMT improved STZ-induced hyperglycemia, nearly normalizing glucose levels, with partially restored pancreatic islet number and size, whereas simple BM cell infusion without preirradiation had no effects. In post-BMT mice, most islets were located near pancreatic ducts and substantial numbers of bromodeoxyuridine-positive cells were detected in islets and ducts. Importantly, green fluorescence protein-positive, i.e. BM-derived, cells were detected around islets and were CD45 positive but not insulin positive. Then to examine whether BM-derived cell mobilization contributes to this process, we used Nos3(-/-) mice as a model of impaired BM-derived cell mobilization. In streptozotocin-treated Nos3(-/-) mice, the effects of BMT on blood glucose, islet number, bromodeoxyuridine-positive cells in islets, and CD45-positive cells around islets were much smaller than those in streptozotocin-treated Nos3(+/+) controls. A series of BMT experiments using Nos3(+/+) and Nos3(-/-) mice showed hyperglycemia-improving effects of BMT to correlate inversely with the severity of myelosuppression and delay of peripheral white blood cell recovery. Thus, mobilization of BM-derived cells is critical for BMT-induced beta-cell regeneration after injury. The present results suggest that homing of donor BM-derived cells in BM and subsequent mobilization into the injured periphery are required for BMT-induced regeneration of recipient pancreatic beta-cells.

    View details for DOI 10.1210/en.2006-1351

    View details for Web of Science ID 000245810600009

    View details for PubMedID 17255204

  • De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells JOURNAL OF EXPERIMENTAL MEDICINE Tadokoro, Y., Ema, H., Okano, M., Li, E., Nakauchi, H. 2007; 204 (4): 715-722


    DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34(-/low), c-Kit(+), Sca-1(+), lineage marker(-) (CD34(-) KSL) cells, a fraction of mouse bone marrow cells highly enriched in hematopoietic stem cells (HSCs), expressed both Dnmt3a and Dnmt3b. Using retroviral Cre gene transduction, we conditionally disrupted Dnmt3a, Dnmt3b, or both Dnmt3a and Dnmt3b (Dnmt3a/Dnmt3b) in CD34(-) KSL cells purified from mice in which the functional domains of these genes are flanked by two loxP sites. We found that Dnmt3a and Dnmt3b function as de novo DNA methyltransferases during differentiation of hematopoietic cells. Unexpectedly, in vitro colony assays and in vivo transplantation assays showed that both myeloid and lymphoid lineage differentiation potentials were maintained in Dnmt3a-, Dnmt3b-, and Dnmt3a/Dnmt3b-deficient HSCs. However, Dnmt3a/Dnmt3b-deficient HSCs, but not Dnmt3a- or Dnmt3b-deficient HSCs, were incapable of long-term reconstitution in transplantation assays. These findings establish a critical role for DNA methylation by Dnmt3a and Dnmt3b in HSC self-renewal.

    View details for Web of Science ID 000245920600004

    View details for PubMedID 17420264

  • Stable transgene expression in mice generated from retrovirally transduced embryonic stem cells MOLECULAR THERAPY Hamanaka, S., Nabekura, T., Otsu, M., Yoshida, H., Nagata, M., Usui, J., Takahashi, S., Nagasawa, T., Nakauchi, H., Onodera, M. 2007; 15 (3): 560-565


    Silencing of transduced genes hampers production of transgenic mice using retroviral vectors. We show stable expression of the enhanced green fluorescent protein (EGFP) gene in chimeric mice generated from retrovirally transduced embryonic stem cells. The vector was a murine stem cell virus-typed retroviral vector (GCDsap) in which the long terminal repeat and primer-binding site were derived from a PCC4 cell-passaged myeloproliferative sarcoma virus and the endogenous retrovirus dl587rev, respectively. To increase the viral titer, the vector was packaged with vesicular stomatitis virus G protein, which allowed concentration of the virus into pellets followed by resuspension in serum-free medium. In chimeric mice, EGFP was detected in various tissues including hematopoietic cells, neurons, cardiac muscle, and intestine. Furthermore, high expression was maintained in the progeny of these mice, suggesting successful germline transmission of active proviruses. Although the proportion of EGFP-expressing cells and the mean intensity of EGFP expression varied among tissues and mice, 100% of peripheral blood leukocytes expressed EGFP in mice carrying a single provirus copy, as well as in their progeny. Therefore, the gene transfer system described here provides a useful tool not only to generate transgenic animals but also to manipulate human embryonic stem cells..

    View details for DOI 10.1038/

    View details for Web of Science ID 000244405700020

    View details for PubMedID 17180117

  • A human mutant CD4 molecule resistant to HIV-1 binding restores helper T-lymphocyte functions in murine CD4-deficient mice EXPERIMENTAL AND MOLECULAR MEDICINE Kim, D., Satoko, T., Shinohara, N., Nakauchi, H. 2007; 39 (1): 1-7


    CD4 is a cell surface glycoprotein that acts as a co-receptor for the T cell antigen receptor by binding to a non-polymorphic portion of MHC molecules. CD4 also functions as a receptor for human immunodeficiency virus type-I (HIV-1) because the viral envelope glycoprotein gp120 binds to CD4 with a high affinity. We have previously demonstrated that introduction of mutations into CD4 abolished the binding of gp120 and prevented HIV-1 from entering cells and spreading. However, whether introduction of such mutations into CD4 causes decreased binding to MHC and loss of function is yet to be determined. We generated transgenic mouse lines by injecting a mutant human CD4 (muthCD4) gene under a murine CD4 enhancer/promoter to ensure tissue and stage specific expression. To exclude the influence of endogenous murine CD4, transgenic mice were crossed with murine CD4-targeted mice to produce muthCD4 transgenic mice lacking endogenous CD4 (muthCD4TG/KO mice). In these mice, T lymphocytes expressing muthCD4 expanded and matured in the thymus and were present in the spleen and lymph nodes. They also activated B cells to mount an antibody response to a T-dependent antigen. The results from this study suggest that a human variant of CD4 modified to be resistant to HIV-1 binding can rescue the signaling for T cell development in the thymus in vivo, having helper T cell functions. Thus, further characterization of muthCD4 molecules should open the way to new HIV treatment modalities.

    View details for Web of Science ID 000244793400001

    View details for PubMedID 17334223

  • Bmi1 cooperates with Dnmt1-associated protein 1 in gene silencing BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Negishi, M., Saraya, A., Miyagi, S., Nagao, K., Inagaki, Y., Nishikawa, M., Tajima, S., Koseki, H., Tsuda, H., Takasaki, Y., Nakauchi, H., Iwama, A. 2007; 353 (4): 992-998


    Polycomb group (PcG) proteins are involved in gene silencing through chromatin modifications. Among polycomb repressive complexes (PRCs), PRC1 exhibits H2A-K119 ubiquitin E3 ligase activity. However, the molecular mechanisms underlying PRC1-mediated gene silencing remain largely obscure. In this study, we found that Bmi1 directly interacts with Dnmt-associated protein 1 (Dmap1), which has been characterized to associate with the maintenance DNA methyltransferase, Dnmt1. Bmi1 was demonstrated to form a ternary complex with Dmap1 and Dnmt1 with Dmap1 in the central position. Chromatin immunoprecipitations confirmed the ternary complex formation within the context of the PRC1 at the Bmi1 target loci. Loss of Dmap1 binding to the Bmi1 target loci was tightly associated with derepressed gene expression in Bmi1-/- cells. Dmap1 knockdown exhibited the same impact as Bmi1 knockout did on the expression of Bmi1 targets, including Hox genes. Collectively, our findings suggest that Bmi1 incorporates Dmap1 in polycomb gene silencing.

    View details for DOI 10.1016/j.bbrc.2006.12.166

    View details for Web of Science ID 000243859600024

    View details for PubMedID 17214966

  • Lnk negatively regulates self-renewal of hematopoietic stem cells by modifying thrombopoietin-mediated signal transduction PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Seita, J., Ema, H., Ooehara, J., Yamazaki, S., Tadokoro, Y., Yamasaki, A., Eto, K., Takaki, S., Takatsu, K., Nakauchi, H. 2007; 104 (7): 2349-2354


    One of the central tasks of stem cell biology is to understand the molecular mechanisms that control self-renewal in stem cells. Several cytokines are implicated as crucial regulators of hematopoietic stem cells (HSCs), but little is known about intracellular signaling for HSC self-renewal. To address this issue, we attempted to clarify how self-renewal potential is enhanced in HSCs without the adaptor molecule Lnk, as in Lnk-deficient mice HSCs are expanded in number >10-fold because of their increased self-renewal potential. We show that Lnk negatively regulates self-renewal of HSCs by modifying thrombopoietin (TPO)-mediated signal transduction. Single-cell cultures showed that Lnk-deficient HSCs are hypersensitive to TPO. Competitive repopulation revealed that long-term repopulating activity increases in Lnk-deficient HSCs, but not in WT HSCs, when these cells are cultured in the presence of TPO with or without stem cell factor. Single-cell transplantation of each of the paired daughter cells indicated that a combination of stem cell factor and TPO efficiently induces symmetrical self-renewal division in Lnk-deficient HSCs but not in WT HSCs. Newly developed single-cell immunostaining demonstrated significant enhancement of both p38 MAPK inactivation and STAT5 and Akt activation in Lnk-deficient HSCs after stimulation with TPO. Our results suggest that a balance in positive and negative signals downstream from the TPO signal plays a role in the regulation of the probability of self-renewal in HSCs. In general, likewise, the fate of stem cells may be determined by combinational changes in multiple signal transduction pathways.

    View details for DOI 10.1073/pnas.0606238104

    View details for Web of Science ID 000244438500056

    View details for PubMedID 17284614

  • Unique composition of polycomb repressive complex 1 in hematopoietic stem cells INTERNATIONAL JOURNAL OF HEMATOLOGY Kato, Y., Koseki, H., Vidal, M., Nakauchi, H., Iwama, A. 2007; 85 (2): 179-181

    View details for DOI 10.1532/IJH97.06235

    View details for Web of Science ID 000249507500020

    View details for PubMedID 17322001

  • Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation NATURE GENETICS Choudhury, A. R., Ju, Z., Djojosubroto, M. W., Schienke, A., Lechel, A., Schaetzlein, S., Jiang, H., Stepczynska, A., Wang, C., Buer, J., Lee, H., von Zglinicki, T., Ganser, A., Schirmacher, P., Nakauchi, H., Rudolph, K. L. 2007; 39 (1): 99-105


    Telomere shortening limits the proliferative lifespan of human cells by activation of DNA damage pathways, including upregulation of the cell cycle inhibitor p21 (encoded by Cdkn1a, also known as Cip1 and Waf1)) (refs. 1-5). Telomere shortening in response to mutation of the gene encoding telomerase is associated with impaired organ maintenance and shortened lifespan in humans and in mice. The in vivo function of p21 in the context of telomere dysfunction is unknown. Here we show that deletion of p21 prolongs the lifespan of telomerase-deficient mice with dysfunctional telomeres. p21 deletion improved hematolymphopoiesis and the maintenance of intestinal epithelia without rescuing telomere function. Moreover, deletion of p21 rescued proliferation of intestinal progenitor cells and improved the repopulation capacity and self-renewal of hematopoietic stem cells from mice with dysfunctional telomeres. In these mice, apoptotic responses remained intact, and p21 deletion did not accelerate chromosomal instability or cancer formation. This study provides experimental evidence that telomere dysfunction induces p21-dependent checkpoints in vivo that can limit longevity at the organismal level.

    View details for DOI 10.1038/ng1937

    View details for Web of Science ID 000243136500024

    View details for PubMedID 17143283

  • Cytokine signaling, lipid raft clustering, and HSC hibernation 6th Biennial International Symposium and Workshop on Hematopoietic Stem Cells Yamazaki, S., Iwama, A., Morita, Y., Eto, K., Ema, H., Nakauchi, H. BLACKWELL PUBLISHING. 2007: 54–63


    Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche in a noncycling state and enter the cell cycle at long intervals. This unique property of HSCs is reminiscent of hibernation in mammals. However, little is known about inter- and intracellular signaling mechanisms underlying this unique property of HSCs. This is largely due to the paucity of HSCs making application of traditional signal transduction assays difficult. To address these issues, we have developed a novel assay based on in-droplet single-cell staining and quantitative fluorescence imaging analysis. Using this assay system, we demonstrate that freshly isolated HSCs from the BM niche lack lipid raft clustering, exhibit repression of the AKT-FOXO signaling pathway, and express abundant p57(Kip2) cyclin-dependent kinase inhibitor. Lipid raft clustering induced by cytokines was essential for HSC re-entry into the cell cycle. Conversely, inhibition of lipid raft clustering caused sustained nuclear accumulation of FOXO transcription factors and induced HSC hibernation ex vivo. Among niche signals examined, transforming growth factor-beta (TGF-beta) efficiently inhibited lipid raft clustering and induced p57(Kip2) expression, leading to HSC hibernation. These data uncover a critical role for lipid rafts in HSC fate decision and establish the role of TGF-beta as a niche signal in control of HSC hibernation in the BM niche.

    View details for DOI 10.1196/annals.1392.017

    View details for Web of Science ID 000248195300005

    View details for PubMedID 17442772

  • Non-side-population hematopoietic stem cells in mouse bone marrow BLOOD Morita, Y., Ema, H., Yamazaki, S., Nakauchi, H. 2006; 108 (8): 2850-2856


    Most hematopoietic stem cells (HSCs) are assumed to reside in the so-called side population (SP) in adult mouse bone marrow (BM). We report the coexistence of non-SP HSCs that do not significantly differ from SP HSCs in numbers, capacities, and cell-cycle states. When stained with Hoechst 33342 dye, the CD34(-/low) c-Kit(+)Sca-1(+)lineage marker(-) (CD34(-)KSL) cell population, highly enriched in mouse HSCs, was almost equally divided into the SP and the main population (MP) that represents non-SP cells. Competitive repopulation assays with single or 30 SP- or MP-CD34(-)KSL cells found similar degrees of repopulating activity and frequencies of repopulating cells for these populations. Secondary transplantation detected self-renewal capacity in both populations. SP analysis of BM cells from primary recipient mice suggested that the SP and MP phenotypes are interconvertible. Cell-cycle analyses revealed that CD34(-)KSL cells were in a quiescent state and showed uniform cell-cycle kinetics, regardless of whether they were in the SP or MP. Bcrp-1 expression was similarly detected in SP- and MP-CD34(-)KSL cells, suggesting that the SP phenotype is regulated not only by Bcrp-1, but also by other factors. The SP phenotype does not specify all HSCs; its identity with stem cell function thus is unlikely.

    View details for DOI 10.1182/blood-2006-03-010207

    View details for Web of Science ID 000241131700057

    View details for PubMedID 16804114

  • Differential impact of Ink4a and Arf on hematopoietic stem cells and their bone marrow microenvironment in Bmi1-deficient mice JOURNAL OF EXPERIMENTAL MEDICINE Oguro, H., Iwama, A., Morita, Y., Kamijo, T., van Lohuizen, M., Nakauchi, H. 2006; 203 (10): 2247-2253


    The polycomb group (PcG) protein Bmi1 plays an essential role in the self-renewal of hematopoietic and neural stem cells. Derepression of the Ink4a/Arf gene locus has been largely attributed to Bmi1-deficient phenotypes in the nervous system. However, its role in hematopoietic stem cell (HSC) self-renewal remained undetermined. In this study, we show that derepressed p16(Ink4a) and p19(Arf) in Bmi1-deficient mice were tightly associated with a loss of self-renewing HSCs. The deletion of both Ink4a and Arf genes substantially restored the self-renewal capacity of Bmi1(-/-) HSCs. Thus, Bmi1 regulates HSCs by acting as a critical failsafe against the p16(Ink4a)- and p19(Arf)-dependent premature loss of HSCs. We further identified a novel role for Bmi1 in the organization of a functional bone marrow (BM) microenvironment. The BM microenvironment in Bmi1(-/-) mice appeared severely defective in supporting hematopoiesis. The deletion of both Ink4a and Arf genes did not considerably restore the impaired BM microenvironment, leading to a sustained postnatal HSC depletion in Bmi1(-/-)Ink4a-Arf(-/-) mice. Our findings unveil a differential role of derepressed Ink4a and Arf on HSCs and their BM microenvironment in Bmi1-deficient mice. Collectively, Bmi1 regulates self-renewing HSCs in both cell-autonomous and nonautonomous manners.

    View details for DOI 10.1084/jem.20052477

    View details for Web of Science ID 000240951400012

    View details for PubMedID 16954369

  • Putative "stemness" gene Jam-b is not required for maintenance of stem cell state in embryonic, neural, or hematopoietic stem cells MOLECULAR AND CELLULAR BIOLOGY Sakaguchi, T., Nishimoto, M., Miyagi, S., Iwama, A., Morita, Y., Iwamori, N., Nakauchi, H., Kiyonari, H., Muramatsu, M., Okuda, A. 2006; 26 (17): 6557-6570


    Many genes have been identified that are specifically expressed in multiple types of stem cells in their undifferentiated state. It is generally assumed that at least some of these putative "stemness" genes are involved in maintaining properties that are common to all stem cells. We compared gene expression profiles between undifferentiated and differentiated embryonic stem cells (ESCs) using DNA microarrays. We identified several genes with much greater signal in undifferentiated ESCs than in their differentiated derivatives, among them the putative stemness gene encoding junctional adhesion molecule B (Jam-B gene). However, in spite of the specific expression in undifferentiated ESCs, Jam-B mutant ESCs had normal morphology and pluripotency. Furthermore, Jam-B homozygous mutant mice are fertile and have no overt developmental defects. Moreover, we found that neural and hematopoietic stem cells recovered from Jam-B mutant mice are not impaired in their ability to self-renew and differentiate. These results demonstrate that Jam-B is dispensable for normal mouse development and stem cell identity in embryonic, neural, and hematopoietic stem cells.

    View details for DOI 10.1128/MCB.00729-06

    View details for Web of Science ID 000239848800021

    View details for PubMedID 16914739

  • Cytokine signals modulated via lipid rafts mimic niche signals and induce hibernation in hematopoietic stem cells EMBO JOURNAL Yamazaki, S., Iwama, A., Takayanagi, S., Morita, Y., Eto, K., Ema, H., Nakauchi, H. 2006; 25 (15): 3515-3523


    Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche in a noncycling state and enter the cell cycle at long intervals. However, little is known about inter- and intracellular signaling mechanisms underlying this unique property of HSCs. Here, we show that lipid raft clustering is a key event in the regulation of HSC dormancy. Freshly isolated HSCs from the BM niche lack lipid raft clustering, exhibit repression of the AKT-FOXO signaling pathway, and express abundant p57(Kip2) cyclin-dependent kinase inhibitor. Lipid raft clustering induced by cytokines is essential for HSC re-entry into the cell cycle. Conversely, inhibition of lipid raft clustering caused sustained nuclear accumulation of FOXO transcription factors and induced HSC hibernation ex vivo. These data establish a critical role for lipid rafts in regulating the cell cycle, the survival, and the entry into apoptosis of HSCs and uncover a striking similarity in HSC hibernation and Caenorhabditis elegans dauer formation.

    View details for Web of Science ID 000239626000008

    View details for PubMedID 16858398

  • Discordant developmental waves of angioblasts and hemangioblasts in the early gastrulating mouse embryo DEVELOPMENT Furuta, C., Ema, H., Takayanagi, S., Ogaeri, T., Okamura, D., Matsui, Y., Nakauchi, H. 2006; 133 (14): 2771-2779


    Vasculogenesis and hematopoiesis are thought to arise in hemangioblasts, the common progenitors of cells in vessels and in blood. This scheme was challenged by kinetic analysis of vascular endothelial and hematopoietic progenitors in early gastrulating mouse embryos. The OP-9 co-culture system with a combination of cytokines permitted the detection of endothelial progenitors, as well as stroma-dependent hematopoietic progenitors. Endothelial progenitors were detected as early as embryonic day (E) 5.50, after which time their numbers increased. Stroma-dependent hematopoietic progenitors were detected at E6.75, the time point when hemangioblasts reportedly emerge. Colony-forming units in culture (CFU-c), most likely generated from stroma-dependent hematopoietic progenitors via contact with the microenvironment, were detected at E7.50, concomitant with the onset of primitive hematopoiesis in the yolk sac. The presence of nucleated erythrocytes and the expression of an embryonic-type globin in erythroid colonies derived from stroma-dependent hematopoietic progenitors and from CFU-c support the notion that these progenitors coordinately establish primitive hematopoiesis. Using Oct3/4 promoter-driven GFP transgenic mice, early endothelial progenitors, stroma-dependent hematopoietic progenitors, and CFU-c were all shown to express the Oct3/4 transcription factor. Among Oct3/4-positive cells, both endothelial and hematopoietic progenitors were present in the CD31-positive fraction, leaving a subset of endothelial progenitors in the CD31-negative fraction. These data imply that Oct3/4-positive mesoderm gives rise to CD31-negative angioblasts, CD31-positive angiboblasts and CD31-positive hemangioblasts. We propose a distinct developmental pathway in which the angioblast lineage directly diverges from mesoderm prior to and independent of hemangioblast development.

    View details for DOI 10.1242/dev.02440

    View details for Web of Science ID 000238475500016

    View details for PubMedID 16794034

  • Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties HEPATOLOGY Chiba, T., Kita, K., Zheng, Y., Yokosuka, O., Saisho, H., Iwama, A., Nakauchi, H., Taniguchi, H. 2006; 44 (1): 240-251


    Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti-apoptotic properties compared with those of non-SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 x 10(3) SP cells were sufficient for tumor formation, whereas an injection of 1 x 10(6) non-SP cells did not initiate tumors. Re-analysis of SP cell-derived tumors showed that SP cells generated both SP and non-SP cells and tumor-initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called "stemness genes" were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy.

    View details for DOI 10.1002/hep.21227

    View details for Web of Science ID 000238690900027

    View details for PubMedID 16799977

  • Genetic marking of hematopoietic stem and endothelial cells: identification of the Tmtsp gene encoding a novel cell surface protein with the thrombospondin-1 domain BLOOD Takayanagi, S., Hiroyarna, T., Yamazaki, S., Nakajima, T., Morita, Y., Usui, J., Eto, K., Motohashi, T., Shiomi, K., Keino-Masu, K., Masu, M., Oike, Y., Mori, S., Yoshida, N., Iwama, A., Nakauchi, H. 2006; 107 (11): 4317-4325


    Using an in silico database search, we identified a novel gene encoding a cell surface molecule with a thrombospondin domain, and designated the gene as transmembrane molecule with thrombospondin module (Tmtsp). Expression profiling of Tmtsp using specific monoclonal antibodies and Venus, a variant of yellow fluorescent protein knock-in mice in the Tmtsp locus, demonstrated its specific expression in hematopoietic and endothelial cells. In lymphohematopoietic cells, Tmtsp was predominantly expressed in hematopoietic stem and progenitor cells, and the level of expression gradually declined as the cells differentiated. Venus expression faithfully traced the expression of Tmtsp, and the level of Venus expression correlated well to the in vitro hematopoietic activity as well as the in vivo bone marrow repopulating capacity. Notably, Venus expression marked the development of definitive hematopoiesis in both the extraembryonic yolk sac and the intraembryonic aorta-gonad-mesonephros (AGM) region and, in combination with CD41, strikingly promoted the enrichment of developing progenitors in the CD41(+)Venus(high) fraction at embryonic day 10.5 (E10.5). In this context, Tmtsp is a novel marker gene for primitive hematopoietic cells and endothelial cells, and Tmtsp(Venus/)(+) mice would serve as a valuable mouse model for the analysis of both embryonic and adult hematopoiesis, as well as for vascular biology.

    View details for Web of Science ID 000237877300025

    View details for PubMedID 16455951

  • A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sakaguchi, M., Shingo, T., Shimazaki, T., Okano, H. J., Shiwa, M., Ishibashi, S., Oguro, H., Ninomiya, M., Kadoya, T., Horie, H., Shibuya, A., Mizusawa, H., Poirier, F., Nakauchi, H., Sawamoto, K., Okano, H. 2006; 103 (18): 7112-7117


    In the subventricular zone of the adult mammalian forebrain, neural stem cells (NSCs) reside and proliferate to generate young neurons. We screened factors that promoted the proliferation of NSCs in vitro by a recently developed proteomics technique, the ProteinChip system. In this screen, we identified a soluble carbohydrate-binding protein, Galectin-1, as a candidate. We show herein that Galectin-1 is expressed in a subset of slowly dividing subventricular zone astrocytes, which includes the NSCs. Based on results from intraventricular infusion experiments and phenotypic analyses of knockout mice, we demonstrate that Galectin-1 is an endogenous factor that promotes the proliferation of NSCs in the adult brain.

    View details for DOI 10.1073/pnas.0508793103

    View details for Web of Science ID 000237399900059

    View details for PubMedID 16636291

  • Identification and differentiation of hepatic stem cells during liver development FRONTIERS IN BIOSCIENCE-LANDMARK Kamiya, A., Gonzalez, F. J., Nakauchi, H. 2006; 11: 1302-1310


    Stem cells responsible for maintenance and repair of tissues are found in a number of organs. The liver's remarkable capacity to regenerate after hepatectomy or chemical-induced injury does not involve proliferation of stem cells. However, recent studies suggest that liver stem cells exist in both embryonic and adult livers. Using fluorescence-activated cell sorting and a culture system in which primitive hepatic progenitor cells form colonies, a novel class of cells with the marker profile c-Met(+)CD49f(+/low)c-Kit(-)CD45(-)TER119(-) was found in the developing liver. This class apparently represents the population of cells that form colonies containing distinct hepatocytes and cholangiocytes. When cells in this class are transplanted into the spleen or liver of mice subjected to liver injury, the cells migrate and differentiate into liver parenchymal cells and cholangiocytes that are morphologically and functionally indistinguishable from their native counterparts. During mid-gestation, hematopoietic cells migrate into the liver from a region bounded by aorta, gonad, and mesonephros and produce oncostatin M (OSM). In combination with glucocorticoid hormones, OSM induces maturation of liver stem and progenitor cells, including those of the c-Met(+)CD49f(+/low)c-Kit(-)CD45(-)TER119(-) class. The ability to manipulate the proliferation and differentiation of liver stem cells in vitro will greatly aid in analyzing mechanisms of liver development and offers promise in stem cell therapy of liver diseases.

    View details for Web of Science ID 000234180200012

    View details for PubMedID 16368517

  • A role for PKC theta in outside-in alpha(IIb)beta(3) signaling JOURNAL OF THROMBOSIS AND HAEMOSTASIS Soriani, A., Moran, B., de Virgilio, M., Kawakami, T., Altman, A., Lowell, C., Eto, K., Shattil, S. J. 2006; 4 (3): 648-655


    Fibrinogen binding to platelets triggers alpha(IIb)beta3-dependent outside-in signals that promote actin rearrangements and cell spreading. Studies with chemical inhibitors or activators have implicated protein kinase C (PKC) in alpha(IIb)beta3 function. However, the role of individual PKC isoforms is poorly understood. Biochemical and genetic approaches were used to determine whether PKCtheta is involved in alpha(IIb)beta3 signaling. PKCtheta was constitutively associated with alpha(IIb)beta3 in human and murine platelets. Fibrinogen binding to alpha(IIb)beta3 stimulated the association of PKCtheta with tyrosine kinases Btk and Syk, and tyrosine phosphorylation of PKCtheta, Btk and the actin regulator, Wiskott-Aldrich syndrome protein (WASP). Mouse platelets deficient in PKCtheta or Btk failed to spread on fibrinogen. Furthermore, PKCtheta was required for phosphorylation of WASP-interacting protein on Ser-488, an event that has been linked to WASP activation of the Arp2/3 complex and actin polymerization in lymphocytes. Neither PKCtheta nor Btk were required for agonist-induced inside-out signaling and fibrinogen binding to alpha(IIb)beta3. Thus, PKCtheta is a newly identified, essential member of a dynamic outside-in signaling complex that includes Btk and that couples alpha(IIb)beta3 to the actin cytoskeleton.

    View details for Web of Science ID 000235168900030

    View details for PubMedID 16460447

  • Potent vaccine therapy with dendritic cells genetically modified by the gene-silencing-resistant retroviral vector GCDNsap MOLECULAR THERAPY Nabekura, T., Otsu, M., Nagasawa, T., Nakauchi, H., Onodera, M. 2006; 13 (2): 301-309


    Dendritic cells (DCs) genetically modified to express tumor-associated antigens (TAAs) would be promising tools in cancer immunotherapy. However, the use of retroviral vectors for such modifications is still a challenge because of low transduction efficiency and gene silencing in DCs. We have established an efficient method to prepare such DCs by in vitro differentiation of hematopoietic progenitor cells transduced with chicken ovalbumin (OVA) cDNA via the gene-silencing-resistant retroviral vector GCDNsap packaged in vesicular stomatitis virus G protein. When c-KIT(+)/lineage(-) cells were transduced with OVA followed by expansion and differentiation, more than 90% of mature DCs expressed the transgene. Mice inoculated with those cells completely rejected the OVA-expressing tumor E.G7-OVA, and the anti-tumor effects were stronger than those observed in mice inoculated with the same number of OVA peptide-pulsed DCs. The mice harbored more cytotoxic T lymphocytes (CTLs) against E.G7-OVA and produced antibody against OVA, suggesting the generation of multiple CTLs recognizing different OVA epitopes and OVA-specific CD4(+) T cells. Successive inoculations of the transduced DCs in a therapeutic setting eradicated preexisting E.G7-OVA and prevented the progression of retransplanted tumors. Thus, this vaccine therapy may represent a potent immunotherapeutic approach for various malignant tumors that express suitable TAAs.

    View details for DOI 10.1016/j.ymthe.2005.09.021

    View details for Web of Science ID 000235122100008

    View details for PubMedID 16311073

  • Adult mouse hematopoietic stem cells: purification and single-cell assays NATURE PROTOCOLS Ema, H., Morita, Y., Yamazaki, S., Matsubara, A., Seita, J., Tadokoro, Y., Kondo, H., Takano, H., Nakauchi, H. 2006; 1 (6): 2979-2987


    Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34-KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.

    View details for DOI 10.1038/nprot.2006.447

    View details for Web of Science ID 000251155700057

    View details for PubMedID 17406558

  • Endomucin, a CD34-like sialomucin, marks hematopoietic stem cells throughout development JOURNAL OF EXPERIMENTAL MEDICINE Matsubara, A., Iwama, A., Yamazaki, S., Furuta, C., Hirasawa, R., Morita, Y., Osawa, M., Motohashi, T., Eto, K., Ema, H., Kitamura, T., VESTWEBER, D., Nakauchi, H. 2005; 202 (11): 1483-1492


    To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34(-)c-Kit(+)Sca-1(+)Lin(-) (CD34(-)KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)-HSCs were exclusively present in the Endomucin(+)CD34(-)KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45(-)CD41(+)Endomucin(+) fraction at day 10.5 of gestation (E10.5) and in the CD45(+)CD41(+)Endomucin(+) fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny.

    View details for DOI 10.1084/jem.20051325

    View details for Web of Science ID 000233753900005

    View details for PubMedID 16314436

  • Identification of immature podocyte specific antigen using retrovirus-mediated gene transfer and cell sorting. Clinical and experimental nephrology Usui, J., Osawa, M., Yamazaki, S., Morita, Y., Koyama, A., Nakauchi, H. 2005; 9 (4): 292-296


    For identifying an antigen recognizable by a monoclonal antibody (mAb), the cDNA cloning method using expression cDNA library has become increasingly popular. For analysis of the mAb clone 2E3, which recognizes the cell membrane surface of immature podocytes, we developed an expression cloning strategy that identified the targeted antigen using retrovirus-mediated gene transfer and enrichment by cell sorting.In this experiment, the NIH3T3 cell line was infected by a retrovirally amplified cDNA library derived from the same fetal murine kidneys that provided the immunized antigen. Infected NIH3T3 staining for mAb clone 2E3 was concentrated by cell sorting, followed by identification of the integrated gene.The infected cells reacted with mAb were highly enriched by two rounds of cell sorting. As a result of sequencing the inserted gene from the enriched cells, we isolated the low-affinity nerve growth factor receptor (NGFR) gene. Furthermore, the NIH3T3 cell line that was enforced by recovery of NGFR cDNA reacted with mAb clone 2E3.Monoclonal antibodies recognizing cell membrane surface molecules are essential tools for immunohistochemistry and flow cytometry analysis, and the application of a combination of retrovirus-mediated gene transfer and cell sorting is beneficial for identifying the targeted antigen.

    View details for PubMedID 16362155

  • The transcriptional landscape of the mammalian genome SCIENCE Carninci, P., Kasukawa, T., Katayama, S., Gough, J., Frith, M. C., Maeda, N., Oyama, R., Ravasi, T., Lenhard, B., Wells, C., Kodzius, R., Shimokawa, K., Bajic, V. B., Brenner, S. E., Batalov, S., Forrest, A. R., Zavolan, M., Davis, M. J., Wilming, L. G., Aidinis, V., Allen, J. E., Ambesi-Impiombato, X., Apweiler, R., Aturaliya, R. N., Bailey, T. L., Bansal, M., Baxter, L., Beisel, K. W., Bersano, T., Bono, H., Chalk, A. M., Chiu, K. P., Choudhary, V., Christoffels, A., Clutterbuck, D. R., Crowe, M. L., Dalla, E., Dalrymple, B. P., de Bono, B., Della Gatta, G., di Bernardo, D., Down, T., Engstrom, P., Fagiolini, M., Faulkner, G., Fletcher, C. F., Fukushima, T., Furuno, M., Futaki, S., Gariboldi, M., Georgii-Hemming, P., Gingeras, T. R., Gojobori, T., Green, R. E., Gustincich, S., Harbers, M., Hayashi, Y., Hensch, T. K., Hirokawa, N., Hill, D., Huminiecki, L., Iacono, M., Ikeo, K., Iwama, A., Ishikawa, T., Jakt, M., Kanapin, A., Katoh, M., Kawasawa, Y., Kelso, J., Kitamura, H., Kitano, H., Kollias, G., Krishnan, S. P., Kruger, A., Kummerfeld, S. K., Kurochkin, I. V., Lareau, L. F., Lazarevic, D., Lipovich, L., Liu, J., Liuni, S., McWilliam, S., Babu, M. M., Madera, M., Marchionni, L., Matsuda, H., Matsuzawa, S., Miki, H., Mignone, F., Miyake, S., Morris, K., Mottagui-Tabar, S., Mulder, N., Nakano, N., Nakauchi, H., Ng, P., Nilsson, R., Nishiguchi, S., Nishikawa, S., Nori, F., Ohara, O., Okazaki, Y., Orlando, V., Pang, K. C., Pavan, W. J., Pavesi, G., Pesole, G., Petrovsky, N., Piazza, S., Reed, J., Reid, J. F., Ring, B. Z., RINGWALD, M., Rost, B., Ruan, Y., Salzberg, S. L., Sandelin, A., Schneider, C., Schonbach, C., Sekiguchi, K., Semple, C. A., Seno, S., Sessa, L., Sheng, Y., Shibata, Y., Shimada, H., Shimada, K., Silva, D., Sinclair, B., Sperling, S., Stupka, E., Sugiura, K., Sultana, R., Takenaka, Y., Taki, K., Tammoja, K., Tan, S. L., Tang, S., Taylor, M. S., Tegner, J., Teichmann, S. A., Ueda, H. R., van Nimwegen, E., Verardo, R., Wei, C. L., Yagi, K., Yamanishi, H., Zabarovsky, E., Zhu, S., Zimmer, A., Hide, W., Bult, C., Grimmond, S. M., Teasdale, R. D., Liu, E. T., Brusic, V., Quackenbush, J., Wahlestedt, C., Mattick, J. S., Hume, D. A., Kai, C., Sasaki, D., Tomaru, Y., Fukuda, S., Kanamori-Katayama, M., Suzuki, M., Aoki, J., Arakawa, T., Iida, J., Imamura, K., Itoh, M., Kato, T., Kawaji, H., Kawagashira, N., Kawashima, T., Kojima, M., Kondo, S., Konno, H., Nakano, K., Ninomiya, N., Nishio, T., Okada, M., Plessy, C., Shibata, K., Shiraki, T., Suzuki, S., Tagami, M., Waki, K., Watahiki, A., Okamura-Oho, Y., Suzuki, H., Kawai, J., Hayashizaki, Y. 2005; 309 (5740): 1559-1563


    This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

    View details for DOI 10.1126/science.1112014

    View details for Web of Science ID 000231715000049

    View details for PubMedID 16141072

  • Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis JOURNAL OF EXPERIMENTAL MEDICINE Kato, Y., Iwama, A., Tadokoro, Y., Shimoda, K., Minoguchi, M., Akira, S., Tanaka, M., Miyajima, A., Kitamura, T., Nakauchi, H. 2005; 202 (1): 169-179


    Although the concept of a leukemic stem cell system has recently been well accepted, its nature and the underlying molecular mechanisms remain obscure. Constitutive activation of signal transducers and activators of transcription 3 (STAT3) and STAT5 is frequently detected in various hematopoietic tumors. To evaluate their role in normal and leukemic stem cells, we took advantage of constitutively active STAT mutants to activate STAT signaling selectively in hematopoietic stem cells (HSCs). Activation of STAT5 in CD34- c-Kit+ Sca-1+ lineage marker- (CD34- KSL) HSCs led to a drastic expansion of multipotential progenitors and promoted HSC self-renewal ex vivo. In sharp contrast, STAT3 was demonstrated to be dispensable for the HSC maintenance in vivo, and its activation facilitated lineage commitment of HSCs in vitro. In a mouse model of myeloproliferative disease (MPD), sustained STAT5 activation in CD34- KSL HSCs but not in CD34+ KSL multipotential progenitors induced fatal MPD, indicating that the capacity of STAT5 to promote self-renewal of hematopoietic stem cells is crucial to MPD development. Our findings collectively establish a specific role for STAT5 in self-renewal of normal as well as leukemic stem cells.

    View details for DOI 10.1084/jem.20042541

    View details for Web of Science ID 000230215100016

    View details for PubMedID 15998795

  • In vivo haematopoietic potential of human neural stem cells BRITISH JOURNAL OF HAEMATOLOGY Almeida-Porada, G., Crapnell, K., Porada, C., Benoit, B., Nakauchi, H., Quesenberry, P., Zanjani, E. D. 2005; 130 (2): 276-283


    The fetal sheep model was used to compare the in vivo haematopoietic potential of human neural stem cells (NSC) versus bone marrow (BM)-derived haematopoietic stem cells (HSC). To this end, sheep were transplanted with either 8 x 10(5) NSC (n = 11) or HSC, CD34(+)Lin(-) (n = 5), and subsequently analysed for haematopoietic chimaerism. While HSC-transplanted sheep displayed robust donor-derived haematopoiesis starting at less than 2 months post-transplant, NSC recipients exhibited haematopoietic engraftment at much later time points. Nevertheless, chimaerism persisted in both groups throughout the course of this study. Transplantation of secondary recipients with human CD45(+)/HLA-DR(+) cells from the BM of NSC primary recipients at 14 and 16 months post-transplant demonstrated that long-term engrafting HSC were present in these animals. At 6 months post-transplant, both NSC- and HSC-transplanted sheep were mobilised with granulocyte colony-stimulating factor. In contrast to HSC-transplanted animals, levels of human blood cells in peripheral blood of NSC-transplanted sheep remained low throughout mobilisation. Our results show that, although human NSC were able to give rise to multilineage haematopoiesis in our model, the levels, timing of blood cell production and the ability to respond to cytokine mobilisation were different, suggesting that human NSCs latent haematopoietic potential is inherently different from that of true HSC.

    View details for DOI 10.1111/j.1365-2141.2005.05588.x

    View details for Web of Science ID 000230333000014

    View details for PubMedID 16029457

  • Parvovirus nonstructural proteins induce an epigenetic modification through histone acetylation in host genes and revert tumor malignancy to benignancy JOURNAL OF VIROLOGY Iseki, H., Shimizukawa, R., Sugiyama, F., Kunita, S., Iwama, A., Onodera, M., Nakauchi, H., Yagami, K. 2005; 79 (14): 8886-8893


    Several malignant tumor cells become apoptotic and revert to the benign phenotype upon parvovirus infection. Recently, we demonstrated that the rat parvovirus RPV/UT also induces apoptosis in the rat thymic lymphoma cell line C58(NT)D. However, a minority of cells that escaped apoptosis showed properties different from the parental cells, such as resistance to apoptosis, enhanced cell adherence, and suppressed tumorigenicity. The present study was performed to determine the molecular mechanism of parvovirus-induced phenotypic modification, including oncosuppression. We demonstrated that the nonstructural (NS) proteins of RPV/UT induced apoptosis in C58(NT)D cells and suppressed tumor growth in vivo. Interestingly, NS proteins induced the expression of ciliary neurotrophic factor receptor alpha, which is up-regulated in revertant cell clones, and enhanced histone acetylation of its gene. These results indicate that parvoviral NS regulate host gene expression through histone acetylation, suggesting a possible mechanism of oncosuppression.

    View details for DOI 10.1128/JVI.79.14.8886-8893.2005

    View details for Web of Science ID 000230216300022

    View details for PubMedID 15994782

  • Isolation of murine hematopoietic stem cells and progenitor cells. Current protocols in immunology / edited by John E. Coligan ... [et al.] Ema, H., Morita, Y., Nakauchi, H., Matsuzaki, Y. 2005; Chapter 22: Unit 22B 1-?


    Protocols for the purification of CD34-KSL cells, CD34+KSL cells, SP cells, and Tip-SP CD34-KSL cells are described. Mouse hematopoietic stem cells are highly enriched in the CD34-KSL and SP fractions. These two populations overlap one another. A nearly homogenous population of HSCs can be obtained by isolating Tip-SP CD34-KSL cells. One of the earliest progenitor cell populations is CD34+KSL cells.

    View details for DOI 10.1002/0471142735.im22b01s67

    View details for PubMedID 18432947

  • Musculin/MyoR is expressed in kidney side population cells and can regulate their function JOURNAL OF CELL BIOLOGY Hishikawa, K., Marumo, T., Miura, S., Nakanishi, A., Matsuzaki, Y., Shibata, K., Ichiyanagi, T., Kohike, H., Komori, T., TAKAHASHI, I., Takase, O., Imai, N., Yoshikawa, M., Inowa, T., Hayashi, M., NAKAKI, T., Nakauchi, H., Okano, H., Fujita, T. 2005; 169 (6): 921-928


    Musculin/MyoR is a new member of basic helix-loop-helix transcription factors, and its expression is limited to skeletal muscle precursors. Here, we report that musculin/MyoR is expressed in adult kidney side population (SP) cells and can regulate their function. SP phenotype can be used to purify stem cell-rich fractions. Microarray analysis clarified that musculin/MyoR was exclusively expressed in kidney SP cells, and the cells resided in the renal interstitial space. Musculin/MyoR-positive cells were decreased in acute renal failure, but infusion of kidney SP cells increased musculin/MyoR-positive cells and improved renal function. Kidney SP cells in reversible acute renal failure expressed a high level of renoprotective factors and leukemia inhibitory factor (LIF), but not in irreversible chronic renal failure. In cultured kidney SP cells, LIF stimulated gene expression of renoprotective factors, and down-regulation of musculin/MyoR augmented LIF-induced gene expression. Our results suggest that musculin/MyoR may play important roles not only in developmental processes but also in regenerative processes in adult tissue.

    View details for DOI 10.1083/jcb.200412167

    View details for Web of Science ID 000229930300009

    View details for PubMedID 15967813

  • Quantification of self-renewal capacity in single hematopoietic stem cells from normal and lnk-deficient mice DEVELOPMENTAL CELL Ema, H., Sudo, K., Seita, J., Matsubara, A., Morita, Y., Osawa, M., Takatsu, K., Takaki, S., Nakauchi, H. 2005; 8 (6): 907-914


    Despite being a hallmark of hematopoietic stem cells (HSCs), HSC self-renewal has never been quantitatively assessed. Establishment of a clonal and quantitative assay for HSC function permitted demonstration that adult mouse HSCs are significantly heterogeneous in degree of multilineage repopulation and that higher repopulating potential reflects higher self-renewal activity. An HSC with high repopulating potential could regenerate approximately 1000 HSCs, whereas the repopulating activity of regenerated HSCs on average was significantly reduced, indicating extensive but limited self-renewal capacity in HSCs. Comparisons of wild-type mice with mutant mice deficient in the signal adaptor molecule Lnk showed that not only HSC numbers but also the self-renewal capacity of some HSCs are markedly increased when Lnk function is lost. Lnk appears to control HSC numbers by negatively regulating HSC self-renewal signaling.

    View details for Web of Science ID 000230006200014

    View details for PubMedID 15935779

  • Epigenetic regulation of hematopoietic stem cell self-renewal by Polycomb group genes INTERNATIONAL JOURNAL OF HEMATOLOGY Iwama, A., Oguro, H., Negishi, M., Kato, Y., Nakauchi, H. 2005; 81 (4): 294-300


    Polycomb group (PcG) genes are involved in the maintenance of cellular memory through epigenetic chromatin modifications. Recent studies have implicated a role for PcG genes in the self-renewal of hematopoietic stem cells (HSCs), a process in which cellular memory is maintained through cell division. Among the PcG genes, Bmi-1 plays a central role in the inheritance of stemness, and its forced expression promotes HSC self-renewal. These findings highlight the importance of epigenetic regulation in HSC self-renewal and identify PcG genes as potential targets for therapeutic HSC manipulation.

    View details for DOI 10.1532/IJH97.05011

    View details for Web of Science ID 000230467400004

    View details for PubMedID 15914357

  • Leukemia inhibitory factor induces multi-lineage differentiation of adult stem-like cells in kidney via kidney-specific cadherin 16 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hishikawa, K., Marumo, T., Miura, S., Nakanishi, A., Matsuzaki, Y., Shibata, K., Kohike, H., Komori, T., Hayashi, M., NAKAKI, T., Nakauchi, H., Okano, H., Fujita, T. 2005; 328 (1): 288-291


    Side population (SP) is reported to be a stem cell-rich population. In the presence of leukemia inhibitory factor (LIF), cultured kidney SP cells differentiated into multi-lineage in collagen gel but not in synthesized polymer that has no cell adhesion factor. In cultured kidney SP cells, gene expression of kidney-specific cadherin 16 was specifically upregulated in collagen gel but not in synthesized polymer. Moreover, decreasing cadherin 16 expression using siRNA abolished LIF-induced multi-lineage differentiation of kidney SP in collagen gel. These results indicated that LIF induced multi-lineage differentiation of adult stem-like cells in kidney via cadherin 16.

    View details for DOI 10.1016/j.bbrc.2004.12.167

    View details for Web of Science ID 000226799600042

    View details for PubMedID 15670782

  • Polycomb gene product Bmi-1 regulates stem cell self-renewal Workshop on Promises and Challenges of Regenerative Medicine Nakauchi, H., Oguro, H., Negishi, M., Iwama, A. SPRINGER-VERLAG BERLIN. 2005: 85–100


    The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSCs). However, the role of each component of PcG complex in HSCs and the impact of forced expression of PcG genes on stem cell self-renewal remain to be elucidated. To address these issues, we performed both loss-of-function and gain-of-function analysis on various PcG proteins. Expression analysis revealed that not only Bmi-1 but also other PcG genes are predominantly expressed in HSCs. Loss-of-function analyses, however, demonstrated that absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal, indicating a central role for Bmi-1, but not the other components, in the maintenance of HSC self-renewal. Over-expression analysis of PcG genes also confirmed an important role of Bmi-1 in HSC self-renewal. Our findings indicate that the expression level of Bmi-1 is the critical determinant for the self-renewal capacity of HSCs. These findings uncover novel aspects of stem cell regulation exerted through epigenetic modifications by the PcG proteins.

    View details for Web of Science ID 000231034100006

    View details for PubMedID 16080288

  • An in vivo assay for retrovirally transduced human peripheral T lymphocytes using nonobese diabetic/severe combined immunodeficiency mice EXPERIMENTAL HEMATOLOGY Kaneko, S., Nagasawa, T., Nakauchi, H., Onodera, M. 2005; 33 (1): 35-41


    Availability of a mouse model to analyze human peripheral lymphocytes genetically modified with retroviral vectors would be useful in T-cell-directed gene transfer studies. To address this issue, we assessed the ability of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to maintain such cells in their peripheral blood.Human peripheral lymphocytes stimulated with recombinant human interleukin-2 (rhIL-2) and anti-CD3 and CD28 antibodies were transduced with the enhanced green fluorescent protein (EGFP) gene using the retroviral vector GCsap(MSCV) and then transplanted into NOD/SCID mice at 1 x 10(8) cells per mouse.Transplanted human peripheral lymphocytes survived and expressed EGFP in the mice over the 6- to 8-week posttransplant period without any signs of graft-vs-host disease. Of importance was that these cells remained at the G(0)/G(1) stage and again proliferated in response to cytokines when cultured in vitro. Interestingly, the mice in which the transduced T lymphocytes remained at the resting stage clearly elucidated the superiority of the murine stem cell virus (MSCV) LTR to maintain the transgene expression by nonproliferating T lymphocytes over the Moloney murine leukemia virus (MoMLV)- and myeloproliferative sarcoma virus (MPSV)-derived LTRs, which was obscure in in vitro culture where the transduced lymphocytes was being stimulated with rhIL-2.The mouse model and GCsap(MSCV) vector described herein comprise a simple and reliable in vivo assay system for studies of gene and cell therapies employing human peripheral lymphocytes.

    View details for DOI 10.1016/j.exphem.2004.10.006

    View details for Web of Science ID 000226815400005

    View details for PubMedID 15661396

  • Role of Dok-1 and Dok-2 in myeloid homeostasis and suppression of leukemia JOURNAL OF EXPERIMENTAL MEDICINE Yasuda, T., Shirakata, M., Iwama, A., Ishii, A., Ebihara, Y., Osawa, M., Honda, K., Shinohara, H., Sudo, K., Tsuji, K., Nakauchi, H., Iwakura, Y., Hirai, H., Oda, H., Yamamoto, T., Yamanashi, Y. 2004; 200 (12): 1681-1687


    Dok-1 and Dok-2 are closely related rasGAP-associated docking proteins expressed preferentially in hematopoietic cells. Although they are phosphorylated upon activation of many protein tyrosine kinases (PTKs), including those coupled with cytokine receptors and oncogenic PTKs like Bcr-Abl, their physiological roles are largely unidentified. Here, we generated mice lacking Dok-1 and/or Dok-2, which included the double-deficient mice succumbed to myeloproliferative disease resembling human chronic myelogenous leukemia (CML) and chronic myelomonocytic leukemia. The double-deficient mice displayed medullary and extramedullary hyperplasia of granulocyte/macrophage progenitors with leukemic potential, and their myeloid cells showed hyperproliferation and hypo-apoptosis upon treatment and deprivation of cytokines, respectively. Consistently, the mutant myeloid cells showed enhanced Erk and Akt activation upon cytokine stimulation. Moreover, loss of Dok-1 and/or Dok-2 induced blastic transformation of chronic phase CML-like disease in mice carrying the bcr-abl gene, a cause of CML. These findings demonstrate that Dok-1 and Dok-2 are key negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia.

    View details for DOI 10.1084/jem.20041247

    View details for Web of Science ID 000226030700017

    View details for PubMedID 15611294

  • Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1 IMMUNITY Iwama, A., Oguro, H., Negishi, M., Kato, Y., Morita, Y., Tsukui, H., Ema, H., Kamijo, T., Katoh-Fukui, Y., Koseki, H., van Lohuizen, M., Nakauchi, H. 2004; 21 (6): 843-851


    The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSC) from loss-of-function analysis. Here, we demonstrate that increased expression of Bmi-1 promotes HSC self-renewal. Forced expression of Bmi-1 enhanced symmetrical cell division of HSCs and mediated a higher probability of inheritance of stemness through cell division. Correspondingly, forced expression of Bmi-1, but not the other PcG genes, led to a striking ex vivo expansion of multipotential progenitors and marked augmentation of HSC repopulating capacity in vivo. Loss-of-function analyses revealed that among PcG genes, absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal. Our findings define Bmi-1 as a central player in HSC self-renewal and demonstrate that Bmi-1 is a target for therapeutic manipulation of HSCs.

    View details for Web of Science ID 000225901600011

    View details for PubMedID 15589172

  • Isolation and clonal characterization of hematopoietic and liver stem cells. Cornea Nakauchi, H. 2004; 23 (8): S2-7


    Prospective isolation of stem cells is essential to understanding the mechanisms that control their proliferation and differentiation.Using 9 monoclonal antibodies and fluorescence-activated cell sorting (FACS), we have succeeded in prospectively identifying hematopoietic stem cells (HSCs) in adult mouse bone marrow. Mouse HSCs were exclusively enriched in CD34 negative, c-Kit Sca-1 Lineage Marker (CD34 KSL) cells representing 0.004% of bone marrow (BM) mononuclear cells. When single CD34-KSL cells were transplanted individually into a lethally irradiated mouse, 25% of the recipient mice survived and showed long-term reconstitution of the BM, providing evidence for multipotency and a self-renewal capacity of HSCs. Using a similar approach, we also prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewal capability in the c-Met CD49f c-Kit CD45 Ter119 fraction of cells isolated from day 13.5 fetal mouse liver. On cell transplantation, these cells differentiated into hepatocytes and cholangiocytes. As an alternative to the antibody based stem cell isolation, Hoechst33342 staining is useful. To understand the mechanism responsible for SP phenotype, we performed an expression cloning and identified bcrp-1/ABCG2 gene, a member of ATP binding-cassette (ABC) transporter family. Bcrp-1 is almost exclusively expressed in CD34 KSL cells among blood cells; however their expression in other tissue specific stem cells remains to be studied.With the use of FACS and monoclonal antibodies, hematopoietic and liver stem cells were prospectively isolated and characterized. HSCs could also be purified by Hoechst 33342 staining. By expression cloning, we identify bcrp-1/ABCG2 transporter as a molecule responsible for SP phenotype. Elucidation of the physiological role of bcrp-1/ABCG2 in HSCs may provide us with clues to understand the molecular mechanisms of stem cell self-renewal and differentiation.

    View details for PubMedID 15448473

  • Mac-1(low) early myeloid cells in the bone marrow-derived SP fraction migrate into injured skeletal muscle and participate in muscle regeneration BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Ojima, K., Uezumi, A., Miyoshi, H., Masuda, S., Morita, Y., Fukase, A., Hattori, A., Nakauchi, H., Miyagoe-Suzuki, Y., Takeda, S. 2004; 321 (4): 1050-1061


    Recent studies have shown that bone marrow (BM) cells, including the BM side population (BM-SP) cells that enrich hematopoietic stem cells (HSCs), are incorporated into skeletal muscle during regeneration, but it is not clear how and what kinds of BM cells contribute to muscle fiber regeneration. We found that a large number of SP cells migrated from BM to muscles following injury in BM-transplanted mice. These BM-derived SP cells in regenerating muscles expressed different surface markers from those of HSCs and could not reconstitute the mouse blood system. BM-derived SP/Mac-1(low) cells increased in number in regenerating muscles following injury. Importantly, our co-culture studies with activated satellite cells revealed that this fraction carried significant potential for myogenic differentiation. By contrast, mature inflammatory (Mac-1(high)) cells showed negligible myogenic activities. Further, these BM-derived SP/Mac-1(low) cells gave rise to mononucleate myocytes, indicating that their myogenesis was not caused by stochastic fusion with host myogenic cells, although they required cell-to-cell contact with myogenic cells for muscle differentiation. Taken together, our data suggest that neither HSCs nor mature inflammatory cells, but Mac-1(low) early myeloid cells in the BM-derived SP fraction, play an important role in regenerating skeletal muscles.

    View details for DOI 10.1016/j.bbrc.2004.07.069

    View details for Web of Science ID 000223381100045

    View details for PubMedID 15358135

  • Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting DIABETES Atsushi, S., Nakauchi, H., Taniguchi, H. 2004; 53 (8): 2143-2152


    During pancreatic development, neogenesis, and regeneration, stem cells might act as a central player to generate endocrine, acinar, and duct cells. Although these cells are well known as pancreatic stem cells (PSCs), indisputable proof of their existence has not been reported. Identification of phenotypic markers for PSCs leads to their prospective isolation and precise characterization to clear whether stem cells exist in the pancreas. By combining flow cytometry and clonal analysis, we show here that a possible pancreatic stem or progenitor cell candidate that resides in the developing and adult mouse pancreas expresses the receptor for the hepatocyte growth factor (HGF) c-Met, but does not express hematopoietic and vascular endothelial antigens such as CD45, TER119, c-Kit, and Flk-1. These cells formed clonal colonies in vitro and differentiated into multiple pancreatic lineage cells from single cells. Some of them could largely expand with self-renewing cell divisions in culture, and, following cell transplantation, they differentiated into pancreatic endocrine and acinar cells in vivo. Furthermore, they produced cells expressing multiple markers of nonpancreatic organs including liver, stomach, and intestine in vitro. Our data strongly suggest that c-Met/HGF signaling plays an important role in stem/progenitor cell function in both developing and adult pancreas. By using this antigen, PSCs could be isolated prospectively, enabling a detailed investigation of stem cell markers and application toward regenerative therapies for diabetes.

    View details for Web of Science ID 000223027000031

    View details for PubMedID 15277399

  • The Sox-2 regulatory regions display their activities in two distinct types of multipotent stem cells MOLECULAR AND CELLULAR BIOLOGY Miyagi, S., Saito, T., Mizutani, K. I., Masuyama, N., Gotoh, Y., Iwanta, A., Nakauchi, H., Masui, S., Niwa, H., Nishimoto, M., Muramatsu, M., Okuda, A. 2004; 24 (10): 4207-4220


    The Sox-2 gene is expressed in embryonic stem (ES) cells and neural stem cells. Two transcription enhancer regions, Sox-2 regulatory region 1 (SRR1) and SRR2, were described previously based on their activities in ES cells. Here, we demonstrate that these regulatory regions also exert their activities in neural stem cells. Moreover, our data reveal that, as in ES cells, both SRR1 and SRR2 show their activities rather specifically in multipotent neural stem or progenitor cells but cease to function in differentiated cells, such as postmitotic neurons. Systematic deletion and mutation analyses showed that the same or at least overlapping DNA elements of SRR2 are involved in its activity in both ES and neural stem or progenitor cells. Thus, SRR2 is the first example of an enhancer in which a single regulatory core sequence is involved in multipotent-state-specific expression in two different stem cells, i.e., ES and neural stem cells.

    View details for Web of Science ID 000221440900012

    View details for PubMedID 15121842

  • Liver repopulation by c-met-positive stem/progenitor cells isolated from the developing rat liver HEPATO-GASTROENTEROLOGY Suzuki, A., Zheng, Y. W., Fukao, K., Nakauchi, H., Taniguchi, H. 2004; 51 (56): 423-426


    Self-renewing stem cells responsible for tissue or organ development and regeneration have been recently described. To isolate such cells using flow cytometry, it should be required to find molecules expressing on their cell surfaces. We have previously reported that, on cells fulfilling the criteria for hepatic stem cells, the hepatocyte growth factor receptor c-Met is expressed specifically in the developing mouse liver. In this study, to determine whether c-Met is an essential marker for hepatic stem cells in other animal strains, we examined the potential for in vivo liver-repopulation in sorted fetal rat-derived c-Met+ cells using the retrorsine model.Using flow cytometry and monoclonal antibodies for c-Met and leukocyte common antigen CD45, fetal rat liver cells were fractionated according to the expression of these molecules. Then, cells in each cell subpopulation were sorted and transplanted into the retrorsine-treated adult rats with two-third hepatectomy. At 9 months post transplant, frequency of liver-repopulation was examined by qualitative and quantitative analyses.When we transplanted c-Met+ CD45- sorted cells, many donor-derived cells formed colonies that included mature hepatocytes expressing albumin and containing abundant glycogen in their cytoplasm. In contrast, c-Met- cells and CD45+ cells could not repopulate damaged recipient livers.High enrichment of liver-repopulating cells was conducted by sorting of c-Met+ cells from the developing rat liver. This result suggests that c-Met/HGF interaction plays a crucial role for stem cell growth, differentiation, and self-renewal in rat liver organogenesis. Since the c-Met is also expressed in the fetal mouse-derived hepatic stem cells, this molecule could be expected to be an essential marker for such cell population in the various animal strains, including human.

    View details for Web of Science ID 000220642500023

    View details for PubMedID 15086173

  • Asymmetric division and lineage commitment at the level of hematopoietic stem cells: Inference from differentiation in daughter cell and granddaughter cell pairs JOURNAL OF EXPERIMENTAL MEDICINE Takano, H., Ema, H., Sudo, K., Nakauchi, H. 2004; 199 (3): 295-302


    How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34-/low, c-Kit+, Sca-1+, and lineage marker- (CD34-KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34-KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors.

    View details for DOI 10.1084/jem.20030929

    View details for Web of Science ID 000188780000001

    View details for PubMedID 14744992

  • Conversion of biliary system to pancreatic tissue in Hes1-deficient mice NATURE GENETICS Sumazaki, R., Shiojiri, N., Isoyama, S., Masu, M., Keino-Masu, K., Osawa, M., Nakauchi, H., Kageyama, R., Matsui, A. 2004; 36 (1): 83-87


    The biliary system, pancreas and liver all develop from the nearby foregut at almost the same time in mammals. The molecular mechanisms that determine the identity of each organ in this complex area are unknown. Hes1 encodes the basic helix-loop-helix protein Hes1 (ref. 1), which represses positive basic helix-loop-helix genes such as Neurog3 (ref. 3). Expression of Hes1 is controlled by the evolutionarily conserved Notch pathway. Hes1 operates as a general negative regulator of endodermal endocrine differentiation, and defects in Notch signaling lead to accelerated pancreatic endocrine differentiation. Mutations in JAG1, encoding a Notch ligand, cause the Alagille syndrome in humans, characterized by poor development of the biliary system, suggesting that the Notch pathway is also involved in normal biliary development. Here we show that Hes1 is expressed in the extrahepatic biliary epithelium throughout development and that Hes1-deficient mice have gallbladder agenesis and severe hypoplasia of extrahepatic bile ducts. Biliary epithelium in Hes1-/- mice ectopically expresses the proendocrine gene Neurog3 (refs. 12,13), differentiates into endocrine and exocrine cells and forms acini and islet-like structures in the mutant bile ducts. Thus, biliary epithelium has the potential for pancreatic differentiation and Hes1 determines biliary organogenesis by preventing the pancreatic differentiation program, probably by directly repressing transcription of Neurog3.

    View details for DOI 10.1038/ng1273

    View details for Web of Science ID 000187666800018

    View details for PubMedID 14702043

  • "Homing to niche," a new criterion for hematopoietic stem cells? IMMUNITY Ema, H., Nakauchi, H. 2004; 20 (1): 1-2


    By combining cell surface staining with fluorochrome-conjugated monoclonal antibodies and Hoechst 33342 dye supravital staining, Matsuzaki et al. have succeeded in enriching hematopoietic stem cells (HSCs) essentially to homogeneity. When single-cell transplantation analysis was performed using the isolated cells, over 95% of the recipient mice showed long-term multilineage engraftment. The work demonstrates unexpectedly high marrow seeding efficiency of HSCs and proposes high marrow homing capacity as a new criterion for HSCs.

    View details for Web of Science ID 000221442200001

    View details for PubMedID 14738758

  • CD226 (DNAM-1) is involved in lymphocyte function-associated antigen 1 costimulatory signal for naive T cell differentiation and proliferation JOURNAL OF EXPERIMENTAL MEDICINE Shibuya, K., Shirakawa, J., Kameyama, T., Honda, S., Tahara-Hanaoka, S., Miyamoto, A., Onodera, M., Sumida, T., Nakauchi, H., Miyoshi, H., Shibuya, A. 2003; 198 (12): 1829-1839


    Upon antigen recognition by the T cell receptor, lymphocyte function-associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12-independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

    View details for DOI 10.1084/jem.20030958

    View details for Web of Science ID 000187449400008

    View details for PubMedID 14676297

  • Self-renewal and lineage restriction of hematopoietic stem cells CURRENT OPINION IN GENETICS & DEVELOPMENT Ema, H., Nakauchi, H. 2003; 13 (5): 508-512


    Over the past decade, the purification and characterization of hematopoietic stem cells have ascertained their presence at the clonal level although they had hitherto existed conceptually. Now we have begun to understand their functions in molecular terms. Several important works indicative of such a new era in stem cell biology have been published recently. In particular, Bmi1, which belongs to the Polycomb group of genes, has been implicated as one of the basic molecules to maintain the proliferation capacity in hematopoietic stem cells. We need to seek other similarly important molecules for their functions. Perhaps studying interactions among genes is one of the most exciting subjects in stem cell research.

    View details for DOI 10.1016/j.gde.2003.08.011

    View details for Web of Science ID 000186015100011

    View details for PubMedID 14550417

  • CD226 mediates platelet and megakaryocytic cell adhesion to vascular endothelial cells JOURNAL OF BIOLOGICAL CHEMISTRY Kojima, H., Kanada, H., Shimizu, S., Kasama, E., Shibuya, K., Nakauchi, H., Nagasawa, T., Shibuya, A. 2003; 278 (38): 36748-36753


    Platelet adhesion to vascular endothelial cells is a pathophysiologically relevant cell-to-cell interaction. However, the mechanisms underlying this cellular interaction are incompletely understood. In search of the ligand for CD226 adhesion molecule expressed on platelets, we found that human umbilical vein endothelial cells (HUVEC) express significant amount of putative CD226 ligand. We demonstrated that thrombin-activated, but not resting, platelets bind to intact HUVEC. Anti-CD226 monoclonal antibody specifically inhibited the binding, indicating that CD226 mediates the intercellular binding between thrombin-activated platelets and HUVEC. We also demonstrated that platelet activation with thrombin induces tyrosine phosphorylation of CD226 as well as CD226-mediated platelet adhesion. Moreover, experiments using mutant transfectants suggested that the tyrosine at residue 322 of CD226 plays an important role for its adhesive function. CD226 was also expressed on primary megakaryocytes and megakaryocytic cell lines. Anti-CD226 monoclonal antibody inhibited binding of megakaryocytic cell lines to HUVEC. Taken together, these results reveal a novel mechanism for adhesion of platelets and megakaryocytic cells to vascular endothelial cells.

    View details for DOI 10.1074/jbc.M300702200

    View details for Web of Science ID 000185318300107

    View details for PubMedID 12847109

  • Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation JOURNAL OF EXPERIMENTAL MEDICINE Yotsumoto, K., Okoshi, Y., Shibuya, K., Yamazaki, S., Tahara-Hanaoka, S., Honda, S., Osawa, M., Kuroiwa, A., Matsuda, Y., Tenen, D. G., Iwama, A., Nakauchi, H., Shibuya, A. 2003; 198 (2): 223-233


    Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

    View details for DOI 10.1084/jem.20021825

    View details for Web of Science ID 000184368200005

    View details for PubMedID 12874256

  • Full reconstitution of hematopoietic system by murine umbilical cord blood TRANSPLANTATION Migishima, F., Oikawa, A., Kondo, S., Ema, H., Morita, Y., Nakauchi, H., Yokoyama, M., Song, S. Y., Nishijima, M., Okabe, M., Shinohara, N. 2003; 75 (11): 1820-1826


    Murine umbilical cord blood cells (UCBCs) were studied for their ability to reconstitute the hematopoietic system.On average, 150 microL of cord blood per fetus containing 1.2 to 2 x 10(4) nucleated cells were collected from day 18.5 fetal umbilical cord, and 3 to 6 x 10(3) cells per fetus were obtained after separation by gradient centrifugation.Although lineage marker-, c-Kit+, and Sca-1+ cells were detectable among UCBCs, cells designated to be in the side population (SP) by Hoechst 33342 staining were hardly detectable within this population; the frequency of cells of this phenotype was less than 1 of 105. Instead, the lineage marker-, c-Kit+, and Sca-1- population contained a considerable number of SP cells. Nevertheless, UCBCs obtained from fetuses of green fluorescent protein-transgenic mice successfully reconstituted the blood cells of lethally irradiated recipients. Fluorescent cells could be readily detected in every blood cell lineage and among immature cell populations. Furthermore, fluorescent SP cells sorted from the recipient bone marrow cells could also reconstitute the blood cells in the secondary recipients, indicating that UCBCs also replenished bone marrow stem cells.Murine UCBC could fully reconstitute the hematopoietic system of lethally irradiated recipients including hematopoietic stem cells in bone marrow.

    View details for DOI 10.1097/01.TP.0000065295.99190.03

    View details for Web of Science ID 000183684900008

    View details for PubMedID 12811240

  • Role for growth factors and extracellular matrix in controlling differentiation of prospectively isolated hepatic stem cells DEVELOPMENT Suzuki, A., Iwama, A., Miyashita, H., Nakauchi, H., Taniguchi, H. 2003; 130 (11): 2513-2524


    In liver development, a number of growth factors (GFs) and components of the extracellular matrix (ECMs) lead to differentiation of liver parenchymal cells. As the liver contains many cell types, specifically investigating their functional effects on hepatic stem cell populations is difficult. Prospective isolation and clonal assays for hepatic stem cells enable the examination of direct effects of GFs and ECMs on this rare cell fraction. Using previously purified cells that fulfill the criteria for hepatic stem cells, we examined how GFs and ECMs regulate differentiation in the developing liver. We show here that hepatocyte growth factor (HGF) induced early transition of albumin (ALB)-negative stem cells to ALB-positive hepatic precursors resembling hepatoblasts and then oncostatin M (OSM) promoted their differentiation to tryptophan-2, 3-dioxygenase (TO)-positive mature hepatocytes. During this transition, ECMs were necessary for the differentiation of stem cells and precursors, but their effects were only supportive. In the first step of stem cell differentiation induced by HGF, the expression of CCAAT/enhancer binding protein (C/EBP), a basic leucine zipper transcription factor, changed dramatically. When C/EBP function was inhibited in stem cells, they stopped differentiating to hepatocyte-lineage cells and proliferated actively. These are the first findings to illustrate the mechanism of hepatic stem cell differentiation in liver development.

    View details for DOI 10.1242/dev.00459

    View details for Web of Science ID 000183623400020

    View details for PubMedID 12702664

  • Targeting a complex transcriptome: The construction of the mouse full-length cDNA encyclopedia GENOME RESEARCH Carninci, P., Waki, K., Shiraki, T., Konno, H., Shibata, K., Itoh, M., Aizawa, K., Arakawa, T., Ishii, Y., Sasaki, D., Bono, H., Kondo, S., Sugahara, Y., Saito, R., Osato, N., Fukuda, S., Sato, K., Watahiki, A., Hirozane-Kishikawa, T., Nakamura, M., Shibata, Y., Yasunishi, A., Kikuchi, N., Yoshiki, A., Kusakabe, M., Gustincich, S., Beisel, K., Pavan, W., Aidinis, V., Nakagawara, A., Held, W. A., Iwata, H., Kono, T., Nakauchi, H., Lyons, P., Wells, C., Hume, D. A., Fagiolini, M., Hensch, T. K., Brinkmeier, M., Camper, S., Hirota, J., Mombaerts, P., Muramatsu, M., Okazaki, Y., Kawai, J., Hayashizaki, Y. 2003; 13 (6B): 1273-1289


    We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

    View details for DOI 10.1101/gr.1119703

    View details for Web of Science ID 000183680800004

    View details for PubMedID 12819125

  • Glucagon-like peptide 1 (1-37) converts intestinal epithelial cells into insulin-producing cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Suzuki, A., Nakauchi, H., Taniguchi, H. 2003; 100 (9): 5034-5039


    Glucagon-like peptide (GLP) 1 is produced through posttranslational processing of proglucagon and acts as a regulator of various homeostatic events. Among its analogs, however, the function of GLP-1-(1-37), synthesized in small amounts in the pancreas, has been unclear. Here, we find that GLP-1-(1-37) induces insulin production in developing and, to a lesser extent, adult intestinal epithelial cells in vitro and in vivo, a process mediated by up-regulation of the Notch-related gene ngn3 and its downstream targets, which are involved in pancreatic endocrine differentiation. These cells became responsive to glucose challenge in vitro and reverse insulin-dependent diabetes after implantation into diabetic mice. Our findings suggest that efficient induction of insulin production in intestinal epithelial cells by GLP-1-(1-37) could represent a new therapeutic approach to diabetes mellitus.

    View details for DOI 10.1073/pnas.0936260100

    View details for Web of Science ID 000182612600015

    View details for PubMedID 12702762

  • Successful multilineage engraftment of human cord blood cells in pigs after in utero transplantation. TRANSPLANTATION Fujiki, Y., Fukawa, K., Kameyama, K., Kudo, O., Onodera, M., Nakamura, Y., Yagami, K., Shiina, Y., Hamada, H., Shibuya, A., Nakauchi, H. 2003; 75 (7): 916-922


    Successful engraftment of human hematopoietic stem and progenitor cells (HSPCs) in a large animal may serve not only as a model to study human hematopoiesis but also as a bioreactor to expand human HSPCs in vivo. The aim of this study was to accomplish xenotransplantation of human HSPCs into pig.Total mononuclear or CD34-positive HSPCs obtained from human cord blood were xenotransplanted percutaneously under an ultrasonographic guidance into preimmune pig fetuses. Peripheral blood and bone marrow (BM) cells of recipient pigs were collected and analyzed for the presence of human cells by a polymerase chain reaction to detect human specific Alu sequence on DNA extracted from those cells. Fluorescence-activated cell sorting (FACS) analysis was also performed to detect human hematopoietic cells.Transplantation of human cord blood cells into pig fetuses aged less than 52 days postcoitus resulted in a good engraftment rate. In one case, engraftment was detected up to 315 days posttransplantation by polymerase chain reaction. Human hematopoietic cells were detectable also by FACS in peripheral blood and BM. Furthermore, human CD34+ HSPCs were also observed in the BM of recipients. Those CD34+ cells in BM were sorted by FACS and subjected to further analyses. First, in vitro colony formation assay resulted in formations of multilineage colonies. Second, when they were transplanted into an immunodeficient mouse they were engrafted in the mouse.These data indicate an engraftment of human HSPCs in pig BM. In utero transplantation of human HSPCs into a preimmune pig fetus is useful to establish a pig reproducing human hematopoiesis.

    View details for DOI 10.1097/01.TP.0000057243.12110.7C

    View details for Web of Science ID 000182338500002

    View details for PubMedID 12698074

  • HES-1 preserves, purified hematopoietic stem cells ex vivo and accumulates side population cells in vivo BLOOD Kunisato, A., Chiba, S., Nakagami-Yamaguchi, E., Kumano, K., Saito, T., Masuda, S., Yamaguchi, T., Osawa, M., Kageyama, R., Nakauchi, H., Nishikawa, M., Hirai, H. 2003; 101 (5): 1777-1783


    Mouse long-term hematopoietic reconstituting cells exist in the c-Kit+Sca-1+Lin- (KSL) cell population; among them, CD34(low/-) cells represent the most highly purified population of hematopoietic stem cells in the adult bone marrow. Here, we demonstrate that retrovirus-mediated transduction of CD34(low/-)c-Kit+Sca-1+Lin- (34-KSL) cells with the HES-1 gene, which encodes a basic helix-loop-helix transcription factor functioning downstream of the Notch receptor, and is a key molecule for the growth phase of neural stem cells in the embryo, preserves the long-term reconstituting activity of these cells in vitro. We also show that cells derived from the HES-1-transduced 34-KSL population produce progenies characterized by negative Hoechst dye staining, which defines the side population, and by CD34(low/-) profile in the bone marrow KSL population in each recipient mouse at ratios 3.5- and 7.8-fold those produced by nontransduced 34-KSL-derived competitor cells. We conclude that HES-1 preserves the long-term reconstituting hematopoietic activity of 34-KSL stem cells ex vivo. Up-regulation of HES-1 protein in the 34-KSL population before unnecessary cell division, that is, without retrovirus transduction, may represent a potent approach to absolute expansion of hematopoietic stem cells.

    View details for DOI 10.1182/blood-2002-07-2051

    View details for Web of Science ID 000181136200018

    View details for PubMedID 12406868

  • In vitro production of functionally mature hepatocytes from prospectively isolated hepatic stem cells Annual Meeting of the Japanese-Society-for-Regenerative-Medicine Suzuki, A., Nakauchi, H., Taniguchi, H. COGNIZANT COMMUNICATION CORP. 2003: 469–73


    Hepatocyte transplantation and artificial organ hepatic support require a number of functionally mature hepatocytes. However, their growth activity and functional behaviors are much smaller in culture after isolation from the liver. We examined whether continuously differentiating hepatocytes from multipotent hepatic stem cells that were isolated by using flow cytometry and propagated clonally in culture could be a source of clinical application. They actually gave rise to cells that were functionally equal to mature hepatocytes found in the adult liver, which secreted albumin into culture medium and metabolized harmful ammonium into urea. These data suggest that stem cell-derived hepatocytes are a useful cell source for developing therapeutic strategies, such as cell transplantation, gene therapy, and artificial liver organ to treat various liver disorders.

    View details for Web of Science ID 000184760100003

    View details for PubMedID 12953920

  • Cell cycle dependence of boron uptake from two boron compounds used for clinical neutron capture therapy CANCER LETTERS Yoshida, F., Matsumura, A., Shibata, Y., Yamamoto, T., Nakauchi, H., Okumura, M., Nose, T. 2002; 187 (1-2): 135-141


    In neutron capture therapy, it is important that the boron is selectively uptaken by tumor cells. In the present study, we used flow cytometry to sort the cells in the G0/G1 phase and those in the G2/M phase, and the boron concentration in each fraction was measured with inductively coupled plasma atomic emission spectroscopy. The results revealed that sodium borocaptate and boronophenylalanine (BPA), were associated with higher rates of boron uptake in the G2/M than in the G0/G1 phase. However, the difference was more prominent in the case of BPA. The G2/M:G0/G1 ratio decreased as a function of exposure time in BPA containing culture medium, thereby indicating the cell cycle dependency of BPA uptake. Such heterogeneity of boron uptake by tumor cells should be considered for microdosimetry.

    View details for Web of Science ID 000179424400018

    View details for PubMedID 12359361

  • Identification and propagation of liver stem cells SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Suzuki, A., Nakauchi, H. 2002; 13 (6): 455-461


    Although the liver has been known for its enormous regenerative capacity, little is known about the mechanisms responsible for such regeneration.To provide evidence for the existence of liver stem cell, using FACS and single cell-based assays, cells with multi-lineage differentiation potential and self-renewal capability have been prospectively identified. These cells could be clonally propagated in culture where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When the cells clonally expanded in vitro were transplanted into mouse, they morphologically and functionally differentiated into hepatocytes and cholangiocytes. Furthermore, these cells differentiated into pancreatic acinar cells or intestinal epithelial cells upon transplantation into pancreas or duodenal wall. Manipulation of self-renewing liver stem cells may provide new insight into therapies for diseases of the digestive system.

    View details for DOI 10.1016/S1084-9521(02)00134-9

    View details for Web of Science ID 000179927800011

    View details for PubMedID 12468247

  • Erythroid expansion mediated by the Gfi-1B zinc finger protein: role in normal hematopoiesis BLOOD Osawa, M., Yamaguchi, T., Nakamura, Y., Kaneko, S., Onodera, M., Sawada, K., Jegalian, A., Wu, H., Nakauchi, H., Iwama, A. 2002; 100 (8): 2769-2777


    In the search for genes expressed in hematopoietic stem cells, we identified that the expression of Gfi-1B (growth factor independence-1B) is highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and 6 zinc finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, we analyzed its function in human hematopoiesis. Enforced expression of Gfi-1B in human CD34(+) hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression of Gfi-1B did not promote erythroid commitment, but enhanced proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biologic effects of Gfi-1B were mediated through its zinc finger domain, but not by the SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription was dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression.

    View details for Web of Science ID 000178519100017

    View details for PubMedID 12351384

  • Essential and instructive roles of GATA factors in eosinophil development JOURNAL OF EXPERIMENTAL MEDICINE Hirasawa, R., Shimizu, R., Takahashi, S., Osawa, M., Takayanagi, S., Kato, Y., Onodera, M., Minegishi, N., Yamamoto, M., Fukao, K., Taniguchi, H., Nakauchi, H., Iwama, A. 2002; 195 (11): 1379-1386


    GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1-deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.

    View details for DOI 10.1084/jem.20020170

    View details for Web of Science ID 000176121800002

    View details for PubMedID 12045236

  • Reciprocal roles for CCAAT/enhancer binding protein (C/EBP) and PU.1 transcription factors in Langerhans cell commitment JOURNAL OF EXPERIMENTAL MEDICINE Iwama, A., Osawa, M., Hirasawa, R., Uchiyama, N., Kaneko, S., Onodera, M., Shibuya, K., Shibuya, A., Vinson, C., Tenen, D. G., Nakauchi, H. 2002; 195 (5): 547-558


    Myeloid progenitor cells give rise to a variety of progenies including dendritic cells. However, the mechanism controlling the diversification of myeloid progenitors into each progeny is largely unknown. PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system. However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members. Using high titer--retroviral infection, we demonstrate that a dominant-negative C/EBP completely blocked the granulocyte--macrophage commitment of human myeloid progenitors. Alternatively, Langerhans cell (LC) commitment was markedly facilitated in the absence of tumor necrosis factor (TNF)alpha, a strong inducer of LC development, whereas expression of wild-type C/EBP in myeloid progenitors promoted granulocytic differentiation, and completely inhibited TNFalpha-dependent LC development. On the other hand, expression of wild-type PU.1 in myeloid progenitors triggered LC development in the absence of TNFalpha, and its instructive effect was canceled by coexpressed C/EBP. Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized.

    View details for Web of Science ID 000176110300003

    View details for PubMedID 11877478

  • Bone marrow chimerism prevents atherosclerosis in arterial walls of mice deficient in apolipoprotein E ATHEROSCLEROSIS Sakai, Y., Kim, D. K., Iwasa, S., Liang, J. X., Watanabe, T., Onodera, M., Nakauchi, H. 2002; 161 (1): 27-34


    apolipoprotein E (apoE) is a key regulator in cholesterol-rich lipoprotein metabolism. Inherited deficiency of this protein results in type III hyperlipoproteinemia in humans. ApoE, especially that derived from macrophages, can efficiently protect against development of atherosclerotic lesion. To use stem cell gene therapy or mini-transplant in treating abnormal lipid metabolism and preventing atherosclerosis, a minimal level of bone marrow chimerism must be determined.lethally irradiated apoE deficient mice (12-16 weeks of age) fed on normal chow were transplanted with normal bone marrow cells (C57BL/6.Ly5.1) mixed with those of apoE deficient mice (C57BL/6.Ly5.2) at various ratios. Plasma cholesterol levels were determined every 3 weeks for up to 42 weeks. Areas of atherosclerotic lesion in the aortas were quantified 6 months post-transplant. Plasma apoE was measured by Western blot analysis.bone marrow transplantation (BMT) in apoE (-/-) mice resulted in a detectable level of plasma apoE as determined by Western blot analysis. The plasma cholesterol levels in mice with > or = 60% chimerism were normalized by 6 weeks post-transplant. Mice with < or = 40% chimerism showed significant reductions, but not normalization, in the plasma cholesterol levels even at 42 weeks posttransplant. However, atherosclerotic areas observed in 10%-chimeric mice were significantly smaller than those in control mice (P<0.01). Immunohistochemical studies in 10%-chimeric mice revealed foam cells derived from donor marrow (apoE (+/+)) and expressed immunoreactive apoE in the atherosclerotic lesion. The positive signals by Western blot analysis were represented in the plasma of up to 8% of the chimeric mice.chimerism of 10%, the minimum level analyzed, was sufficient to reduce the severity of atherosclerosis, although the plasma cholesterol levels were not completely normalized. The results indicate that stem cell gene therapy and mini-transplant may provide possible therapeutic approaches to treat patients with abnormal lipid metabolism and atherosclerosis.

    View details for Web of Science ID 000174505700003

    View details for PubMedID 11882314

  • Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver JOURNAL OF CELL BIOLOGY Suzuki, A., Zheng, Y. W., Kaneko, S., Onodera, M., Fukao, K., Nakauchi, H., Taniguchi, H. 2002; 156 (1): 173-184


    Using flow cytometry and single cell-based assays, we prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewing capability. These cells could be clonally propagated in culture where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When cells that expanded in vitro were transplanted into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes with reconstitution of hepatocyte and bile duct structures. Furthermore, these cells differentiated into pancreatic ductal and acinar cells or intestinal epithelial cells when transplanted into pancreas or duodenal wall. These data indicate that self-renewing pluripotent stem cells persist in the developing mouse liver and that such cells can be induced to become cells of other organs of endodermal origin under appropriate microenvironment. Manipulation of hepatic stem cells may provide new insight into therapies for diseases of the digestive system.

    View details for Web of Science ID 000173256300017

    View details for PubMedID 11781341

  • Lentiviral vector-mediated transduction of murine CD34(-) hematopoietic stem cells EXPERIMENTAL HEMATOLOGY Tahara-Hanaoka, S., Sudo, K., Ema, H., Miyoshi, H., Nakauchi, H. 2002; 30 (1): 11-17


    Efficient gene transfer into murine hematopoietic stem cells (HSCs) provides a powerful tool for exploring hematopoietic stem cell biology. In this study, we evaluated the efficiency of lentiviral vector-mediated gene transfer into murine CD34(-/low)c-Kit(+)Sca-1(+)Lin(-) (CD34(-) KSL) cells that are highly enriched for HSCs.FACS-sorted CD34(-) KSL cells were transduced with the vesicular stomatitis virus G glycoprotein-pseudotyped HIV-1-based lentiviral vector containing the green fluorescent protein (GFP) gene under the control of the cytomegalovirus promoter, and then 50 transduced cells were transplanted into lethally irradiated mice. Transduction efficiency was assessed by FACS analysis for GFP expression in peripheral blood (PB) cells. FACS-sorted GFP(+) KSL bone marrow (BM) cells from primary recipients were used for secondary transplantation, and GFP expression in PB cells of reconstituted mice was analyzed by FACS.GFP expression was detected in PB cells of all primary recipients (n = 10) at an average of 40% (range 26-58%) when the lentiviral vector containing the woodchuck hepatitis virus posttranscriptional regulatory element was used. GFP(+) cells were found in multilineage cells in PB, BM, spleen, and thymus for at least 8 months posttransplantation. In secondary recipients, donor-derived GFP(+) KSL BM cells could reconstitute and GFP expression was detected in both myeloid and lymphoid cells in PB.Our results indicate that lentiviral vectors can efficiently transduce highly enriched murine HSCs and sustain long-term expression of the transgene in the multilineage differentiated progeny in reconstituted mice.

    View details for Web of Science ID 000173693700004

    View details for PubMedID 11823032

  • Chronological changes of boron uptake and its relationship to the cell cycle. An in vitro study of different cell lines RESEARCH AND DEVELOPMENT IN NEUTRON CAPTURE THERAPY Yoshida, F., Matsumura, A., Shibata, Y., Yamamoto, T., Zhang, T., Nakauchi, H., Okumura, M., Nose, T. 2002: 857-860
  • Establishment of clonal colony-forming assay system for pancreatic stem/progenitor cells 9th Annual Meeting of the Society-for-Cell-Therapy (SCT) Suzuki, A., Oyama, K., Fukao, K., Nakauchi, H., Taniguchi, H. COGNIZANT COMMUNICATION CORP. 2002: 451–53


    Pluripotent stem cells found in a number of organs are usually in small cell populations. However, under adaptive stimulation, they enter the stage of growth and differentiation to compensate for the loss of differentiated cells. To analyze stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are strongly required. In this study, we established a colony-forming assay system for pancreatic stem/progenitor cells in vitro. In this culture condition, they received signals for growth and differentiation, and formed clonal colonies including pancreatic endocrine-lineage cells, such as alpha and beta cells. By combining this culture system with flow cytometric cell sorting, pancreatic stem/progenitor cells will be enriched, and their potential can be analyzed precisely in single cell-based experiments.

    View details for Web of Science ID 000178321600010

    View details for PubMedID 12382672

  • Evidence for hepatocyte differentiation from embryonic stem cells in vitro 9th Annual Meeting of the Society-for-Cell-Therapy (SCT) Miyashita, H., Suzuki, A., Fukao, K., Nakauchi, H., Taniguchi, H. COGNIZANT COMMUNICATION CORP. 2002: 429–34


    We confirmed hepatocyte differentiation from embryonic stem (ES) cells in vitro. RT-PCR analysis revealed that a broad range of hepatic gene expression was observed in ES cells differentiated through formation of embryoid bodies (EBs) and its attachment culture. Quantitative PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of ES cells. The presence of albumin-producing cells in the peripheral region of attached EBs was confirmed by immunocytochemical analysis. Future experiments will reveal the molecules that induce hepatocyte differentiation from ES cells in vitro. This research will provide systems for the investigation of mechanisms in liver development and establish a method of ES cell-based therapy for liver diseases.

    View details for Web of Science ID 000178321600007

    View details for PubMedID 12382669

  • A transmembrane trap method for efficient cloning of genes encoding proteins possessing transmembrane domain BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Miyoshi, S., Motohashi, T., Nakamura, Y., Osawa, M., Hiroyama, T., Kim, D. K., Tokumoto, Y., Nakauchi, H. 2001; 289 (5): 1192-1198


    To facilitate searching for genes encoding cell membrane proteins, we developed a method for isolating cDNAs that contain sequences for hydrophobic transmembrane runs. This cloning strategy, termed the "transmembrane (TM) trap method," utilizes a vector that directs the cell surface expression of mouse CD4 fusion protein when an insert encoding hydrophobic transmembrane sequences is cloned in-frame with correct orientation. We applied this novel method to isolation of cytokine receptor cDNAs. Our strategy enabled efficient isolation of relatively rare species encoding receptors such as IL-2Rgamma, IL-3Rbeta, IL-4Ralpha, IL-5Ralpha, and IL-6Ralpha. This method also could be used to isolate cDNAs for intracellular molecules with a transmembrane region, e.g., bcl-2. These results indicate that the TM trap method provides an efficient cloning strategy for identification of various families of genes encoding proteins with one or more transmembrane regions.

    View details for DOI 10.1006/bbrc.2001.6097

    View details for Web of Science ID 000173406500043

    View details for PubMedID 11741319

  • Molecular cloning and characterization of mouse Tspan-3, a novel member of the tetraspanin superfamily, expressed on resting dendritic cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Tokoro, Y., Shibuya, K., Osawa, M., Tahara-Hanaoka, S., Iwama, A., Kitamura, T., Nakauchi, H., Shibuya, A. 2001; 288 (1): 178-183


    Dendritic cells (DCs) are the most potent antigen-presenting cells and play an essential role for triggering T-cell-mediated immune responses. In search for novel cell surface molecules expressed on DCs involved in T cell priming by representational differential analysis, we identified a mouse homologue of Tspan-3 (mTspan-3), a novel member of the tetraspanin superfamily. The mTspan-3 consists of four hydrophobic, putative transmembrane regions, forming a small and a large extracellular loop, with short intracellular amino and carboxil tails. Although the mTspan-3 is expressed on a variety of immune cell types including resting DCs, its expression on DCs is downregulated during activation induced by cross-linking CD40 with anti-CD40 monoclonal antibody. These results suggest that mTspan-3 may be involved in the function of DCs in association with T cell stimulation.

    View details for DOI 10.1006/bbrc.2001.5742

    View details for Web of Science ID 000171813400027

    View details for PubMedID 11594770

  • Fc alpha/mu receptor is a single gene-family member closely related to polymeric immunoglobulin receptor encoded on Chromosome 1 IMMUNOGENETICS Shimizu, Y., Honda, S., Yotsumoto, K., Tahara-Hanaoka, S., Eyre, H. J., Sutherland, G. R., Endo, Y., Shibuya, K., Koyama, A., Nakauchi, H., Shibuya, A. 2001; 53 (8): 709-711

    View details for Web of Science ID 000172962300012

    View details for PubMedID 11797105

  • The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype NATURE MEDICINE Zhou, S., Schuetz, J. D., Bunting, K. D., Colapietro, A. M., Sampath, J., Morris, J. J., Lagutina, I., Grosveld, G. C., Osawa, M., Nakauchi, H., Sorrentino, B. P. 2001; 7 (9): 1028-1034


    Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.

    View details for Web of Science ID 000170853300028

    View details for PubMedID 11533706

  • Increased cell surface expression of C-terminal truncated erythropoietin receptors in polycythemia EUROPEAN JOURNAL OF HAEMATOLOGY Motohashi, T., Nakamura, Y., Osawa, M., Hiroyama, T., Iwama, A., Shibuya, A., Nakauchi, H. 2001; 67 (2): 88-93


    Primary familial and congenital polycythemia (PFCP) is a disorder characterized by an increased number of erythrocytes despite normal blood oxygen pressure and a normal serum erythropoietin (EPO) level. Recent studies revealed that erythroid progenitor cells from certain individuals with PFCP express various forms of EPO receptor (EPOR) truncated at the terminal carboxyl site (EPOR-TTC(PFCP)). EPOR-TTC(PFCP) can transmit EPO-mediated proliferative signals more efficiently than can full-length EPOR (EPOR-F), at least partly because of defective recruitment of SHP-1 phosphatase to these receptors. In agreement with previous studies, Ba/F3 transfectants expressing EPOR-TTC(PFCP) showed higher proliferative responses to EPO. In those transfectants, we found that EPOR-TTC(PFCP) was expressed more abundantly on the cell surface than was EPOR-F. This tendency was confirmed by a transient-expression experiment using COS7 cells. Since expression levels of EPOR protein were not significantly different among these transfectants, differences in cell surface expression were likely dependent on post-translational mechanism(s). In addition to defective recruitment of SHP-1 to EPOR-TTC(PFCP), more efficient transport and expression on the cell surface appear to serve as mechanisms responsible for increased EPO-responsiveness of erythroid progenitor cells in PFCP.

    View details for Web of Science ID 000171975900004

    View details for PubMedID 11722595

  • Fractalkine shares signal sequence with TARC: Gene structures and expression profiles of two chemokine genes GENOMICS Hiroyama, T., Iwama, A., Nakamura, Y., Nakauchi, H. 2001; 75 (1-3): 3-5


    In the process of cloning the gene (Scyd1) encoding the mouse CX3C chemokine fractalkine, we identified a novel cDNA that encodes a chimeric molecule termed fracTARC. This molecule is a variant form of the mouse CC chemokine, TARC (for thymus- and activation-regulated chemokine), bearing the fractalkine signal sequence instead of its own. Analysis of the genomic organization of the two genes revealed that Scyd1 and Scya17, encoding TARC, are tightly linked on chromosome 8 and that fracTARC is generated by alternative splicing of the two genes. Among tissues in which Scyd1 mRNA is expressed, fracTARC mRNA is selectively expressed in brain and kidney, indicating that fracTARC mRNA is generated by tissue-specific alternative splicing under the control of the Scyd1 promoter. On the other hand, Scya17 and the fracTARC gene are reciprocally expressed in thymus, brain, lung, and kidney and are never expressed in the same tissue. These expression profiles indicate that tissue specificity of Scya17 is precisely regulated by two independent mechanisms, one by transcription from its own promoter and the other from the promoter of Scyd1 followed by tissue-specific alternative splicing. These data provide evidence for a novel mechanism that controls gene expression of two independent genes of the same family. Such a mechanism may also operate in other genes that are tightly linked on the same chromosome.

    View details for Web of Science ID 000169795100002

    View details for PubMedID 11472060

  • A novel Fc receptor for IgA and IgM is expressed on both hematopoietic and non-hematopoietic tissues EUROPEAN JOURNAL OF IMMUNOLOGY Sakamoto, N., Shibuya, K., Shimizu, Y., Yotsumoto, K., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Nakauchi, H., Shibuya, A. 2001; 31 (5): 1310-1316


    By contrast to well-defined Fc gamma and Fc epsilon receptors, the structural and functional characteristics of Fc mu receptor are unclear. We have recently described a novel mouse Fc receptor, designated Fc alpha/mu receptor, and its human homologue, which bind both IgM and IgA. Here we show that the Fc alpha/mu receptor is expressed on mature, but not immature, B lymphocytes and acquires the ability to bind IgM and IgA antibodies after stimulation of B lymphocytes. Moreover, stimulation with phorbol 12-myristate 13-acetate increased endocytosis of IgM-coated microparticles mediated by the Fc alpha/mu receptor expressed on pro-B cell line Ba/F3 cells. We also show that the Fc alpha/mu receptor is expressed in secondary lymphoid organs, such as lymph node and appendix, kidney and intestine, suggesting an important role of the receptor for immunity in these organs.

    View details for Web of Science ID 000168814900003

    View details for PubMedID 11465087

  • Detection of fetal HPCs in maternal circulation after delivery TRANSFUSION Osada, H., Doi, S., Fukushima, T., Nakauchi, H., Seki, K., Sekiya, S. 2001; 41 (4): 499-503


    Circulation of mature fetal blood cells in the maternal blood for a certain postpartum period has been verified, but detailed study of the fetal HPCs has not been reported. The objective of this study was to evaluate the frequency and clearance of these cells in the peripheral blood of puerperal women.PBMNCs from 15 puerperal women who gave birth to male infants were cultured in semi-solid medium containing hematopoietic stimulating factors. Colonies formed in the medium were individually characterized, collected, and subjected to PCR amplification of the SRY gene on Y chromosome to confirm fetal origin.The mean numbers of fetal progenitor cell colonies isolated per mL of maternal blood were 1.63, 2.48, 0.56, 0.12, and 0 on the day of delivery, at 4 days, 1 month, 6 months, and 1 year after delivery, respectively. There was no difference in the ratio of fetal versus maternal colonies between erythroid and granulocyte/macrophage lineages.The present study demonstrated that a significant number of fetal HPCs circulate in the maternal blood for a duration of at least 6 months after delivery.

    View details for Web of Science ID 000168296800013

    View details for PubMedID 11316901

  • Hepatic stem/progenitor cells with high proliferative potential in liver organ formation 18th World Congress of the Transplantation-Society Suzuki, A., Zheng, Y. W., Fukao, K., Nakauchi, H., Taniguchi, H. ELSEVIER SCIENCE INC. 2001: 585–86

    View details for Web of Science ID 000167629900276

    View details for PubMedID 11266969

  • Clonal expansion of hepatic stem/progenitor cells following flow cytometric cell sorting 8th Annual Meeting of the Society-for-Cell-Therapy (SCT) Suzuki, A., Zheng, Y. W., Fukao, K., Nakauchi, H., Taniguchi, H. COGNIZANT COMMUNICATION CORP. 2001: 393–96


    Although hepatic stem cells are believed to exist and play a critical role in developing and regenerating liver, little is known about their cell surface specificity or differentiation capabilities. To make prospective studies of hepatic stem cells possible, we established an in vitro culture system for identification and characterization of hepatic stem/progenitor cells. By combining this culture system with fluorescence activated cell sorting (FACS), a population of cells that were capable of forming large colonies and providing their descendants for relative longer period was isolated from fetal mouse livers. These data suggest that hepatic stem/progenitor cells with high proliferative potential are existent in the developing mouse liver, and that they are enriched by using flow cytometry.

    View details for Web of Science ID 000170544500008

    View details for PubMedID 11549060

  • Prospective identification and enrichment of hepatic stem cells in the liver by flowcytometric cell sorting 5th Internation Symposium on Tissue Engineering for Therapeutic Use Taniguchi, H., Suzuki, A., Zheng, Y., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE BV. 2001: 53–58
  • Identification of Fc alpha/mu receptor expressed on B lymphocytes and macrophages CREST International Symposium on Immunoglobulin-like Receptors Shibuya, A., Shibuya, K., Shimizu, Y., Yotsumoto, K., Nakauchi, H. SPRINGER-VERLAG TOKYO. 2001: 79–84
  • Simplified retroviral vector GCsap with murine stem cell virus long terminal repeat allows high and continued expression of enhanced green fluorescent protein by human hematopoietic progenitors engrafted in nonobese diabetic/severe combined immunodeficient mice HUMAN GENE THERAPY Kaneko, S., Onodera, M., Fujiki, Y., Nagasawa, T., Nakauchi, H. 2001; 12 (1): 35-44


    Despite efforts toward improvements in retrovirus-mediated gene transfer, stable high-level expression of a therapeutic gene in human hematopoietic stem cells remains a great challenge. We have evaluated the efficiency of different viral long terminal repeats (LTRs) in long-term expression of a transgene in vivo, using severe combined immunodeficiency (SCID)-repopulating cell assays. Vectors used were variants of the simplified retroviral vector GCsap with the different LTRs of Moloney murine leukemia virus (MLV), myeloproliferative sarcoma virus (MPSV), and murine stem cell virus (MSCV). The enhanced green fluorescent protein (EGFP) gene was used as a marker to assess levels of transduction efficiency. CD34+ cells isolated from human cord blood were transduced by exposure to virus-containing supernatants on fibronectin fragments and in the presence of stem cell factor, interleukin 6, Flt-3 ligand, and thrombopoietin, and then transplanted into nonobese diabetic/SCID mice. Engraftment of human cells highly expressing EGFP, with differentiation along multiple cell lineages, was demonstrated for up to 18 weeks posttransplant, although the three different vectors showed different transduction frequencies (MLV, <0.1-33.2%; MPSV, <0.1-22.8%; MSCV, 0.3-51.7%). Of importance is that high-level transduction frequencies in human progenitor cells were also confirmed by colony-forming cell assays using bone marrow from transplanted mice, in which EGFP-expressing, highly proliferative potential colonies were observed by fluorescence microscopy. In these mice the vector carrying the MSCV LTR generated more EGFP-expressing human cells than did either of the other two constructs, indicating that GCsap carrying the MSCV LTR may be an efficient tool for stem cell gene therapy.

    View details for Web of Science ID 000166401500004

    View details for PubMedID 11177540

  • Quantitative assessment of the stem cell self-renewal capacity 3rd International Conference on Hematopoietic Stem Cells: Genetics and Medicine Nakauchi, H., Sudo, K., Ema, H. NEW YORK ACAD SCIENCES. 2001: 18–25


    Little is known about the manner in which hematopoietic stem cells (HSCs) self-renew. To address this issue, we used a serum-free single-cell culture, followed by transplantation of cultured cells into lethally irradiated mice. CD34-negative or low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) cells are highly enriched for murine bone marrow HSCs. Successful long-term reconstitution with a single CD34-KSL cell enabled us to study in vitro self-renewal of HSC at clonal level. Using this clonal cell transplantation system, we examined the effect of various cytokines on CD34-KSL cells. Among the cytokines examined, stem cell factor (SCF) and thrombopoietin (TPO) were minimum cytokines to induce cell division of CD34-KSL cells most efficiently. Similarly, multilineage repopulating activity was detected in the cells derived from a significant portion of single cells after culture in the presence of TPO and SCF. However, SCF + IL-3, SCF + IL-6, or SCF + IL-11 + FL appeared to be less effective for self-renewal of HSCs. The activity of HSCs as indicated by repopulation unit (RU) remaining after culture with SCF and TPO was not so different from that of freshly isolated HSCs. However, there was a substantial loss of HSC number in these cultured cells. Taken together, this study provides definitive proof that one HSC can generate at least one HSC in vitro.

    View details for Web of Science ID 000172028500003

    View details for PubMedID 11458506

  • Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver BLOOD Hsu, H. C., Ema, H., Osawa, M., Nakamura, Y., Suda, T., Nakauchi, H. 2000; 96 (12): 3757-3762


    Tie-2 receptor tyrosine kinase expressed in endothelial and hematopoietic cells is believed to play a role in both angiogenesis and hematopoiesis during development of the mouse embryo. This article addressed whether Tie-2 is expressed on fetal liver hematopoietic stem cells (HSCs) at day 14 of gestation. With the use of anti-Tie-2 monoclonal antibody, its expression was detected in approximately 7% of an HSC population of Kit-positive, Sca-1-positive, lineage-negative or -low, and AA4.1-positive (KSLA) cells. These Tie-2-positive KSLA (T(+) KSLA) cells represent 0.01% to 0.02% of fetal liver cells. In vitro colony and in vivo competitive repopulation assays were performed for T(+) KSLA cells and Tie-2-negative KSLA (T(-) KSLA) cells. In the presence of stem cell factor, interleukin-3, and erythropoietin, 80% of T(+) KSLA cells formed colonies in vitro, compared with 40% of T(-) KSLA cells. Long-term multilineage repopulating cells were detected in T(+) KSLA cells, but not in T(-) KSLA cells. An in vivo limiting dilution analysis revealed that at least 1 of 8 T(+) KSLA cells were such repopulating cells. The successful secondary transplantation initiated with a limited number of T(+) KSLA cells suggests that these cells have self-renewal potential. In addition, engraftment of T(+) KSLA cells in conditioned newborn mice indicates that these HSCs can be adapted equally by the adult and newborn hematopoietic environments. The data suggest that T(+) KSLA cells represent HSCs in the murine fetal liver. (Blood. 2000;96:3757-3762)

    View details for Web of Science ID 000165514000013

    View details for PubMedID 11090057

  • Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver HEPATOLOGY Suzuki, A., Zheng, Y. W., Kondo, R., Kusakabe, M., Takada, Y., Fukao, K., Nakauchi, H., Taniguchi, H. 2000; 32 (6): 1230-1239


    Stem cells responsible for tissue maintenance and repair are found in a number of organs. However, hepatic stem cells assumed to play a key role in liver development and regeneration remain to be well characterized. To address this issue, we set up a culture system in which primitive hepatic progenitor cells formed colonies. By combining this culture system with fluorescence-activated cell sorting (FACS), cells forming colonies containing distinct hepatocytes and cholangiocytes were identified in the fetal mouse liver. These cells express both CD49f and CD29 (alpha6 and beta1 integrin subunits), but do not mark for hematopoietic antigens such as CD45, TER119, and c-Kit. When transplanted into the spleen, these cells migrated to the recipient liver and differentiated into liver parenchymal cells. Our data demonstrate that hepatic progenitor cells are enriched by FACS and suggest approaches to supplanting organ allografting and improving artificial-organ hepatic support.

    View details for DOI 10.1053/jhep.2000.20349

    View details for Web of Science ID 000165560300007

    View details for PubMedID 11093729

  • In vitro self-renewal division of hematopoietic stem cells JOURNAL OF EXPERIMENTAL MEDICINE Ema, H., Takano, H., Sudo, K., Nakauchi, H. 2000; 192 (9): 1281-1288


    Little is known about how hematopoietic stem cells (HSCs) self-renew. We studied the regeneration of HSCs in culture. Effects of various cytokines on cell division of CD34(-/low) c-Kit(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) bone marrow cells of the mouse were first evaluated in serum-free single cell culture. We then performed a competitive repopulation assay on divided cells to ask if such cell division involved self-renewal of HSCs. In the presence of stem cell factor (SCF), thrombopoietin (TPO) induced a first cell division of CD34(-)KSL cells more efficiently than did interleukin (IL)-3 or IL-6. Multilineage repopulating cells were detected in a significant proportion of cells derived from single cells in culture with TPO and SCF, although this culture condition led to a substantial decrease in HSC number. These regenerated repopulating cells could be further transplanted into secondary recipients. When paired daughter cells were separately studied, one of a pair gave rise to repopulating cells with self-renewal potential, suggesting asymmetric self-renewal division. This study provides evidence that one HSC regenerates at least one HSC in culture.

    View details for Web of Science ID 000165471100007

    View details for PubMedID 11067877

  • Age-associated characteristics of murine hematopoietic stem cells JOURNAL OF EXPERIMENTAL MEDICINE Sudo, K., Ema, H., Morita, Y., Nakauchi, H. 2000; 192 (9): 1273-1280


    Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34(-/low)c-Kit(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34(-)KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34(-)KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34(-)KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

    View details for Web of Science ID 000165471100006

    View details for PubMedID 11067876

  • Fc alpha/mu receptor mediates endocytosis of IgM-coated microbes NATURE IMMUNOLOGY Shibuya, A., Sakamoto, N., Shimizu, Y., Shibuya, K., Osawa, M., Hiroyama, T., Eyre, H. J., Sutherland, G. R., Endo, Y., Fujita, T., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Phillips, J. H., Lanier, L. L., Nakauchi, H. 2000; 1 (5): 441-446


    IgM is the first antibody to be produced in a humoral immune response and plays an important role in the primary stages of immunity. Here we describe a mouse Fc receptor, designated Fc alpha/microR, and its human homolog, that bind both IgM and IgA with intermediate or high affinity. Fc alpha/microR is constitutively expressed on the majority of B lymphocytes and macrophages. Cross-linking Fc alpha/microR expressed on a pro-B cell line Ba/F3 transfectant with soluble IgM or IgM-coated microparticles induced internalization of the receptor. Fc alpha/microR also mediated primary B lymphocyte endocytosis of IgM-coated Staphylococcus aureus. Thus, Fc alpha/microR is involved in the primary stages of the immune response to microbes.

    View details for Web of Science ID 000165076100018

    View details for PubMedID 11062505

  • Effects of combined growth factors on clonal growth and albumin secretion of murine fetal hepatocytes in low density culture 6th Congress of the Asian-Society-of-Transplantation Zheng, Y. W., Taniguchi, H., Suzuki, A., Takada, Y., Fukunaga, K., Seino, K., Yuzawa, K., Otsuka, M., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 2000: 2372–73

    View details for Web of Science ID 000166001400422

    View details for PubMedID 11120205

  • Clonal colony formation of hepatic stem/progenitor cells enhanced by embryonic fibroblast conditioning medium 6th Congress of the Asian-Society-of-Transplantation Suzuki, A., Taniguchi, H., Zheng, Y. W., Takada, Y., Fukunaga, K., Seino, K., Yazawa, K., Otsuka, M., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 2000: 2328–30

    View details for Web of Science ID 000166001400403

    View details for PubMedID 11120186

  • Proliferative and functional ability of transplanted murine neonatal hepatocytes in adult livers 6th Congress of the Asian-Society-of-Transplantation Suzuki, A., Taniguchi, H., Zheng, Y. W., Takada, Y., Fukunaga, K., Seino, K., Yazawa, K., Otsuka, M., Yoshiki, A., Kusakabe, M., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 2000: 2370–71

    View details for Web of Science ID 000166001400421

    View details for PubMedID 11120204

  • Effects of four extracellular matrices associated with growth factors on clonal culture and proliferation of murine fetal hepatocytes 6th Congress of the Asian-Society-of-Transplantation Zheng, Y. W., Taniguchi, H., Suzuki, A., Takada, Y., Fukunaga, K., Seino, K., Yuzawa, K., Otsuka, M., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 2000: 2498–99

    View details for Web of Science ID 000166001400482

    View details for PubMedID 11120265

  • Molecular cloning and chromosomal mapping of a novel five-span transmembrane protein gene, M83 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Motohashi, T., Miyoshi, S., Osawa, M., Eyre, H. J., Sutherland, G. R., Matsuda, Y., Nakamura, Y., Shibuya, A., Iwama, A., Nakauchi, H. 2000; 276 (1): 244-250


    In an attempt to identify novel transmembrane molecules expressed on hematopoietic cells, we identified a novel transmembrane protein gene, M83. Cloning of the full-length cDNAs of human and mouse M83 revealed that M83 encodes a type I transmembrane protein with a region containing five hydrophobic segments within the C-terminal part of the protein, suggesting that M83 is a five-span transmembrane molecule. The M83 protein was expressed on the cell surface as a glycosylated protein with a molecular mass of 84 kDa. The M83 gene was localized to human chromosome 16p13.3, mouse chromosome 17B1, and rat chromosome 10q12.3 distal. In human, M83 mRNA was highly expressed in placenta, pancreas, and lymphohematopoietic tissues including peripheral blood, spleen, and bone marrow. Among hematopoietic cells, it was highly expressed in resting T lymphocytes and was downregulated by cell activation, suggestive of its biological role related to the T cell resting status.

    View details for DOI 10.1006/bbrc.2000.3409

    View details for Web of Science ID 000089388100042

    View details for PubMedID 11006113

  • Characterization of the mouse interleukin-13 receptor alpha 1 gene IMMUNOGENETICS Osawa, M., Miyoshi, S., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Hiroyama, T., Motohashi, T., Nakamura, Y., Iwama, A., Nakauchi, H. 2000; 51 (11): 974-981


    Interleukin (IL)-13 is a pleiotropic immune regulatory cytokine that shares structural and biological characteristics with IL-4. The receptor for IL-13 is comprised of the IL-4 receptor alpha (IL-4Ralpha) subunit and a low-affinity IL-13-binding subunit, IL-13Ralpha1. An additional receptor, IL-13Ralpha2, binds to IL-13 with high affinity, but lacks the cytoplasmic domain for signaling. In this study, we isolated the mouse IL-13Ralpha1 gene (Il13ra1) of approximately 56 kb that spans the entire coding region. The mouse Il13ra1 gene is composed of 11 exons, and shows striking similarity in genomic structure to the previously reported class I cytokine receptor genes. Motifs characteristic of the cytokine receptor family are similarly organized on the genome, including conserved cysteines, a WSxWS motif, and Box1, indicating closely related genetic evolution of the cytokine receptor superfamily. Alternative mRNA splicings were demonstrated to generate variant transcripts that encode soluble IL-13Ralpha1. The mouse Ill13ra1 gene was mapped to the proximal region of the mouse X chromosome, and was closely linked to the DXPas3 locus by interspecific backcross analysis. Il13ra1 mRNA was co-expressed with I14ra mRNA in mouse myeloid and natural killer cells on which IL-13 has been known to act, whereas the Il13ra2 mRNA was not detected in these cells, indicating that IL-13Ralpha1 is the major component of the IL-13 receptor complex in lymphohematopoietic cells.

    View details for Web of Science ID 000089339200010

    View details for PubMedID 11003391

  • Clonogenic colony-forming ability of flow cytometrically isolated hepatic progenitor cells in the murine fetal liver 6th Annual Meeting of the American-Society-for-Neural-Transplantation-and-Repair (ASNTR) Taniguchi, H., Kondo, R., Suzuki, A., Zheng, Y. W., Takada, Y., Fukunaga, K., Seino, K., Yuzawa, K., Otsuka, M., Fukao, K., Nakauchi, H. COGNIZANT COMMUNICATION CORP. 2000: 697–700


    Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45 TER119 cells (the non-blood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45TER119- cells in the murine fetal liver.

    View details for Web of Science ID 000165702700017

    View details for PubMedID 11144968

  • New type of matrix support for bone marrow cell cultures: In vitro culture and in vivo transplantation experiments ASAIO JOURNAL Tun, T., Miyoshi, H., Ema, H., Nakauchi, H., Ohshima, N. 2000; 46 (5): 522-526


    A new type of bone marrow cell culture system was developed by using a highly porous substrate matrix, i.e., porous polyvinyl formal (PVF) resin. Murine bone marrow (BM) cells were cultured without the use of exogenous growth factors in a three-dimensional matrix support made of collagen coated porous PVF resin. To examine the optimal conditions for highest stromal cell density, short-term and long-term in vitro culture experiments using PVF were performed. In the short-term culture experiments, it was found that cubes of PVF (10 x 10 x 2 mm and 130 microm in pore size) coated with type I collagen with a seeding density of 2x10(7) BM cells offered the most appropriate culture conditions. In the long-term cultures, BM cells in PVF maintained their viability for up to 6 weeks. In another series of re-inoculation experiments, freshly isolated BM cells were inoculated onto the already developed stromal layer. In this study, a higher cell density of the stromal layer was obtained in the PVF culture compared with those in the control dish culture. Based upon the results of in vitro experiments, in vivo transplantation studies were also performed. Histologic examinations of the subcutaneously transplanted PVF with stroma revealed host derived hematopoiesis inside the PVF matrix. Moreover, survival of approximately 15% of the transplanted BM cells that were cultured in PVF were confirmed in X-ray irradiated recipients. From these results, it is suggested that PVF resin is a promising three-dimensional substrate for BM cell culture and that it can maintain hematopoietic stem cells or progenitor cells after transplantation.

    View details for Web of Science ID 000089329900002

    View details for PubMedID 11016499

  • Induction of M-phase arrest and apoptosis after HIV-1 Vpr expression through uncoupling of nuclear and centrosomal cycle in HeLa cells EXPERIMENTAL CELL RESEARCH Watanabe, N., Yamaguchi, T., Akimoto, Y., Rattner, J. B., Hirano, H., Nakauchi, H. 2000; 258 (2): 261-269


    The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces cell cycle arrest in the G2 phase of the cell cycle followed by apoptosis. The mechanism of the arrest is unknown but the arrest is believed to facilitate viral replication. In the present study, we have established cell lines that allow conditional expression of Vpr, and have examined the mechanism of cell death following Vpr expression. We found that cells expressing Vpr enter M phase after long G2 arrest but formed aberrant multipolar spindles that were incapable of completing karyokinesis or cytokinesis. This abnormality provided the basis for apoptosis, which always followed in these cells. The multipolar spindles formed in response to abnormal centrosomal duplication that occurred during the G2 arrest but did not occur in cells arrested in G2 by irradiation. Thus, the expression of Vpr appears to be responsible for abnormal centrosome duplication, which in turn contributes in part to the rapid cell death following HIV-1 infection.

    View details for DOI 10.1006/excr.2000.4908

    View details for Web of Science ID 000088529900004

    View details for PubMedID 10896777

  • Dominant expansion of human T cells in non-obese diabetic/severe combined immunodeficiency mice implanted with human bone fragments EXPERIMENTAL HEMATOLOGY Fujiki, Y., Onodera, M., Yamaguchi, T., Osawa, M., Sudo, K., Hamada, H., Ema, H., Shibuya, A., Takiguchi, M., Kubo, T., Nakauchi, H. 2000; 28 (7): 792-801


    To establish an in vivo animal model in which human T cells develop and function normally, a step toward developing new vaccines or chemical compounds that modulate immune functions and toward understanding T-cell immunity in humans.Human bone fragments were implanted into non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice. The presence of human blood cells in the peripheral blood of these mice was monitored periodically by immunostaining and fluorescence-activated cell sorting.After implantation of bone fragments, dominant expansion of human T lymphocytes, rather than myeloid and B cells, was observed over a 3-month period. In some cases, the proportion of human T cells rose to 40% of the peripheral blood mononuclear cells. These T cells showed CD4/CD8 ratios similar to those observed in human peripheral blood lymphocytes and had a broad repertoire of rearranged T-cell receptor genes. Graft-versus-host reaction was not noted in any organ analyzed. To assess the suitability of NOD/SCID mice implanted with human bone fragments (hu-bone-NOD/SCID mice) as an in vivo model for HIV infection, the mice were infected with a T-lymphotropic strain of HIV-1 (NL4-3) at 7 weeks posttransplant. Serum p24 gag was detected at 2 weeks after inoculation, after which total CD4-positive cell numbers declined, as seen clinically in patients infected with HIV.Although the precise mechanism is yet to be determined by which predominant expansion of human T cells occurs in hu-bone-NOD/SCID mice, such mice appear likely to serve as a useful and versatile model for studies involving human T-cell immunity.

    View details for Web of Science ID 000087892300009

    View details for PubMedID 10907641

  • Long-term culture of fetal liver cells using a three-dimensional porous polymer substrate ASAIO JOURNAL Miyoshi, H., Ehashi, T., Ema, H., Hsu, H. C., Nakauchi, H., Ohshima, N. 2000; 46 (4): 397-402


    To develop a bioartificial liver, long-term culture of fetal liver cells over a month's time was performed under three different culture conditions, i.e., stationary cultures and shaken-flask cultures, both by using a substratum made of porous polyvinyl formal (PVF) resin and conventional monolayer dish cultures as controls. Time course changes in cell numbers and albumin secretion were evaluated in cultures using Williams' E medium (WE) or minimum essential medium alpha (aMEM) supplemented with serum and hormones. In the WE medium, the numbers of fetal liver cells in all culture conditions gradually decreased with time, and albumin secretion rates rapidly decreased. In the stationary cultures using PVF, however, a significant increase in albumin secretion was observed after two weeks of culture. When cells were cultured in aMEM, the fetal liver cells exhibited sufficient proliferation in stationary and monolayer cultures, although albumin secretion rates per single cell were lower than those in WE. On the basis of these results, another series of culture experiments were performed, in which aMEM was used for the first 10 days to encourage cell proliferation, and the medium was changed to WE afterward. In these cultures, albumin secretion rates in the stationary cultures dramatically increased after the medium exchanges and were maintained at these high levels throughout the remaining culture period.

    View details for Web of Science ID 000090139100006

    View details for PubMedID 10926134

  • Molecular cloning and characterization of CRLM-2, a novel type I cytokine receptor preferentially expressed in hematopoietic cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hiroyama, T., Iwama, A., Morita, Y., Nakamura, Y., Shibuya, A., Nakauchi, H. 2000; 272 (1): 224-229


    A murine expressed sequence tag (EST) showing homology with erythropoietin receptor (EPOR) was identified in the EST database. Cloning of the full-length cDNA revealed a 359 amino acid novel type I cytokine receptor, designated cytokine receptor like molecule-2 (CRLM-2). While CRLM-2 lacks typical WSXWS motif, it has a significant homology with EPOR, IL-2 receptor beta and gamma, and IL-9 receptor alpha. The murine CRLM-2 gene is composed of 8 exons, and an alternative mRNA splicing generates a variant transcript encoding a soluble CRLM-2. CRLM-2 is preferentially expressed in hematopoietic cells, particularly in hematopoietic progenitors and myeloid cells. Furthermore, CRLM-2 is constitutively associated with JAK2, a well-known tyrosine kinase that transmits signals from cytokine receptors. These data strongly suggest that CRLM-2 is a novel cytokine receptor involved in the regulation of hematopoietic system.

    View details for Web of Science ID 000087378900038

    View details for PubMedID 10872831

  • Expansion of hematopoietic stem cells in the developing liver of a mouse embryo BLOOD Ema, H., Nakauchi, H. 2000; 95 (7): 2284-2288


    The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 x 10(5) adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential.

    View details for Web of Science ID 000086130100017

    View details for PubMedID 10733497

  • Usefulness of flow-cytometric cell sorting for enrichment of hepatic stem and progenitor cells in the liver 35th Annual Congress of the Japan-Society-for-Transplantation Taniguchi, H., Suzuki, A., Zheng, Y., Kondo, R., Takada, Y., Fukunaga, K., Seino, K., Yuzawa, K., Otsuka, M., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 2000: 249–51

    View details for Web of Science ID 000085928900005

    View details for PubMedID 10715407

  • A role for pref-1 and HES-1 in thymocyte development. Journal of immunology Kaneta, M., Osawa, M., Sudo, K., Nakauchi, H., Farr, A. G., Takahama, Y. 2000; 164 (1): 256-264


    T lymphocyte development requires a series of interactions between developing thymocytes and thymic epithelial (TE) cells. In this paper we show that TE cells in the developing thymus express Pref-1, a Delta-like cell-surface molecule. In fetal thymus organ cultures (FTOC), thymocyte cellularity was increased by the exogenous dimeric Pref-1 fusion protein, but was reduced by the soluble Pref-1 monomer or anti-Pref-1 Ab. Dimeric Pref-1 in FTOC also increased thymocyte expression of the HES-1 transcription factor. Thymocyte cellularity was increased in FTOC repopulated with immature thymocytes overexpressing HES-1, whereas FTOC from HES-1-deficient mice were hypocellular and unresponsive to the Pref-1 dimer. We detected no effects of either Pref-1 or HES-1 on developmental choice among thymocyte lineages. These results indicate that Pref-1 expressed by TE cells and HES-1 expressed by thymocytes are critically involved in supporting thymocyte cellularity.

    View details for PubMedID 10605019

  • A role for Pref-1 and HES-1 in thymocyte development JOURNAL OF IMMUNOLOGY Kaneta, M., Osawa, M., Osawa, M., Sudo, K., Nakauchi, H., Farr, A. G., Takahama, Y. 2000; 164 (1): 256-264
  • Isolation and characterization of CD34-low/negative mouse hematopoietic stem cells 5th Keio-University International Symposium for Life Sciences and Medicine Nakauchi, H., Osawa, M., Sudo, K., Ema, H. SPRINGER-VERLAG TOKYO. 2000: 95–103
  • Differential level in co-down-modulation of CD4 and CXCR4 primed by HIV-1 gp120 in response to phorbol ester, PMA, among HIV-1 isolates MICROBIOLOGY AND IMMUNOLOGY Tahara-Hanaoka, S., Ushijima, Y., Tarui, H., Wada, M., Hara, T., Imanishi, S., Yamaguchi, T., Hattori, T., Nakauchi, H., Koito, A. 2000; 44 (6): 489-498


    HIV-1 enters cells through interacting with cell surface molecules such as CD4 and chemokine receptors. We generated recombinant soluble gp120s derived from T-cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) HIV-1 strains using a baculovirus expression system and investigated the association of CD4-gp120 complex with the chemokine receptor and/or other surface molecule(s). For monitoring the co-down-modulations of the CD4-gp120 complex, a cytoplasmic domain deletion mutant (tailless CD4), which is not capable of undergoing down-modulation by itself in response to phorbol ester PMA, was used. Our studies revealed both cell-type and HIV-1 strain-specific differences. We found that T-tropic gp120s were capable of priming co-down-modulation with tailless CD4 by interacting with CXCR4, whereas M-tropic SF162 gp120 could not after PMA treatment even in the presence of CCR5. Among the T-tropic HIV-1 envelopes, IIIB gp120 was the most potent. Furthermore, the ability of gp120 to prime the PMA induced co-down-modulation of tailless CD4 appeared to be dependent on the concentration of the principal coreceptor CXCR4. Nevertheless, the observation that IIIB gp120 strongly primed tailless CD4 co-down-modulation on human osteosarcoma HOS cells that express undetectable levels of surface CXCR4 raised the possibility that membrane component(s) other than those recently identified can be involved in down-modulation of the CD4/gp120 complexes.

    View details for Web of Science ID 000087433800009

    View details for PubMedID 10941932

  • Physical and functional association of LFA-1 with DNAM-1 adhesion molecule IMMUNITY Shibuya, K., Lanier, L. L., Phillips, J. H., Ochs, H. D., Shimizu, K., Nakayama, E., Nakauchi, H., Shibuya, A. 1999; 11 (5): 615-623


    Whereas ligation of the DNAM-1 adhesion molecule triggers cytotoxicity mediated by normal NK and T cells, this function was defective in NK cell clones from leukocyte adhesion deficiency syndrome. However, genetic reconstitution of cell surface expression of LFA-1 restored the ability of DNAM-1 to initiate anti-DNAM-1 mAb-induced cytotoxicity, indicating a functional relationship between DNAM-1 and LFA-1. Further studies demonstrated that LFA-1 physically associates with DNAM-1 in NK cells and anti-CD3 mAb stimulated T cells, for which serine phosphorylation of DNAM-1 plays a critical role. In addition, cross-linking of LFA-1 induces tyrosine phosphorylation of DNAM-1, for which the Fyn protein tyrosine kinase is responsible. These results indicate that DNAM-1 is involved in the LFA-1-mediated intracellular signals.

    View details for Web of Science ID 000083952200013

    View details for PubMedID 10591186

  • Microchimerism in Japanese women patients with systemic sclerosis LANCET Murata, H., Nakauchi, H., Sumida, T. 1999; 354 (9174): 220-220


    To examine whether microchimerism occurs in Japanese women patients with systemic sclerosis, we analysed the Y-chromosome in DNA by PCR. There was no significant difference between patients and healthy people, suggesting that microchimerism may not be the sole pathogenesis in Japanese women with systemic sclerosis.

    View details for Web of Science ID 000081504200018

    View details for PubMedID 10421308

  • Single-cell analysis of mitochondrial DNA in patients and a carrier of the tRNA(Leu(UUR)) gene mutation JOURNAL OF INHERITED METABOLIC DISEASE Saitoh, S., Momoi, M. Y., Yamagata, T., Nakauchi, H., Nihei, K., Fujii, M. 1999; 22 (5): 608-614


    We examined heteroplasmy of mutated mitochondrial DNA in single peripheral lymphocytes derived from 4 individuals carrying the nt 3243 A-to-G mutation, including two patients with MELAS, a patient with cardiomyopathy, deafness and diabetes mellitus, and the asymptomatic mother of one of the MELAS patients. In these subjects, all lymphocytes examined were heteroplasmic to different degrees, with a wider range of heteroplasmy evident in the symptomatic patients than in the healthy carrier.

    View details for Web of Science ID 000080750600005

    View details for PubMedID 10399093

  • Murine natural killer cells contribute to the granulomatous reaction caused by mycobacterial cell walls PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Apostolou, I., Takahama, Y., Belmant, C., Kawano, T., Huerre, M., Marchal, G., Cui, J., Taniguchi, M., Nakauchi, H., Fournie, J. J., Kourilsky, P., Gachelin, G. 1999; 96 (9): 5141-5146


    Mice injected with deproteinized cell walls prepared from the strain H37rv of Mycobacterium tuberculosis develop a granuloma-like lesion in which NKT cells are predominant. NKT cells play a primary role in the granulomatous response, because the latter does not occur in Jalpha281(-/-) mice, which miss NKT cells. The glycolipidic fraction of the cell walls is responsible for the recruitment of NKT cells; the recruiting activity is associated with fractions containing phosphatidylinositolmannosides. These results define a powerful experimental set up for studying the in vivo induction of NKT cell responses to microbial components.

    View details for Web of Science ID 000080130200074

    View details for PubMedID 10220432

  • A novel stromal cell-dependent B lymphoid stem-like cell line that induces immunoglobulin gene rearrangement JOURNAL OF BIOCHEMISTRY Matsuda, K., Koguma, M., Okuyama, R., Nakazawa, T., Matsuzaki, Y., Nakauchi, H., Yanai, N., Terasaki, T., Obinata, M. 1999; 125 (3): 602-612


    A stroma-dependent B lymphoid cell line (B31-1) has been established by coculturing sorted stem cells on a novel bone marrow stromal cell line (TBR31-1). B31-1 cells express B220, but do not express other B lymphoid differentiation markers including CD43, heat stable antigen (HSA), or surface immunoglobulin (Ig) M (sIgM), and their Ig heavy chain (IgH) gene loci are germ-line in configuration. The addition of interleukin (IL)-7 or coculture with another stromal cell line, ST2, induces D-J rearrangement of the IgH gene and B lymphocyte differentiation markers. B31-1 cells restore an in vivo repopulation activity to lethally irradiated mice, and the repopulated cells differentiate to HSA+ pre-B cells.Continuous coculture results in two distinct populations, B220(-) c-Kit+ cells and B220(+) c-Kit+ cells; B220(-) c-Kit+ cells are self-renewed and differentiate to B220(+) c-Kit+ cells, while B220(+) c-Kit+ cells produce only B220(+) c-Kit+ cells. Both B220(-) and B220(+) cells similarly express the IgH germ-line transcript (Imu), mRNAs for recombinase (TdT, Rag-1, and Rag-2), and lymphoid-specific transcription factors (Pax-5, EBF, E12/E47, Oct-2, and Ikaros), but the DNA binding activity of Pax-5, EBF, Oct-2, and E2A are low in B220(-) cells and while high in B220(+) cells. These results suggest the existence of at least two active states in the IgH locus before the induction of IgH gene rearrangement during B lymphopoietic development.

    View details for Web of Science ID 000079234500023

    View details for PubMedID 10050050

  • Evidence for the presence of hepatic stem cells in the murine fetal liver XVIIth World Congress of the Transplantation-Society Taniguchi, H., Kondo, R., Suzuki, A., Zheng, Y., Ito, S., Takada, Y., Fukunaga, K., Seino, K., Yuzawa, K., Otsuka, M., Fukao, K., Yoshiki, A., Kusakabe, M., Nakauchi, H. ELSEVIER SCIENCE INC. 1999: 454–54

    View details for Web of Science ID 000078960600195

    View details for PubMedID 10083186

  • Human immunodeficiency virus type 1 Vpr modifies cell proliferation via multiple pathways MICROBIOLOGY AND IMMUNOLOGY Yamaguchi, T., Watanabe, N., Nakauchi, H., Koito, A. 1999; 43 (5): 437-447


    Vpr, one of the accessory molecules of HIV-1, has been demonstrated to arrest the cell cycle at the G2 phase. This Vpr-mediated cell cycle arrest is implicated to have an important role in the viral life cycle. In the present study, we quantitate the extent of Vpr-mediated cell cycle arrest with the use of a bicistronic vector consisting of a vpr gene and a green fluorescence protein sequence. Using this system, we examined the effect of several Vprs on cell cycle progression and growth of cells from different species quantitatively. We found that Vpr from the T-cell line-adapted HIV-1SF2 strain (Vpr2) could not significantly induce G2 arrest in HeLa cells but was able to induce it in 293T cells. However, strong inhibition of cell proliferation in HeLa cells as well as in 293T cells was observed by Vpr2. This ability of Vpr2 to inhibit cell proliferation without G2 arrest was also observed when expressed in monkey cell line. Analyses of chimeric Vprs revealed that this species-non-specific growth inhibitory activity of Vpr was not mediated solely by the C-terminal region of Vpr. These results indicated that the growth inhibitory activity of Vpr is independent of its G2 arresting activity. In addition, the species-non-specific nature of this activity suggests that Vpr has a novel mechanism to retard cell proliferation by influencing basic cellular functions.

    View details for Web of Science ID 000080239600007

    View details for PubMedID 10449250

  • Further characterization of CD34-low/negative mouse hematopoietic stem cells 2nd International Symposium and Workshop on Hematopoietic Stem Cells Nakauchi, H., Takano, H., Ema, H., Osawa, M. NEW YORK ACAD SCIENCES. 1999: 57–70


    We have previously reported that in adult mouse bone marrow, CD34low/- c-kit+ Sca-1+ lineage markers negative (Lin-) (CD34-KSL) cells represent hematopoietic stem cells with long-term marrow repopulating ability whereas CD34+ c-kit+ Sca-1+ Lin- (CD34+KSL) cells are progenitors with short-term reconstitution capacity. To further characterize cells in those two populations, relative expression of various genes were examined by reverse transcriptase polymerase chain reaction (RT-PCR). In CD34-KSL cells, none of the genes studied was found to be expressed with the exception of GATA-2, IL-1R alpha, IL-2R gamma, AIC-2B, c-kit, EPO-R, and c-mpl. In contrast, expression of GATA-1 and all cytokine receptor genes examined except IL-2R beta, IL-7R alpha and IL-9R alpha were found in CD34+KSL. The difference between these two populations was also shown in single cell culture analysis of these cells. When cells were clone-sorted and cultured in the presence of SCF, IL-3 and EPO, CD34-KSL cells required much more time to undergo the first cell division than CD34+KSL cells. Dormancy and random fashion of cell division by CD34-KSL cells were also evident by the analysis of the second cell division, which was found to be delayed and unsynchronous compared with CD34+KSL cells. Clonal culture analysis showed that CD34-KSL cells were more potent in proliferation and multilineage differentiation capacities than CD34+KSL cells. In a paired-daughter cell experiment, 75% of CD34-KSL and 50% of CD34+KSL paired-daughter-derived colonies were nonidentical with wide variety of lineage combinations. Taken together, these data support our previous notion that CD34-KSL cells are at higher rank in hematopoietic hierarchy than CD34+KSL cells. In addition, our results using highly enriched stem cell population directly obtained from mouse bone marrow support the proposed stochastic nature of lineage commitment.

    View details for Web of Science ID 000081273400007

    View details for PubMedID 10372111

  • Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells STEM CELLS Kim, D. K., Fujiki, Y., Fukushima, T., Ema, H., Shibuya, A., Nakauchi, H. 1999; 17 (5): 286-294


    Although the hematopoietic activities of human CD34+ bone marrow (BM) and cord blood (CB) cells have been well characterized, the phenotype of nonobese-diabetic severe combined immunodeficient (NOD/SCID) mice repopulating cells (SRCs) in CB and BM has not yet been fully examined. To address this issue, various hematopoietic activities were compared in terms of total and CD34+ CB and BM cells. Clonal culture of fluorescence-activated cell sorter (FACS) CD34+ CB and BM cells revealed a higher incidence of colony-forming cells with greater proliferation capacity in CB over BM CD34+ cells. CB CD34+ cells also demonstrated higher secondary plating efficiency over BM cells. In addition, we demonstrated that mice transplanted with CB mononuclear cells (MNCs) showed significantly higher levels of chimerism than those transplanted with BM MNCs. However, recipients of FACS-sorted CD34+ CB cells showed significantly lower levels of chimerism than those that received total CB MNCs, suggesting a role of facilitating cells in the CD34- cell population. To further analyze the role of CD34- cells, the NOD/SCID repopulating ability of FACS-sorted CB CD34-c-kit+Lin- and CD34-c-kit-Lin- cells were examined. However, SRCs were not detected in those cells. Taken together, these data suggest that CB is a better source of hematopoietic stem cells and that there are cells in the CD34- fraction that facilitate repopulation of hematopoiesis in the NOD/SCID environment.

    View details for Web of Science ID 000083022600005

    View details for PubMedID 10527463

  • A mouse carrying genetic defect in the choice between T and B lymphocytes JOURNAL OF IMMUNOLOGY Tokoro, Y., Sugawara, T., Yaginuma, H., Nakauchi, H., TERHORST, C., Wang, B. P., Takahama, Y. 1998; 161 (9): 4591-4598


    Transgenic mice with human CD3epsilon gene have been shown to exhibit early arrest of T cell development in the thymus. The present study shows that, instead of T cells, B cells are generated in the thymus of a line, tg epsilon26, of the human CD3epsilon transgenic mice. The accumulation of mature B cells in the thymus was found only in tg epsilon26 mice, not in other human CD3epsilon transgenic mouse lines or other T cell-deficient mice, including CD3-epsilon knockout mice and TCR-beta/TCR-delta double knockout mice. Hanging drop-mediated transfer into 2-deoxyguanosine-treated thymus lobes showed that lymphoid progenitor cells rather than thymus stromal cells were responsible for abnormal B cell development in tg epsilon26 thymus, and that tg epsilon26 fetal liver cells were destined to become B cells in normal thymus even in the presence of normal progenitor cells undergoing T cell development. These results indicate that lymphoid progenitor cells in tg epsilon26 mice are genetically defective in thymic choice between T cells and B cells, generating B cells even in normal thymus environment. Interestingly, tg epsilon26 thymocytes expressed GATA-3 and TCF-1, but not LEF-1 and PEBP-2alpha, among T cell-specific transcription factors that are involved in early T cell development, indicating that GATA-3 and TCF-1 expressed during thymocyte development do not necessarily determine the cell fate into T cell lineage. Thus, tg epsilon26 mice provide a novel mouse model in that lineage choice between T and B lymphocytes is genetically defective.

    View details for Web of Science ID 000076581100021

    View details for PubMedID 9794386

  • Is donor-derived long-term and multilineage hematopoiesis established after liver transplantation? 5th Congress of the Asian-Society-of-Transplantation (CAST) Taniguchi, H., Sugioka, A., Morita, M., Hasumi, A., Takada, Y., Fukunaga, K., Yuzawa, K., Otsuka, M., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 1998: 3865–66

    View details for Web of Science ID 000077128200410

    View details for PubMedID 9838691

  • Impaired erythropoiesis in transgenic mice overexpressing a truncated erythropoietin receptor EXPERIMENTAL HEMATOLOGY Nakamura, Y., Takano, H., Osawa, M., Tomita, T., Kim, D. K., Kojima, M., Motohashi, T., Miyoshi, S., Hiroyama, T., Tokumoto, Y., Nakauchi, H. 1998; 26 (12): 1105-1110


    Erythropoietin (EPO), one of the pivotal regulators of erythrocyte production, transmits signals through the EPO receptor (EPOR). We have previously reported that human bone marrow (BM) cells express two dominant forms of the EPOR, one full-length and one truncated (EPOR-F and EPOR-T). Experiments with a cell line have shown that the EPOR-T acts as a dominant-negative regulator of EPOR-F-mediated signals. Its role in erythropoiesis in vivo, however, has yet to be clarified. Here we show the presence in mouse BM of a truncated form of the EPOR that is essentially the same as EPOR-T in humans. To investigate its role in vivo, we generated transgenic mice overexpressing mouse EPOR-T (EPOR-T-Tg mice). As a result, two independent EPOR-T-Tg lines were established. One line revealed mild anemia, but another line did not. When anemia was induced experimentally in these mice, however, both lines showed apparently poor recovery resulting in higher mortality than wild-type control mice. The impaired erythropoiesis found in these mice thus strongly suggests the EPOR-T's role as a negative regulator of erythropoiesis in vivo.

    View details for Web of Science ID 000076783700001

    View details for PubMedID 9808048

  • Identification of Bach2 as a B-cell-specific partner for small Maf proteins that negatively regulate the immunoglobulin heavy chain gene 3 ' enhancer EMBO JOURNAL Muto, A., Hoshino, H., MADISEN, L., Yanai, N., Obinata, M., Karasuyama, H., Hayashi, M., Nakauchi, H., Yamamoto, M., Groudine, M., Igarashi, K. 1998; 17 (19): 5734-5743


    Maf family transcription factors are important regulators in various differentiation systems. Putative Maf recognition elements (MAREs) are found in the 3' enhancer region of the immunoglobulin heavy chain (IgH) gene. These elements are bound in B-cell extracts by a heterodimeric protein complex containing both Bach2 and a small Maf protein. Analysis of normal hematopoietic cells revealed that Bach2 is specifically expressed in B cells. Bach2 is abundantly expressed in the early stages of B-cell differentiation and turned off in terminally differentiated cells. Bach2 acts together with MafK as a negative effector of the IgH 3' enhancer and binds to the co-repressor SMRT (silencing mediator of retinoid and thyroid receptor). Hence the Bach2-small-Maf heterodimer may represent the first example of a B-cell lineage, and of a developmental stage-restricted negative effector of the MARE in the IgH 3' enhancer region.

    View details for Web of Science ID 000076463200023

    View details for PubMedID 9755173

  • An improved retroviral gene transfer technique demonstrates inhibition of CD4(-)CD8(-) thymocyte development by kinase-inactive ZAP-70 JOURNAL OF IMMUNOLOGY Sugawara, T., Di Bartolo, V., Miyazaki, T., Nakauchi, H., Acuto, O., Takahama, Y. 1998; 161 (6): 2888-2894


    ZAP-70 is a Syk family tyrosine kinase that plays an essential role in initiating TCR signals. Deficiency in ZAP-70 causes a defect in the development at CD4+CD8+ thymocytes due to defective TCR-mediated positive and negative selection. Using a newly devised retrovirus gene transfer and an efficient green fluorescence protein detection technique in fetal thymus organ cultures, the present study shows that forced expression in developing thymocytes of a catalytically inactive mutant of ZAP-70, but not wild-type ZAP-70, inhibits T cell development at the earlier CD4-CD8- stage. The ZAP-70 mutant blocked the generation of CD4+CD8+ thymocytes even in the absence of endogenous ZAP-70. Thus, the present results demonstrate a novel technique for gene transfer into developing T cells and suggest that ZAP-70/Syk family tyrosine kinases are involved in the signals inducing the generation of CD4+CD8+ thymocytes.

    View details for Web of Science ID 000075864600030

    View details for PubMedID 9743350

  • Hematopoietic stem cells: Are they CD34-positive or CD34-negative? NATURE MEDICINE Nakauchi, H. 1998; 4 (9): 1009-1010

    View details for Web of Science ID 000075804100030

    View details for PubMedID 9734390

  • Engraftment of human myelodysplastic syndrome derived cell line in transgenic severe combined immunodeficient (TG-SCID) mice expressing human GM-CSF and IL-3 EUROPEAN JOURNAL OF HAEMATOLOGY Kim, D. K., Kojima, M., Fukushima, T., Miyasaka, M., Nakauchi, H. 1998; 61 (2): 93-99


    A transgenic SCID (TG-SCID) mouse expressing the human cytokines interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been generated with the aim of making a model system allowing the in vivo proliferation of human hematopoietic cells. Using TG-SCID mice expressing high levels (30-35 ng/ml in the serum) of human GM-CSF and IL-3, we attempted to engraft a human myeloid leukemia cell line, F-36P, derived from a myelodysplastic syndrome (MDS) patient. When F-36P cells were transferred intravenously into sublethally irradiated TG-SCID mice, extensive proliferation of F-36P cells was found 4-6 wk later. Successful engraftment, however, required the preadministration of a monoclonal antibody to mouse interleukin-2 receptor (IL-2R) beta chain, neutralizing NK activity. Surprisingly, all the transplanted TG-SCID mice engrafted with F-36P cells developed hind leg paralysis approximately 6 wk after transfer. Histological analysis demonstrated extensive invasion and formation of osteolytic lesions by the F-36P cells in the vertebrae. These data indicate that transgenic SCID mice expressing human IL-3 and GM-CSF provide a useful system for the study of human leukemia. In addition, NK cells appear to play an important role in rejection of human cells.

    View details for Web of Science ID 000075129400004

    View details for PubMedID 9714520

  • Multivalent DNA binding complex generated by small Maf and Bach1 as a possible biochemical basis for beta-globin locus control region complex JOURNAL OF BIOLOGICAL CHEMISTRY Igarashi, K., Hoshino, H., Muto, A., Suwabe, N., Nishikawa, S., Nakauchi, H., Yamamoto, M. 1998; 273 (19): 11783-11790


    The human beta-globin locus control region (LCR) is required to properly regulate chromatin domain opening, replication timing, and globin gene activation. The LCR contains multiple NF-E2 sites (Maf recognition elements, MAREs) that allow the binding of various basic leucine zipper (bZip) proteins like p45 NF-E2, Nrf1, Nrf2, Bach1, and Bach2, in some cases as obligate heterodimers with a small Maf protein. In addition to the bZip domain, the Bach proteins bear a BTB/POZ domain, which has been implicated in the regulation of chromatin structure. We show here that Bach1 is highly expressed in hematopoietic cells and constitutes one of the two MARE-binding activities in murine erythroleukemic (MEL) cells. We further demonstrate that Bach1/MafK heterodimers interact with each other through the BTB domain, generating a multimeric and multivalent DNA binding complex. These results strongly implicate Bach1/MafK heterodimer as an architectural transcription factor that mediates interactions among multiple MAREs. Such a factor could then provide a model for assembly of the theoretical beta-globin LCR "holocomplex. " Other BTB domain proteins have already been demonstrated to be involved in remodeling chromatin, and thus this class of proteins likely promote the formation of nucleoprotein complexes required to establish the architecture of regulatory domains.

    View details for Web of Science ID 000073536700056

    View details for PubMedID 9565602

  • Multicolor flow-cytometric, morphologic, and clonogenic analysis of marrow CD10-positive cells in children with leukemia in remission or nonmalignant diseases JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY Fukushima, T., Sumazaki, R., Koike, K., Tsuchida, M., Matsui, A., Nakauchi, H. 1998; 20 (3): 222-228


    To characterize nonleukemic CD10-positive cells in the marrows of children with leukemia in remission or benign conditions.Seventeen children with acute leukemia in complete remission, 12 with solid tumors, and 17 with benign blood diseases were included in this study. Bone marrow cells were analyzed by multicolor flow-cytometry and polymerase chain reaction (PCR) to detect immunoglobulin gene rearrangement. The CD10-positive cells were purely sorted and examined by light microscopy and single cell hemopoietic progenitor assay.In patients with acute leukemia, CD10-positive cells were present in higher proportion after completion of therapy than during chemotherapy. They were also higher in the patients of preschool age than in the older age group with benign blood diseases and solid tumors. These CD10-positive cells were morphologically compatible with immature lymphocytes but some blast-like cells also occurred in this population. Most CD10-positive cells coexpressed CD19 and HLA-DR, although only 10 to 30% coexpressed CD20 and CD34. Although some CD10-positive cells expressed CD34, they did not make any colonies. PCR analysis did not show monoclonal bands in CD10-positive bone marrow cells in any patients in remission.Marrow CD10-positive cells possess immature B-lymphocyte phenotype and are present in higher proportion in the marrows of children with acute leukemia in continuous complete remission after completion of therapy and children of preschool age than school-age children with benign diseases or solid tumors without marrow involvement. The clonality of these cells was excluded by PCR, which is a distinct point from CD10-positive ALL blasts.

    View details for Web of Science ID 000074032300007

    View details for PubMedID 9628433

  • Expression and function of c-Met, a receptor for hepatocyte growth factor, during T-cell development SCANDINAVIAN JOURNAL OF IMMUNOLOGY Tamura, S., Sugawara, T., Tokoro, Y., Taniguchi, H., Fukao, K., Nakauchi, H., Takahama, Y. 1998; 47 (4): 296-301


    The c-Met oncoprotein is a cell-surface receptor for hepatocyte growth factor (HGF). Signals through HGF and c-Met have been appreciated for their crucial roles in the development of many cell types, including liver cells. The present study examined whether c-Met is expressed in the thymus and whether c-Met/HGF signals can regulate T-cell development in the thymus. We have found that mRNA transcripts encoding c-Met are expressed in mouse thymus. The c-Met transcripts were expressed at higher levels in fetal and neonatal thymus than in adult thymus, and were mostly expressed by lymphoid cells rather than by stromal cells. Interestingly, the addition of HGF to fetal thymus organ cultures increased the generation of mature T cells expressing high levels of T-cell antigen receptors. These results indicate that c-Met is expressed in the thymus during early ontogeny, and that c-Met/HGF signals can promote T-cell development.

    View details for Web of Science ID 000073124000002

    View details for PubMedID 9600310

  • Alternative promoters regulate transcription of the mouse GATA-2 gene JOURNAL OF BIOLOGICAL CHEMISTRY Minegishi, N., Ohta, J., Suwabe, N., Nakauchi, H., Ishihara, H., Hayashi, N., Yamamoto, M. 1998; 273 (6): 3625-3634


    Transcription factor GATA-2 has been shown to be a key regulator in hematopoietic progenitor cells. To elucidate how the expression of the GATA-2 gene is controlled, we isolated the mouse GATA-2 (mGATA-2) gene. Transcription of mGATA-2 mRNAs was found to initiate from two distinct first exons, both of which encode entirely untranslated regions, while the remaining five exons are shared by each of the two divergent mRNAs. Reverse transcriptase-polymerase chain reaction analysis revealed that GATA-2 mRNA initiated at the upstream first exon (IS) in Sca-1+/c-kit+ hematopoietic progenitor cells, whereas mRNA that initiates at the downstream first exon (IG) is expressed in all tissues and cell lines that express GATA-2. While the structure of the IG exon/promoter shows high similarity to those of the Xenopus and human GATA-2 genes, the IS exon/promoter has not been described previously. When we examined the regulation contributing to IS transcription using transient transfection assays, we found that sequences lying between -79 and -61 are critical for the cell type-specific activity of the IS promoter. DNase I footprinting experiments and electrophoretic mobility shift assays demonstrated the binding of transcription factors to this region. These data indicate that the proximal 80 base pair region of IS promoter is important for the generation of cell type-specific expression of mGATA-2 from the IS exon.

    View details for Web of Science ID 000071822300076

    View details for PubMedID 9452491

  • Pertussis toxin can replace T cell receptor signals that induce positive selection of CD8 T cells EUROPEAN JOURNAL OF IMMUNOLOGY Takahama, Y., Tokoro, Y., Sugawara, T., Negishi, I., Nakauchi, H. 1997; 27 (12): 3318-3331


    CD4+ helper T lymphocytes and CD8+ killer T lymphocytes are both generated in the thymus from common precursor cells expressing CD4 and CD8. The development of immature CD4 CD8+ thymocytes into mature 'single-positive' T cells requires T cell antigen-receptor (TCR)-mediated positive selection signals. Although it is known that the recognition specificity of TCR expressed by CD4+ CD8+ thymocytes determines their fate to become either CD4+ or CD8+ T cells, the molecular signals that direct precursor thymocytes to become CD4+ and CD8+ T cells are unclear. By using ZAP-70 mutant thymus organ cultures in which T cell development is arrested at the CD4+ CD8+ thymocyte stage, the present study shows that distinct biochemical treatments can selectively restore the generation of mature CD4+ and CD8+ T cells, bypassing TCR-induced positive selection signals. The combination of phorbol ester and ionomycin selectively restores the generation of CD4+ CD8- TCR(high) cells, consistent with previous results. On the other hand, we find that the generation of CD4- CD8+ TCR(high) cells is selectively induced by pertussis toxin. Interestingly, the signals generated by pertussis toxin, which increase Notch expression, can dominate the signals by phorbol ester and ionomycin, steering thymocyte development to CD8 lineage. These results indicate that distinct biochemical signals replace TCR signals that selectively induce positive selection of CD4+ and CD8+ T cells, and that biochemical treatment can manipulate the development and choice of CD4+ and CD8+ T cells.

    View details for Web of Science ID 000071409200030

    View details for PubMedID 9464820

  • Human immunodeficiency virus type 2 envelope glycoprotein binds to CD8 as well as to CD4 molecules on human T cells JOURNAL OF VIROLOGY Kaneko, H., Neoh, L. P., Takeda, N., Akimoto, H., Hishikawa, T., Hashimoto, H., Hirose, S., Karaki, S., Takiguchi, M., Nakauchi, H., Kaneko, Y., Yamamoto, N., Sekigawa, I. 1997; 71 (11): 8918-8922


    We report here that human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (gp105), but not HIV-1 gp120, can bind to CD8 molecules as well as to CD4 molecules on human T cells. This phenomenon may lead to differences in the life cycles of HIV-1 and HIV-2, and it may be related to the differences in disease manifestations of HIV-1 and HIV-2 infection, including longer survival of HIV-2-infected patients.

    View details for Web of Science ID A1997YB14300103

    View details for PubMedID 9343259

  • The proportion of fetal nucleated red blood cells in maternal blood: Estimation by FACS analysis PRENATAL DIAGNOSIS Sohda, S., Arinami, T., Hamada, H., Nakauchi, H., Hamaguchi, H., Kubo, T. 1997; 17 (8): 743-752


    The purpose of this study was to determine the proportion of fetal nucleated red blood cells (NRBCs) among enriched NRBCs and to evaluate the effectiveness of enriching NRBCs in maternal blood using fluorescence-activated cell sorting (FACS) to separate NRBCs. The origin of enriched NRBCs was determined using fluorescence in situ hybridization (FISH) methods. Y-specific signals were observed in 4.6 +/- 1.5 per cent of the enriched cells from 14 of 16 (87.5 per cent) pregnant women who gave birth to boys. In this series, the specificity of the fetal sex diagnosis was 100 per cent, the sensitivity 88 per cent, and the negative predictive value 86 per cent. Fetal NRBCs are present in maternal blood and FACS has the potential to enrich fetal NRBCs. Fetal cells were estimated to be enriched more than 10,000-fold in the first trimester and more than 100-fold in the third trimester. Average frequencies of fetal cells in maternal blood were 8.1 x 10(-5) and 1.6 x 10(-5) in the first trimester and the second/third trimesters. However, most of the NRBCs in maternal blood are maternal in origin.

    View details for Web of Science ID A1997XQ91500007

    View details for PubMedID 9267898

  • Constant delivery of proinsulin by encapsulation of transfected cells JOURNAL OF SURGICAL RESEARCH Taniguchi, H., Fukao, K., Nakauchi, H. 1997; 70 (1): 41-45


    Gene therapy is a potentially excellent approach for the treatment of diabetes instead of pancreas and islet transplantation. However, one difficulty involved in gene therapy for diabetes is a control of insulin/proinsulin production by the cells transfected with insulin cDNA. The purpose of this study is to examine whether control of the proliferation of transfected cells by encapsulation is a feasible approach for the constant delivery of proinsulin to avoid a life-threatening hypoglycemic state. Previously, we established a mouse fibroblast Ltk- cells transfected with human insulin cDNA producing human proinsulin (91 ng/24 hr/10(6) cells). These cells were encapsulated with semipermeable 5% agarose gel and proinsulin production was examined by in vitro long-term culture system. Intraperitoneal implantation into streptozocin (STZ)-induced diabetic mice was performed to investigate in vivo function of the encapsulated cells. The data from the in vitro study demonstrated that encapsulation of 2 x 10(6) transfectants enabled the stable production of proinsulin for over 80 days (204.4 +/- 5.18 ng/ml/day). Implantation of the encapsulated 2 x 10(7) transfectants improved the hyperglycemic state of diabetic mice for 30 days on the mean value of blood glucose concentration (n = 20). Histological analysis revealed pericapsular inflammation at 30 days after implantation and this may result in malfunction of encapsulated cells. Constant production and delivery of proinsulin could be achieved by encapsulating the human insulin cDNA-transfected cells using 5% agarose. Control of the proliferation of transfected cells appears to be an important factor for constant delivery of human proinsulin toward gene therapy of diabetes mellitus.

    View details for Web of Science ID A1997XL33700007

    View details for PubMedID 9228925

  • Isolation of a cDNA encoding a tyrosine kinase expressed in murine skin EXPERIMENTAL DERMATOLOGY Kawachi, Y., Nakauchi, H., Otsuka, E. 1997; 6 (3): 140-146


    Tyrosine phosphorylation is widely recognized as playing an important role in cell differentiation, proliferation and carcinogenesis. We used the polymerase chain reaction (PCR) method to identify protein tyrosine kinases that were expressed in the skin. Mixed oligonucleotide probes were used to amplify and screen neonatal murine skin mRNA for clones encoding amino acid contiguities, the conservation of which is characteristic of the protein tyrosine kinase family. When the PCR products were sequenced, a novel clone encoding protein tyrosine kinase, PTK70, was identified. A full-length cDNA was isolated from a mouse thymus cDNA library. The nucleotide and deduced amino acid sequence showed that it featured src-homology (SH) 2 domain, SH3 domain and kinase domain like other src family protein tyrosine kinases, but lacked the N-terminal myristylation site and C-terminal tyrosine residue. Although the mRNA of PTK70 was detected in various tissues ubiquitously, the degree of its expression differed among tissues. Murine skin is one in which PTK70 was expressed strongly, with its expression being much stronger in the epidermis and in the cell line derived from murine keratinocytes than in those from melanoma or fibroblast cell lines. These evidences suggest that PTK70 may be involved in proliferation or differentiation of keratinocytes in the skin.

    View details for Web of Science ID A1997XK97100006

    View details for PubMedID 9226137

  • In vivo analysis of Fas antigen-mediated apoptosis: Effects of agonistic anti-mouse Fas mAb on thymus, spleen and liver INTERNATIONAL IMMUNOLOGY Nishimura, Y., HIRABAYASHI, Y., Matsuzaki, Y., Musette, P., Ishii, A., Nakauchi, H., Inoue, T., Yonehara, S. 1997; 9 (2): 307-316


    Fas antigen (Fas/CD95) is a cell surface receptor protein that mediates apoptosis-inducing signals. To analyze the function of Fas in vivo, we examined the effects of agonistic anti-Fas antibodies in mice. The i.p. administration of the hamster anti-mouse Fas mAb, RK-8, which induced apoptosis both in vivo and in vitro, did not kill adult mice, whereas those given the another hamster anti-mouse Fas mAb, Jo2, rapidly died of fulminant hepatitis with hemorrhage. Histological analyses of mice given RK-8 indicated severe damage of the thymus, and moderate damage of the spleen and liver. Most of the thymocytes and some hepatocytes underwent apoptosis within 1 day of administration. Flow cytometry revealed that CD4+ T cells were more sensitive to Fas-mediated apoptosis than CD8+ T cells. At day 7 after administration, the thymus was atrophied. These in vivo effects of RK-8 were transient; the thymus was regenerated, and the liver and spleen were apparently normal 1 month after injection. The administration of RK-8 into newborn mice caused severe damage of the liver and thymus. Most of the hepatocytes died and jaundice was induced. The newborn mice died within 1 week. Most hepatocytes of newborn mice may be more sensitive to apoptosis-inducing signals through Fas than those of adult mice. These results indicated that functional Fas, which introduces the death signal in vivo, is expressed on thymocytes, CD4+ splenocytes, and some adult and most newborn mouse hepatocytes.

    View details for Web of Science ID A1997WJ65100011

    View details for PubMedID 9040012

  • Role of bcl-2 in the development of lymphoid cells from the hematopoietic stem cell BLOOD Matsuzaki, Y., Nakayama, K., Nakayama, K., Tomita, T., Isoda, M., Loh, D. Y., Nakauchi, H. 1997; 89 (3): 853-862


    To investigate the role of bcl-2 in lymphohematopoiesis, a long-term bone marrow reconstitution system was established. Transplantation of 1,000 c-Kit+ Sca-1+ and lineage markers negative cells from bcl-2-l-mouse bone marrow resulted in long-term reconstitution of nonlymphoid cells. However, T cells were totally absent and B-lymphocyte development was severely impaired at a very early stage of differentiation in the chimeric mouse. On the other hand, transplantation of day 14 fetal liver cells from bcl-2-l-mice resulted in generation of both T and B cells in the recipient, albeit transiently. These data suggest that bcl-2 plays a critical role in the development of lymphoid progenitor cells from the hematopoietic stem cell (HSC), but is not essential for the development of nonlymphoid cells and the self-renewal of HSC. In addition, lymphopoiesis from fetal liver HSC appears to be less dependent on bcl-2 than adult bone marrow HSC.

    View details for Web of Science ID A1997WG07300013

    View details for PubMedID 9028316

  • Characterization of hematopoietic stem cells in the adult liver XVI International Congress of the Transplantation-Society Taniguchi, H., Sugioka, A., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 1997: 1212–13

    View details for Web of Science ID A1997WM12700544

    View details for PubMedID 9123278

  • Extent of chimerism determines the mode of tolerance in mixed bone marrow chimeras XVI International Congress of the Transplantation-Society Taniguchi, H., Yuzawa, K., Takada, Y., Otsuka, M., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 1997: 1193–95

    View details for Web of Science ID A1997WM12700534

    View details for PubMedID 9123268

  • B220 expression by T lymphoid progenitor cells in mouse fetal liver JOURNAL OF IMMUNOLOGY Sagara, S., Sugaya, K., Tokoro, Y., Tanaka, S., Takano, H., Kodama, H., Nakauchi, H., Takahama, Y. 1997; 158 (2): 666-676


    The present study has characterized T lymphoid progenitor cells that reside in mouse fetal liver. Day 14 fetal liver contains progenitor cells that can differentiate into mature T cells upon being transferred into the thymus by hanging drop cultures. Fractionation of fetal liver cells indicated that T progenitor cells were confined in TER119- CD45+ FcR(low) cells. To our surprise, B220+ rather than B220- fraction in TER119- CD45+ FcR(low) fetal liver cells exhibited efficient progenitor activity generating T cells. Progenitor activity by the B220+ fetal liver cells was restricted to T cells, B cells, and macrophages at frequency approximately 1/10, approximately 1/10, and approximately 1/20, respectively, of isolated B220+ cells. B220+ fetal liver cells did not contain detectable D-J rearrangement of TCR-beta gene and were c-kit+ IL-7R+ Thy-1- CD3- CD4(low) CD8- CD25- CD44+. B220+ fetal liver cells expressed mRNAs encoding TCR-beta, pT alpha, Ig alpha, and VpreB. Interestingly, TCR beta-chains were expressed by B220+ fetal liver cells in the VDJ-rearranged TCR-beta-transgenic mice, indicating that TCR-beta transcription and B220 expression are activated simultaneously by the transgenic B220+ fetal liver cells. These results indicate that B220 is expressed by fetal liver lymphoid progenitor cells that can become T cells, and suggest that lymphoid progenitor cells in fetal liver concurrently undergo T- and B-specific molecular events within a single cell.

    View details for Web of Science ID A1997WC63800018

    View details for PubMedID 8992982

  • Control of proinsulin production by sense-anti-sense regulation in response to glucocorticoids 3rd Annual Meeting of the Society-for-Cell-Therapy Taniguchi, H., Hirochika, R., Fukao, K., Nakauchi, H. PERGAMON-ELSEVIER SCIENCE LTD. 1996: S55–S57


    One difficulty involved in gene therapy for diabetes is a control of proinsulin production by the cells transfected with insulin cDNA. The introduction of a feedback mechanism to control the expression of the introduced gene based on the host's need for insulin is one possible treatment approach. To control proinsulin production at a transcriptional level, we introduced a glucocorticoid responsive promoter in the 3' region of insulin cDNA in reverse orientation (pBCMGS-neo-Ins-invMMTV) so that antisense insulin mRNA is produced in response to glucocorticoids. When fibroblasts transfected with pBCMGS-neo-Ins-invMMTV were cultured with 1 x 10(-5) M dexamethasone, two of nine clones showed a 10-20% reduction in proinsulin production. On the other hand, all clones of the cells transfected with a control vector containing human insulin cDNA (pBCMGS-neo-Ins) showed an 20-80% increase of proinsulin production when cultured with dexamethazone because of the increase of protein synthesis by glucocorticoids. These data indicated that antisense insulin mRNA effectively suppressed the transcription of insulin cDNA in response to glucocorticoids. This sense-antisense regulation system may make it feasible to induce a feedback mechanism to control proinsulin based on the blood glucose concentration.

    View details for Web of Science ID A1996VK18900017

    View details for PubMedID 8889233

  • Phorbol ester and calcium ionophore can replace TCR signals that induce positive selection of CD4 T cells JOURNAL OF IMMUNOLOGY Takahama, Y., Nakauchi, H. 1996; 157 (4): 1508-1513


    Positive selection of immature thymocytes is a developmental process in which TCR ligation by low avidity interaction induces the generation of mature T cells. However, biochemical signals that can induce positive selection have been unclear. By using TCR-alpha beta- mutant thymus cultures, the present study shows that direct stimulation of intracellular signals by PMA and calcium ionophore ionomycin can induce the generation of mature CD4+8- T cells, bypassing TCR-induced positive selection signals. Interestingly, the concentrations of phorbol ester that induced positive selection were more restricted than those that induced mature T cell activation. Moreover, the combination of phorbol ester and ionomycin restored the generation of CD4+8- T cells in class II MHC- thymus cultures, but did not induce the generation of CD4-8+ T cells in class I MHC- thymus cultures. These results identify that the combination of protein kinase C activation and calcium elevation is the biochemical signal that can induce positive selection of CD4+ T cells.

    View details for Web of Science ID A1996VH12400023

    View details for PubMedID 8759732

  • Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell SCIENCE Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. 1996; 273 (5272): 242-245


    Hematopoietic stem cells (HSCs) supply all blood cells throughout life by making use of their self-renewal and multilineage differentiation capabilities. A monoclonal antibody raised to the mouse homolog of CD34 (mCD34) was used to purify mouse HSCs to near homogeneity. Unlike in humans, primitive adult mouse bone marrow HSCs were detected in the mCD34 low to negative fraction. Injection of a single mCD34(lo/-), c-Kit+, Sca-1(+), lineage markers negative (Lin-) cell resulted in long-term reconstitution of the lymphohematopoietic system in 21 percent of recipients. Thus, the purified HSC population should enable analysis of the self-renewal and multilineage differentiation of individual HSCs.

    View details for Web of Science ID A1996UW78700042

    View details for PubMedID 8662508

  • Conditions for the successful engraftment of hepatocyte progenitors injected into the spleen 4th International Congress of the Asian-Transplantation-Society Taniguchi, H., Yoshiki, A., Kusakabe, M., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 1996: 1855–56

    View details for Web of Science ID A1996UR78100317

    View details for PubMedID 8658916

  • Role of c-jun in the inhibition of erythropoietin receptor-mediated apoptosis BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Shimizu, R., Komatsu, N., Nakamura, Y., Nakauchi, H., Nakabeppu, Y., Miura, Y. 1996; 222 (1): 1-6


    Human bone marrow cells express both a truncated and full-length form of the erythropoietin receptor (EpoR-T and EpoR-F, respectively). Transfection experiments using the murine interleukin (IL)-3-dependent cell line, Ba/F3, revealed that the cells coexpressing EpoR-F and EpoR-T (Ba/F3-FT) were more likely to undergo programmed cell death (apoptosis) than cells expressing EpoR-F (Ba/F3-FF), even in the presence of erythropoietin (Epo). When Ba/F3-FF cells were stimulated with Epo or IL-3, rapid induction of c-myc, c-fos, c-jun and junB genes was observed. A similar effect was also seen in IL-3 stimulated Ba/F3-Ft cells. However, in Ba/F3-FT cells expression of the c-jun gene was not induced by Epo stimulation. Administration of Epo could prevent apoptosis induced by IL-3 deprivation in Ba/F3-FT cells expressing ectopic c-Jun protein. These results indicate that induction of c-Jun through the Epo signaling pathway has an important role in the inhibition of apoptosis.

    View details for Web of Science ID A1996UJ62500001

    View details for PubMedID 8630050

  • In vivo self-renewal of c-Kit(+) Sca-1(+) Lin(low/-) hemopoietic stem cells JOURNAL OF IMMUNOLOGY Osawa, M., Nakamura, K., Nishi, N., Takahashi, N., Tokuomoto, Y., Inoue, H., Nakauchi, H. 1996; 156 (9): 3207-3214


    The long term marrow-repopulating ability of mouse bone marrow c-Kit+ Sca-1+ Lin(low/-) cells was studied. Injection of as few as 100 c-Kit+ Sca-1+ Lin(low/-) cells could rescue a lethally irradiated recipient mouse and reconstitute both myeloid and lymphoid cells in their peripheral blood for at least 10 mo. When limiting dilution analysis was performed in the presence of host-derived rescue cells, as low as five c-Kit+ Sca-1+ Lin(low/-) cells were shown to be capable of repopulating lymphohemopoietic cells. Subsequently, we examined whether c-Kit+ Sca-1+ Lin(low/-) cells had a capacity for self-renewal in vivo. After transplantation of 500 c-Kit+ Sca-1+ Lin(low/-) Ly-5.1+ cells into lethally irradiated Ly-5 congenic mice, the expansion of cells with the same phenotypes as the injected cells was monitored. By day 28 post-transplantation, more than 50,000 donor type c-Kit+ Sca-1+ Lin(low/-) Ly-5.1+ cells were found in the spleen and over 18,000 cells were found in bone marrow. The expansion in spleen, however, was transient in that by day 60 cells of the donor phenotype were found only in bone marrow. The c-Kit+ Sca-1+ Lin(low/-) LY-5.1+ cells expanded in spleen or bone marrow contained as many high proliferative potential colony-forming cells as the originally injected cells. They also contained cells with LTRA, but the frequency appeared to be less compared with naive c-Kit+ Sca-1+ Lin(low/-) cells. These data provide evidence that c-Kit+ Sca-1+ Lin(low/-) cells in bone marrow are capable of multilineage differentiation as well as self-renewal and that spleen is a primary site of stem cell expansion after transplantation.

    View details for Web of Science ID A1996UF74200016

    View details for PubMedID 8617942

  • IL-7 supports D-J but not V-DJ rearrangement of TCR-beta gene in fetal liver progenitor cells JOURNAL OF IMMUNOLOGY Tsuda, S., Rieke, S., Hashimoto, Y., Nakauchi, H., Takahama, Y. 1996; 156 (9): 3233-3242


    The rearrangement of TCR-beta gene, one of the earliest events in T cell development, consists of two consecutive steps: D-J rearrangement and V-DJ rearrangement. The present study examined the signals supporting D-J beta and V-DJ beta rearrangements during early T cell development from progenitor cells that reside in fetal liver. We have found that there is an interval of 1 to 2 days between D-J beta and V-DJ beta rearrangements during the early T cell development from fetal liver progenitor cells in deoxyguanosine-treated thymus lobes. We have also found that IL-7, a cytokine expressed in the subcapsular area of the thymus, can promote D-J beta rearrangement of fetal liver progenitor cells, and that anti-IL-7 and anti-IL-7R Abs inhibit the D-J beta rearrangement and further T cell development of fetal liver progenitor cells in the thymus environment. Interestingly, unlike the thymus environment, IL-7 alone was not capable of supporting V-DJ beta rearrangement in the fetal liver cell cultures. These results indicate that D-J beta rearrangement during fetal liver-derived early T cell development is supported in the thymus by IL-7. Furthermore, the present results demonstrate that IL-7, supporting D-J beta rearrangement, does not promote V-DJ beta rearrangement of fetal liver progenitor cells, suggesting that intrathymic molecules promoting V-DJ beta rearrangement are distinct from IL-7 that supports the D-J beta rearrangement.

    View details for Web of Science ID A1996UF74200019

    View details for PubMedID 8617945

  • Apoptosis during an early stage of nephrogenesis induces renal hypoplasia in bcl-2-deficient mice AMERICAN JOURNAL OF PATHOLOGY Nagata, M., Nakauchi, H., Nakayama, K. I., Nakayama, K., Loh, D., Watanabe, T. 1996; 148 (5): 1601-1611


    Renal development in bcl-2-deficient mice was monitored to examine the temporal and spatial function of this gene during nephrogenesis in vivo. Extensive apoptosis occurred during abnormal nephrogenesis in bcl-2-deficient mice. In embryos and newborn mice, the sequence of morphological events was monitored by morphology in conjunction with morphometry, and bcl-2 -/-, bcl-2 +/-, and bcl-2 +/+ mice were compared. In bcl-2 -/- mice, initial induction of nephrons was detected by embryonic day 13 (E-13) as normal. Then, apoptotic cells became five times more frequent at E-13 to E-16 with a significant reduction (1/5) in nephron number at E-17 to E-19 in bcl-2 -/- mice compared with bcl-2 +/+ mice. No morphological difference was evident between bcl-2 +/- mice and bcl-2 +/+ mice by morphometry. Apoptotic cells were found mainly among the mesenchyme and less frequently in tubuli. Little apoptosis among ureteric buds was noted. In bcl-2 -/- mice at E-17 to E-19, inactive branching and insufficient convolution of ureteric buds were accompanied by fulminant apoptosis in the mesenchyme. Neonatal bcl-2 -/- mice lacked the nephrogenic zone, exhibiting renal hypoplasia. Thus, bcl-2 seems to inhibit apoptosis in renal stem cells during the induction of nephrons in vivo.

    View details for Web of Science ID A1996UJ47300027

    View details for PubMedID 8623928

  • CD3-induced apoptosis of CD4(+)CD8(+) thymocytes in the absence of clonotypic T cell antigen receptor EUROPEAN JOURNAL OF IMMUNOLOGY Tokoro, Y., Tsuda, S., Tanaka, S., Nakauchi, H., Takahama, Y. 1996; 26 (5): 1012-1017


    Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4+CD8+ double positive thymocyte stage. Immature CD4+CD8+ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4+CD8+ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4+CD8+ thymocytes can occur even in TCR alpha- mutant mice which do not express the TCR alpha beta/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4+CD8+ thymocytes in TCR alpha- mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4+CD8+ thymocytes from TCR alpha- mice are not associated with clonotypic TCR chains, including TCR beta. Thus, apoptosis of CD4+CD8+ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4+CD8+ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.

    View details for Web of Science ID A1996UT53100008

    View details for PubMedID 8647161

  • Long-term and multilineage bone marrow reconstitution in normal untreated recipients TRANSPLANTATION PROCEEDINGS Taniguchi, H., Yuzawa, K., Otsuka, M., Fukao, K., Nakauchi, H. 1996; 28 (2): 1042-1044

    View details for Web of Science ID A1996UG38300264

    View details for PubMedID 8623221

  • Loss of ganciclovir sensitivity by exclusion of thymidine kinase gene from transplanted proinsulin-producing fibroblasts as a gene therapy model for diabetes GENE THERAPY Yoshimoto, K., Murakami, R., Moritani, M., Ohta, M., Iwahana, H., Nakauchi, H., Itakura, M. 1996; 3 (3): 230-234


    To establish a practical method of somatic gene therapy, we aimed to develop a regulatory system at the cellular level using a suicide vector. We introduced the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene into the human proinsulin-producing Ltk-cells and examined whether ganciclovir (GCV) administration could control proinsulin production in vivo. The cells transfected with the HSV-tk gene showed more than 100-fold increase in sensitivity to GCV compared with the parent cells. Analysis of blood glucose in diabetic nude mice with transplanted cells showed that proinsulin production by these cells was strongly suppressed by GCV treatment in vivo as reflected by the reversal to hyperglycemia. However, in the in vivo experiment, the plasmid containing the HSV-tk gene was spontaneously lost from the transplanted cells in one of six cases resulting in the resistance to GCV as reflected by the persistent hypoglycemia and increased tumor size. This system of HSV-tk and administration of GCV may be applicable to gene therapy as a suicide vector, but the system of stable expression of the HSV-tk gene must be established.

    View details for Web of Science ID A1996UC65700007

    View details for PubMedID 8646554

  • Presence of hematopoietic stem cells in the adult liver NATURE MEDICINE Taniguchi, H., Toyoshima, T., Fukao, K., Nakauchi, H. 1996; 2 (2): 198-203


    Recently, cases have been reported in which a mixed chimeric state of blood cells is established after liver transplantation. Because the established chimerism may have aided in the induction of donor-specific tolerance, the mechanism responsible for this chimerism is of clinical importance. To establish this, we examined cells in adult mouse liver and identified the presence of c-kit+ Sca-1+ Lin(lo/-) cells. These cells were capable of forming in vivo as well as in vitro colonies. Furthermore, the cells could reconstitute bone marrow of lethally irradiated recipient mice for at least 12 months. These data obtained from the mouse study strongly suggest that hematopoietic stem cells residing in the donor liver are responsible for mixed chimerism and maintenance of tolerance after liver transplantation.

    View details for Web of Science ID A1996TU06000040

    View details for PubMedID 8574965

  • Role of a truncated erythropoietin receptor for erythroid differentiation BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Nakamura, Y., Tokumoto, Y., Nakauchi, H. 1996; 218 (1): 205-209


    Erythropoietin (EPO) is a cytokine that regulates erythropoiesis through the EPO receptor (EPOR). We reported previously that erythroid progenitor cells express both a full-length and a truncated form of EPOR (EPOR-F and EPOR-Tph). EPOR-Tph cannot transmit growth signals and acts as a dominant negative regulator against EPOR-F-mediated signals for cell survival and growth. Upon transfection of EPOR-F in a cell line, Ba/F3, beta-globin accumulation, which is considered to be a marker of erythroid-differentiation, is inducible in the transformants. We show here that the co-expression of EPOR-Tph in EPOR-F-transformants does not inhibit and rather upregulates the beta-globin induction while inhibiting survival and growth of the transformants. These data suggest that, in contrast to survival and growth signals, EPOR-Tph acts as a positive regulator for erythroid-differentiation signals in erythroid progenitor cells.

    View details for Web of Science ID A1996TP75400036

    View details for PubMedID 8573132

  • Characterization of the mouse CD8 beta chain-encoding gene promoter region IMMUNOGENETICS Kawachi, Y., Otsuka, F., Nakauchi, H. 1996; 44 (5): 358-365


    We identified a regulatory region of the mouse CD8 beta chain-encoding gene (CD8b) promoter. The CD8b 5' upstream sequence could not drive the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene without T-cell receptor or SV40 enhancer elements. The results of transient transfection assays indicated that the dominant transcription-activating element within the CD8b-promoter is located at -45 to -40 base pairs (CCGCCC) from the transcriptional initiation site. Elimination of this element, by deletion or specific point mutation, significantly reduced transcriptional activity from this promoter. The sequence of this core region corresponds to a GC box motif known to act as a binding site for a ubiquitously expressed transcriptional activator, Sp1. However, the promoter activity appeared to be T-cell-specific, and the gel retardation assay using the core sequence as a probe revealed formation of complexes with multiple nuclear factors, one of them being specific to T lineage cells. These data suggest that the CD8b promoter requires a cis-acting element as well as several nuclear factors for full-range, tissue-specific transcription.

    View details for Web of Science ID A1996VG28500005

    View details for PubMedID 8781121



    Although allogeneic bone marrow (BM) transplantation is an effective way to induce donor-specific tolerance, its clinical application is hampered because of the risk associated with vigorous myeloablative pretransplant conditioning. One approach to overcome this problem is to establish a lower chimeric state by mild myeloablation. It is not clear, however, whether there is a threshold in the extent of chimerism that is required for tolerance induction. In this study, we establish a mixed BM chimera system to examine the correlation between the reconstitution ratio of BM chimerism, donor-reactive T cell deletion, and skin graft acceptance using I-E alpha transgenic C57BL/6 mice as the BM and skin graft donors and Ly5 congenic C57BL/6 mice as the recipients. In this system, the class II MHC molecule I-E was the transplantation Ag, and the extent of I-E-reactive T cell deletion was determined by flow cytometry using a mAb specific for the V beta 11 TCR. The degree of BM chimerism was measured by examining the expression of donor-derived Ly5.2 and host-derived Ly5.1 on peripheral blood cells. Transplantation of I-E+ transgenic donor BM cells resulted in deletion of V beta 11+CD4+ T cells in recipient's PBL, and the extent of deletion was proportional to the degree of chimerism. When mice of different degrees of chimerism were tested for skin graft survival, we found that recipient mice with > 30% chimerism could accept skin grafts from I-E+ donor mice, whereas those with < 10% chimerism showed prolonged but not permanent graft survival. These findings revealed the sequence of events for induction of tolerance. First, the degree of BM chimerism determines the number of I-E+ cells in the thymus, which then elicits negative selection of I-E-reactive T cells in a form of clonal deletion. The extent of T cell deletion ultimately determines the mode of tolerance. These data provide experimental evidence for the potential use of partial chimerism by bone marrow transplantation for the induction of donor-specific tolerance in clinical settings.

    View details for Web of Science ID A1995TJ63100023

    View details for PubMedID 7499847

  • Developmental defects of lymphoid cells in Jak3 kinase-deficient mice IMMUNITY Park, S. Y., Saijo, K., Takahashi, T., Osawa, M., Arase, H., Hirayama, N., Miyake, K., Nakauchi, H., Shirasawa, T., Saito, T. 1995; 3 (6): 771-782


    Jak3 is a tyrosine kinase mediating cytokine receptor signaling through the association with the common gamma chain of the cytokine receptors such as IL-2, IL-4, IL-7, IL-9, and IL-15. Unlike other members of the Jak family, the expression of Jak3 is highly restricted in hematopoietic cells. To elucidate in vivo function of Jak3, Jak3-deficient mice were generated by homologous recombination. Mice homozygous for Jak3 null mutation showed severe defects, specifically in lymphoid cells. B cell precursors in bone marrow, thymocytes, and both T and B cells in the spleen drastically decreased, although these defects were significantly recovered as aging occurred. Peripheral lymph nodes, NK cells, dendritic epidermal T cells, and intestinal intraepithelial gamma delta T cells were absent. Normal number of hematopoietic stem cells in bone marrow from Jak3-deficient mice and the similar capability to generate myeloid and erythroid colonies as wild-type mice indicated specific defects in lymphoid stem cells. Furthermore, the abnormal architecture of lymphoid organs suggested the involvement of Jak3 in the function of epithelial cells. T cells developed in the mutant mice did not respond to either IL-2, IL-4, or IL-7. These findings establish the crucial role of Jak3 in the development of lymphoid cells.

    View details for Web of Science ID A1995TM02300011

    View details for PubMedID 8777722



    We have established a cell culture system without stromal cells that allows the CD34+ hematopoietic progenitor cells (HPC) to differentiate into natural killer (NK) cells. CD34+Lin (CD3, CD16, CD56)- cells were purified using fluorescence-activated cell sorting from normal adult bone marrow (BM) and cultured for 28 days in medium supplemented with interleukin-2 (IL-2) and stem cell factor (SCF). NK (CD3-CD16-CD56+) cells were generated in a dose-dependent manner in response to SCF. NK cells originated from CD34+CD33+Lin- cells, but they were barely detectable in cultures of CD34+CD33-Lin- cells. However, on addition of IL-3, an induced differentiation of NK cells from CD34+CD33-Lin- cells was observed, although at a lower frequency. Supplementing of the cell cultures with SCF alone or both SCF and IL-3 for the first 7 days followed by IL-2 for the next 21 days is essential for production of NK cells from CD34+CD33+Lin- cells and from CD34+CD33-Lin- cells, respectively. These data provide direct evidence that NK cells arise from CD34+HPC and show the minimum lymphokine requirement for their differentiation.

    View details for Web of Science ID A1995RD28800021

    View details for PubMedID 7540065

  • ACTIVITY AND EXPRESSION OF MURINE SMALL MAF FAMILY PROTEIN MAFK JOURNAL OF BIOLOGICAL CHEMISTRY Igarashi, K., Itoh, K., Motohashi, H., Hayashi, N., MATUZAKI, Y., Nakauchi, H., Nishizawa, M., Yamamoto, M. 1995; 270 (13): 7615-7624


    Transcription factor NF-E2 is believed to be crucial for the regulation of erythroid-specific gene transcription. The three small Maf family proteins (MafF, MafG, and MafK), which are closely related to c-Maf proto-oncoprotein, constitute half of NF-E2 activity by virtue of forming heterodimers with the large, tissue-restricted subunit of NF-E2 (p45). We isolated cDNA clones encoding the murine small Maf family protein MafK and characterized the structure, activity, and expression profile of MafK mRNA. Functional analyses demonstrate that MafK binds to consensus NF-E2 sites in the absence of p45 in vitro and represses transcription of NF-E2 site-dependent reporter genes in transient transfection assays, while p45 introduced into cells alone does not effectively bind to DNA and does not affect transcription. In the presence of p45, MafK confers site-specific DNA binding activity to p45, and p45 in turn mediates transcriptional activation with its amino-terminal proline-rich domain. mRNA for MafK is expressed in fractions enriched for hematopoietic stem cells as well as erythroid cells, suggesting that MafK plays an important regulatory role in hematopoiesis.

    View details for Web of Science ID A1995QQ43100087

    View details for PubMedID 7706310

  • EVIDENCE FOR THE PRESENCE OF HEMATOPOIETIC STEM-CELLS IN THE ADULT LIVER XVth World Congress of the Transplantation-Society Taniguchi, H., Toyoshima, T., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 1995: 196–99

    View details for Web of Science ID A1995QJ19900068

    View details for PubMedID 7533387

  • WT1 AS A NEW PROGNOSTIC FACTOR AND A NEW MARKER FOR THE DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE-LEUKEMIA BLOOD Inoue, K., Sugiyama, H., Ogawa, H., Nakagawa, M., Yamagami, T., Miwa, H., Kita, K., Hiraoka, A., Masaoka, T., Nasu, K., Kyo, T., Dohy, H., Nakauchi, H., Ishidate, T., Akiyama, T., Kishimoto, T. 1994; 84 (9): 3071-3079


    The WT1 gene encoding a zinc finger polypeptide is a tumor suppressor gene that plays a key role in the carcinogenesis of Wilms' tumor. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine relative levels of WT1 gene expression (defined in K562 cells as 1.00) in 45 patients with acute myelogenous leukemia (AML), 22 with acute lymphocytic leukemia (ALL), 6 with acute mixed lineage leukemia (AMLL), 23 with chronic myelogenous leukemia (CML), and 24 with non-Hodgkin's lymphoma. Significant levels of WT1 gene were expressed in all leukemia patients and for CML the levels increased as the clinical phase progressed. In striking contrast with acute leukemia, the levels of WT1 gene expression for NHL were significantly lower or even undetectable. Clear correlation was observed between the relative levels of WT1 gene expression (< 0.6 v > or = 0.6) and the prognosis for acute leukemia (AML, ALL, and AMLL). Patients with less than 0.6 levels had significantly higher rates of complete remission (CR), disease-free survival, and overall survival than those with > or = 0.6 levels, whereas CR could not be induced in any of the 7 patients with acute leukemia having greater than 1.0 levels of WT1 gene expression. The quantitation of the WT1 gene expression made it possible to detect minimal residual disease (MRD) in acute leukemia regardless of the presence or absence of tumor-specific DNA markers. Continuous monitoring of the WT1 mRNA was performed for 9 patients with acute leukemia. In 4 patients, MRD was detected 2 to 8 months before clinical relapse became apparent. In 2 other patients, the WT1 mRNA gradually increased after discontinuation of chemotherapy. No MRD was detected in the remaining 3 patients with AML who received intensive induction and consolidation therapy. Simultaneous monitoring of MRD by RT-PCR using primers for specific DNA markers in 3 patients (2 AML-M3 with PML/RAR alpha, and 1 AML-M2 with AML1/ETO) among these 9 patients detected MRD comparable with that obtained from quantitation of WT1 gene expression. In a patient with acute promyelocytic leukemia, the limits of leukemic cell detection by RT-PCR using either WT1 or promyelocytic leukemia/retinoic acid receptor-alpha gene primers were 10(-3) to 10(-4) and 10(-4) for bone marrow, and 10(-5) and 10(-4) for peripheral blood, respectively. Therefore, we conclude that WT1 is a new prognostic factor and a new marker for the detection of MRD in acute leukemia.

    View details for Web of Science ID A1994PP02100031

    View details for PubMedID 7949179



    The Rh blood group system was recognized as one of the most complex polymorphisms in human. Whether or not the Rh antigens are present on human leukocytes is still an unresolved question. The expression of the Rh gene at the mRNA was analyzed in purified populations of human leukocytes by using reverse transcription and polymerase chain reaction (RT-PCR) followed by Southern blot analysis. The PCR products of the 5'-terminal region showed a single band as expected in the CD19+TCR-1 and CD14+ cells but doubtfully in the CD13+CD71- and CD190-TCR-1+ cells. On the other hand, in all these cells, the PCR products of the 3' region exhibited multiple additional bands migrating ahead of the band as expected, which showed distinctly different sets of bands in each cell. The additional bands appeared to consist of RhPI- and RhPII-cDNA splicing isoforms. These results indicated that the expression of the Rh gene is not restricted to human erythroid lineage. Additionally, it was suggested that different transcription initiation sites might be utilized preferentially for Rh gene expression.

    View details for Web of Science ID A1994PB55300044

    View details for PubMedID 8060332

  • Identification of a novel cDNA clone encoding protein tyrosine kinase in murine skin. journal of dermatology Kawachi, Y., Nakauchi, H., Otsuka, F. 1994; 21 (8): 533-538


    Tyrosine phosphorylation is widely recognized as playing important roles in cell differentiation, proliferation, and carcinogenesis. We have used the polymerase chain reaction (PCR) method to identify protein tyrosine kinases that are expressed in the skin. Mixed oligonucleotide probes were used to amplify and screen a neonatal murine skin cDNA pool for clones encoding amino acid contiguities whose conservation is characteristic of the protein tyrosine kinase family. When the PCR products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named tks (for tyrosine kinase identified from skin). Sequence homology comparison showed that the tks gene is homologous to the src and fes/fps families. Northern blotting using PCR products of tks as a probe revealed that the mRNA of tks is detected ubiquitously and weakly in other tissues such as brain, lung, liver, thymus and kidney. This fact suggests that the tks gene is expressed in widely distributed cell types.

    View details for PubMedID 7962951

  • USE OF CYTOKINES FOR EFFICIENT INTRODUCTION OF FOREIGN GENES INTO THE HEMATOPOIETIC STEM-CELL 3rd Congress of the Asian-Society-of-Transplantation Taniguchi, H., Osawa, M., Watanabe, Y., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 1994: 1972–74

    View details for Web of Science ID A1994PB86700056

    View details for PubMedID 7520617

  • HEMATOPOIETIC STEM-CELL CHIMERISM - RELATIONSHIPS BETWEEN DEGREE OF CHIMERISM, T-CELL CLONAL DELETION, AND GRAFT-SURVIVAL 3rd Congress of the Asian-Society-of-Transplantation Taniguchi, H., Jinzenji, Y., Fukao, K., Nakauchi, H. ELSEVIER SCIENCE INC. 1994: 1966–68

    View details for Web of Science ID A1994PB86700053

    View details for PubMedID 8066636


    View details for Web of Science ID A1994NH01000044

    View details for PubMedID 8160019

  • GLYCOSPHINGOLIPIDS OF RAT T-CELLS - PREDOMINANCE OF ASIALO-GM1 AND GD1C BIOCHEMISTRY Nohara, K., Nakauchi, H., Spiegel, S. 1994; 33 (15): 4661-4666


    Glycosphingolipids play an important role in the immune response, yet their compositions in T and B cells which mediate cellular and humoral immunity, respectively, have not been elucidated. In this study, characteristic features of glycosphingolipids in rat T lineage cells were revealed by comparing the gangliosides and neutral glycolipids of spleen T- and beta-cell-enriched fractions and thymocytes. In T cells, GD1c(NeuGc,NeuGc), a unique ganglioside synthesized through asialo-GM1 (GA1), was the predominant ganglioside as previously found in thymocytes [Nohara, K., Suzuki, M., Inagaki, F., & Kaya, K. (1991) J. Biochem. (Tokyo) 110, 274-278], and the amount was much higher than in thymocytes. In addition, three other GA1-derived gangliosides were detected in T cells and identified as GM1b(NeuAc), GM1b(NeuGc), and GD1 alpha(NeuAc,NeuAc). In contrast, GD1 alpha(NeuAc,NeuAc) was not discernible in thymocytes, although gangliosides corresponding to GM1b(NeuAc) and GM1b(NeuGc) were detected. The neutral glycolipids of T cells contained almost exclusively GA1, while thymocytes contained much lower amounts. The predominance of these GA1-derived gangliosides was confirmed as a singular feature of T lineage cells by comparison with gangliosides of spleen B-cell-enriched fractions which mainly consisted of gangliosides synthesized through GM3 and GM1. Furthermore, the unique structures, which contain the GM1 core and the extended modification of the lacto series, alpha Gal-LacNAc-GM1, alpha Gal-(LacNAc)2-GM1, and sialyl-LacNAc-GM1, were found in B-cell-enriched fractions. Unexpectedly, the neutral glycolipid composition of the thymocytes resembled that of the B-cell enriched fraction rather than that of the T cells.

    View details for Web of Science ID A1994NH49300029

    View details for PubMedID 8161523

  • RETROVIRUS-MEDIATED GENE-TRANSFER OF HUMAN PYRUVATE-KINASE (PK) CDNA INTO MURINE HEMATOPOIETIC-CELLS - IMPLICATIONS FOR GENE-THERAPY OF HUMAN PK DEFICIENCY BLOOD Tani, K., Yoshikubo, T., Ikebuchi, K., Takahashi, K., Tsuchiya, T., Takahashi, S., Shimane, M., Ogura, H., Tojo, A., Ozawa, K., Takahara, Y., Nakauchi, H., Markowitz, D., Bank, A., Asano, S. 1994; 83 (8): 2305-2310


    With the advent of recent molecular studies, nonspherocytic hemolytic anemia caused by red blood cell pyruvate kinase (PK) deficiency is now considered to be caused by a structural mutation of the PK-LR gene. Because PK deficiency is a monogenic disorder, the introduction of the normal PK gene into a patient's bone marrow stem cells should cure the disorder. To study the feasibility of gene therapy for PK deficiency, we first constructed the PK retrovirus pMNSM-hPK using human liver-type PK (LPK) cDNA and obtained a producer cell line of E86/AmPK. By using the supernatant of this virus-producer cell, we transduced NIH/3T3 cells, mouse leukemic cells (NFS60, FDCP-2), and human leukemic cells (K562, HEL). The expression of human LPK enzyme activity was ascertained from the retrovirally transduced NIH/3T3 cells. Northern blot analysis demonstrated the expression of the human LPK mRNA in each transduced cell line. Furthermore, bone marrow stem cells (c-kit+, Lin-, Thy-1lo) sorted by fluorescence-activated cell sorting were also transduced by the producer cells in the presence of interleukin-3 and interleukin-6, and were transplanted into lethally irradiated C57BL/6 mice. Polymerase chain reaction analysis demonstrated the expression of human LPK mRNA in both the peripheral blood and hematopoietic organs on day 30 and on day 135 of bone marrow transplantation.

    View details for Web of Science ID A1994NF96700036

    View details for PubMedID 8161797