Administrative Appointments


  • Director, Baxter Laboratory for Stem Cell Biology (2000 - Present)
  • Associate Program Director, Predoctoral Training, Developmental Biology and Neonatology Training Grant (2008 - Present)
  • Member, Institute for Stem Cell Biology and Regenerative Medicine (2004 - Present)
  • Faculty Affiliate, Bio-X Program (2005 - Present)
  • Faculty Affiliate, Stanford Cardiovascular Institute (2003 - Present)
  • Chair, Department of Molecular Pharmacology (1997 - 2002)

Honors & Awards


  • Board Member, Harvard Board of Overseer’s (2004-2010)
  • Irving Weinstein Foundation Award for Outstanding Innovations in Science, AACR (2011)
  • Fulbright Senior Specialist, Institut Pasteur, Paris (2007)
  • Council Member, Institute of Medicine (IOM) (2003-2009)
  • Plenary Lecturer, 400th Pontifical Academy, The Vatican (2003)
  • Honorary Doctorate, University of Nijmegen, Holland (2003)
  • Rolf-Sammet-Fonds Visiting Professorship, University of Frankfurt (2003)
  • President, International Society of Differentiation (2002-2004)
  • Council Member, American Society for Cell Biology (2002-2004)
  • Director's Lectureship, NIH (2000)
  • Board Member, American Society for Gene Therapy (1999-2002)
  • Donald E. and Delia B. Baxter Endowed Professorship, Stanford University (1999)
  • Merit Award, National Institute of Health (1995-2005)
  • Council Member, National Institute on Aging, NIH (1996-2000)
  • Elected Fellow, American Academy of Arts and Sciences (1996)
  • Elected Member, Institute of Medicine, National Academy of Arts and Sciences (1995)
  • FASEB Excellence in Science Award, FASEB (1999)
  • Yvette Mayent-Rothschild Visiting Professor, Instituts Curie and Pasteur, Paris (1995)
  • Nobel Forum Lectureship, Karolinska Institute, Sweden (1995)
  • President, American Society for Developmental Biology (1994-1995)
  • Senior Career Recognition Award, Women in Cell Biology, ASCB (1992)
  • Elected Fellow, American Association for the Advancement of Science (1991)

Boards, Advisory Committees, Professional Organizations


  • Senior Editor, Differentiation (2005 - Present)
  • Associate Editor, FASEB Journal (2005 - Present)
  • Senior Editor, Genes to Cells (1994 - Present)

Professional Education


  • Ph.D., Harvard University (1975)
  • M.A., Harvard University (1970)
  • B.A., University of York (1969)

Current Research and Scholarly Interests


How do cells maintain or alter quiescent, proliferative, or differentiated states? Once a cell becomes specialized for function in a particular tissue, that differentiated state is stable, yet the molecular mechanisms that control the expression of its characteristic repertoire of genes are largely dynamic. Our research is directed at understanding this apparent paradox and elucidating the nature of cell stability and cell plasticity. By perturbing the intracellular or extracellular milieu, we are probing the regulatory network that determines cell fate and how it can be altered. This knowledge is key to our understanding of nuclear reprogramming and how to enlist cells for therapeutic purposes. We are probing the fundamental regulatory mechanisms using cell fusion and dedifferentiation. We also focus on dedicated stem cells that exist in our muscle tissues to learn what goes awry as we age or suffer from genetic muscle disorders. For example, we have identified skeletal muscle stem cells and have discovered novel small molecules and niche proteins that enhance their function and expand their numbers, which is crucial for muscle regeneration. We have also determined a new role for telomeres in Duchenne muscular dystrophy, which provides novel insights into the development of the disease and treatment for it. We are developing the use of somatic cells and stem cells for therapeutic purposes - regenerative medicine.

Journal Articles


  • Rejuvenation of the muscle stem cell population restores strength to injured aged muscles. Nature medicine Cosgrove, B. D., Gilbert, P. M., Porpiglia, E., Mourkioti, F., Lee, S. P., Corbel, S. Y., Llewellyn, M. E., Delp, S. L., Blau, H. M. 2014; 20 (3): 255-264

    Abstract

    The elderly often suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging owing in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of MuSCs from aged mice are intrinsically defective relative to MuSCs from young mice, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation. This deficiency is correlated with a higher incidence of cells that express senescence markers and is due to elevated activity of the p38α and p38β mitogen-activated kinase pathway. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the MuSC population from aged mice to transient inhibition of p38α and p38β in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional MuSC population from aged mice, rejuvenating its potential for regeneration and serial transplantation as well as strengthening of damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy for the elderly.

    View details for DOI 10.1038/nm.3464

    View details for PubMedID 24531378

  • Early role for IL-6 signalling during generation of induced pluripotent stem cells revealed by heterokaryon RNA-Seq. Nature cell biology Brady, J. J., Li, M., Suthram, S., Jiang, H., Wong, W. H., Blau, H. M. 2013; 15 (10): 1244-1252

    Abstract

    Molecular insights into somatic cell reprogramming to induced pluripotent stem cells (iPS) would aid regenerative medicine, but are difficult to elucidate in iPS because of their heterogeneity, as relatively few cells undergo reprogramming (0.1-1%; refs , ). To identify early acting regulators, we capitalized on non-dividing heterokaryons (mouse embryonic stem cells fused to human fibroblasts), in which reprogramming towards pluripotency is efficient and rapid, enabling the identification of transient regulators required at the onset. We used bi-species transcriptome-wide RNA-seq to quantify transcriptional changes in the human somatic nucleus during reprogramming towards pluripotency in heterokaryons. During heterokaryon reprogramming, the cytokine interleukin 6 (IL6), which is not detectable at significant levels in embryonic stem cells, was induced 50-fold. A 4-day culture with IL6 at the onset of iPS reprogramming replaced stably transduced oncogenic c-Myc such that transduction of only Oct4, Klf4 and Sox2 was required. IL6 also activated another Jak/Stat target, the serine/threonine kinase gene Pim1, which accounted for the IL6-mediated twofold increase in iPS frequency. In contrast, LIF, another induced GP130 ligand, failed to increase iPS frequency or activate c-Myc or Pim1, thereby revealing a differential role for the two Jak/Stat inducers in iPS generation. These findings demonstrate the power of heterokaryon bi-species global RNA-seq to identify early acting regulators of reprogramming, for example, extrinsic replacements for stably transduced transcription factors such as the potent oncogene c-Myc.

    View details for DOI 10.1038/ncb2835

    View details for PubMedID 23995732

  • Role of telomere dysfunction in cardiac failure in Duchenne muscular dystrophy NATURE CELL BIOLOGY Mourkioti, F., Kustan, J., Kraft, P., Day, J. W., Zhao, M., Kost-Alimova, M., Protopopov, A., DePinho, R. A., Bernstein, D., Meeker, A. K., Blau, H. M. 2013; 15 (8): 895-U300

    Abstract

    Duchenne muscular dystrophy (DMD), the most common inherited muscular dystrophy of childhood, leads to death due to cardiorespiratory failure. Paradoxically, mdx mice with the same genetic deficiency of dystrophin exhibit minimal cardiac dysfunction, impeding the development of therapies. We postulated that the difference between mdx and DMD might result from differences in telomere lengths in mice and humans. We show here that, like DMD patients, mice that lack dystrophin and have shortened telomeres (mdx/mTR(KO)) develop severe functional cardiac deficits including ventricular dilation, contractile and conductance dysfunction, and accelerated mortality. These cardiac defects are accompanied by telomere erosion, mitochondrial fragmentation and increased oxidative stress. Treatment with antioxidants significantly retards the onset of cardiac dysfunction and death of mdx/mTR(KO) mice. In corroboration, all four of the DMD patients analysed had 45% shorter telomeres in their cardiomyocytes relative to age- and sex-matched controls. We propose that the demands of contraction in the absence of dystrophin coupled with increased oxidative stress conspire to accelerate telomere erosion culminating in cardiac failure and death. These findings provide strong support for a link between telomere length and dystrophin deficiency in the etiology of dilated cardiomyopathy in DMD and suggest preventive interventions.

    View details for DOI 10.1038/ncb2790

    View details for Web of Science ID 000322570900006

    View details for PubMedID 23831727

  • Redefining differentiation: Reshaping our ends NATURE CELL BIOLOGY Blau, H. M. 2012; 14 (6): 558-558

    View details for DOI 10.1038/ncb2506

    View details for Web of Science ID 000304599900002

    View details for PubMedID 22643873

  • DNA Demethylation Dynamics CELL Bhutani, N., Burns, D. M., Blau, H. M. 2011; 146 (6): 866-872

    Abstract

    The discovery of cytosine hydroxymethylation (5hmC) suggested a simple means of demethylating DNA and activating genes. Further experiments, however, unearthed an unexpectedly complex process, entailing both passive and active mechanisms of DNA demethylation by the ten-eleven translocation (TET) and AID/APOBEC families of enzymes. The consensus emerging from these studies is that removal of cytosine methylation in mammalian cells can occur by DNA repair. These reports highlight that in certain contexts, DNA methylation is not fixed but dynamic, requiring continuous regulation.

    View details for DOI 10.1016/j.cell.2011.08.042

    View details for Web of Science ID 000295258100010

    View details for PubMedID 21925312

  • Short Telomeres and Stem Cell Exhaustion Model Duchenne Muscular Dystrophy in mdx/mTR Mice CELL Sacco, A., Mourkioti, F., Tran, R., Choi, J., Llewellyn, M., Kraft, P., Shkreli, M., Delp, S., Pomerantz, J. H., Artandi, S. E., Blau, H. M. 2010; 143 (7): 1059-1071

    Abstract

    In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure of MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.

    View details for DOI 10.1016/j.cell.2010.11.039

    View details for Web of Science ID 000285625400005

    View details for PubMedID 21145579

  • Substrate Elasticity Regulates Skeletal Muscle Stem Cell Self-Renewal in Culture SCIENCE Gilbert, P. M., Havenstrite, K. L., Magnusson, K. E., Sacco, A., Leonardi, N. A., Kraft, P., Nguyen, N. K., Thrun, S., Lutolf, M. P., Blau, H. M. 2010; 329 (5995): 1078-1081

    Abstract

    Stem cells that naturally reside in adult tissues, such as muscle stem cells (MuSCs), exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to recapitulate key biophysical and biochemical niche features in conjunction with a highly automated single-cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes (approximately 10(6) kilopascals), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12 kilopascals) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by noninvasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle-wasting diseases.

    View details for DOI 10.1126/science.1191035

    View details for Web of Science ID 000281253500041

    View details for PubMedID 20647425

  • Transient Inactivation of Rb and ARF Yields Regenerative Cells from Postmitotic Mammalian Muscle CELL STEM CELL Pajcini, K. V., Corbel, S. Y., Sage, J., Pomerantz, J. H., Blau, H. M. 2010; 7 (2): 198-213

    Abstract

    An outstanding biological question is why tissue regeneration in mammals is limited, whereas urodele amphibians and teleost fish regenerate major structures, largely by cell cycle reentry. Upon inactivation of Rb, proliferation of postmitotic urodele skeletal muscle is induced, whereas in mammalian muscle this mechanism does not exist. We postulated that a tumor suppressor present in mammals but absent in regenerative vertebrates, the Ink4a product ARF (alternative reading frame), is a regeneration suppressor. Concomitant inactivation of Arf and Rb led to mammalian muscle cell cycle reentry, loss of differentiation properties, and upregulation of cytokinetic machinery. Single postmitotic myocytes were isolated by laser micro-dissection-catapulting, and transient suppression of Arf and Rb yielded myoblast colonies that retained the ability to differentiate and fuse into myofibers upon transplantation in vivo. These results show that differentiation of mammalian cells is reversed by inactivation of Arf and Rb and support the hypothesis that Arf evolved at the expense of regeneration.

    View details for DOI 10.1016/j.stem.2010.05.022

    View details for Web of Science ID 000281107400012

    View details for PubMedID 20682446

  • Reprogramming towards pluripotency requires AID-dependent DNA demethylation NATURE Bhutani, N., Brady, J. J., Damian, M., Sacco, A., Corbel, S. Y., Blau, H. M. 2010; 463 (7284): 1042-U57

    Abstract

    Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

    View details for DOI 10.1038/nature08752

    View details for Web of Science ID 000275108400028

    View details for PubMedID 20027182

  • Tumor suppressors: enhancers or suppressors of regeneration? DEVELOPMENT Pomerantz, J. H., Blau, H. M. 2013; 140 (12): 2502-2512

    Abstract

    Tumor suppressors are so named because cancers occur in their absence, but these genes also have important functions in development, metabolism and tissue homeostasis. Here, we discuss known and potential functions of tumor suppressor genes during tissue regeneration, focusing on the evolutionarily conserved tumor suppressors pRb1, p53, Pten and Hippo. We propose that their activity is essential for tissue regeneration. This is in contrast to suggestions that tumor suppression is a trade-off for regenerative capacity. We also hypothesize that certain aspects of tumor suppressor pathways inhibit regenerative processes in mammals, and that transient targeted modification of these pathways could be fruitfully exploited to enhance processes that are important to regenerative medicine.

    View details for DOI 10.1242/dev.084210

    View details for Web of Science ID 000319655800010

    View details for PubMedID 23715544

  • A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells FASEB JOURNAL Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. 2013; 27 (3): 1107-1113

    Abstract

    Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.

    View details for DOI 10.1096/fj.12-222125

    View details for Web of Science ID 000315585200024

    View details for PubMedID 23212122

  • An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Oliver, S. L., Brady, J. J., Sommer, M. H., Reichelt, M., Sung, P., Blau, H. M., Arvin, A. M. 2013; 110 (5): 1911-1916

    Abstract

    Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.

    View details for DOI 10.1073/pnas.1216985110

    View details for Web of Science ID 000314558100067

    View details for PubMedID 23322733

  • Protein-Engineered Biomaterials to Generate Human Skeletal Muscle Mimics ADVANCED HEALTHCARE MATERIALS Sengupta, D., Gilbert, P. M., Johnson, K. J., Blau, H. M., Heilshorn, S. C. 2012; 1 (6): 785-789

    View details for DOI 10.1002/adhm.201200195

    View details for Web of Science ID 000315120500014

    View details for PubMedID 23184832

  • Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB FASEB JOURNAL Banfi, A., von Degenfeld, G., Gianni-Barrera, R., Reginato, S., Merchant, M. J., McDonald, D. M., Blau, H. M. 2012; 26 (6): 2486-2497

    Abstract

    Therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, yet clinical trials have thus far failed to show efficacy. As a result, limb amputation remains a common outcome for muscle ischemia due to severe atherosclerotic disease, with an overall incidence of 100 per million people in the United States per year. A challenge has been that the angiogenic master regulator vascular endothelial growth factor (VEGF) induces dysfunctional vessels, if expressed outside of a narrow dosage window. We tested the hypothesis that codelivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, could induce normal angiogenesis in skeletal muscle irrespective of VEGF levels. Coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Notably, in an ischemic hindlimb model, single-vector expression led to efficient growth of collateral arteries, revascularization, increased blood flow, and reduced tissue damage. Furthermore, these results were confirmed in a clinically applicable gene therapy approach by adenoviral-mediated delivery of the bicistronic vector. We conclude that coordinated expression of VEGF and PDGF-BB via a single vector constitutes a novel strategy for harnessing the potency of VEGF to induce safe and efficacious angiogenesis.

    View details for DOI 10.1096/fj.11-197400

    View details for Web of Science ID 000305017200025

    View details for PubMedID 22391130

  • A single cell bioengineering approach to elucidate mechanisms of adult stem cell self-renewal INTEGRATIVE BIOLOGY Gilbert, P. M., Corbel, S., Doyonnas, R., Havenstrite, K., Magnusson, K. E., Blau, H. M. 2012; 4 (4): 360-367

    Abstract

    The goal of regenerative medicine is to restore form and function to damaged and aging tissues. Adult stem cells, present in tissues such as skeletal muscle, comprise a reservoir of cells with a remarkable capacity to proliferate and repair tissue damage. Muscle stem cells, known as satellite cells, reside in a quiescent state in an anatomically distinct compartment, or niche, ensheathed between the membrane of the myofiber and the basal lamina. Recently, procedures for isolating satellite cells were developed and experiments testing their function upon transplantation into muscles revealed an extraordinary potential to contribute to muscle fibers and access and replenish the satellite cell compartment. However, these properties are rapidly lost once satellite cells are plated in culture. Accordingly, elucidating the role of extrinsic factors in controlling muscle stem cell fate, in particular self-renewal, is critical. Through careful design of bioengineered culture platforms, analysis of specific proteins presented to stem cells is possible. Critical to the success of the approach is single cell analysis, as more rapidly proliferating progenitors may mask the behavior of stem cells that proliferate slowly. Bioengineering approaches provide a potent means of gaining insight into the role of extrinsic factors in the stem cell microenvironment on stem cell function and the mechanisms that control their diverse fates. Ultimately, the multidisciplinary approach presented here will lead to novel therapeutic strategies for degenerative diseases.

    View details for DOI 10.1039/c2ib00148a

    View details for Web of Science ID 000302017100002

    View details for PubMedID 22327505

  • Structure-function analysis of varicella-zoster virus glycoprotein H identifies domain-specific roles for fusion and skin tropism PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vleck, S. E., Oliver, S. L., Brady, J. J., Blau, H. M., Rajamani, J., Sommer, M. H., Arvin, A. M. 2011; 108 (45): 18412-18417

    Abstract

    Enveloped viruses require membrane fusion for cell entry and replication. For herpesviruses, this event is governed by the multiprotein core complex of conserved glycoproteins (g)B and gH/gL. The recent crystal structures of gH/gL from herpes simplex virus 2, pseudorabies virus, and Epstein-Barr virus revealed distinct domains that, surprisingly, do not resemble known viral fusogens. Varicella-zoster virus (VZV) causes chicken pox and shingles. VZV is an ?-herpesvirus closely related to herpes simplex virus 2, enabling prediction of the VZV gH structure by homology modeling. We have defined specific roles for each gH domain in VZV replication and pathogenesis using structure-based site-directed mutagenesis of gH. The distal tip of domain (D)I was important for skin tropism, entry, and fusion. DII helices and a conserved disulfide bond were essential for gH structure and VZV replication. An essential (724)CXXC(727) motif was critical for DIII structural stability and membrane fusion. This assignment of domain-dependent mechanisms to VZV gH links elements of the glycoprotein structure to function in herpesvirus replication and virulence.

    View details for DOI 10.1073/pnas.1111333108

    View details for Web of Science ID 000296700000053

    View details for PubMedID 22025718

  • MicroRNA programs in normal and aberrant stem and progenitor cells GENOME RESEARCH Arnold, C. P., Tan, R., Zhou, B., Yue, S., Schaffert, S., Biggs, J. R., Doyonnas, R., Lo, M., Perry, J. M., Renault, V. M., Sacco, A., Somervaille, T., Viatour, P., Brunet, A., Cleary, M. L., Li, L., Sage, J., Zhang, D., Blau, H. M., Chen, C., Chen, C. 2011; 21 (5): 798-810

    Abstract

    Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ?22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

    View details for DOI 10.1101/gr.111385.110

    View details for Web of Science ID 000290088000018

    View details for PubMedID 21451113

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

    Abstract

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

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

    View details for Web of Science ID 000289807600012

    View details for PubMedID 21357764

  • Engineering a stem cell house into a home STEM CELL RESEARCH & THERAPY Gilbert, P. M., Blau, H. M. 2011; 2

    Abstract

    In the body, tissue homeostasis is established and maintained by resident tissue-specific adult stem cells (aSCs). Through preservation of bidirectional communications with the surrounding niche and integration of biophysical and biochemical cues, aSCs actively direct the regeneration of aged, injured and diseased tissues. Currently, the ability to guide the behavior and fate of aSCs in the body or in culture after prospective isolation is hindered by our poor comprehension of niche composition and the regulation it imposes. Two-and three-dimensional biomaterials approaches permit systematic analysis of putative niche elements as well as screening approaches to identify novel regulatory mechanisms governing stem cell fate. The marriage of stem cell biology with creative bioengineering technology has the potential to expand our basic understanding of stem cell regulation imposed by the niche and to develop novel regenerative medicine applications.

    View details for DOI 10.1186/scrt44

    View details for Web of Science ID 000292884900001

    View details for PubMedID 21345268

  • Re"evolutionary" Regenerative Medicine JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Blau, H. M., Pomerantz, J. H. 2011; 305 (1): 87-88

    View details for DOI 10.1001/jama.2010.1938

    View details for Web of Science ID 000285924100028

    View details for PubMedID 21177496

  • Toxoplasma secreting Cre recombinase for analysis of host-parasite interactions NATURE METHODS Koshy, A. A., Fouts, A. E., Lodoen, M. B., Alkan, O., Blau, H. M., Boothroyd, J. C. 2010; 7 (4): 307-309

    Abstract

    We describe a Toxoplasma gondii strain that will permit the use of site-specific recombination to study the host-parasite interactions of this organism. This Toxoplasma strain efficiently injects a Cre fusion protein into host cells. In a Cre-reporter cell line, a single parasite invasion induced Cre-mediated recombination in 95% of infected host cells. By infecting Cre-reporter mice with these parasites, we also monitored host-cell infection in vivo.

    View details for DOI 10.1038/NMETH.1438

    View details for Web of Science ID 000276150600022

    View details for PubMedID 20208532

  • Designing materials to direct stem-cell fate NATURE Lutolf, M. P., Gilbert, P. M., Blau, H. M. 2009; 462 (7272): 433-441

    Abstract

    Proper tissue function and regeneration rely on robust spatial and temporal control of biophysical and biochemical microenvironmental cues through mechanisms that remain poorly understood. Biomaterials are rapidly being developed to display and deliver stem-cell-regulatory signals in a precise and near-physiological fashion, and serve as powerful artificial microenvironments in which to study and instruct stem-cell fate both in culture and in vivo. Further synergism of cell biological and biomaterials technologies promises to have a profound impact on stem-cell biology and provide insights that will advance stem-cell-based clinical approaches to tissue regeneration.

    View details for DOI 10.1038/nature08602

    View details for Web of Science ID 000272144200031

    View details for PubMedID 19940913

  • Artificial Stem Cell Niches ADVANCED MATERIALS Lutolf, M. P., Blau, H. M. 2009; 21 (32-33): 3255-3268

    Abstract

    Stem cells are characterized by their dual ability to reproduce themselves (self-renew) and specialize (differentiate), yielding a plethora of daughter cells that maintain and regenerate tissues. In contrast to their embryonic counterparts, adult stem cells retain their unique functions only if they are in intimate contact with an instructive microenvironment, termed stem cell niche. In these niches, stem cells integrate a complex array of molecular signals that, in concert with induced cell-intrinsic regulatory networks, control their function and balance their numbers in response to physiologic demands. This progress report provides a perspective on how advanced materials technologies could be used (i) to engineer and systematically analyze specific aspects of functional stem cells niches in a controlled fashion in vitro and (ii) to target stem cell niches in vivo. Such "artificial niches" constitute potent tools for elucidating stem cell regulatory mechanisms with the capacity to directly impact the development of novel therapeutic strategies for tissue regeneration.

    View details for DOI 10.1002/adma.200802582

    View details for Web of Science ID 000269936900005

    View details for PubMedID 20882496

  • A home away from home: Challenges and opportunities in engineering in vitro muscle satellite cell niches DIFFERENTIATION Cosgrove, B. D., Sacco, A., Gilbert, P. M., Blau, H. M. 2009; 78 (2-3): 185-194

    Abstract

    Satellite cells are skeletal muscle stem cells with a principal role in postnatal skeletal muscle regeneration. Satellite cells, like many tissue-specific adult stem cells, reside in a quiescent state in an instructive, anatomically defined niche. The satellite cell niche constitutes a distinct membrane-enclosed compartment within the muscle fiber, containing a diversity of biochemical and biophysical signals that influence satellite cell function. A major limitation to the study and clinical utility of satellite cells is that upon removal from the muscle fiber and plating in traditional plastic tissue culture platforms, their muscle stem cell properties are rapidly lost. Clearly, the maintenance of stem cell function is critically dependent on in vivo niche signals, highlighting the need to create novel in vitro microenvironments that allow for the maintenance and propagation of satellite cells while retaining their potential to function as muscle stem cells. Here, we discuss how emerging biomaterials technologies offer great promise for engineering in vitro microenvironments to meet these challenges. In engineered biomaterials, signaling molecules can be presented in a manner that more closely mimics cell-cell and cell-matrix interactions, and matrices can be fabricated with diverse rigidities that approximate in vivo tissues. The development of in vitro microenvironments in which niche features can be systematically modulated will be instrumental not only to future insights into muscle stem cell biology and therapeutic approaches to muscle diseases and muscle wasting with aging, but also will provide a paradigm for the analysis of numerous adult tissue-specific stem cells.

    View details for DOI 10.1016/j.diff.2009.08.004

    View details for Web of Science ID 000274532300017

    View details for PubMedID 19751902

  • Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional FASEB JOURNAL Palermo, A., Doyonnas, R., Bhutani, N., Pomerantz, J., Alkan, O., Blau, H. M. 2009; 23 (5): 1431-1440

    Abstract

    An understanding of nuclear reprogramming is fundamental to the use of cells in regenerative medicine. Due to technological obstacles, the time course and extent of reprogramming of cells following fusion has not been assessed to date. Here, we show that hundreds of genes are activated or repressed within hours of fusion of human keratinocytes and mouse muscle cells in heterokaryons, and extensive changes are observed within 4 days. This study was made possible by the development of a broadly applicable approach, species-specific transcriptome amplification (SSTA), which enables global resolution of transcripts derived from the nuclei of two species, even when the proportions of species-specific transcripts are highly skewed. Remarkably, either phenotype can be dominant; an excess of primary keratinocytes leads to activation of the keratinocyte program in muscle cells and the converse is true when muscle cells are in excess. We conclude that nuclear reprogramming in heterokaryons is rapid, extensive, bidirectional, and dictated by the balance of regulators contributed by the cell types.

    View details for DOI 10.1096/fj.08-122903

    View details for Web of Science ID 000266651700019

    View details for PubMedID 19141533

  • Reprogramming to a muscle fate by fusion recapitulates differentiation JOURNAL OF CELL SCIENCE Pomerantz, J. H., Mukherjee, S., Palermo, A. T., Blau, H. M. 2009; 122 (7): 1045-1053

    Abstract

    Fusion of mammalian cells to form stable, non-dividing heterokaryons results in nuclear reprogramming without the exchange of genetic material. In this report, we show that reprogramming in somatic cell heterokaryons involves activation of the canonical skeletal muscle transcription factors as well as contraction-excitation genes. Thus, the effect of heterokaryon formation on gene expression is to induce a recapitulation of differentiation. Heterokaryons formed with a relatively refractory cell type, the hepatocyte cell line HepG2, revealed the importance of both MyoD expression and other unidentified cytoplasmic components, neither of which are sufficient for efficient muscle gene activation, but are synergistic. We provide evidence that de-repression by transient histone deacetylase inhibition can induce MyoD expression and increase the extent and efficiency of muscle gene transcription. Taken together, the results suggest that understanding the mechanistic basis, using a combination of approaches, and taking into account cell history, will facilitate an increase in the efficiency and fidelity of conversion from one differentiated phenotype to another desired cell type. Inherent advantages of the heterokaryon system merit further investigation in the pursuit of directed cloning.

    View details for DOI 10.1242/jcs.041376

    View details for Web of Science ID 000264342000019

    View details for PubMedID 19295131

  • Imaging beta-galactosidase activity in vivo using sequential reporter-enzyme luminescence. Methods in molecular biology (Clifton, N.J.) von Degenfeld, G., Wehrman, T. S., Blau, H. M. 2009; 574: 249-259

    Abstract

    Bioluminescence using the reporter enzyme firefly luciferase (Fluc) and the substrate luciferin enables non-invasive optical imaging of living animals with extremely high sensitivity. This type of analysis enables studies of gene expression, tumor growth, and cell migration over time in live animals that were previously not possible. However, a major limitation of this system is that Fluc activity is restricted to the intracellular environment, which precludes important applications of in vivo imaging such as antibody labeling, or serum protein monitoring. In order to expand the application of bioluminescence imaging to other enzymes, we characterized a sequential reporter-enzyme luminescence (SRL) technology for the in vivo detection of beta-galactosidase (beta-gal) activity. The substrate is a "caged" D-luciferin conjugate that must first be cleaved by beta-gal before it can be catalyzed by Fluc in the final, light-emitting step. Hence, luminescence is dependent on and correlates with beta-gal activity. A variety of experiments were performed in order to validate the system and explore potential new applications. We were able to visualize non-invasively over time constitutive beta-gal activity in engineered cells, as well as inducible tissue-specific beta-gal expression in transgenic mice. Since beta-gal, unlike Fluc, retains full activity outside of cells, we were able to show that antibodies conjugated to the recombinant beta-gal enzyme could be used to detect and localize endogenous cells and extracellular antigens in vivo. In addition, we developed a low-affinity beta-gal complementation system that enables inducible, reversible protein interactions to be monitored in real time in vivo, for example, sequential responses to agonists and antagonists of G-protein-coupled receptors (GPCRs). Thus, using SRL, the exquisite luminescent properties of Fluc can be combined with the advantages of another enzyme. Other substrates have been described that extend the scope to endogenous enzymes, such as cytochromes or caspases, potentially enabling additional unprecedented applications.

    View details for DOI 10.1007/978-1-60327-321-3_20

    View details for PubMedID 19685314

  • Perturbation of single hematopoietic stem cell fates in artificial niches INTEGRATIVE BIOLOGY Lutolf, M. P., Doyonnas, R., Havenstrite, K., Koleckar, K., Blau, H. M. 2009; 1 (1): 59-69

    Abstract

    Hematopoietic stem cells (HSCs) are capable of extensive self-renewal in vivo and are successfully employed clinically to treat hematopoietic malignancies, yet are in limited supply as in culture this self-renewal capacity is lost. Using an approach at the interface of stem cell biology and bioengineering, here we describe a novel platform of hydrogel microwell arrays for assessing the effects of either secreted or tethered proteins characteristic of the in vivo microenvironment, or niche, on HSC fate in vitro. Time-lapse microscopic analyses of single cells were crucial to overcoming inevitable heterogeneity of FACS-enriched HSCs. A reduction in proliferation kinetics or an increase in asynchronous division of single HSCs in microwells in response to specific proteins (Wnt3a and N-Cadherin) correlated well with subsequent serial long-term blood reconstitution in mice in vivo. Single cells that divided once in the presence of a given protein were capable of in vivo reconstitution, providing evidence of self-renewal divisions of HSCs in vitro. These results validate the hydrogel microwell platform as a broadly applicable paradigm for dissecting the regulatory role of specific signals within a complex stem cell niche.

    View details for DOI 10.1039/b815718a

    View details for Web of Science ID 000266978200007

    View details for PubMedID 20023792

  • Self-renewal and expansion of single transplanted muscle stem cells NATURE Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. 2008; 456 (7221): 502-506

    Abstract

    Adult muscle satellite cells have a principal role in postnatal skeletal muscle growth and regeneration. Satellite cells reside as quiescent cells underneath the basal lamina that surrounds muscle fibres and respond to damage by giving rise to transient amplifying cells (progenitors) and myoblasts that fuse with myofibres. Recent experiments showed that, in contrast to cultured myoblasts, satellite cells freshly isolated or satellite cells derived from the transplantation of one intact myofibre contribute robustly to muscle repair. However, because satellite cells are known to be heterogeneous, clonal analysis is required to demonstrate stem cell function. Here we show that when a single luciferase-expressing muscle stem cell is transplanted into the muscle of mice it is capable of extensive proliferation, contributes to muscle fibres, and Pax7(+)luciferase(+) mononucleated cells can be readily re-isolated, providing evidence of muscle stem cell self-renewal. In addition, we show using in vivo bioluminescence imaging that the dynamics of muscle stem cell behaviour during muscle repair can be followed in a manner not possible using traditional retrospective histological analyses. By imaging luciferase activity, real-time quantitative and kinetic analyses show that donor-derived muscle stem cells proliferate and engraft rapidly after injection until homeostasis is reached. On injury, donor-derived mononucleated cells generate massive waves of cell proliferation. Together, these results show that the progeny of a single luciferase-expressing muscle stem cell can both self-renew and differentiate after transplantation in mice, providing new evidence at the clonal level that self-renewal is an autonomous property of a single adult muscle stem cell.

    View details for DOI 10.1038/nature07384

    View details for Web of Science ID 000261170500038

    View details for PubMedID 18806774

  • Cell therapies for muscular dystrophy NEW ENGLAND JOURNAL OF MEDICINE Blau, H. M. 2008; 359 (13): 1403-1405

    View details for Web of Science ID 000259440900015

    View details for PubMedID 18815403

  • Extensive fusion of haematopoietic cells with Purkinje neurons in response to chronic inflammation NATURE CELL BIOLOGY Johansson, C. B., Youssef, S., Koleckar, K., Holbrook, C., Doyonnas, R., Corbel, S. Y., Steinman, L., Rossi, F. M., Blau, H. M. 2008; 10 (5): 575-583

    Abstract

    Transplanted bone marrow-derived cells (BMDCs) have been reported to fuse with cells of diverse tissues, but the extremely low frequency of fusion has led to the view that such events are biologically insignificant. Nonetheless, in mice with a lethal recessive liver disease (tyrosinaemia), transplantation of wild-type BMDCs restored liver function by cell fusion and prevented death, indicating that cell fusion can have beneficial effects. Here we report that chronic inflammation resulting from severe dermatitis or autoimmune encephalitis leads to robust fusion of BMDCs with Purkinje neurons and formation of hundreds of binucleate heterokaryons per cerebellum, a 10-100-fold higher frequency than previously reported. Single haematopoietic stem-cell transplants showed that the fusogenic cell is from the haematopoietic lineage and parabiosis experiments revealed that fusion can occur without irradiation. Transplantation of rat bone marrow into mice led to activation of dormant rat Purkinje neuron-specific genes in BMDC nuclei after fusion with mouse Purkinje neurons, consistent with nuclear reprogramming. The precise neurological role of these heterokaryons awaits elucidation, but their frequency in brain after inflammation is clearly much higher than previously appreciated.

    View details for DOI 10.1038/ncb1720

    View details for Web of Science ID 000255502400014

    View details for PubMedID 18425116

  • Myoblasts and macrophages share molecular components that contribute to cell-cell fusion JOURNAL OF CELL BIOLOGY Pajcini, K. V., Pomerantz, J. H., Alkan, O., Doyonnas, R., Blau, H. M. 2008; 180 (5): 1005-1019

    Abstract

    Cell-cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and beta(1)-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells.

    View details for Web of Science ID 000254081100015

    View details for PubMedID 18332221

  • Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells HEPATOLOGY Streetz, K. L., Doyonnas, R., Grimm, D., Jenkins, D. D., Fuess, S., Perryman, S., Lin, J., Trautwein, C., Shizuru, J., Blau, H., Sylvester, K. G., Kay, M. A. 2008; 47 (2): 706-718

    Abstract

    The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT.The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure.

    View details for DOI 10.1002/hep.22012

    View details for Web of Science ID 000252939500040

    View details for PubMedID 18220289

  • A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals FASEB JOURNAL von Degenfeld, G., Wehrman, T. S., Hammer, M. M., Blau, H. M. 2007; 21 (14): 3819-3826

    Abstract

    G-protein coupled receptors (GPCRs) are a versatile and ubiquitous family of membrane receptors that transmit extracellular signals to mammalian cells and constitute the most important class of drug targets. Yet, sensitive and specific methods are lacking that would allow quantitative comparisons of pharmacologic properties of these receptors in physiological or pathological settings in live animals. We sought to overcome these limitations by employing low affinity, reversible beta-galactosidase complementation to quantify GPCR activation via interaction with beta-arrestin. A panel of cell lines was engineered expressing different GPCRs together with the reporter system. In vitro evaluation revealed highly sensitive, dynamic, and specific assessment of GPCR agonists and antagonists. Following implantation of the cells into mice, it was possible for the first time to monitor pharmacological GPCR activation and inhibition in their physiological context by noninvasive bioluminescence imaging in living animals. This technology has unique advantages that enable novel applications in the functional investigation of GPCR modulation in live animals in biological research and drug discovery.

    View details for DOI 10.1096/fj.07-9597com

    View details for Web of Science ID 000251283500006

    View details for PubMedID 17942828

  • A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization FASEB JOURNAL Hammer, M. M., Wehrman, T. S., Blau, H. M. 2007; 21 (14): 3827-3834

    Abstract

    G-protein-coupled receptor (GPCR) signaling is involved in a wide range of physiological processes and diseases, and around one-half of currently used drugs target GPCRs. Assays for the signaling of GPCRs have suffered from drawbacks, including low signal-to-noise, temporally transient signals, and difficulty in applying a single assay to a wide range of GPCRs. We have developed a set of assays for G-protein-coupled receptor signaling based on beta-galactosidase enzyme complementation in live mammalian cells. We previously described an assay for GPCR activation by monitoring the binding of beta-arrestin to the receptor. Here we describe a second assay that monitors the internalization of GPCRs to endosomes, an event that follows receptor activation and is critical in desensitizing and resensitizing the receptor. We show that both assays display high signal-to-noise ratios with low variability and are quantitative for a wide range of GPCRs. EC50s obtained with these assays closely match results reported in the literature. Finally, we show that these assays are readily adapted to high-throughput chemical screens. Thus, these two assays for monitoring G-protein-coupled receptor activation and internalization should prove valuable in basic biological studies as well as in high-throughput screens.

    View details for DOI 10.1096/fj.07-8777com

    View details for Web of Science ID 000251283500007

    View details for PubMedID 17942829

  • Anne McLaren (1927-2007) - Obituary DIFFERENTIATION Blau, H. M. 2007; 75 (10): 899-901
  • Increased host neuronal survival and motor function in BMT parkinsonian mice: Involvement of immunosuppression JOURNAL OF COMPARATIVE NEUROLOGY Keshet, G. I., Tolwani, R. J., Trejo, A., Kraft, P., Doyonnas, R., Clayberger, C., Weimann, J. M., Blau, H. M. 2007; 504 (6): 690-701

    Abstract

    We examined the potential of bone marrow transplantation (BMT) to rescue dopaminergic neurons in a mouse model of Parkinson's disease (PD). A BMT from mice transgenic for green fluorescent protein (GFP(+)) given either before or after administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) led to the accumulation of transplanted adult GFP(+) bone-marrow-derived cells (BMDC) in the substantia nigra, where dopaminergic neurodegeneration occurs in PD. Post-BMT, mice exposed to MPTP had substantially greater numbers of endogenous tyrosine hydroxylase-positive neuronal cell bodies in the substantia nigra and increased dopamine transporter-positive projections into the striatum compared to controls. Moreover, motor function was restored to normal within 1 month post-MPTP in BMT-treated mice assayed by a rotarod behavioral test. The effect of BMT on PD was indirect, as no evidence of BMDC fusion with or transdifferentiation into dopaminergic neurons was observed. BMDC activated by BMT or associated factors could play a trophic role in rescuing damaged cells. Alternatively, the beneficial effects of BMT are due to immunosuppression reflected by a reduction in the proportion of T-cells and a reduction of T-cell proliferation in BMT mice. These findings highlight that when immunosuppression is required for transplantation studies, the amelioration of symptoms may not be due to the transplant itself. Further, they suggest that the immune system plays a role in the development of characteristics typical of PD.

    View details for DOI 10.1002/cne.21483

    View details for Web of Science ID 000249616400007

    View details for PubMedID 17722033

  • Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes NATURE CHEMICAL BIOLOGY Blum, G., von Degenfeld, G., Merchant, M. J., Blau, H. M., Bogyo, M. 2007; 3 (10): 668-677

    Abstract

    We have generated a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit a fluorescent signal only after covalently modifying a specific protease target. After intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive, whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to identify specific proteases and to correlate their activity with whole-body images. Finally, we demonstrate that these probes can be used to monitor small-molecule inhibition of protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo.

    View details for Web of Science ID 000249642700017

    View details for PubMedID 17828252

  • Noggin suppression enhances in vitro osteogenesis and accelerates in vivo bone formation JOURNAL OF BIOLOGICAL CHEMISTRY Wan, D. C., Pomerantz, J. H., Brunet, L. J., Kim, J., Chou, Y., Wu, B. M., Harland, R., Blau, H. M., Longaker, M. T. 2007; 282 (36): 26450-26459

    Abstract

    Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.

    View details for DOI 10.1074/jbc.M703282200

    View details for Web of Science ID 000249239600050

    View details for PubMedID 17609215

  • Localization of vascular response to VEGF is not dependent on heparin binding FASEB JOURNAL Springer, M. L., Banfi, A., Ye, J., von Degenfeld, G., Kraft, P. E., Saini, S. A., Kapasi, N. K., Blau, H. M. 2007; 21 (9): 2074-2085

    Abstract

    The major vascular endothelial growth factor (VEGF) isoforms are splice variants from a single gene that differ in their extent of heparin affinity due to the absence of the heparin binding domain in the smallest isoform (mouse VEGF120, human VEGF121). A long-held assumption that has guided the use of VEGF isoforms clinically has been that their differences in heparin binding dictate their ability to diffuse through tissue, with VEGF121 moving most freely and that the distribution of recombinant VEGF would have therapeutically relevant consequences. To test this assumption, we delivered the genes encoding these isoforms by myoblast-mediated gene transfer, a means of delivering genes to highly localized sites within muscle. Surprisingly, all isoforms induced comparable extremely localized physiological effects. Significantly, irrespective of the isoform delivered, the vessels passing within several micrometers of muscle fibers expressing VEGF displayed sharply delineated changes in morphology. The induction of capillary wrapping around VEGF-producing fibers, and of vascular malformations in the muscle at high levels, did not differ among isoforms. These results indicate that heparin binding is not essential for the localization of VEGF in adult tissue and suggest that the preferential delivery of VEGF121 cDNA for clinical applications may not have a physiological basis.

    View details for DOI 10.1096/fj.06-7700com

    View details for Web of Science ID 000247500300017

    View details for PubMedID 17325231

  • Active tissue-specific DNA demethylation conferred by somatic cell nuclei in stable heterokaryons PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, F., Pomerantz, J. H., Sen, G., Palermo, A. T., Blau, H. M. 2007; 104 (11): 4395-4400

    Abstract

    DNA methylation is among the most stable epigenetic marks, ensuring tissue-specific gene expression in a heritable manner throughout development. Here we report that differentiated mesodermal somatic cells can confer tissue-specific changes in DNA methylation on epidermal progenitor cells after fusion in stable multinucleate heterokaryons. Myogenic factors alter regulatory regions of genes in keratinocyte cell nuclei, demethylating and activating a muscle-specific gene and methylating and silencing a keratinocyte-specific gene. Because these changes occur in the absence of DNA replication or cell division, they are mediated by an active mechanism. Thus, the capacity to transfer epigenetic changes to other nuclei is not limited to embryonic stem cells and oocytes but is also a property of highly specialized mammalian somatic cells. These results suggest the possibility of directing the reprogramming of readily available postnatal human progenitor cells toward specific tissue cell types.

    View details for DOI 10.1073/pnas.0700181104

    View details for Web of Science ID 000244972700029

    View details for PubMedID 17360535

  • In vivo optical bioluminescence imaging of collagen-supported cardiac cell grafts JOURNAL OF HEART AND LUNG TRANSPLANTATION Kutschka, I., Chen, I. Y., Kofidis, T., von Degenfeld, G., Sheikh, A. Y., Hendry, S. L., Hoyt, G., Pearl, J., Blau, H. M., Gambhir, S. S., Robbins, R. C. 2007; 26 (3): 273-280

    Abstract

    Histology-based survival assessment of cell grafts does not allow for in vivo follow-up. In this study we introduce two new experimental models for longitudinal in vivo survival studies of cardiac cell grafts using optical bioluminescence imaging.H9c2 cardiomyoblasts expressing both firefly luciferase (fluc) and green fluorescent protein (GFP) reporter genes were implanted into Lewis rats. In Model 1, H9c2-fluc-IRES-GFP cells (0.5 x 10(6)) were implanted into a cryoinjured abdominal wall muscle. Cells were injected using either liquid collagen (Matrigel [MG]) or phosphate-buffered saline (PBS) suspension. Cell survival was evaluated in vivo using bioluminescence imaging on days 1, 5 and 10 post-operatively. In model 2, rats underwent ligation of the left anterior descending (LAD) artery. The donor hearts were harvested, and the infarcted region was restored ex situ using 1 x 10(6) H9c2-fluc-IRES-GFP cells seeded in collagen matrix (Gelfoam [GF]) or suspended in PBS (n = 8/group). Hearts were then transplanted into the abdomen of syngeneic recipients. Optical bioluminescence imaging was performed on Days 1, 5, 8 and 14 post-operatively. After 4 weeks, immunohistologic studies were performed.For model 1, at day 5, bioluminescence signals were markedly higher for the H9c2/MG group (449 +/- 129 photons/second x 10(3)) compared with the H9c2/PBS group (137 +/- 82 photons/second x 10(3)) (p < 0.05). For model 2, bioluminescence signals were significantly (p < 0.04) higher in the H9c2/GF group compared with plain cell injection on days 5 (534 +/- 115 vs 219 +/- 34) and 8 (274 +/- 34 vs 180 +/- 23). Data were in accordance with GFP immunohistology.Optical bioluminescence is a powerful method for assessment of cardiac cell graft survival in vivo. Collagen matrices support early survival of cardiomyoblasts after transplantation into injured musculature.

    View details for Web of Science ID 000244979000010

    View details for PubMedID 17346630

  • Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors NEURON Wehrman, T., He, X., Raab, B., Dukipatti, A., Blau, H., Garcia, K. C. 2007; 53 (1): 25-38

    Abstract

    Nerve growth factor engages two structurally distinct transmembrane receptors, TrkA and p75, which have been proposed to create a "high-affinity" NGF binding site through formation of a ternary TrkA/NGF/p75 complex. To define a structural basis for the high-affinity site, we have determined the three-dimensional structure of a complete extracellular domain of TrkA complexed with NGF. The complex reveals a crab-shaped homodimeric TrkA structure, but a mechanism for p75 coordination is not obvious. We investigated the heterodimerization of membrane-bound TrkA and p75, on intact mammalian cells, using a beta-gal protein-protein interaction system. We find that NGF dimerizes TrkA and that p75 exists on the cell surface as a preformed oligomer that is not dissociated by NGF. We find no evidence for a direct TrkA/p75 interaction. We propose that TrkA and p75 likely communicate through convergence of downstream signaling pathways and/or shared adaptor molecules, rather than through direct extracellular interactions.

    View details for DOI 10.1016/j.neuron.2006.09.034

    View details for Web of Science ID 000245126500006

    View details for PubMedID 17196528

  • System for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wehrman, T. S., Raab, W. J., Casipit, C. L., Doyonnas, R., Pomerantz, J. H., Blau, H. M. 2006; 103 (50): 19063-19068

    Abstract

    The orphan receptor tyrosine kinase ErbB2 is activated by each of the EGFR family members upon ligand binding. However, difficulties monitoring the dynamic interactions of the membrane receptors have hindered the elucidation of the mechanism of ErbB2 activation. We have engineered a system to monitor protein-protein interactions in intact mammalian cells such that different sets of protein interactions can be quantitatively compared. Application of this system to the interactions of the EGFR family showed that ErbB2 interacts stably with the EGFR and ErbB3, but fails to spontaneously homooligomerize. The widely used anti-cancer antibody Herceptin was found to effectively inhibit the interaction of the EGFR and ErbB2 but not to interfere with the interaction of ErbB2-ErbB3. Treatment of cells expressing EGFR and ErbB2 with Herceptin results in increased EGFR homooligomerization in the presence of EGF and a subsequent rapid internalization and down-regulation of the EGFR. In summary, the protein interaction system described here enabled the characterization of ErbB2 interactions within the biological context of the plasma membrane and provides insight into the mechanism of Herceptin action on cells overexpressing ErbB2.

    View details for DOI 10.1073/pnas.0605218103

    View details for Web of Science ID 000242884200035

    View details for PubMedID 17148612

  • Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia FASEB JOURNAL von Degenfeld, G., Banfi, A., Springer, M. L., Wagner, R. A., Jacobi, J., Ozawa, C. R., Merchant, M. J., Cooke, J. P., Blau, H. M. 2006; 20 (14): 2657-?

    Abstract

    The critical role of vascular endothelial growth factor (VEGF) expression levels in developmental angiogenesis is well established. Nonetheless, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and VEGF delivery to patients has been disappointing. Here, we demonstrate the existence of both lower and upper threshold levels of microenvironmental VEGF concentrations for the induction of therapeutic vessel growth in ischemia. In the ischemic hind limb, implantation of myoblasts transduced to express VEGF164 at different levels per cell increased blood flow only moderately, and vascular leakage and aberrant preangiomatous vessels were always induced. When the same total dose was uniformly distributed by implanting a monoclonal population derived from a single VEGF-expressing myoblast, blood flow was fully restored to nonischemic levels, collateral growth was induced, and ischemic damage was prevented. Hemangiomas were avoided and only normal, pericyte-covered vessels were induced persisting over 15 mo. Surprisingly, clones uniformly expressing either lower or higher VEGF levels failed to provide any functional benefit. A biphasic effect of VEGF dose on vessel number and diameter was found. Blood flow was only improved if vessels were increased both in size and in number. Microenvironmental VEGF concentrations determine efficacy and safety in a therapeutic setting.

    View details for DOI 10.1096/fj.06-6568fje

    View details for Web of Science ID 000242490700056

    View details for PubMedID 17095533

  • Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts CIRCULATION Kutschka, I., Kofidis, T., Chen, I. Y., von Degenfeld, G., Zwierzchoniewska, M., Hoyt, G., Arai, T., Lebl, D. R., Hendry, S. L., Sheikh, A. Y., Cooke, D. T., Connolly, A., Blau, H. M., Gambhir, S. S., Robbins, R. C. 2006; 114: I174-I180

    Abstract

    Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human Bcl-2 (hBcl-2) has been shown to attenuate apoptosis in the experimental setting. Therefore, we studied the potential benefit of hBcl-2 transgene expression on the survival of cardiomyoblast grafts in ischemic rat hearts.H9c2 rat cardiomyoblasts were genetically modified to express both firefly luciferase and green fluorescent protein (mH9c2). The cells were then transduced with adenovirus carrying hBcl-2 (AdCMVhBcl-2/mH9c2). Lewis rats underwent ligation of the left anterior descending artery (LAD) to induce a sizable left ventricular (LV) infarct. Hearts were explanted and the infarcted region was restored using collagen matrix (CM) seeded with 1x10(6) mH9c2 cells (n=9) or AdCMVhBcl-2/mH9c2 cells (n=9). Control animals received CM alone (n=6) or no infarct (n=6). Restored hearts were transplanted into the abdomen of syngeneic recipients in a "working heart" model. Cell survival was evaluated using optical bioluminescence imaging on days 1, 5, 8, 14, and 28 after surgery. The left heart function was assessed 4 weeks postoperatively using echocardiography and magnetic resonance imaging. During 4 weeks after surgery, the optical imaging signal for the AdCMVhBCL2/mH9c2 group was significantly (P<0.05) higher than that of the mH9c2-control group. Both grafts led to better fractional shortening (AdCMVhBcl-2/mH9c2: 0.21+/-0.03; mH9c2: 0.21+/-0.04; control: 0.15+/-0.03; P=0.04) and ejection fraction (AdCMVhBcl-2/mH9c2: 47.0+/-6.2; mH9c2: 48.7+/-6.1; control: 34.3+/-6.0; P=0.02) compared with controls. Importantly, no malignant cells were found in postmortem histology.Transduction of mH9c2 cardiomyoblasts with AdCMVhBcl-2 increased graft survival in ischemic rat myocardium without causing malignancies. Both AdCMVhBcl-2/mH9c2 and mH9c2 grafts improved LV function.

    View details for DOI 10.1161/CIRCULATIONAHA.105.001370

    View details for Web of Science ID 000238688200029

    View details for PubMedID 16820569

  • Collagen matrices enhance survival of transplanted cardiomyoblasts and contribute to functional improvement of ischemic rat hearts CIRCULATION Kutschka, I., Chen, I. Y., Kofidis, T., Arai, T., von Degenfeld, G., Sheikh, A. Y., Hendry, S. L., Pearl, J., Hoyt, G., Sista, R., Yang, P. C., Blau, H. M., Gambhir, S. S., Robbins, R. C. 2006; 114: I167-I173

    Abstract

    Cardiac cell transplantation is limited by poor graft viability. We aimed to enhance the survival of transplanted cardiomyoblasts using growth factor-supplemented collagen matrices.H9c2 cardiomyoblasts were lentivirally transduced to express firefly luciferase and green fluorescent protein (GFP). Lewis rats underwent ligation of the left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Hearts (n=9/group) were harvested and restored ex vivo with 1 x 10(6) genetically labeled H9c2 cells either in (1) saline-suspension, or seeded onto (2) collagen-matrix (Gelfoam [GF];), (3) GF/Matrigel (GF/MG), (4) GF/MG/VEGF (10 microg/mL), or (5) GF/MG/FGF (10 microg/mL). Hearts were then abdominally transplanted into syngeneic recipients (working heart model). Controls (n=6/group) underwent infarction followed by GF implantation or saline injection. Cell survival was evaluated using optical bioluminescence on days 1, 5, 8, 14, and 28 postoperatively. At 4 weeks, fractional shortening and ejection fraction were determined using echocardiography and magnetic resonance imaging, respectively. Graft characteristics were assessed by immunohistology. Bioluminescence signals on days 5, 8, and 14 were higher for GF-based grafts compared with plain H9c2 injections (P<0.03). Signals were higher for GF/MG grafts compared with GF alone (P<0.02). GFP-positive, spindle-shaped H9c2 cells were found integrated in the infarct border zones at day 28. Left ventricular (LV) function of hearts implanted with collagen-based grafts was better compared with controls (P<0.05). Vascular endothelial growth factor or fibroblast growth factor did not further improve graft survival or heart function.Collagen matrices enhance early survival of H9c2 cardiomyoblasts after transplantation into ischemic hearts and lead to improved LV function. Further optimization of the graft design should make restoration of large myocardial infarctions by tissue engineering approaches effective.

    View details for DOI 10.1161/CIRCULATIONAHA.105.001297

    View details for Web of Science ID 000238688200028

    View details for PubMedID 16820568

  • A brief history of RNAi: the silence of the genes FASEB JOURNAL Sen, G. L., Blau, H. M. 2006; 20 (9): 1293-1299

    Abstract

    The use of the RNA interference (RNAi) pathway to eliminate gene products has greatly facilitated the understanding of gene function. Behind this remarkable pathway is an intricate network of proteins that ensures the degradation of the target mRNA. In this review, we explore the history of RNAi as well as highlighting recent discoveries.

    View details for DOI 10.1096/fj.06-6014rev

    View details for Web of Science ID 000240266000006

    View details for PubMedID 16816104

  • Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence NATURE METHODS Wehrman, T. S., von Degenfeld, G., Krutzik, P., Nolan, G. P., Blau, H. M. 2006; 3 (4): 295-301

    Abstract

    We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of beta-galactosidase (beta-gal) activity. The substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered cells and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of beta-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant beta-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of beta-gal permits bioluminescent imaging applications that previously were not possible.

    View details for DOI 10.1038/NMETH868

    View details for Web of Science ID 000236501400017

    View details for PubMedID 16554835

  • IGF-I increases bone marrow contribution to adult skeletal muscle and enhances the fusion of myelomonocytic precursors JOURNAL OF CELL BIOLOGY Sacco, A., Doyonnas, R., LaBarge, M. A., Hammer, M. M., Kraft, P., Blau, H. M. 2005; 171 (3): 483-492

    Abstract

    Muscle damage has been shown to enhance the contribution of bone marrow-derived cells (BMDCs) to regenerating skeletal muscle. One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC). However, the molecular components responsible for this injury-related response remain largely unknown. In this paper, we show that delivery of insulin-like growth factor I (IGF-I) to adult skeletal muscle by three different methods-plasmid electroporation, injection of genetically engineered myoblasts, and recombinant protein injection-increases the integration of BMDCs up to fourfold. To investigate the underlying mechanism, we developed an in vitro fusion assay in which co-cultures of MMCs and myotubes were exposed to IGF-I. The number of fusion events was substantially augmented by IGF-I, independent of its effect on cell survival. These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

    View details for DOI 10.1083/jcb.200506123

    View details for Web of Science ID 000233113400013

    View details for PubMedID 16275752

  • Optimizing techniques for tracking transplanted stem cells in vivo STEM CELLS Brazelton, T. R., Blau, H. M. 2005; 23 (9): 1251-1265

    Abstract

    The potential for bone marrow-derived cells (BMDCs) to contribute to nonhematopoietic tissues has generated considerable debate in recent years. Causes for the controversies include disparities in the techniques used to track engraftment of BMDCs, inappropriate tissue preparation, a lack of appropriate positive and negative controls, and basic misunderstandings about how to properly collect and interpret images from epifluorescent and confocal microscopes. Our laboratory was among the first to use bone marrow transplants from transgenic mice constitutively expressing enhanced green fluorescent protein (GFP) to study the ability of BMDCs to give rise to nonhematopoietic tissue types, a system that is now in widespread use. During our 6 years of experience using GFP, as well as beta-galactosidase and the Y chromosome, to track BMDCs in vivo, we have identified many difficulties and have developed techniques to resolve them. We discuss several of these methods, and, in particular, we describe ratiometric analysis techniques for improving detection of transplanted cells derived from genetically modified bone marrow. Finally, to help resolve reported discrepancies regarding the frequency with which BMDCs contribute to skeletal myofibers, we demonstrate that the pattern of highly autofluorescent myofibers in skeletal muscle is clearly distinct from that of GFP-expressing myofibers and describe how unambiguous conclusions can be drawn from such data.

    View details for Web of Science ID 000232672700005

    View details for PubMedID 16109764

  • mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage DIFFERENTIATION Sen, G. L., Wehrman, T. S., Blau, H. M. 2005; 73 (6): 287-293

    Abstract

    RNA interference constitutes a major means of eliminating mRNAs, yet how the small interfering RNAs (siRNA) within the RNA-induced silencing complex (RISC) finds its homologous target in the cell remains unknown. An attractive hypothesis is that RNA interference is linked to translation which allows RISC ready access to every translated mRNA. To test whether translation could direct siRNAs to mRNAs, chemical and biological inhibitors of translation and their effects on mRNA cleavage were tested. Our results show that mRNA degradation by siRNAs is not dependent on mRNA translation.

    View details for Web of Science ID 000231506500003

    View details for PubMedID 16138829

  • Enzymatic detection of protein translocation NATURE METHODS Wehrman, T. S., Casipit, C. L., Gewertz, N. M., Blau, H. M. 2005; 2 (7): 521-527

    Abstract

    Fundamental to eukaryotic cell signaling is the regulation of protein function by directed localization. Detection of these events has been largely qualitative owing to the limitations of existing technologies. Here we describe a method for quantitatively assessing protein translocation using proximity-induced enzyme complementation. The complementation assay for protein translocation (CAPT) is derived from beta-galactosidase and comprises one enzyme fragment, omega, which is localized to a particular subcellular region, and a small complementing peptide, alpha, which is fused to the protein of interest. The concentration of alpha in the immediate vicinity of omega correlates with the amount of enzyme activity obtained in a dose- and time-dependent manner, thus acting as a genetically encoded biosensor for local protein concentration. Using CAPT, inducible protein movement from the cytosol to the nucleus or plasma membrane was quantitatively monitored in multiwell format and in live mammalian cells by flow cytometry.

    View details for DOI 10.1038/NMETH771

    View details for Web of Science ID 000230165700016

    View details for PubMedID 15973423

  • Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies NATURE CELL BIOLOGY Sen, G. L., Blau, H. M. 2005; 7 (6): 633-U28

    Abstract

    RNA interference (RNAi) is an important means of eliminating mRNAs, but the intracellular location of RNA-induced silencing complex (RISC) remains unknown. We show here that Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centres that are known as cytoplasmic bodies. The localization of Argonaute 2 in decay centres is not altered by the presence or absence of small interfering RNAs or their targeted mRNAs. However, RNA is required for the integrity of cytoplasmic bodies because RNase eliminates Argonaute 2 localization. In addition, Argonaute 1, another Argonaute family member, is concentrated in cytoplasmic bodies. These results provide new insight into the mechanism of RNAi function.

    View details for DOI 10.1038/ncb1265

    View details for Web of Science ID 000229562100018

    View details for PubMedID 15908945

  • Critical role of microenvironmental factors in angiogenesis. Current atherosclerosis reports Banfi, A., von Degenfeld, G., Blau, H. M. 2005; 7 (3): 227-234

    Abstract

    Therapeutic angiogenesis, which entails the induction of new blood vessels by the delivery of angiogenic growth factors, is a highly attractive approach to the treatment of ischemic diseases. However, it is becoming increasingly clear that this is not easily achieved, as the effects of angiogenic growth factors can differ markedly depending on the timing of their expression, on the shape of the concentration gradients they form in vivo, and the inter-actions between endothelial cells and pericytes they induce. In fact, the same dose of vascular endothelial growth factor can induce stable, nonleaky, pericyte-covered normal capillaries or aberrant vascular structures that develop into hemangiomas. This difference in outcome can be due solely to the spatial characteristics of the delivery method. If delivery allows a homogeneous spatial distribution of VEGF in the microenvironment around each producing cell, angiogenesis can be therapeutic, whereas if the total dose is the average of diverse spatial levels, aberrant angiogenesis cannot be avoided. To achieve therapeutic angiogenesis, a means of regulating the microenvironmental levels of angiogenic factors will be critical to the generation of effective new treatment strategies.

    View details for PubMedID 15811258

  • Overexpression of dimethylarginine dimethylaminohydrolase reduces tissue asymmetric dimethylarginine levels and enhances angiogenesis CIRCULATION Jacobi, J., Sydow, K., von Degenfeld, G., Zhang, Y., Dayoub, H., Wang, B. Y., Patterson, A. J., Kimoto, M., Blau, H. M., Cooke, J. P. 2005; 111 (11): 1431-1438

    Abstract

    This study was designed to determine whether overexpression of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) could enhance angiogenesis by reducing levels of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA).In DDAH1 transgenic (TG) and wild-type mice (each n=42), the role of DDAH overexpression on angiogenesis was studied by use of the disk angiogenesis system and a murine model of hindlimb ischemia (each n=21). After surgery, animals were treated with either PBS or the NOS inhibitors ADMA or N(omega)-nitro-L-arginine methyl ester (L-NAME; each 250 micromol x kg(-1) x d(-1)) by use of osmotic minipumps (each n=7). L-NAME was chosen to study an inhibitor that is not degraded by DDAH. Neovascularization in the disk angiogenesis system was impaired by both NOS inhibitors; however, TG animals were resistant to the effects of ADMA on neovascularization. Similarly, TG mice were more resistant to the inhibitory effect of ADMA on angioadaptation (angiogenesis and arteriogenesis) after hindlimb ischemia, as assessed by fluorescent microsphere studies and postmortem microangiograms. Enhanced neovascularization and limb perfusion in TG mice were associated with reduced plasma and tissue ADMA levels and enhanced tissue NOS enzyme activity.We describe a novel mechanism by which DDAH regulates postnatal neovascularization. Therapeutic manipulation of DDAH expression or activity may represent a novel approach to restore tissue perfusion.

    View details for DOI 10.1161/01.CIR.0000158487.80483.09

    View details for Web of Science ID 000227805700012

    View details for PubMedID 15781754

  • Bone marrow contribution to skeletal muscle: A physiological response to stress DEVELOPMENTAL BIOLOGY Palermo, A. T., LaBarge, M. A., Doyonnas, R., Pomerantz, J., Blau, H. M. 2005; 279 (2): 336-344

    Abstract

    Adult bone marrow-derived stem cells (BMDC) have been shown to contribute to numerous tissues after transplantation into a new host. However, whether the participation of these cells is part of the normal response to injury remains a matter of debate. Using parabiotically joined pairs of genetically labeled and wildtype mice, we show here that irradiation-induced damage of the target tissue, injection of bone marrow into the circulation, and immunological perturbation that are consequences of bone marrow transplantation are not necessary for bone marrow contribution to myofibers. Moreover, severe toxin-induced damage is not a prerequisite, as BMDC contribution to muscle is enhanced in response to increased muscle activity resulting from muscle overloading or forced exercise. Indeed, these two forms of muscle stress result in much more rapid contribution (within 1 month) than voluntary running (6 months). These results indicate that BMDC contribute to myofibers in response to physiologic stresses encountered by healthy organisms throughout life.

    View details for DOI 10.1016/j.ydbio.2004.12.024

    View details for Web of Science ID 000227487200005

    View details for PubMedID 15733662

  • Hematopoietic contribution to skeletal muscle regeneration by myelomonocytic precursors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Doyonnas, R., LaBarge, M. A., Sacco, A., Charlton, C., Blau, H. M. 2004; 101 (37): 13507-13512

    Abstract

    Adult bone marrow-derived cells can participate in muscle regeneration after bone marrow transplantation. In recent studies a single hematopoietic stem cell (HSC) was shown to give rise to cells that not only reconstituted all of the lineages of the blood, but also contributed to mature muscle fibers. However, the relevant HSC derivative with this potential has not yet been definitively identified. Here we use fluorescence-activated cell sorter-based protocols to test distinct hematopoietic fractions and show that only fractions containing c-kit(+) immature myelomonocytic precursors are capable of contributing to muscle fibers after i.m. injection. Although these cells belong to the myeloid lineage, they do not include mature CD11b(+) myelomonocytic cells, such as macrophages. Of the four sources of mature macrophages tested that were derived either from monocytic culture, bone marrow, peripheral blood after granulocyte colony-stimulating factor mobilization, or injured muscle, none contributed to muscle. In addition, after transplantation of bone marrow isolated from CD11b-Cre-transgenic mice into the Cre-reporter strain (Z/EG), no GFP myofibers were detected, demonstrating that macrophages expressing CD11b do not fuse with myofibers. Irrespective of the underlying mechanisms, these data suggest that the HSC derivatives that integrate into regenerating muscle fibers exist in the pool of hematopoietic cells known as myelomonocytic progenitors.

    View details for DOI 10.1073/pnas.0405361101

    View details for Web of Science ID 000223917900022

    View details for PubMedID 15353585

  • Nuclear reprogramming: A key to stem cell function in regenerative medicine NATURE CELL BIOLOGY Pomerantz, J., Blau, H. M. 2004; 6 (9): 810-816

    Abstract

    The goal of regenerative medicine is to restore form and function to damaged tissues. One potential therapeutic approach involves the use of autologous cells derived from the bone marrow (bone marrow-derived cells, BMDCs). Advances in nuclear transplantation, experimental heterokaryon formation and the observed plasticity of gene expression and phenotype reported in multiple phyla provide evidence for nuclear plasticity. Recent observations have extended these findings to show that endogenous cells within the bone marrow have the capacity to incorporate into defective tissues and be reprogrammed. Irrespective of the mechanism, the potential for new gene expression patterns by BMDCs in recipient tissues holds promise for developing cellular therapies for both proliferative and post-mitotic tissues.

    View details for DOI 10.1038/ncb0904-810

    View details for Web of Science ID 000223750300010

    View details for PubMedID 15340448

  • Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis JOURNAL OF CLINICAL INVESTIGATION Ozawa, C. R., Banfi, A., Glazer, N. L., Thurston, G., Springer, M. L., Kraft, P. E., McDonald, D. M., Blau, H. M. 2004; 113 (4): 516-527

    Abstract

    Use of long-term constitutive expression of VEGF for therapeutic angiogenesis may be limited by the growth of abnormal blood vessels and hemangiomas. We investigated the relationship between VEGF dosage and the morphology and function of newly formed blood vessels by implanting retrovirally transduced myoblasts that constitutively express VEGF164 into muscles of adult mice. Reducing VEGF dosage by decreasing the total number of VEGF myoblasts implanted did not prevent vascular abnormalities. However, when clonal populations of myoblasts homogeneously expressing different levels of VEGF were implanted, a threshold between normal and aberrant angiogenesis was found. Clonal myoblasts that expressed low to medium levels of VEGF induced growth of stable, pericyte-coated capillaries of uniform size that were not leaky and became VEGF independent, as shown by treatment with the potent VEGF blocker VEGF-TrapR1R2. In contrast, clones that expressed high levels of VEGF induced hemangiomas. Remarkably, when different clonal populations were mixed, even a small proportion of cells with high production of VEGF was sufficient to cause hemangioma growth. These results show for the first time to our knowledge that the key determinant of whether VEGF-induced angiogenesis is normal or aberrant is the microenvironmental amount of growth factor secreted, rather than the overall dose. Long-term continuous delivery of VEGF, when maintained below a threshold microenvironmental level, can lead to normal angiogenesis without other exogenous growth factors.

    View details for DOI 10.1172/JCI200418420

    View details for Web of Science ID 000189008000007

    View details for PubMedID 14966561

  • Discordant effects of a soluble VEGF receptor on wound healing and angiogenesis GENE THERAPY Jacobi, J., Tam, B. Y., Sundram, U., von Degenfeld, G., Blau, H. M., Kuo, C. J., Cooke, J. P. 2004; 11 (3): 302-309

    Abstract

    Soluble receptors to vascular endothelial growth factor (VEGF) can inhibit its angiogenic effect. Since angiogenesis is involved in wound repair, we hypothesized that adenovirus-mediated gene transfer of a soluble form of VEGF receptor 2 (Flk-1) would attenuate wound healing in mice. C57Bl/6J and genetically diabetic (db/db) mice (each n=20) received intravenous (i.v.) injections of recombinant adenoviruses (10(9) PFU) encoding the ligand-binding ectodomain of VEGF receptor 2 (Flk-1) or cDNA encoding the murine IgG2alpha Fc fragment (each n=10). At 4 days after gene transfer, two full-thickness skin wounds (0.8 cm) were created on the dorsum of each animal. Wound closure was measured over 9-14 days after which wounds were resected for histological analysis. Prior to killing, fluorescent microspheres were systemically injected for quantitation of wound vascularity. Single i.v. injections of adenoviruses encoding soluble Flk-1 significantly decreased wound angiogenesis in both wild-type and diabetic mice. Fluorescence microscopy revealed a 2.0-fold (wild type) and 2.9-fold (diabetic) reduction in wound vascularity in Flk-1-treated animals (p<0.05). Impairment of angiogenesis was confirmed by CD31 immunohistochemistry. Interestingly, despite significant reductions in wound vascularity, wound closure was not grossly delayed. Our data indicates that while VEGF function is essential for optimal wound angiogenesis, it is not required for wound closure.

    View details for DOI 10.1038/sj.gt.3302162

    View details for Web of Science ID 000188329600011

    View details for PubMedID 14737090

  • Restriction enzyme-generated siRNA (REGS) vectors and libraries NATURE GENETICS Sen, G., Wehrman, T. S., MYERS, J. W., Blau, H. M. 2004; 36 (2): 183-189

    Abstract

    Small interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme-generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4 x 10(5) clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.

    View details for DOI 10.1038/ng1288

    View details for Web of Science ID 000188542700022

    View details for PubMedID 14704668

  • Restriction enzyme-generated siRNA (REGS) vectors and libraries. Nature Genetics Sen, G., Wehrman TS, Myers JW, Blau HM. 2004; 36 (2): 183-9
  • Nuclear reprogramming: a key to stem cell function in regenerative medicine. Nature Cell Biology Pomerantz, J., Blau, HM 2004; 6 (9): 810-816
  • Hematopoietic contribution to skeletal muscle regeneration by myelomonocytic precursors. PNAS Doyonnas R, LaBarge MA, Sacco A, Charlton C, Blau HM 2004; 100 (37): 13507-13512
  • Contribution of hematopoietic stem cells to skeletal muscle NATURE MEDICINE Corbel, S. Y., Lee, A., Yi, L., Duenas, J., Brazelton, T. R., Blau, H. M., Rossi, F. M. 2003; 9 (12): 1528-1532

    Abstract

    Cells from adult bone marrow participate in the regeneration of damaged skeletal myofibers. However, the relationship of these cells with the various hematopoietic and nonhematopoietic cell types found in bone marrow is still unclear. Here we show that the progeny of a single cell can both reconstitute the hematopoietic system and contribute to muscle regeneration. Integration of bone marrow cells into myofibers occurs spontaneously at low frequency and increases with muscle damage. Thus, classically defined single hematopoietic stem cells can give rise to both blood and muscle.

    View details for DOI 10.1038/nm959

    View details for Web of Science ID 000186845400037

    View details for PubMedID 14625543

  • Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant NATURE CELL BIOLOGY Weimann, J. M., Johansson, C. B., Trejo, A., Blau, H. M. 2003; 5 (11): 959-966

    Abstract

    Heterokaryons are the product of cell fusion without subsequent nuclear or chromosome loss. Decades of research using Sendai-virus or polyethylene glycol (PEG)-mediated fusion in tissue culture showed that the terminally differentiated state of a cell could be altered. But whether stable non-dividing heterokaryons could occur in animals has remained unclear. Here, we show that green fluorescent protein (GFP)-positive bone-marrow-derived cells (BMDCs) contribute to adult mouse Purkinje neurons through cell fusion. The formation of heterokaryons increases in a linear manner over 1.5 years and seems to be stable. The dominant Purkinje neurons caused the BMDC nuclei within the resulting heterokaryons to enlarge, exhibit dispersed chromatin and activate a Purkinje neuron-specific transgene, L7-GFP. The observed reprogrammed heterokaryons that form in brain may provide insights into gene regulation associated with cell-fate plasticity.

    View details for DOI 10.1038/ncb1053

    View details for Web of Science ID 000186273100011

    View details for PubMedID 14562057

  • Significant differences among skeletal muscles in the incorporation of bone marrow-derived cells DEVELOPMENTAL BIOLOGY Brazelton, T. R., Nystrom, M., Blau, H. M. 2003; 262 (1): 64-74

    Abstract

    While numerous reports indicate that adult bone marrow-derived cells can contribute to nonhematopoietic tissues in vivo in adult mice, the generally low frequency of these events has made it difficult to study the molecular and cellular pathways involved. Here, we show a 1000-fold range in the frequency with which diverse skeletal muscles incorporate adult bone marrow-derived cells in adult mice. Most striking was the finding of one specific muscle, the panniculus carnosus, in which up to 5% of myofibers incorporated bone marrow-derived cells over a 16- month period in the absence of experimentally induced selective pressure. These results suggest that muscles differ physiologically, establishing the panniculus carnosus as an assay for identifying the key regulators, such as trophic, homing, and differentiation factors, as well as the relevant cells within the bone marrow that are capable of circulating throughout the periphery and contributing to adult, nonhematopoietic tissues, such as skeletal muscle. Finally, the 5% incorporation of adult stem cells into skeletal muscle is the highest reported to date in the absence of experimentally induced selective pressure and is at a level that may be consistent with improving the function of defective muscle tissue.

    View details for DOI 10.1016/S0012-1606(03)00357-9

    View details for Web of Science ID 000185616400005

    View details for PubMedID 14512018

  • Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis BRITISH JOURNAL OF PHARMACOLOGY von Degenfeld, G., Banfi, A., Springer, M. L., Blau, H. M. 2003; 140 (4): 620-626

    Abstract

    Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as VEGF and FGF. Clinical trials are underway in patients with peripheral vascular and coronary heart disease. However, increasing evidence indicates that the new vasculature needs to be stabilized to avoid deleterious effects such as edema and hemangioma formation. Moreover, a major challenge is to induce new vessels that persist following cessation of the angiogenic stimulus. Mature vessels may be generated by modulating timing and dosage of growth factor expression, or by combination of 'growth' factors with 'maturation' factors like PDGF-BB, angiopoietin-1 or TGF-beta. Myoblast-mediated gene transfer has unique characteristics that make it a useful tool for studying promising novel approaches to therapeutic angiogenesis. It affords robust and long-lasting expression, and can be considered as a relatively rapid form of 'adult transgenesis' in muscle. The combined insertion of different gene constructs into single myoblasts and their progeny allows the simultaneous expression of different 'growth' and 'maturation' factors within the same cell in vivo. The additional insertion of a reporter gene makes it possible to analyze the phenotype of the vessels surrounding the transgenic muscle fibers into which the myoblasts have fused. The effects of timing and duration of gene expression can be studied by using tetracycline-inducible constructs, and dosage effects by selecting subpopulations consistently expressing distinct levels of growth factors. Finally, the autologous cell-based approach using transduced myoblasts could be an alternative gene delivery system for therapeutic angiogenesis in patients, avoiding the toxicities seen with some viral vectors.

    View details for DOI 10.1038/sj.bjp.0705492

    View details for Web of Science ID 000186240600005

    View details for PubMedID 14534145

  • Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle MOLECULAR THERAPY Springer, M. L., Ozawa, C. R., Banfi, A., Kraft, P. E., Ip, T. K., Brazelton, T. R., Blau, H. M. 2003; 7 (4): 441-449

    Abstract

    We have shown previously that implantation of myoblasts constitutively expressing the VEGF-A gene into nonischemic mouse skeletal muscle leads to overgrowth of capillary-like blood vessels and hemangioma formation. These aberrant effects occurred directly at the implantation site. We show here that these regions result from angiogenic capillary growth and involve a change in capillary growth pattern and that smooth muscle-coated vessels similar to arterioles form directly adjacent to the implantation site. Myoblasts genetically engineered to produce VEGF were implanted into mouse leg muscles. Implantation sites were surrounded by a zone of dense capillary-sized vessels, around which was a second zone of muscle containing larger, smooth-muscle-covered vessels but few capillaries, and an outer zone of muscle exhibiting normal capillary density. The lack of capillaries in the middle region suggests that the preexisting capillaries adjacent to the implantation site underwent enlargement and/or fusion and recruited a smooth muscle coat. Capillaries at the implantation site were frequently wrapped around VEGF-producing muscle fibers and were continuous with the circulation and were not observed to include bone-marrow-derived endothelial cells. In contrast with the distant arteriogenesis resulting from VEGF delivery described in previous studies, we report here that highly localized arterioles also form adjacent to the site of delivery.

    View details for DOI 10.1016/S1525-0016(03)00010-8

    View details for Web of Science ID 000182448100007

    View details for PubMedID 12727106

  • Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Weimann, J. M., Charlton, C. A., Brazelton, T. R., Hackman, R. C., Blau, H. M. 2003; 100 (4): 2088-2093

    Abstract

    We show here that cells within human adult bone marrow can contribute to cells in the adult human brain. Cerebellar tissues from female patients with hematologic malignancies, who had received chemotherapy, radiation, and a bone marrow transplant, were analyzed. Brain samples were obtained at autopsy from female patients who received male (sex-mismatched) or female (sex-matched, control) bone marrow transplants. Cerebella were evaluated in 10-microm-thick, formaldehyde-fixed, paraffin-embedded sections that encompassed up to approximately 50% of a human Purkinje nucleus. A total of 5,860 Purkinje cells from sex-mismatched females and 3,202 Purkinje cells from sex-matched females were screened for Y chromosomes by epifluorescence. Confocal laser scanning microscopy allowed definitive identification of the sex chromosomes within the morphologically distinct Purkinje cells. In the brains of females who received male bone marrow, four Purkinje neurons were found that contained an X and a Y chromosome and two other Purkinje neurons contained more than a diploid number of sex chromosomes. No Y chromosomes were detected in the brains of sex-matched controls. The total frequency of male bone marrow contribution to female Purkinje cells approximated 0.1%. This study demonstrates that although during human development Purkinje neurons are no longer generated after birth, cells within the bone marrow can contribute to these CNS neurons even in adulthood. The underlying mechanism may be caused either by generation de novo of Purkinje neurons from bone marrow-derived cells or by fusion of marrow-derived cells with existing recipient Purkinje neurons.

    View details for DOI 10.1073/pnas.0337659100

    View details for Web of Science ID 000181073000115

    View details for PubMedID 12576546

  • Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant. Nature Cell Biology Weimann JM, Johansson CB, Trejo A, Blau HM 2003; 5 (11): 959 - 966
  • Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains. PNAS Weimann JM, Charlton CA, Brazelton TR, Hackman RC, Blau HM 2003; 100 (4): 2088-93
  • Something in the eye of the beholder SCIENCE Blau, H., Brazelton, T., Keshet, G., Rossi, F. 2002; 298 (5592): 361-362

    View details for Web of Science ID 000178510400020

    View details for PubMedID 12392027

  • RIP2, a checkpoint in myogenic differentiation MOLECULAR AND CELLULAR BIOLOGY Munz, B., Hildt, E., Springer, M. L., Blau, H. M. 2002; 22 (16): 5879-5886

    Abstract

    Using a subtractive cDNA library hybridization approach, we found that receptor interacting protein 2 (RIP2), a tumor necrosis factor receptor 1 (TNFR-1)-associated factor, is a novel early-acting gene that decreases markedly in expression during myogenic differentiation. RIP2 consists of three domains: an amino-terminal kinase domain, an intermediate domain, and a carboxy-terminal caspase activation and recruitment domain (CARD). In some cell types, RIP2 has been shown to be a potent inducer of apoptosis and an activator of NF-kappa B. To analyze the function of RIP2 during differentiation, we transduced C2C12 myoblasts with retroviral vectors to constitutively produce RIP2 at high levels. When cultured in growth medium, these cells did not show an enhanced rate of proliferation compared to controls. When switched to differentiation medium, however, they continued to proliferate, whereas control cells withdrew from the cell cycle, showed increased expression of differentiation markers such as myogenin, and began to differentiate into multinucleated myotubes. The complete RIP2 protein appeared to be necessary to inhibit myogenic differentiation, since two different deletion mutants lacking either the amino-terminal kinase domain or the carboxy-terminal CARD had no effect. A mutant deficient in kinase activity, however, had effects similar to wild-type RIP2, indicating that phosphorylation was not essential to the function of RIP2. Furthermore, RIP proteins appeared to be important during myogenic differentiation in vivo, as we detected a marked decrease in expression of the RIP2 homolog RIP in several muscle tissues of the dystrophic mdx mouse, a model for continuous muscle degeneration and regeneration. We conclude that RIP proteins can act independently of TNFR-1 stimulation by ligand to modulate downstream signaling pathways, such as activation of NF-kappa B. These results implicate RIP2 in a previously unrecognized role: a checkpoint for myogenic proliferation and differentiation.

    View details for DOI 10.1128/MCB.22.16.5879-5886.2002

    View details for Web of Science ID 000177089200023

    View details for PubMedID 12138198

  • VEGF gene delivery for treatment of ischemic cardiovascular disease TRENDS IN CARDIOVASCULAR MEDICINE Koransky, M. L., Robbins, R. C., Blau, H. M. 2002; 12 (3): 108-114

    Abstract

    There are increasing numbers of patients with ischemic myocardial disease not amenable to traditional methods of revascularization. These patients may benefit from new research into the use of naturally occurring angiogenic compounds, such as vascular endothelial growth factor (VEGF) for re-establishing blood flow into regions of hibernating myocardium. Animal studies and human clinical trials evaluating VEGF demonstrate increases in myocardial perfusion after treatment, with some patients reporting improvement in anginal symptoms. Further research into the ideal form of VEGF therapy (protein, plasmid, or adenoviral) and delivery method (intracoronary, intramyocardial, or epicardial) seems justified.

    View details for Web of Science ID 000175505900003

    View details for PubMedID 12007735

  • Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation CELL MOTILITY AND THE CYTOSKELETON Springer, M. L., Ozawa, C. R., Blau, H. M. 2002; 51 (4): 177-186

    Abstract

    alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle myoblasts or skeletal muscle fibers. However, both primary myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary myoblasts. Within one day after implantation of primary myoblasts into mouse skeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, both neonatal primary myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.

    View details for DOI 10.1002/cm.10022

    View details for Web of Science ID 000175264900002

    View details for PubMedID 11977092

  • Protein-protein interactions monitored in mammalian cells via complementation of beta-lactamase enzyme fragments PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wehrman, T., Kleaveland, B., Her, J. H., Balint, R. F., Blau, H. M. 2002; 99 (6): 3469-3474

    Abstract

    We have defined inactive alpha and omega fragments of beta-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the alpha fragment increased complemented enzyme activity by up to 4 orders of magnitude. beta-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date.

    View details for DOI 10.1073/pnas.062043699

    View details for Web of Science ID 000174511000023

    View details for PubMedID 11904411

  • Gene delivery to muscle. Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.] Springer, M. L., Rando, T. A., Blau, H. M. 2002; Chapter 13: Unit13 4-?

    Abstract

    The delivery of genes to skeletal muscle by myoblast implantation, DNA injection, or viral transduction has therapeutic applications for human neuromuscular and systemic disorders, many of which are now represented by transgenic or "knockout" mouse models. This unit describes the isolation and retroviral transduction of mouse myoblasts, the injection of myoblasts and plasmid DNA into mouse muscle, and histological methods for analyzing the recipient muscle. A procedure describing the injection of plasmid DNA into muscle with or without electric charge is also included.

    View details for DOI 10.1002/0471142905.hg1304s31

    View details for PubMedID 18428326

  • Myoblast-mediated gene transfer for therapeutic angiogenesis GENE THERAPY METHODS Banfi, A., Springer, M. L., Blau, H. M. 2002; 346: 145-157

    View details for Web of Science ID 000174462100009

    View details for PubMedID 11883066

  • The evolving concept of a stem cell: Entity or function? CELL Blau, H. M., Brazelton, T. R., Weimann, J. M. 2001; 105 (7): 829-841

    View details for Web of Science ID 000169664300004

    View details for PubMedID 11439179

  • Not the usual suspects: the unexpected sources of tissue regeneration JOURNAL OF CLINICAL INVESTIGATION Springer, M. L., Brazelton, T. R., Blau, H. M. 2001; 107 (11): 1355-1356

    View details for Web of Science ID 000169160700005

    View details for PubMedID 11390416

  • Laminin-induced change in conformation of preexisting alpha 7 beta 1 integrin signals secondary myofiber formation DEVELOPMENTAL BIOLOGY Blanco-Bose, W. E., Blau, H. M. 2001; 233 (1): 148-160

    Abstract

    Two distinct populations of myoblasts, distinguishable by alpha7 integrin expression have been hypothesized to give rise to two phases of myofiber formation in embryonic limb development. We show here that alpha7 integrin is detectable far earlier than previously reported on both "primary" and "secondary" lineage myoblasts and myofibers. An antibody (1211) that recognizes an intracellular epitope allowed detection of alpha7 integrin previously missed using an antibody (H36) that recognizes an extracellular epitope. We found that when myoblasts were isolated and cultured from different developmental stages, H36 only detected alpha7 integrin that was in direct contact with its ligand, laminin. Moreover, alpha7 integrin detection by H36 was reversible and highly localized to subcellular points of contact between myoblasts and laminin-coated 2.8-microm microspheres. Prior to secondary myofiber formation in limb embryogenesis, laminin was present but not in close proximity to clusters of primary myofibers that expressed alpha7 integrin detected by antibody 1211 using deconvolution microscopy. These results suggest that the timing of the interaction of preexisting alpha7 integrin with its ligand, laminin, is a major determinant of allosteric changes that result in an activated form of alpha7 integrin capable of transducing signals from the extracellular matrix commensurate with secondary myofiber formation.

    View details for Web of Science ID 000168500100012

    View details for PubMedID 11319864

  • Purification of mouse primary myoblasts based on alpha 7 integrin expression EXPERIMENTAL CELL RESEARCH Blanco-Bose, W. E., Yao, C. C., Kramer, R. H., Blau, H. M. 2001; 265 (2): 212-220

    Abstract

    Fundamental insights have come from the study of myogenesis. Primary myoblasts isolated directly from muscle tissue more closely approximate myogenesis than established cell lines. However, contamination of primary muscle cultures with nonmyogenic cells can complicate the results. To overcome this problem, we previously described a method for myoblast purification based on novel culture conditions (T. A. Rando and H. M. Blau, 1994, J. Cell Biol. 125, 1275--1287). Here we report a refinement of this method that leads directly to an enriched population of mouse primary myoblasts, within significantly fewer population doublings. The method described here avoids using adhesion as a criterion for selection. This advance capitalizes on the ability of the antibody CA5.5 to recognize alpha 7 integrin, a muscle-specific cell surface antigen. Enrichment of myoblasts to greater than 95% of the cell population can be achieved by a single round of flow cytometry or magnetic bead separation. This is the first description of a mouse myoblast purification method based on a cell-type-specific antigen. The ease of this procedure for isolating primary myoblasts should expand the opportunities for (1) using these cells in cell transplantation studies in animal models of human disease, (2) isolating and characterizing mutant myoblasts from transgenic animals, and (3) allowing in vitro studies of molecules that regulate muscle cell growth, differentiation, and neoplasia.

    View details for Web of Science ID 000168434400004

    View details for PubMedID 11302686

  • In vivo monitoring of myoblast transplantation into rat myocardium. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation Koransky, M. L., Ip, T. K., Wu, S., Cao, Y., Berry, G., Contag, C., Blau, H., Robbins, R. 2001; 20 (2): 188-189

    View details for PubMedID 11250320

  • From marrow to brain: Expression of neuronal phenotypes in adult mice SCIENCE Brazelton, T. R., Rossi, F. M., Keshet, G. I., Blau, H. M. 2000; 290 (5497): 1775-1779

    Abstract

    After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton neurofilament, and class III beta-tubulin) and were able to activate the transcription factor cAMP response element-binding protein (CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates a remarkable plasticity of adult tissues with potential clinical applications.

    View details for Web of Science ID 000165632400046

    View details for PubMedID 11099418

  • Transcriptional control: Rheostat converted to on/off switch MOLECULAR CELL Rossi, F. M., Kringstein, A. M., Spicher, A., Guicherit, O. M., Blau, H. M. 2000; 6 (3): 723-728

    Abstract

    Individual cells translate concentration gradients of extracellular factors into all-or-none threshold responses leading to discrete patterns of gene expression. Signaling cascades account for some but not all such threshold responses, suggesting the existence of additional mechanisms. Here we show that all-or-none responses can be generated at a transcriptional level. A graded rheostat mechanism obtained when either transactivators or transrepressors are present is converted to an on/off switch when these factors compete for the same DNA regulatory element. Hill coefficients of dose-response curves confirm that the synergistic responses generated by each factor alone are additive, obviating the need for feedback loops. We postulate that regulatory networks of competing transcription factors prevalent in cells and organisms are crucial for establishing true molecular on/off switches.

    View details for Web of Science ID 000089578600021

    View details for PubMedID 11030351

  • VEGF gene delivery to myocardium - Deleterious effects of unregulated expression CIRCULATION Lee, R. J., Springer, M. L., Blanco-Bose, W. E., Shaw, R., Ursell, P. C., Blau, H. M. 2000; 102 (8): 898-901

    Abstract

    Vascular endothelial growth factor (VEGF) is being investigated for therapeutic angiogenesis in ischemic myocardium. Primarily, transient delivery systems have been tested. The goal of this study was to investigate the effects of continuous expression of VEGF in myocardium by use of myoblast-mediated delivery.Primary murine myoblasts (5 x 10(5) cells in 10 microL of PBS with 0.5% BSA) expressing both the murine VEGF gene and the beta-galactosidase (beta-gal) gene from a retroviral promoter were implanted in the ventricular wall of immunodeficient mice (n=11) via a subdiaphragmatic approach. Control immunodeficient mice (n=12) were injected with the same number of myoblasts expressing only the beta-gal gene. Between days 14 and 16, surviving mice were euthanized and the hearts processed for histology. In the experimental group, 11 of 11 mice demonstrated failure to thrive by day 13; 5 deaths occurred between days 8 and 15. There were no complications in the control mice. Histochemistry documented successful implantation of myoblasts (positive beta-gal reaction product) in 6 of 6 surviving experimental mice and 12 of 12 controls. Histology disclosed intramural vascular tumors resembling hemangiomas in the VEGF-myoblast-injected myocardium in 6 of 6 surviving mice. beta-Gal-expressing cells were present at the site of the vascular tumors. Immunohistochemistry localized abundant endothelial nitric oxide synthase and CD31 (platelet and endothelial cell adhesion molecule) within the lesion, consistent with the presence of endothelial cells.In this model, unregulated continuous expression of VEGF is associated with (1) a high rate of failure to thrive/death and (2) formation of endothelial cell-derived intramural vascular tumors in the implantation site. These results underscore the importance of regulating VEGF expression for therapeutic angiogenesis.

    View details for Web of Science ID 000088863200015

    View details for PubMedID 10952959

  • Neural cell adhesion molecule (NCAM) and myoblast fusion DEVELOPMENTAL BIOLOGY Charlton, C. A., Mohler, W. A., Blau, H. M. 2000; 221 (1): 112-119

    Abstract

    Considerable evidence points to an involvement of neural cell adhesion molecule (NCAM) in myoblast fusion. Changes in the level of NCAM expression, isoform specificity, and localization in muscle cells and tissues correspond to key morphogenetic events during muscle differentiation and repair. Furthermore, anti-NCAM antibodies have been shown by others to reduce the rate of myoblast fusion, whereas overexpression of NCAM cDNAs increases the rate of myoblast fusion compared to controls. In this study we have used a novel fusion assay based on intracistronic complementation of lacZ, in combination with fluorescent X-gal histochemistry and immunocytochemistry to assess levels of NCAM expression in individual muscle cells. Our results indicate that a substantial proportion of newly fused myoblasts have NCAM expression levels unchanged from the levels of the surrounding unfused population suggesting that increased expression of NCAM is not required for wild-type myoblasts to fuse. Moreover, pure populations of primary myoblasts isolated from mice homozygous null for NCAM and therefore lacking the molecule, when placed in differentiation medium, consistently fused to form contractile myotubes with kinetics equivalent to wild-type primary myoblasts. We conclude that the increase in expression of NCAM, although typically observed during myogenesis, is not essential to myoblast fusion to form myotubes.

    View details for Web of Science ID 000086873800009

    View details for PubMedID 10772795

  • Interaction blues: protein interactions monitored in live mammalian cells by beta-galactosidase complementation TRENDS IN CELL BIOLOGY Rossi, F. M., Blakely, B. T., Blau, H. M. 2000; 10 (3): 119-122

    View details for Web of Science ID 000085420200008

    View details for PubMedID 10675906

  • Epidermal growth factor receptor dimerization monitored in live cells NATURE BIOTECHNOLOGY Blakely, B. T., Rossi, F. M., Tillotson, B., Palmer, M., Estelles, A., Blau, H. M. 2000; 18 (2): 218-222

    Abstract

    We present a method for monitoring receptor dimerization at the membrane of live cells. Chimeric proteins containing the epidermal growth factor (EGF) receptor extracellular and transmembrane domains fused to weakly complementing beta-galactosidase (beta-gal) deletion mutants were expressed in cells in culture. Treatment of the cells with EGF-like compounds for as little as 15 s resulted in chimeric receptor dimerization detectable as beta-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. beta-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and beta-gal complementation. These results demonstrate that beta-gal complementation provides a rapid, simple, and sensitive assay for protein interactions and for detecting and monitoring the kinetics of receptor dimerization.

    View details for Web of Science ID 000085146900032

    View details for PubMedID 10657132

  • Angiogenesis monitored by perfusion with a space-filling microbead suspension MOLECULAR THERAPY Springer, M. L., Ip, T. K., Blau, H. M. 2000; 1 (1): 82-87

    Abstract

    Numerous laboratories are focusing efforts on delivering gene products to induce or prevent the development of new blood vessels in adults, with the hope of rescuing ischemic tissues, circumventing cardiac bypass surgery, or inhibiting tumor growth. Current approaches to the assessment of vascular continuity involve the introduction of either dyes or fluorescent microspheres to track blood flow. However, dyes and dextrans are subject to leakage when vessels are hyperpermeable, a situation that may occur in studies of tumor vasculature and during efforts to stimulate therapeutic angiogenesis. Furthermore, the microspheres that are used for flow studies do not allow a comprehensive visual analysis of vascular continuity. Here we report a method for the visual assessment of microvascular continuity in mouse muscle under circumstances in which vessels are leaky. The approach involves perfusion of the vasculature with fluorescent beads that are much smaller than those used for flow studies. The suspension behaves like a fluid and completely fills the vessels, yet the beads do not leak from VEGF-permeablized capillaries and remain localized in histological sections. Use of beads with the proper fluorescence emission wavelengths allows immunofluorescent colocalization with vessel-specific markers. We compare this improved method with other methods for tracking vascular continuity involving dextrans and larger beads. This approach should aid in the dynamic study of tumor angiogenesis and the evaluation of efforts to deliver angiogenic factors.

    View details for Web of Science ID 000090018300014

    View details for PubMedID 10933915

  • A novel means of drug delivery: Myoblast-mediated gene therapy and regulatable retroviral vectors ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY Ozawa, C. R., Springer, M. L., Blau, H. M. 2000; 40: 295-317

    Abstract

    A potentially powerful approach to drug delivery in the treatment of disease involves the use of cells to introduce genes encoding therapeutic proteins into the body. Candidate genes for delivery include those encoding secreted factors that could have broad applications ranging from treatment of inherited single-gene deficiencies to acquired disorders of the vasculature or cancer. Myoblasts, the proliferative cell type of skeletal muscle tissues, are potent tools for stable delivery of a gene of interest into the body, as they become an integral part of the muscle into which they are injected, in close proximity to the circulation. The recent development of improved tetracycline-inducible retroviral vectors allows for fine control of recombinant gene expression levels. The combination of ex vivo gene transfer using myoblasts and regulatable retroviral vectors provides a powerful toolbox with which to develop gene therapies for a number of human diseases.

    View details for Web of Science ID 000087216400014

    View details for PubMedID 10836138

  • Monitoring protein-protein interactions in live mammalian cells by beta-galactosidase complementation APPLICATIONS OF CHIMERIC GENES AND HYBRID PROTEINS, PT C Rossi, F. M., Blakely, B. T., Charlton, C. A., Blau, H. M. 2000; 328: 231-251

    View details for Web of Science ID 000166565300015

    View details for PubMedID 11075348

  • Analysis of immune responses to varicella tester viral proteins induced by DNA vaccination ANTIVIRAL RESEARCH Abendroth, A., Slobedman, B., Springer, M. L., Blau, H. M., Arvin, A. M. 1999; 44 (3): 179-192

    Abstract

    In this study we sought to examine the mechanism by which immune responses were induced following intramuscular injection of mice with DNA expression vectors encoding genes of varicella zoster virus (VZV). Both VZV-specific antibody and T cell proliferative responses were induced by immunization with DNA sequences for the immediate early 62 (IE62) and glycoprotein E (gE). The viral proteins were shown to be expressed in non-regenerating, rather than regenerating muscle cells. After primary immunization, muscle cells did not express major histocompatibility complex (MHC) class II transcripts and little inflammatory response was detected at the site of inoculation. Histochemical staining and non-isotopic in situ hybridization demonstrated that a second injection of IE62 plasmid DNA was again associated with protein synthesis in non-regenerating muscle cells but that a marked inflammatory infiltrate was induced in muscle tissue. These cells, but not muscle cells, expressed MHC class II transcripts. Significantly, PCR analyses demonstrated that IE62 DNA localized specifically to local draining lymph nodes following primary DNA immunization by intramuscular inoculation. These experiments indicate that transport of plasmid DNA to sites of antigen presentation in regional lymphoid tissue may play an important role in the initial generation of immune responses and that enhancement by secondary inoculation is mediated by immune cells that traffic to the site of viral protein synthesis in muscle cells.

    View details for Web of Science ID 000084820600004

    View details for PubMedID 10651069

  • The phosphoprotein protein PEA-15 inhibits Fas- but increases TNF-R1-mediated caspase-8 activity and apoptosis DEVELOPMENTAL BIOLOGY Estelles, A., Charlton, C. A., Blau, H. M. 1999; 216 (1): 16-28

    Abstract

    We have characterized a phosphoprotein protein with a death effector domain that has a novel bifunctional role in programmed cell death. The 15-kDa phosphoprotein enriched in astrocytes (PEA-15) inhibits Fas-mediated apoptosis and increases tumor necrosis factor receptor-1 (TNF-R1)-mediated apoptosis in the same cell type in a ligand-dependent manner. Phosphorylation appears to play a role in its differential effects, since point mutations at one or both phosphorylation consensus sites within PEA-15 destroy its effect on Fas-mediated, but not TNF-R1-mediated, apoptosis. Furthermore, the differential effect is evident at the level of caspase-8 activity which is inhibited via Fas activation, but increased via TNF-R1 activation upon PEA-15 expression. These results show that PEA-15 provides a potential mechanism during development for distinguishing between diverse extracellular death-inducing signals that culminate either in apoptosis or in survival.

    View details for Web of Science ID 000084171500002

    View details for PubMedID 10588860

  • Plasticity of cell fate: Insights from heterokaryons SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Blau, H. M., Blakely, B. T. 1999; 10 (3): 267-272

    Abstract

    Experiments with somatic cell hybrids and stable heterokaryons have demonstrated that differentiated cells exhibit a remarkable capacity to change. Heterokaryons have been particularly useful in determining the extent to which the differentiated state of a cell is plastic. Cell fate can be altered by a change in the balance of positive and negative trans-acting regulators. Although a single regulator may be sufficient in certain environments to trigger a change in cell fate, that regulator may be ineffective in other cell contexts where it encounters a different composition of regulators.

    View details for Web of Science ID 000081996700006

    View details for PubMedID 10441538

  • Tet B or not tet B: Advances in tetracycline-inducible gene expression - Commentary PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Blau, H. M., Rossi, F. M. 1999; 96 (3): 797-799

    View details for Web of Science ID 000078484100001

    View details for PubMedID 9927646

  • Highly conserved RNA sequences that are sensors of environmental stress MOLECULAR AND CELLULAR BIOLOGY Spicher, A., Guicherit, O. M., Duret, L., Aslanian, A., Sanjines, E. M., Denko, N. C., Giaccia, A. J., Blau, H. M. 1998; 18 (12): 7371-7382

    Abstract

    The putative function of highly conserved regions (HCRs) within 3' untranslated regions (3'UTRs) as regulatory RNA sequences was efficiently and quantitatively assessed by using modular retroviral vectors. This strategy led to the identification of HCRs that alter gene expression in response to oxidative or mitogenic stress. Databases were screened for UTR sequences of >100 nucleotides that had retained 70% identity over more than 300 million years of evolution. The effects of 10 such HCRs on a standard reporter mRNA or protein were studied. To this end, we developed a modular retroviral vector that can allow for a direct comparison of the effects of different HCRs on gene expression independent of their gene-intrinsic 5'UTR, promoter, protein coding region, or poly(A) sequence. Five of the HCRs tested decreased mRNA steady-state levels 2- to 10-fold relative to controls, presumably by altering mRNA stability. One HCR increased translation, and one decreased translation. Elevated mitogen levels caused four HCRs to increase protein levels twofold. One HCR increased protein levels fourfold in response to hypoxia. Although nonconserved UTR sequences may also have a role, these results provide evidence that sequences that are highly conserved during evolution are good candidates for RNA motifs with posttranscriptional regulatory functions in gene expression.

    View details for Web of Science ID 000077099700049

    View details for PubMedID 9819424

  • Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16 NATURE GENETICS Rossi, F. M., Guicherit, O. M., Spicher, A., Kringstein, A. M., Fatyol, K., Blakely, B. T., Blau, H. M. 1998; 20 (4): 389-393

    Abstract

    Continuous regulation is required to maintain a given cell state or to allow it to change in response to the environment. Studies of the mechanisms underlying such regulation have often been hindered by the inability to control gene expression at will. Among the inducible systems available for regulating gene expression in eukaryotes, the tetracycline (tet) regulatable system has distinct advantages. It is highly specific, non-toxic and non-eukaryotic, and consequently does not have pleiotropic effects on host cell genes. Previously this system also had drawbacks, as it did not extinguish gene expression completely, precluding the study of toxic or growth-inhibitory gene products. We report here the development of a facile reversible tetracycline-inducible retroviral system (designated RetroTet-ART) in which activators and repressors together are expressed in cells. Gene expression can now be actively repressed in the absence of tet and induced in the presence of tet, as we have engineered distinct dimerization domains that allow co-expression of homodimeric tet-regulated transactivators and transrepressors in the same cells, without the formation of non-functional heterodimers. Using this system, we show that growth arrest by the cell cycle inhibitor p16 is reversible and dependent on its continuous expression.

    View details for Web of Science ID 000077199600030

    View details for PubMedID 9843215

  • Graded transcriptional response to different concentrations of a single transactivator PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kringstein, A. M., Rossi, F. M., Hofmann, A., Blau, H. M. 1998; 95 (23): 13670-13675

    Abstract

    Threshold mechanisms of transcriptional activation are thought to be critical for translating continuous gradients of extracellular signals into discrete all-or-none cellular responses, such as mitogenesis and differentiation. Indeed, unequivocal evidence for a graded transcriptional response in which the concentration of inducer directly correlates with the level of gene expression in individual eukaryotic cells is lacking. By using a novel binary tetracycline regulatable retroviral vector system, we observed a graded rather than a threshold mechanism of transcriptional activation in two different model systems. When polyclonal populations of cells were analyzed at the single cell level, a dose-dependent, stepwise increase in expression of the reporter gene, green fluorescent protein (GFP), was observed by fluorescence-activated cell sorting. These data provide evidence that, in addition to the generally observed all-or-none switch, the basal transcription machinery also can respond proportionally to changes in concentration of extracellular inducers and trancriptional activators.

    View details for Web of Science ID 000076997000057

    View details for PubMedID 9811858

  • VEGF gene delivery to muscle: Potential role for vasculogenesis in adults MOLECULAR CELL Springer, M. L., Chen, A. S., Kraft, P. E., Bednarski, M., Blau, H. M. 1998; 2 (5): 549-558

    Abstract

    Constitutive expression of VEGF after implantation of genetically engineered myoblasts into non-ischemic muscle led to an increase in vascular structures. Previously, effects of VEGF delivery to adult muscle have only been reported in ischemic tissues. The resulting vascular structures were reminiscent of those formed during embryonic vasculogenesis, rather than angiogenesis, sprouting from preexisting vessels. Initially, VEGF caused an accumulation of endothelial cells and macrophages, followed by networks of vascular channels and hemangiomas with locally high serum VEGF levels. No effects were evident in adjacent tissue or contralateral legs, where low serum VEGF was detected. These data suggest that the induction by VEGF of angiogenesis or vasculogenesis may be dose-dependent. Furthermore, VEGF expression must be carefully modulated, as overexpression is deleterious.

    View details for Web of Science ID 000077203800003

    View details for PubMedID 9844628

  • Recent advances in inducible gene expression systems CURRENT OPINION IN BIOTECHNOLOGY Rossi, F. M., Blau, H. M. 1998; 9 (5): 451-456

    Abstract

    A means of controlling the level and timing of expression of specific genes in cultured cells or in animals would have broad applications. There has been recent progress in two very promising systems: problems due to the high background expression from tetracycline-responsive promoters have been solved by constructing tetracycline-sensitive transcriptional repressors; and new rapamycin analogues have been isolated that are capable of activating the FK506-inducible system but lack the cytostatic side effects of the original inducers. Both systems now provide opportunities for expressing toxic genes, growth arrest genes, and therapeutic products in a regulated fashion previously not possible.

    View details for Web of Science ID 000076409800002

    View details for PubMedID 9821271

  • Inhibition of solid tumor growth by Fas ligand-expressing myoblasts SOMATIC CELL AND MOLECULAR GENETICS Springer, M. L., Kraft, P. E., Blau, H. M. 1998; 24 (5): 281-289

    Abstract

    A major problem with standard treatments of solid tumors such as chemotherapy is that the effects are not localized to the tumor. As a result, normal tissue function is often severely impaired. Here we show that myoblasts from skeletal muscle that have been engineered with retroviral vectors to express Fas ligand (FasL) have potential as site-specific anti-tumor agents. FasL-expression by myoblasts was previously shown to lead to neutrophil-mediated immunodestruction, both of the cells and the surrounding tissue. Moreover, myoblasts expressing FasL induced apoptosis in Fas-expressing human tumor cells in vitro. These findings led us to investigate the possibility that myoblasts expressing FasL could serve as anti-tumor agents acting by both apoptotic and immunological mechanisms. The C57BL/6 lpr/lpr mouse primary myoblasts either expressing or not expressing murine FasL were co-injected with Fas-positive or Fas-negative human rhabdomyosarcoma cells into the tibialis anterior of immunodeficient mice. After 19-31 days, FasL-expressing myoblasts resulted in a marked accumulation of neutrophils and inhibited tumor growth in every case. By contrast, control myoblasts did not prevent significant tumor growth. The status of Fas expression by the tumor tissue in vivo was confirmed by immunostaining tumor sections with antibodies against Fas. Tumor inhibition was observed regardless of the presence or absence of Fas on the tumor cells, suggesting that in vivo, the induction of a neutrophil response is remarkably potent and sufficient to inhibit tumors.

    View details for Web of Science ID 000084466500003

    View details for PubMedID 10696236

  • The fate of individual myoblasts after transplantation into muscles of DMD patients NATURE MEDICINE Gussoni, E., Blau, H. M., KUNKEL, L. M. 1997; 3 (9): 970-977

    Abstract

    Muscle biopsies from six patients with Duchenne muscular dystrophy (DMD) participating in a myoblast transplantation clinical trial were reexamined using a fluorescence in situ hybridization (FISH)-based method. Donor nuclei were detected in all biopsies analyzed, including nine where no donor myoblasts were previously thought to be present. In three patients, more than 10% of the original number of donor cells were calculated as present 6 months after implantation. Half of the detected donor nuclei were fused into host myofibers, and of these, nearly 50% produced dystrophin. These findings demonstrate that although donor myoblasts have persisted after injection, their microenvironment influences whether they fuse and express dystrophin. Our methodology could be used for developing new approaches to improve myoblast transfer efficacy and for the analysis of future gene- and/or cell-based therapies of numerous genetic disorders.

    View details for Web of Science ID A1997XT84200028

    View details for PubMedID 9288722

  • Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rossi, F., Charlton, C. A., Blau, H. M. 1997; 94 (16): 8405-8410

    Abstract

    We present an approach for monitoring protein-protein interactions within intact eukaryotic cells, which should increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer. Chimeric proteins composed of proteins of interest fused to complementing beta-galactosidase (beta-gal) deletion mutants permit a novel analysis of protein complexes within cells. In this approach, the beta-gal activity resulting from the forced interaction of nonfunctional weakly complementing beta-gal peptides (Deltaalpha and Deltaomega) serves as a measure of the extent of interaction of the non-beta-gal portions of the chimeras. To test this application of lacZ intracistronic complementation, proteins that form a complex in the presence of rapamycin were used. These proteins, FRAP and FKBP12, were synthesized as fusion proteins with Deltaalpha and Deltaomega, respectively. Enzymatic beta-gal activity served to monitor the formation of the rapamycin-induced chimeric FRAP/FKBP12 protein complex in a time- and dose-dependent manner, as assessed by histochemical, biochemical, and fluorescence-activated cell sorting assays. This approach may prove to be a valuable adjunct to in vitro immunoprecipitation and crosslinking methods and in vivo yeast two-hybrid and fluorescence energy transfer systems. It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.

    View details for Web of Science ID A1997XQ12400022

    View details for PubMedID 9237989

  • Fusion competence of myoblasts rendered genetically null for N-cadherin in culture JOURNAL OF CELL BIOLOGY Charlton, C. A., Mohler, W. A., Radice, G. L., HYNES, R. O., Blau, H. M. 1997; 138 (2): 331-336

    Abstract

    Myoblast fusion is essential to muscle tissue development yet remains poorly understood. N-cadherin, like other cell surface adhesion molecules, has been implicated by others in muscle formation based on its pattern of expression and on inhibition of myoblast aggregation and fusion by antibodies or peptide mimics. Mice rendered homozygous null for N-cadherin revealed the general importance of the molecule in early development, but did not test a role in skeletal myogenesis, since the embryos died before muscle formation. To test genetically the proposed role of N-cadherin in myoblast fusion, we successfully obtained N-cadherin null primary myoblasts in culture. Fusion of myoblasts expressing or lacking N-cadherin was found to be equivalent, both in vitro by intracistronic complementation of lacZ and in vivo by injection into the muscles of adult mice. An essential role for N-cadherin in mediating the effects of basic fibroblast growth factor was also excluded. These methods for obtaining genetically homozygous null somatic cells from adult tissues should have broad applications. Here, they demonstrate clearly that the putative fusion molecule, N-cadherin, is not essential for myoblast fusion.

    View details for Web of Science ID A1997XP26700010

    View details for PubMedID 9230075

  • Death of solid tumor cells induced by Fas ligand expressing primary myoblasts SOMATIC CELL AND MOLECULAR GENETICS Hofmann, A., Blau, H. M. 1997; 23 (4): 249-257

    Abstract

    Anticancer therapy for solid tumors suffers from inadequate methods for the localized administration of cytotoxic agents. Fas ligand (FasL) has been reported to be cytotoxic to a variety of cells, including certain tumor cell lines. We therefore postulated that myoblasts could serve as non-transformed gene therapy vehicles for the continuous localized delivery of cytotoxic anticancer agents such as FasL. However, contrary to previous reports, fluorescence activated cell sorting (FACS) analyses revealed that both primary mouse and human myoblasts express Fas, the receptor for FasL. To avoid self-destruction and test the cytotoxic potential of myoblasts, the cells were isolated from mice deficient in Fas (lpr/lpr), the mouse counterpart of human autoimmune lymphoproliferative syndrome (ALPS). These primary mouse myoblasts were transduced with a retroviral vector encoding mouse FasL and expression of a biologically active and soluble form of the molecule was confirmed by the apoptotic demise of cocultured Fas-expressing Jurkat cells, the standard in the field. To test whether the lpr myoblasts expressing FasL could be used in anticancer therapy, human rhabdomyosarcoma derived cell lines were assayed for Fas and then tested in the apoptosis coculture assay. The majority of Fas-expressing muscle tumor cells were rapidly killed. Moreover, FasL expressing myoblasts were remarkably potent; indeed well characterized cytotoxic antibodies to Fas were only 20% as efficient at killing rhabdomyosarcoma cells as FasL expressing myoblasts. These findings together with previous findings suggest that primary myoblasts, defective in Fas but genetically engineered to express FasL, could function as potent anticancer agents for use in the localized destruction of solid tumors in vivo by three synergistic mechanisms: (1) directly via Fas/FasL mediated apoptosis, (2) indirectly via neutrophil infiltration and immunodestruction, and (3) as allogeneic inducers of a bystander effect via B and T cells.

    View details for Web of Science ID 000072792800002

    View details for PubMedID 9542527

  • High-efficiency retroviral infection of primary myoblasts SOMATIC CELL AND MOLECULAR GENETICS Springer, M. L., Blau, H. M. 1997; 23 (3): 203-209

    Abstract

    In the past, it has been hard to introduce genes into primary myoblasts without selection, as they have been very difficult to transfect or infect. We describe conditions under which mouse primary skeletal muscle myoblasts can be infected with retroviral vectors at high efficiency. Infection can be greatly increased by minimizing the time during which cells are exposed to virus, adding a minimal centrifugation step, and supplementing the infection cocktail to mimic more closely primary myoblast growth medium. Under these conditions, one round of exposure to virus results in an infection efficiency of up to 80%, whereas 4-5 rounds of infection over a two day period reproducibly yield an infection efficiency of > 99%. These methods greatly enhance the potential for studying genetically engineered primary myoblasts from any mouse strain, transgenic or knockout, and may have useful application to other primary cell types that are refractory to transfection or infection.

    View details for Web of Science ID A1997XZ59400003

    View details for PubMedID 9330631

  • Myoblast implantation in Duchenne muscular dystrophy: The San Francisco study MUSCLE & NERVE MILLER, R. G., Sharma, K. R., PAVLATH, G. K., Gussoni, E., MYNHIER, M., LANCTOT, A. M., Greco, C. M., Steinman, L., Blau, H. M. 1997; 20 (4): 469-478

    Abstract

    We evaluated myoblast implantation in 10 boys with Duchenne muscular dystrophy (DMD) and absent dystrophin (age 5-10 years) who were implanted with 100 million myoblasts in the anterior tibial muscle of one leg and placebo in the other. Cyclosporine (5 mg/kg/day) was administered for 7 months. Pre- and postimplantation (after 1 and 6 months) muscle biopsies were analyzed. Force generation (tetanic tension and maximum voluntary contraction) was measured monthly in a double-blind design. There was increased force generation in both legs of all boys, probably due to cyclosporine. Using the polymerase chain reaction, evidence of myoblast survival and dystrophin mRNA expression was obtained in 3 patients after 1 month and in 1 patient after 6 months. These studies suggest a salutary effect of cyclosporine upon muscular force generation in Duchenne muscular dystrophy; however, myoblast implantation was not effective in replacing clinically significant amounts of dystrophin in DMD muscle.

    View details for Web of Science ID A1997WR86800010

    View details for PubMedID 9121505

  • Rapid plasmid minipreps in microplate format from culture to gel BIOTECHNIQUES Mohler, W. A., Blau, H. M. 1997; 22 (3): 388-390

    View details for Web of Science ID A1997WM67200001

    View details for PubMedID 9067004

  • Methods for myoblast transplantation METHODS IN CELL BIOLOGY, VOL 52 Rando, T. A., Blau, H. M. 1997; 52: 261-?

    View details for Web of Science ID A1997BJ78C00012

    View details for PubMedID 9379953

  • Genetic analysis of alpha(4) integrin functions in the development of mouse skeletal muscle JOURNAL OF CELL BIOLOGY Yang, J. T., Rando, T. A., Mohler, W. A., Rayburn, H., Blau, H. M., HYNES, R. O. 1996; 135 (3): 829-835

    Abstract

    It has been suggested, on the basis of immunolocalization studies in vivo and antibody blocking experiments in vitro, that alpha 4 integrins interacting with vascular cell adhesion molecule 1 (VCAM-1) are involved in myogenesis and skeletal muscle development. To test this proposal, we generated embryonic stem (ES) cells homozygous null for the gene encoding the alpha 4 subunit and used them to generate chimeric mice. These chimeric mice showed high contributions of alpha 4-null cells in many tissues, including skeletal muscle, and muscles lacking any detectable (< 2%) alpha 4-positive cells did not reveal any gross morphological abnormalities. Furthermore, assays for in vitro myogenesis using either pure cultures of alpha 4-null myoblasts derived from the chimeras or alpha 4-null ES cells showed conclusively that alpha 4 integrins are not essential for muscle cell fusion and differentiation. Taking these results together, we conclude that alpha 4 integrins appear not to play essential roles in normal skeletal muscle development.

    View details for Web of Science ID A1996VR26800023

    View details for PubMedID 8909554

  • Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mohler, W. A., Blau, H. M. 1996; 93 (22): 12423-12427

    Abstract

    Complementing reporter genes provide biological indicators of coincident expression of proteins in cells. We have adapted intracistronic complementation of the Escherichia coli lacZ gene for use in mammalian cells. Enzymatic activity detectable by quantitative biochemical assay, flow cytometry, or microscopy is produced upon convergent expression of two distinct mutant lacZ peptides within single cells, or upon fusion of cells expressing such mutants. A novel fluorescent substrate for beta-galactosidase (Fluor-X-Gal) increases detection and permits simultaneous microscopic visualization of other fluorescent markers. The enzymatic complementation described here should facilitate studies of cell fusion, cell lineage, and signal transduction, by producing activity only when two proteins are expressed at the same time and place in intact cells.

    View details for Web of Science ID A1996VP93700068

    View details for PubMedID 8901597

  • Myoblast-mediated expression of colony stimulating factor-1 (CSF-1) in the cytokine-deficient op/op mouse SOMATIC CELL AND MOLECULAR GENETICS Dhawan, J., Rando, T. A., Elson, S. E., Lee, F., Stanley, E. R., Blau, H. M. 1996; 22 (5): 363-381

    Abstract

    The osteopetrotic (op/op) mouse lacks colony stimulating factor-1 (CSF-1) due to an inactivating mutation in the CSF-1 gene. Intramuscular transplantation of engineered myoblasts was used to introduce CSF-1 into the circulation of op/op mice. The CSF-1 cDNA was introduced into C2C12 mouse myoblasts in culture using retroviral mediated gene transfer. Upon transplantation into the skeletal muscle of mutant mice, physiological levels of the cytokine were achieved systemically and elicited a biological response: circulating monocytes were induced. Howvever, both circulating CSF-1 levels and the induction of monocytes were transient. Analysis of the site of cell transplantation revealed local changes that may account for the transience of serum cytokine levels. Macrophage markers were induced in muscle tissue implanted with CSF-1 expressing myoblasts: c-fms, the CSF-1 receptor as well as the lineage-restricted antigen F4/80. We propose that in addition to CSF-1 clearance by Kupffer cells of the liver, macrophages that accumulated at the site of cell transplantation bound the CSF-1 produced by the muscle cell transplants, precluding the sustained release of this cytokine into the systemic circulation. Our studies also revealed that damage to muscle caused during cell transplantation or by freeze injury resulted in the accumulation of macrophages in op/op mouse muscle tissue. Indeed, op/op mice were fully capable of regenerating injured muscle suggesting the presence of as yet unidentified CSF-1-independent factors capable of generating macrophages that presumably participate in tissue remodeling in this cytokine-deficient mouse.

    View details for Web of Science ID A1996WK59300002

    View details for PubMedID 9039846

  • Defective myogenesis in NFB-s mutant associated with a saturable suppression of MYF5 activity SOMATIC CELL AND MOLECULAR GENETICS Rohrer, D. K., Blau, H. M. 1996; 22 (5): 349-361

    Abstract

    Myogenic cell lines have proved to be useful tools for investigating the molecular mechanisms that control cellular differentiation. NFB-s is a mutant myogenic cell line which fails to differentiate in vitro, and can repress differentiation in normal myogenic cells when fused to form heterokaryons. The NFB-s cell line was used here to study the molecular mechanisms underlying such myogenic repression. Using muscle-specific reporter genes, we show that NFB-s cells fail to activate fully the muscle differentiation program at a transcriptional level, although muscle-specific transcription can be enhanced by regulators of differentiation such as pertussis toxin. Paradoxically we find that the myogenic regulator myf5 is expressed at constitutively high levels in NFB-s cells, and retains DNA binding activity. Expression plasmids encoding NFB-derived myf5 cDNA can rescue the myogenic phenotype in NFB-s cells, demonstrating that a threshold level of positive regulators must be reached before the myogenic program is activated. Thus, the dominant negative phenotype does not appear to result from defective myf5, but is due to a dosage-dependent saturable mechanism that interferes with myf5 function. These studies demonstrate that the stoichiometric ratio of positive and negative regulators is critical for determining the myogenic differentiation state.

    View details for Web of Science ID A1996WK59300001

    View details for PubMedID 9039845

  • Spectrophotometric quantitation of tissue culture cell number in any medium BIOTECHNIQUES Mohler, W. A., Charlton, C. A., Blau, H. M. 1996; 21 (2): 260-?

    Abstract

    We have developed a spectrophotometric assay for cell number in suspensions of tissue culture cells. For each cell type tested, absorbance between 650 and 800 nm is linearly dependent upon cell density over a 50-fold range and is independent of the color or composition of the medium in which cells are suspended. A standard curve of absorbance vs. cell density is used to estimate cell number with accuracy and reproducibility superior to hemacytometer counting and with speed and ease surpassing use of a Coulter counter. Less than 5000 cells are needed for this quantitation. The same cells that are counted can be maintained live in culture after the reading is taken, thus allowing the growth of cells to be measured within individual cultures over time. The assay should be readily extended to assays of cell number directly within microplate culture wells. The spectrophotometric assay described here is of significant use in all experiments requiring rapid, accurate measurements of cell number, including determinations of cell doubling time and equal plating of parallel cultures.

    View details for Web of Science ID A1996VA83100020

    View details for PubMedID 8862811

  • Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hofmann, A., Nolan, G. P., Blau, H. M. 1996; 93 (11): 5185-5190

    Abstract

    We describe a single autoregulatory cassette that allows reversible induction of transgene expression in response to tetracycline (tet). This cassette contains all of the necessary components previously described by others on two separate plasmids that are introduced sequentially over a period of months [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. The cassette is introduced using a retrovirus, allowing transfer into cell types that are difficult to transfect. Thus, populations of thousands of cells, rather than a few clones, can be isolated and characterized within weeks. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalovirus minimal promoter, the vector is self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells. Tandem tet operator sequences and the cytomegalovirus minimal promoter drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (lacZ) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented. Northern blot, biochemical, and single cell analyses have all shown that the construct yields low basal levels of gene expression and induction of one to two orders of magnitude. Thus, the current cassette of the retroviral construct (SIN-RetroTet vector) allows rapid delivery of inducible genes and should have broad applications to cultured cells, transgenic animals, and gene therapy.

    View details for Web of Science ID A1996UN25300003

    View details for PubMedID 8643550

  • GENE-THERAPY - A NOVEL FORM OF DRUG-DELIVERY NEW ENGLAND JOURNAL OF MEDICINE Blau, H. M., Springer, M. L. 1995; 333 (18): 1204-1207

    View details for Web of Science ID A1995TB56000008

    View details for PubMedID 7565977

  • THE FATE OF MYOBLASTS FOLLOWING TRANSPLANTATION INTO MATURE MUSCLE EXPERIMENTAL CELL RESEARCH Rando, T. A., PAVLATH, G. K., Blau, H. M. 1995; 220 (2): 383-389

    Abstract

    Cell transplantation has potential benefits for tissue replacement in the the enhancement of tissue regeneration and as cell-mediated gene therapy for systemic diseases. The transplantation of myoblasts into skeletal muscle also allows gene transfer into cells of the host since myoblasts fuse with host fibers thereby forming hybrid myofibers. The success of myoblast transplantation can be determined by a variety of measures, such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression. Each measure is a reflection of the fate of the transplanted cells. In order to compare different measures of transplantation efficacy, we followed the fate of transplanted myoblasts expressing the marker enzyme beta-galactosidase (beta-gal) in two different assays. Two weeks after transplantation, the number of hybrid myofibers was determined histochemically, whereas transgene (beta-gal) expression was measured biochemically. To control for variabilities of transplantation among different animals, we obtained both measurements from each muscle by using alternate cryosections in the two assays. Within each individual muscle, both hybrid fiber number and beta-gal expression were maximal at the site of implantation and diminished in parallel with distance from the site. However, for determining the success of transplantation among groups of muscles, these two measures of efficacy yielded discordant results: the transplants with the highest number of hybrid fibers were not the transplants with the greatest beta-gal activity. Such discrepancies are likely due to regional variations at the transplantation site that arise when cells are introduced into a solid tissue. These results demonstrate the importance of multiple measures of cell fate and transplantation efficacy for studies of cell transplantation and for the application of such studies to cell therapy and cell-mediated gene therapy.

    View details for Web of Science ID A1995RX76400017

    View details for PubMedID 7556447

  • TETRACYCLINE-REGULATED GENE-EXPRESSION FOLLOWING DIRECT GENE-TRANSFER INTO MOUSE SKELETAL-MUSCLE SOMATIC CELL AND MOLECULAR GENETICS Dhawan, J., Rando, T. A., ELSON, S. L., Bujard, H., Blau, H. M. 1995; 21 (4): 233-240

    Abstract

    For most experimental and therapeutic applications of gene transfer, regulation of the timing and level of gene expression is preferable to constitutive gene expression. Among the systems that have been developed for pharmacologically controlled gene expression in mammalian cells, the bacterial tetracycline (tet)-responsive system has the advantage that it is dependent on a drug (tet) that is both highly specific and non-toxic. The tet-responsive system has been previously used to modulate expression of cell cycle regulatory proteins in cultured cells, reporter genes in plants and transgenic mice and reporter genes directly injected into the heart. Here we show that orally or parenterally administered tet regulates expression of tet-responsive plasmids injected directly into mouse skeletal muscle. Reporter gene expression was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn. These data show that skeletal muscle offers an accessible and well characterized target tissue for tet-controlled expression of genes in vivo, suggesting applications to developmental studies and gene therapy.

    View details for Web of Science ID A1995TK30000002

    View details for PubMedID 8525429

  • TRANSIENT IMMUNOSUPPRESSIVE TREATMENT LEADS TO LONG-TERM RETENTION OF ALLOGENEIC MYOBLASTS IN HYBRID MYOFIBERS JOURNAL OF CELL BIOLOGY PAVLATH, G. K., Rando, T. A., Blau, H. M. 1994; 127 (6): 1923-1932

    Abstract

    Normal and genetically engineered skeletal muscle cells (myoblasts) show promise as drug delivery vehicles and as therapeutic agents for treating muscle degeneration in muscular dystrophies. A limitation is the immune response of the host to the transplanted cells. Allogeneic myoblasts are rapidly rejected unless immunosuppressants are administered. However, continuous immunosuppression is associated with significant toxic side effects. Here we test whether immunosuppressive treatment, administered only transiently after allogeneic myoblast transplantation, allows the long-term survival of the transplanted cells in mice. Two immunosuppressive treatments with different modes of action were used: (a) cyclosporine A (CSA); and (b) monoclonal antibodies to intracellular adhesion molecule-1 and leukocyte function-associated molecule-1. The use of myoblasts genetically engineered to express beta-galactosidase allowed quantitation of the survival of allogeneic myoblasts at different times after cessation of the immunosuppressive treatments. Without host immunosuppression, allogeneic myoblasts were rejected from all host strains tested, although the relative time course differed as expected for low and high responder strains. The allogeneic myoblasts initially fused with host myofibers, but these hybrid cells were later destroyed by the massive immunological response of the host. However, transient immunosuppressive treatment prevented the hybrid myofiber destruction and led to their long-term retention. Even four months after the cessation of treatment, the hybrid myofibers persisted and no inflammatory infiltrate was present in the tissue. Such long-term survival indicates that transient immunosuppression may greatly increase the utility of myoblast transplantation as a therapeutic approach to the treatment of muscle and nonmuscle disease.

    View details for Web of Science ID A1994PZ27200014

    View details for PubMedID 7806570

  • SPECIFIC T-CELL RECEPTOR GENE REARRANGEMENTS AT THE SITE OF MUSCLE DEGENERATION IN DUCHENNE MUSCULAR-DYSTROPHY JOURNAL OF IMMUNOLOGY Gussoni, E., PAVLATH, G. K., MILLER, R. G., Panzara, M. A., Powell, M., Blau, H. M., Steinman, L. 1994; 153 (10): 4798-4805

    Abstract

    Mononuclear cells infiltrate degenerating muscles of Duchenne muscular dystrophy (DMD) patients. Using a quantitative PCR, we first characterized the T cells infiltrating muscle biopsies from six DMD patients. High levels of TCR V beta 2 transcripts were observed in DMD muscle tissue. TCR V beta 2 transcripts from seven DMD patients and five controls were sequenced, and the VDJ junctional region analyzed in 166 clones. One specific amino acid motif, RVSG, was found in the third complementary determining region (CDR3) of TCR V beta 2 chains in samples from five DMD patients, but not in controls. A specific immune reaction at the site of tissue degeneration may play an important role in the pathogenesis of DMD.

    View details for Web of Science ID A1994PR22200047

    View details for PubMedID 7963545

  • FAST MYOSIN HEAVY-CHAINS EXPRESSED IN SECONDARY MAMMALIAN MUSCLE-FIBERS AT THE TIME OF THEIR INCEPTION JOURNAL OF CELL SCIENCE Cho, M., Hughes, S. M., KARSCHMIZRACHI, I., Travis, M., Leinwand, L. A., Blau, H. M. 1994; 107: 2361-2371

    Abstract

    Mammalian skeletal muscle is generated by two waves of fiber formation, resulting in primary and secondary fibers. These fibers mature to give rise to several classes of adult muscle fibers with distinct contractile properties. Here we describe fast myosin heavy chain (MyHC) isoforms that are expressed in nascent secondary, but not primary, fibers in the early development of rat and human muscle. These fast MyHCs are distinct from previously described embryonic and neonatal fast MyHCs. To identify these MyHCs, monoclonal antibodies were used whose specificity was determined in western blots of MyHCs on denaturing gels and reactivity with muscle tissue at various stages of development. To facilitate a comparison of our results with those of others obtained using different antibodies or species, we have identified cDNAs that encode the epitopes recognized by our antibodies wherever possible. The results suggest that epitopes characteristic of adult fast MyHCs are expressed very early in muscle fiber development and distinguish newly formed secondary fibers from primary fibers. This marker of secondary fibers, which is detectable at the time of their inception, should prove useful in future studies of the derivation of primary and secondary fibers in mammalian muscle development.

    View details for Web of Science ID A1994PH54500002

    View details for PubMedID 7531198

  • PRIMARY MOUSE MYOBLAST PURIFICATION, CHARACTERIZATION, AND TRANSPLANTATION FOR CELL-MEDIATED GENE-THERAPY JOURNAL OF CELL BIOLOGY Rando, T. A., Blau, H. M. 1994; 125 (6): 1275-1287

    Abstract

    The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity--suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.

    View details for Web of Science ID A1994NT42000008

    View details for PubMedID 8207057

  • MEMBRANE-BOUND NEOMYCIN PHOSPHOTRANSFERASE CONFERS DRUG-RESISTANCE IN MAMMALIAN-CELLS - A MARKER FOR HIGH-EFFICIENCY TARGETING OF GENES ENCODING SECRETED AND CELL-SURFACE PROTEINS SOMATIC CELL AND MOLECULAR GENETICS Mohler, W. A., Blau, H. M. 1994; 20 (3): 153-162

    Abstract

    An efficient method for inactivating genes is the use of silent selectable markers that are expressed only after homologous recombination into the active target gene. However, use of this approach for genes encoding secreted or membrane-anchored proteins may produce hybrid proteins comprising the N-terminal signal sequence from the target gene linked to the protein conferring drug resistance. Such chimeric enzymes will be secreted, precluding selection for drug resistance. To overcome this problem, we tested the possibility of anchoring in the membrane the cytoplasmic neomycin phosphotransferase (NPT). We constructed a fusion gene with a transmembrane domain connecting the N-terminal signal sequence of a membrane-targeted protein and the neo gene. Expression of this gene yielded G418-resistant colonies of C2C12 cells which contained assayable NPT activity. Comparison of enzyme activity in cell extract fractions verified that the active fusion protein was insoluble, presumably through localization to a membrane compartment. Transmembrane neo cassettes should serve as integration-activated markers capable of targeting genes encoding secreted or cell surface proteins.

    View details for Web of Science ID A1994PJ61800002

    View details for PubMedID 7940017

  • TUMOR SUPPRESSION BY RNA FROM THE 3' UNTRANSLATED REGION OF ALPHA-TROPOMYOSIN CELL Rastinejad, F., Conboy, M. J., Rando, T. A., Blau, H. M. 1993; 75 (6): 1107-1117

    Abstract

    NMU2, a nondifferentiating mutant myogenic cell line, gives rise to rhabdomyosarcomas in mice. We show that constitutive expression of RNA from 0.2 kb of the alpha-tropomyosin (Tm) 3' untranslated region (UTR), but not control 3'UTRs, suppresses anchorage-independent growth and tumor formation by NMU2 cells. When beta-galactosidase (beta-gal)-labeled cells were implanted into muscles of adult mouse hindlimbs, Tm 3'UTR expression suppressed the proliferation, invasion, and destruction of muscle tissues characteristic of NMU2. In the rare tumors that developed from Tm 3'UTR transfectants, RNA expression was extinguished. These results suggest that suppression of tumorigenicity is dependent on the continued expression of Tm transcripts lacking a coding region. We conclude that untranslated RNAs can function as regulators (riboregulators) that suppress tumor formation.

    View details for Web of Science ID A1993MM89300009

    View details for PubMedID 7505203

  • MYOBLASTS IN PATTERN-FORMATION AND GENE-THERAPY TRENDS IN GENETICS Blau, H. M., Dhawan, J., PAVLATH, G. K. 1993; 9 (8): 269-274

    Abstract

    The tissues of a multicellular animal are composed of diverse cell types arranged in a precisely organized pattern. Features unique to muscle allow an analysis of pattern formation and maintenance in mammals. The progeny of single cells can be taken full cycle from the animal to the culture dish and back to the animal where they fuse into mature myofibers of the host. These features not only facilitate the use of genetically engineered myoblasts in studies of pattern formation, but also in cell-mediated gene therapy: a novel mode of drug delivery for the treatment of muscle and nonmuscle diseases such as hemophilia, cardiac disease and cancer.

    View details for Web of Science ID A1993LN54200005

    View details for PubMedID 8379006

  • 3 SLOW MYOSIN HEAVY-CHAINS SEQUENTIALLY EXPRESSED IN DEVELOPING MAMMALIAN SKELETAL-MUSCLE DEVELOPMENTAL BIOLOGY Hughes, S. M., Cho, M., KARSCHMIZRACHI, I., Travis, M., Silberstein, L., Leinwand, L. A., Blau, H. M. 1993; 158 (1): 183-199

    Abstract

    Myosin heavy chain (MyHC) isoforms show a striking diversity of expression patterns during mammalian development. Using a set of monoclonal antibodies that recognize different epitopes on myosin heavy chain isoforms we show that there exist in human and rat skeletal muscle at least three isoforms of slow twitch myosin heavy chain. To facilitate a comparison of our results to others obtained using different antibodies or species, we have identified cDNAs encoding the epitopes recognized by the three slow antibodies. Using these reagents, we show that the onset of expression of three slow MyHC isoforms is temporally distinct during early gestation. This result suggests that a sequence of MyHC transitions plays an important role in determining muscle fiber function at fetal, neonatal, and adult stages.

    View details for Web of Science ID A1993LM14800015

    View details for PubMedID 7687223

  • EVIDENCE FOR MYOBLAST-EXTRINSIC REGULATION OF SLOW MYOSIN HEAVY-CHAIN EXPRESSION DURING MUSCLE-FIBER FORMATION IN EMBRYONIC-DEVELOPMENT JOURNAL OF CELL BIOLOGY Cho, M., Webster, S. G., Blau, H. M. 1993; 121 (4): 795-810

    Abstract

    Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or intrinsic "clock." Taken together, these results suggest that, unlike embryonic and neonatal MyHCs, the expression of slow MyHC in vivo at different developmental stages during gestation is not the result of commitment to a distinct myoblast lineage, but is largely determined by the environment.

    View details for Web of Science ID A1993LB63800008

    View details for PubMedID 8491773

  • GENETIC COMPLEMENTATION REVEALS A NOVEL REGULATORY ROLE FOR 3' UNTRANSLATED REGIONS IN GROWTH AND DIFFERENTIATION CELL Rastinejad, F., Blau, H. M. 1993; 72 (6): 903-917

    Abstract

    Differentiated skeletal muscle cells cease dividing and sustain expression of a battery of tissue-specific genes. To identify regulators of growth and differentiation, we used a genetic complementation approach. Following introduction of a cDNA expression library into a differentiation-defective myoblast mutant (NMU2), cDNAs were isolated that activated muscle-specific promoters. The complementing cDNAs were identified as muscle structural genes, troponin I, tropomyosin, and alpha-cardiac actin, and their activity was mapped to the 3' untranslated region (3'UTR). The 3'UTRs augmented the differentiation of wild-type muscle cells. Upon expression in 10T1/2 fibroblasts, proliferation was suppressed, indicating that the effects of the 3'UTRs are not limited to myogenic cells. These data suggest that 3'UTRs of certain differentiation-specific RNAs are trans-acting regulators in a feedback loop that inhibits cell division and promotes differentiation.

    View details for Web of Science ID A1993KU17500011

    View details for PubMedID 8384533

  • MYOBLAST TRANSFER IN DMD - PROBLEMS IN THE INTERPRETATION OF EFFICIENCY - A REPLY MUSCLE & NERVE Blau, H. M., PAVLATH, G. K., Gussoni, E., Steinman, L., MILLER, R. G., Sharma, K. 1992; 15 (10): 1209-1210
  • BETA-ENOLASE IS A MARKER OF HUMAN MYOBLAST HETEROGENEITY PRIOR TO DIFFERENTIATION DEVELOPMENTAL BIOLOGY Peterson, C. A., Cho, M., Rastinejad, F., Blau, H. M. 1992; 151 (2): 626-629

    Abstract

    In this report, we define a muscle-specific marker, beta-enolase, that distinguishes proliferating myoblasts from different stages of development. Enolase exists as multiple isoforms and in the course of cardiac and skeletal muscle development the beta isoform progressively replaces the alpha isoform. In skeletal muscle, this change in gene expression, unlike most developmental changes in myogenic gene expression, is evident in undifferentiated myoblasts. Whereas myoblasts from fetal tissues express alpha-enolase mRNA, beta-enolase is the predominant mRNA expressed by myoblasts from postnatal tissues. Our results are consistent with the idea that distinct precursor myoblasts contribute to the diversity of fiber types characteristic of muscle tissue at different stages of development.

    View details for Web of Science ID A1992HY74000029

    View details for PubMedID 1339335

  • NORMAL DYSTROPHIN TRANSCRIPTS DETECTED IN DUCHENNE MUSCULAR-DYSTROPHY PATIENTS AFTER MYOBLAST TRANSPLANTATION NATURE Gussoni, E., PAVLATH, G. K., LANCTOT, A. M., Sharma, K. R., MILLER, R. G., Steinman, L., Blau, H. M. 1992; 356 (6368): 435-438

    Abstract

    Gene delivery by transplantation of normal myoblasts has been proposed as a treatment of the primary defect, lack of the muscle protein dystrophin, that causes Duchenne muscular dystrophy (DMD), a lethal human muscle degenerative disorder. To test this possibility, we transplanted normal myoblasts from a father or an unaffected sibling into the muscle of eight boys with DMD, and assessed their production of dystrophin. Three patients with deletions in the dystrophin gene expressed normal dystrophin transcripts in muscle biopsy specimens taken from the transplant site one month after myoblast injection. Using the polymerase chain reaction we established that the dystrophin in these biopsies derived from donor myoblast DNA. These results show that transplanted myoblasts persist and produce dystrophin in muscle fibres of DMD patients.

    View details for Web of Science ID A1992HL83000064

    View details for PubMedID 1557125

  • CLONING MUSCLE ISOFORMS OF NEURAL CELL-ADHESION MOLECULE USING AN EPISOMAL SHUTTLE VECTOR SOMATIC CELL AND MOLECULAR GENETICS Pan, L. C., Margolskee, R. F., Blau, H. M. 1992; 18 (2): 163-177

    Abstract

    Three distinct transcripts encoding two phosphatidylinositol (PI) linked isoforms of the neural cell adhesion molecule (NCAM) are induced during the differentiation of C2C12 myoblasts into myotubes. Corresponding NCAM clones were isolated from a mouse muscle cDNA library made in an Epstein-Barr virus shuttle vector that replicates extrachromosomally in human cells. Following transfection with the library, human cells expressing mouse NCAM were enriched using the fluorescence-activated cell sorter. Episomal NCAM clones recovered from sorted cells contain an 18-bp insert between exons 12 and 13. Two other NCAM cDNAs encode identical polypeptides containing a 108-bp insert homologous to the complete MSD1 domain, but differ in their 3' untranslated regions. Induction of MSD1-containing transcripts in advance of myotube formation suggests that muscle-specific NCAMs contribute to myogenesis from the earliest stages of differentiation. Moreover, our studies demonstrate the feasibility of cloning tissue-specific molecules by transfection and expression of cDNA libraries in episomal vectors.

    View details for Web of Science ID A1992HZ10700006

    View details for PubMedID 1315456

  • MUSCLE-FIBER PATTERN IS INDEPENDENT OF CELL LINEAGE IN POSTNATAL RODENT DEVELOPMENT CELL Hughes, S. M., Blau, H. M. 1992; 68 (4): 659-671

    Abstract

    Muscle fibers specialized for fast or slow contraction are arrayed in characteristic patterns within developing limbs. Clones of myoblasts analyzed in vitro express fast and slow myosin isoforms typical of the muscle from which they derive. As a result, it has been suggested that distinct myoblast lineages generate and maintain muscle fiber pattern. We tested this hypothesis in vivo by using a retrovirus to label myoblasts genetically so that the fate of individual clones could be monitored. Both myoblast clones labeled in muscle in situ and clones labeled in tissue culture and then injected into various muscles contribute progeny to all fiber types encountered. Thus, extrinsic signals override the intrinsic commitment of myoblast nuclei to particular programs of gene expression. We conclude that in postnatal development, pattern is not dictated by myoblast lineage.

    View details for Web of Science ID A1992HF44000007

    View details for PubMedID 1531450

  • DIFFERENTIATION REQUIRES CONTINUOUS ACTIVE CONTROL ANNUAL REVIEW OF BIOCHEMISTRY Blau, H. M. 1992; 61: 1213-1230

    Abstract

    The problems posed by differentiation that appear most soluble by a passive control mechanism can readily be solved by an active mechanism. Given the need for plasticity in gene expression in different cell types at different stages, an active mechanism may be advantageous, even essential. It is striking how few changes during differentiation are completely irreversible, the gene rearrangements leading to immunoglobulin expression being one clear exception. Indeed, a prediction of the active-control hypothesis is that any nucleus exposed to the appropriate constellation of proteins at the appropriate concentration should be able to perform functions typical of any given differentiated cell type. In the next decade, the elucidation of novel memory mechanisms, or feedback loops, will substantially increase our understanding of how stable differentiated states can be maintained by continuous active control.

    View details for Web of Science ID A1992JE31500037

    View details for PubMedID 1497309

  • SYSTEMIC DELIVERY OF HUMAN GROWTH-HORMONE BY INJECTION OF GENETICALLY ENGINEERED MYOBLASTS SCIENCE Dhawan, J., Pan, L. C., PAVLATH, G. K., Travis, M. A., LANCTOT, A. M., Blau, H. M. 1991; 254 (5037): 1509-1512

    Abstract

    A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli. These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins.

    View details for Web of Science ID A1991GT90300041

    View details for PubMedID 1962213

  • MODULATION OF MHC CLASS-II ANTIGEN EXPRESSION IN HUMAN MYOBLASTS AFTER TREATMENT WITH IFN-GAMMA NEUROLOGY Mantegazza, R., Hughes, S. M., Mitchell, D., Travis, M., Blau, H. M., Steinman, L. 1991; 41 (7): 1128-1132

    Abstract

    Some investigators have proposed myoblast transfer as a potential therapy for the treatment of Duchenne muscular dystrophy. Little is known about the immunobiology of myoblast transplantation. Transplantation rejection is mediated to a large extent by CD8+ T cells, which recognize alloantigens encoded by class I HLA genes, and by CD4+ T cells, which recognize alloantigens encoded by class II HLA genes. Gamma interferon (IFN-gamma) is a potent inducer of HLA class II molecules as well as beta 2-microglobulin, which is co-expressed with HLA class I. IFN-gamma may be a critical cytokine involved in graft rejection. We purified human myoblasts by flow cytometry and incubated them in vitro for varying time periods with recombinant human IFN-gamma. The inducibility of HLA-DR and -DP molecules raises a note of caution concerning possible rejection phenomenon which might occur following myoblast transplantation.

    View details for Web of Science ID A1991FX12300032

    View details for PubMedID 1906147

  • DIFFERENTIATION REQUIRES CONTINUOUS REGULATION JOURNAL OF CELL BIOLOGY Blau, H. M., Baltimore, D. 1991; 112 (5): 781-783

    View details for Web of Science ID A1991EZ87600001

    View details for PubMedID 1999456

  • Cell lineage in vertebrate development. Current opinion in cell biology Blau, H. M., Hughes, S. M. 1990; 2 (6): 981-985

    View details for PubMedID 2099812

  • ACCELERATED AGE-RELATED DECLINE IN REPLICATIVE LIFE-SPAN OF DUCHENNE MUSCULAR-DYSTROPHY MYOBLASTS - IMPLICATIONS FOR CELL AND GENE-THERAPY SOMATIC CELL AND MOLECULAR GENETICS Webster, C., Blau, H. M. 1990; 16 (6): 557-565

    Abstract

    An assessment of the replicative life-span of myoblasts is of fundamental importance in designing treatment strategies for Duchenne muscular dystrophy (DMD) based on cell or gene therapy. To ascertain myoblast life-span, or the total number of cell divisions of which a myoblast was capable, we serially passaged and counted the progeny of individual myoblasts until they senesced. We compared the life-span of myoblasts from eight DMD patients with controls: three individuals with no known neuromuscular disease, three DMD carriers, and three patients with other muscle degenerative diseases. A decline in replicative capacity was observed with increasing donor age, which was markedly accelerated for DMD relative to control myoblasts. The average myoblast from a 5-year-old control was capable of 56 doublings, or a potential yield of approximately 10(17) cells per cell. By contrast, at 2 years of age, the typical age at clinical onset, only 6% of DMD myoblasts had a life-span of 50 doublings in tissue culture, and by age 7 DMD myoblasts capable of 10 doublings were rare. Our results suggest that the myoblasts (satellite cells) of even the youngest DMD patients have undergone extensive division in an attempt to regenerate degenerating myofibers. These findings have implications for therapeutic intervention in DMD involving genetic engineering and myoblast implantation.

    View details for Web of Science ID A1990ER23200006

    View details for PubMedID 2267630

  • STEROIDS INDUCE ACETYLCHOLINE-RECEPTORS ON CULTURED HUMAN MUSCLE - IMPLICATIONS FOR MYASTHENIA-GRAVIS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kaplan, I., Blakely, B. T., PAVLATH, G. K., Travis, M., Blau, H. M. 1990; 87 (20): 8100-8104

    Abstract

    Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. We show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. Our results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also to a direct effect on muscle. We propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.

    View details for Web of Science ID A1990ED61200064

    View details for PubMedID 2236023

  • NEGATIVE CONTROL OF THE HELIX-LOOP-HELIX FAMILY OF MYOGENIC REGULATORS IN THE NFB MUTANT CELL Peterson, C. A., Gordon, H., Hall, Z. W., Paterson, B. M., Blau, H. M. 1990; 62 (3): 493-502

    Abstract

    We have characterized a nondifferentiating mouse muscle cell line, NFB, that represses the activity of the helix-loop-helix (HLH) family of myogenic regulators, yet expresses sarcomeric actins. The NFB MyoD gene is silent, but can be activated upon transfection of a long terminal region-controlled chicken MyoD cDNA, resulting in myogenesis. When NFB cells are fused with H9c2 rat muscle cells in heterokaryons, the level of rat MyoD transcripts declines. Thus, the stoichiometry of MyoD and the putative repressor controls myogenesis. Although NFB cells express myogenin and Myf-5 transcripts, the activity of these regulators is also repressed:myogenesis is not induced in 10T1/2 fibroblasts and is repressed in L6 muscle cells upon fusion with NFB cells. We conclude that the myogenic HLH regulators are not required for sarcomeric actin gene activation and that myogenesis is subject to dominant-negative control.

    View details for Web of Science ID A1990DU44500011

    View details for PubMedID 1696180

  • MIGRATION OF MYOBLASTS ACROSS BASAL LAMINA DURING SKELETAL-MUSCLE DEVELOPMENT NATURE Hughes, S. M., Blau, H. M. 1990; 345 (6273): 350-353

    Abstract

    Basal lamina is a sheet of extracellular matrix that separates cells into topologically distinct groups during morphogenesis and is thought to form a barrier to cell migration. We have examined whether, during normal muscle development, myoblasts--mononucleate muscle precursor cells--can cross the basal lamina that surrounds each multinucleate muscle fibre. We marked myoblasts in vivo by injecting replication-defective retroviral vectors encoding LacZ into muscle tissue and analysed the fate of their progeny by the expression of beta-galactosidase. A dual labelling method with broad application to retroviral lineage-marking studies was developed to ensure that most clusters of labelled cells were clones derived from a single precursor cell. Most of the myoblasts that were infected at a late stage of rat hindlimb development, when each fibre with its satellite myoblasts is individually encased in a basal lamina sheath, gave rise to clones that contributed to several labelled fibres. Our results show that myoblasts from healthy fibres migrate across basal lamina during normal development and could contribute to the repair of fibres damaged by injury or disease.

    View details for Web of Science ID A1990DF14900068

    View details for PubMedID 2111464

  • EFFECT OF CELL HISTORY ON RESPONSE TO HELIX LOOP HELIX FAMILY OF MYOGENIC REGULATORS NATURE Schafer, B. W., Blakely, B. T., Darlington, G. J., Blau, H. M. 1990; 344 (6265): 454-458

    Abstract

    In multinucleated heterokaryons formed from the fusion of differentiated muscle cells to either hepatocytes or fibroblasts, muscle-specific gene expression is activated, liver-specific gene expression is repressed, and there are changes in the location of the Golgi apparatus. An understanding of the regulatory mechanisms that underlie this plasticity is of particular interest given the stability of the differentiated state in vivo. We have now investigated whether MyoD or myogenin, regulators of muscle-specific gene expression that have a helix-loop-helix motif, can induce the phenotypic conversion observed in heterokaryons. When these regulators were stably or transiently introduced into fibroblasts or hepatocytes by microinjection, transfection or retroviral infection with complementary DNA in expression vectors, fibroblasts expressed muscle-specific genes, whereas hepatocytes did not. However, fusion of hepatocytes stably expressing MyoD to fibroblasts resulted in activation in the heterokaryon of muscle-specific genes of both cell types. These results imply that other regulators, present in fibroblasts but not in hepatocytes, are necessary for the activation of muscle-specific genes, and indicate that the differentiated state of a cell is dictated by its history and a dynamic interaction among the proteins that it contains.

    View details for Web of Science ID A1990CW47800071

    View details for PubMedID 2157160

  • EXPRESSION AND DNA-SEQUENCE ANALYSIS OF A HUMAN-EMBRYONIC SKELETAL-MUSCLE MYOSIN HEAVY-CHAIN GENE NUCLEIC ACIDS RESEARCH KARSCHMIZRACHI, I., Travis, M., Blau, H., Leinwand, L. A. 1989; 17 (15): 6167-6179

    Abstract

    Vertebrate myosin heavy chains (MHC) are represented by multiple genes that are expressed in a spatially and temporally distinct pattern during development. In order to obtain molecular probes for developmentally regulated human MHC isoforms, we used monoclonal antibodies to screen an expression cDNA library constructed from primary human myotube cultures. A 3.4 kb cDNA was isolated that encodes one of the first MHCs to be transcribed in human skeletal muscle development. A portion of the corresponding gene encoding this isoform has also been isolated. Expression of this embryonic MHC is a hallmark of muscle regeneration after birth and is a characteristic marker of human muscular dystrophies. During normal human development, expression is restricted to the embryonic period of development prior to birth. In primary human muscle cell cultures, devoid of other cell types, mRNA accumulation begins as myotubes form, reaches a peak 2 days later and declines to undetectable levels within 10 days. The expression of the protein encoded by the embryonic skeletal MHC gene follows a similar time course, lagging behind the mRNA by approximately two days. Thus, expression of the human embryonic gene is efficiently induced and then repressed in cultured muscle cells, as it is in muscle tissue. The study of the regulation of a human MHC isoform with a central role in muscle development and in muscle regeneration in disease states is therefore amendable to analysis at a molecular level.

    View details for Web of Science ID A1989AK53100023

    View details for PubMedID 2771643

  • INVIVO AGING OF HUMAN-FIBROBLASTS DOES NOT ALTER NUCLEAR PLASTICITY IN HETEROKARYONS SOMATIC CELL AND MOLECULAR GENETICS PAVLATH, G. K., Chiu, C. P., Blau, H. M. 1989; 15 (3): 191-202

    Abstract

    In vivo aging of human fibroblasts altered proliferative properties but not the potential for novel gene expression in response to muscle trans-acting factors. Heterokaryons produced by fusing fibroblasts with muscle cells permitted a dissociation of the effects of aging on cell division and other cell functions. Skin fibroblasts derived from fetal and adult stages of development were distinct cell types based on their doubling time, protein content, cell size, and specific binding of insulin and insulin-like growth factor I. Despite these differences in growth parameters, the two cell types were indistinguishable in heterokaryons. Muscle gene activation occurred in the absence of changes in chromatin structure requiring DNA replication. In addition, the time course, maximal efficiency, and effect of gene dosage on the expression of muscle gene products were similar for heterokaryons containing fetal and adult fibroblasts but distinct for heterokaryons containing keratinocytes. The difference between fibroblasts and keratinocytes in the time course of muscle gene expression is likely to reflect mechanisms of gene activation at the transcriptional level, since the kinetics of muscle protein accumulation paralleled that of muscle transcripts. These results indicate that nuclear plasticity is not altered in fibroblasts by in vivo aging.

    View details for Web of Science ID A1989U835300001

    View details for PubMedID 2471278

  • LOCALIZATION OF MUSCLE GENE-PRODUCTS IN NUCLEAR DOMAINS NATURE PAVLATH, G. K., Rich, K., Webster, S. G., Blau, H. M. 1989; 337 (6207): 570-573

    Abstract

    The localization of gene products is central to the development of cell polarity and pattern specification during embryogenesis. To monitor the distribution of gene products encoded by different nuclei in the same cell in tissue culture, we fused cells of different species to form multinucleated non-dividing heterokaryons. In previous fusion studies, cell-surface antigens and organelles contributed by disparate cell types intermixed within minutes. Using heterokaryons produced with differentiated muscle cells, we demonstrate here that a muscle membrane component, the Golgi apparatus mediating its transport, and a sarcomeric myosin heavy chain are localized in the vicinity of the nuclei responsible for their synthesis. These results provide direct evidence that products (organelle, membrane and structural proteins) derived from individual nuclei can remain localized in myotubes, a finding with implications both for neuromuscular synapse formation and for the carrier state of Duchenne muscular dystrophy.

    View details for Web of Science ID A1989T173700070

    View details for PubMedID 2915707

  • HIERARCHIES OF REGULATORY GENES MAY SPECIFY MAMMALIAN DEVELOPMENT CELL Blau, H. M. 1988; 53 (5): 673-674

    View details for Web of Science ID A1988N799700002

    View details for PubMedID 3286013

  • TUMOR NECROSIS FACTOR INHIBITS HUMAN MYOGENESIS INVITRO MOLECULAR AND CELLULAR BIOLOGY Miller, S. C., Ito, H., Blau, H. M., Torti, F. M. 1988; 8 (6): 2295-2301

    Abstract

    We examined the effects of human recombinant tumor necrosis factor-alpha (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of alpha-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as alpha-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon beta-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.

    View details for Web of Science ID A1988N633800005

    View details for PubMedID 3405207

  • INVIVO SYSTEM FOR CHARACTERIZING CLONAL VARIATION AND TISSUE-SPECIFIC GENE REGULATORY FACTORS BASED ON FUNCTION JOURNAL OF CELL BIOLOGY Hardeman, E. C., Minty, A., BENTONVOSMAN, P., Kedes, L., Blau, H. M. 1988; 106 (4): 1027-1034

    Abstract

    The inducibility of stably transfected alpha-cardiac actin genes differs among L cell clones. We examined the ability of muscle-specific factors to induce the expression of the human muscle alpha-cardiac actin gene promoter when stably transfected into mouse fibroblast L cells. This promoter is transcriptionally active in L cells at a low level, 2-5% of that in transfected muscle cells. Upon fusion with muscle cells to form heterokaryons, expression of the transfected alpha-cardiac actin gene promoter can be induced. However, induction is observed with only 10% of transfected L cell clones and the magnitude of this induction varies between 5- and 50-fold. These properties of the transfected L cell appear to be stably inherited. Our results are consistent with the hypothesis that muscle cells contain factors capable of increasing the transcription of the transfected gene, but that differences among L cell clones, possibly in the site of integration in the genome, determine the extent to which the gene can respond. By fusion into heterokaryons, transfectants with responsive genes can be identified. Such clones should prove useful in determining the basis for clonal variation. In addition, they provide an in vivo system for isolating functionally active tissue-specific transcription factors and the genes that encode them.

    View details for Web of Science ID A1988N183700002

    View details for PubMedID 3162914

  • MUSCLE-CELL COMPONENTS DICTATE HEPATOCYTE GENE-EXPRESSION AND THE DISTRIBUTION OF THE GOLGI-APPARATUS IN HETEROKARYONS GENES & DEVELOPMENT Miller, S. C., PAVLATH, G. K., Blakely, B. T., Blau, H. M. 1988; 2 (3): 330-340

    Abstract

    Major changes in cytoarchitecture and gene expression were induced in short-term heterokaryons. When human hepatocytes were fused with mouse muscle cells, the hepatocyte Golgi apparatus changed from its usual polar location to a uniformly circumnuclear location typical of striated muscle. Human liver albumin ceased to be expressed, and expression of the human muscle cell-surface antigen 5.1H11 was induced without DNA replication or cell division. Coexpression of liver and muscle proteins was rarely observed. These novel findings provide insight into the regulation of gene expression and the targeting and localization of organelles with a central role in cell polarity, intracellular transport, and secretion.

    View details for Web of Science ID A1988P138300006

    View details for PubMedID 3378703

  • FAST MUSCLE-FIBERS ARE PREFERENTIALLY AFFECTED IN DUCHENNE MUSCULAR-DYSTROPHY CELL Webster, C., Silberstein, L., Hays, A. P., Blau, H. M. 1988; 52 (4): 503-513

    Abstract

    We show that Duchenne muscular dystrophy (DMD) selectively affects a subset of skeletal muscle fibers specialized for fast contraction. Muscle fiber types were characterized immunohistochemically with monoclonal antibodies that distinguish isoforms of fetal and adult-fast or adult-slow myosin heavy chain present in the same fiber. Fetal myosin expression increased with patient age and was not due to arrested development but rather to de novo synthesis, which served as a sensitive indicator of muscle regeneration. A subset of fast fibers were the first to degenerate (type IIb). Extensive fast fiber regeneration occurred before slow fibers were affected. These results suggest that the DMD gene product has a specific function in a subpopulation of muscle fibers specialized to respond to the highest frequency of neuronal stimulation with maximal rates of contraction.

    View details for Web of Science ID A1988M352400003

    View details for PubMedID 3342447

  • ISOLATION OF HUMAN MYOBLASTS WITH THE FLUORESCENCE-ACTIVATED CELL SORTER EXPERIMENTAL CELL RESEARCH Webster, C., PAVLATH, G. K., Parks, D. R., Walsh, F. S., Blau, H. M. 1988; 174 (1): 252-265

    Abstract

    We have established procedures for the rapid and efficient purification of human myoblasts using the fluorescence-activated cell sorter. Our approach capitalizes on the specific reaction of monoclonal antibody 5.1H11 with a human muscle cell surface antigen. For each of the five samples analyzed, an enrichment of myoblasts to greater than 99% of the cell population was immediately achieved. Following 3 to 4 weeks of additional growth in vitro, sorted myoblast cultures remained 97% pure. Differentiation of the sorted myoblast cultures, assessed by creatine kinase activity and isozyme content, was comparable to that of pure myoblast cultures obtained by cloning, and was significantly greater than that of mixed fibroblast and myoblast cultures. An average of 10(4) viable myoblasts can be obtained per 0.1 g tissue, each with the potential to undergo approximately 40 cell divisions. Accordingly, if only two-thirds of this proliferative capacity is utilized, the potential yield approximates 10(12) myoblasts, equivalent to 1 kg of cells. Human myogenesis in vitro is no longer limited by cell number and is now amenable to molecular and biochemical analysis on a large scale.+

    View details for Web of Science ID A1988L704600024

    View details for PubMedID 3335226

  • DIFFERENTIAL PATTERNS OF TRANSCRIPT ACCUMULATION DURING HUMAN MYOGENESIS MOLECULAR AND CELLULAR BIOLOGY Gunning, P., Hardeman, E., Wade, R., Ponte, P., Bains, W., Blau, H. M., Kedes, L. 1987; 7 (11): 4100-4114

    Abstract

    We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low- and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major alpha-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.

    View details for Web of Science ID A1987K504300029

    View details for PubMedID 3431550

  • THE PATTERN OF ACTIN EXPRESSION IN HUMAN FIBROBLAST X MOUSE MUSCLE HETEROKARYONS SUGGESTS THAT HUMAN-MUSCLE REGULATORY FACTORS ARE PRODUCED CELL Hardeman, E. C., Chiu, C. P., Minty, A., Blau, H. M. 1986; 47 (1): 123-130

    Abstract

    The expression of previously dormant human muscle genes encoding two major components of the contractile apparatus was activated in multinucleated heterokaryons formed by the fusion of mouse muscle cells and human fibroblasts. The accumulation of human and mouse alpha-cardiac and alpha-skeletal actin transcripts was compared by Northern blot, slot blot, and S1 nuclease assays. The pattern of human transcript accumulation in heterokaryons was quite distinct from that in the mouse muscle cells that induced it, and strikingly similar in time course and relative amounts to that in human primary muscle cultures. In addition, the usual decline in the level of mouse alpha-cardiac actin transcripts was not observed; instead, after fusion with human fibroblasts the levels increased. Our findings suggest that the activated human nuclei in heterokaryons produce their own muscle regulatory factors that alter the expression of mouse muscle genes and direct the expression of the human muscle phenotype.

    View details for Web of Science ID A1986E471700015

    View details for PubMedID 3757033

  • DEVELOPMENTAL PROGRESSION OF MYOSIN GENE-EXPRESSION IN CULTURED MUSCLE-CELLS CELL Silberstein, L., Webster, S. G., Travis, M., Blau, H. M. 1986; 46 (7): 1075-1081

    Abstract

    Myosin heavy chains are encoded by distinct members of a multigene family at different stages of muscle development. Study of the underlying regulatory mechanisms has been hindered because transitions in myosin expression have not been readily attained in tissue culture. Here we show a transition from early (fetal) to late (perinatal/adult) myosins defined by two monoclonal antibodies, F1.652 and N3.36, in the myotubes of mouse C2C12 cells. On day 1 of differentiation, essentially all myosin was early myosin. By day 8, early myosin dropped to 25% of its day 1 value and was replaced by late myosin. The transition occurred without neural contact, connective tissue components, or complex substrates, suggesting that its regulation may be intrinsic to the muscle cell. Our results demonstrate that a developmental progression in myosin gene expression, which occurs rapidly, with high frequency, and under relatively simple conditions, is now amenable to molecular analysis in cultured muscle cells.

    View details for Web of Science ID A1986E242100014

    View details for PubMedID 3530499

  • 2-LEVEL REGULATION OF CARDIAC ACTIN GENE-TRANSCRIPTION - MUSCLE-SPECIFIC MODULATING FACTORS CAN ACCUMULATE BEFORE GENE ACTIVATION MOLECULAR AND CELLULAR BIOLOGY Minty, A., Blau, H., Kedes, L. 1986; 6 (6): 2137-2148

    Abstract

    We have previously proposed that the upstream regions of the human cardiac actin gene contain sequences that interact with muscle-specific factors with direct high-level transcription of this gene in differentiated muscle cells. In this study we showed that these factors already accumulate in the dividing myoblasts of the mouse C2C12 cell line before differentiation of the cells. The endogenous cardiac actin gene in the C2C12 line is expressed only at a low level in myoblasts but at a high level when these cells differentiate into multinucleate myotubes. In contrast, human cardiac actin genes stably introduced into C2C12 cells show high-level expression in both myoblasts and myotubes, indicating that the endogenous cardiac actin gene is repressed in myoblasts by a mechanism which does not affect transfected genes. In a second muscle cell line (the rat L8 cell line), the level of expression of transfected cardiac actin genes increases when these cells differentiate into myotubes, paralleling the expression of the endogenous sarcomeric actin genes. We suggest that the level of transcriptional modulating factors is low in L8 myoblasts and increases when these cells differentiate into myotubes. Our results demonstrate that at least two steps are necessary for high-level cardiac actin gene expression: activation of the gene and subsequent modulation of its transcriptional activity. Furthermore, the results indicate that the two regulatory steps can be dissociated and that the factors involved in modulation are distinct from those involved in gene activation.

    View details for Web of Science ID A1986C580900032

    View details for PubMedID 3785190

  • EXPRESSION OF MUSCLE GENES IN HETEROKARYONS DEPENDS ON GENE DOSAGE JOURNAL OF CELL BIOLOGY PAVLATH, G. K., Blau, H. M. 1986; 102 (1): 124-130

    Abstract

    We report that gene dosage, or the ratio of nuclei from two cell types fused to form a heterokaryon, affects the time course of differentiation-specific gene expression. The rate of appearance of the human muscle antigen, 5.1H11, is significantly faster in heterokaryons with equal or near-equal numbers of mouse muscle and human fibroblast nuclei than in heterokaryons with increased numbers of nuclei from either cell type. By 4 d after fusion, a high frequency of gene expression is evident at all ratios and greater than 75% of heterokaryons express the antigen even when the nonmuscle nuclei greatly outnumber the muscle nuclei. The kinetic differences observed with different nuclear ratios suggest that the concentration of putative trans-acting factors significantly influences the rate of muscle gene expression: a threshold concentration is necessary, but an excess may be inhibitory.

    View details for Web of Science ID A1986AXK3000013

    View details for PubMedID 3941151

  • EVIDENCE FOR DEFECTIVE MYOBLASTS IN DUCHENNE MUSCULAR-DYSTROPHY ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Blau, H. M., Webster, C., PAVLATH, G. K., Chiu, C. P. 1985; 182: 85-110

    View details for Web of Science ID A1985AGS7700007

    View details for PubMedID 4003170

  • PLASTICITY OF THE DIFFERENTIATED STATE SCIENCE Blau, H. M., PAVLATH, G. K., Hardeman, E. C., Chiu, C. P., Silberstein, L., Webster, S. G., Miller, S. C., Webster, C. 1985; 230 (4727): 758-766

    Abstract

    Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level. In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.

    View details for Web of Science ID A1985ATT2700011

    View details for PubMedID 2414846

  • MUSCLE GENE-EXPRESSION IN HETEROKARYONS ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Blau, H. M., Chiu, C. P., PAVLATH, G. K., Webster, C. 1985; 182: 231-247

    View details for Web of Science ID A1985AGS7700020

    View details for PubMedID 4003160

  • CARDIAC ACTIN IS THE MAJOR ACTIN GENE-PRODUCT IN SKELETAL-MUSCLE CELL-DIFFERENTIATION INVITRO MOLECULAR AND CELLULAR BIOLOGY Bains, W., Ponte, P., Blau, H., Kedes, L. 1984; 4 (8): 1449-1453

    Abstract

    We examined the expression of alpha-skeletal, alpha-cardiac, and beta- and gamma-cytoskeletal actin genes in a mouse skeletal muscle cell line (C2C12) during differentiation in vitro. Using isotype-specific cDNA probes, we showed that the alpha-skeletal actin mRNA pool reached only 15% of the level reached in adult skeletal muscle and required several days to attain this peak, which was then stably maintained. However, these cells accumulated a pool of alpha-cardiac actin six times higher than the alpha-skeletal actin mRNA peak within 24 h of the initiation of differentiation. After cells had been cultured for an additional 3 days, this pool declined to 10% of its peak level. In contrast, over 95% of the actin mRNA in adult skeletal muscle coded for alpha-actin. This suggests that C2C12 cells express a pattern of sarcomeric actin genes typical of either muscle development or regeneration and distinct from that seen in mature, adult tissue. Concurrently in the course of differentiation the beta- and gamma-cytoskeletal actin mRNA pools decreased to less than 10% of their levels in proliferating cells. The decreases in beta- and gamma-cytoskeletal actin mRNAs are apparently not coordinately regulated.

    View details for Web of Science ID A1984TD30600003

    View details for PubMedID 6493226

  • ISOLATION AND CHARACTERIZATION OF FULL-LENGTH CDNA CLONES FOR HUMAN ALPHA-ACTIN, BETA-ACTIN AND GAMMA-ACTIN MESSENGER-RNAS - SKELETAL BUT NOT CYTOPLASMIC ACTINS HAVE AN AMINO-TERMINAL CYSTEINE THAT IS SUBSEQUENTLY REMOVED MOLECULAR AND CELLULAR BIOLOGY Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H., Kedes, L. 1983; 3 (5): 787-795

    Abstract

    cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.

    View details for Web of Science ID A1983QN67600005

    View details for PubMedID 6865942

  • ALPHA-SKELETAL AND ALPHA-CARDIAC ACTIN GENES ARE COEXPRESSED IN ADULT HUMAN SKELETAL-MUSCLE AND HEART MOLECULAR AND CELLULAR BIOLOGY Gunning, P., Ponte, P., Blau, H., Kedes, L. 1983; 3 (11): 1985-1995

    Abstract

    We determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes derived from alpha-skeletal, beta- and gamma-actin cDNAs and from an alpha-cardiac actin genomic clone, we showed that 28 of the cDNAs correspond to alpha-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from alpha-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle alpha-cardiac actin cDNAs are derived from transcripts of the cloned alpha-cardiac actin gene. Direct measurements of actin isotype mRNA expression in human skeletal muscle showed that alpha-cardiac actin mRNA is expressed at 5% the level of alpha-skeletal actin. Furthermore, the alpha-cardiac actin gene expressed in skeletal muscle is the same gene which produces alpha-cardiac actin mRNA in the human heart. Of equal surprise, we found that alpha-skeletal actin mRNA accounts for about half of the total actin mRNA in adult heart. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. We conclude that alpha-skeletal and alpha-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (beta and gamma) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, we postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.

    View details for Web of Science ID A1983RP00200012

    View details for PubMedID 6689196

  • HUMAN ACTIN GENES ARE SINGLE COPY FOR ALPHA-SKELETAL AND ALPHA-CARDIAC ACTIN BUT MULTICOPY FOR BETA-CYTOSKELETAL AND GAMMA-CYTOSKELETAL GENES - 3' UNTRANSLATED REGIONS ARE ISOTYPE SPECIFIC BUT ARE CONSERVED IN EVOLUTION MOLECULAR AND CELLULAR BIOLOGY Ponte, P., Gunning, P., Blau, H., Kedes, L. 1983; 3 (10): 1783-1791

    Abstract

    We have constructed isotype-specific subclones from the 3' untranslated regions of alpha-skeletal, alpha-cardiac, beta-cytoskeletal, and gamma-cytoskeletal actin cDNAs. These clones have been used as hybridization probes to assay the number and organization of these actin isotypes in the human genome. Hybridization of these probes to human genomic actin clones (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981; Engel et al., Mol. Cell. Biol. 2:674-684, 1982) has allowed the unambiguous assignment of the genomic clones to isotypically defined actin subfamilies. In addition, only one isotype-specific probe hybridizes to each actin-containing gene, with a single exception. This result suggests that the multiple actin genes in the human genome are not closely linked. Genomic DNA blots probed with these subclones under stringent conditions demonstrate that the alpha-skeletal and alpha-cardiac muscle actin genes are single copy, whereas the cytoskeletal actins, beta and gamma, are present in multiple copies in the human genome. Most of the actin genes of other mammals are cytoplasmic as well. These observations have important implications for the evolution of multigene families.

    View details for Web of Science ID A1983RK29900013

    View details for PubMedID 6646124

  • DEFECTIVE MYOBLASTS IDENTIFIED IN DUCHENNE MUSCULAR-DYSTROPHY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Blau, H. M., Webster, C., PAVLATH, G. K. 1983; 80 (15): 4856-4860

    Abstract

    A defect in the proliferative capacity of satellite cells, mononucleated precursors of mature muscle fibers, was found in clonal analyses of cells cultured from Duchenne muscular dystrophy (DMD) patients. The total yield of myoblasts per gram of muscle biopsy was decreased to 5% of normal. Of the DMD myoblast clones obtained, a large proportion contained a morphological class of flat distended cells that had an increased generation time and ceased to proliferate beyond 100-1,000 cells but could be induced to fuse and form myotubes. The altered muscle phenotype was detected in all cultures from DMD patients but was rarely found among myoblasts of controls. By age 14 yr, it comprised as man as 90% of DMD myoblasts. The remaining DMD myoblast clones, which initially grew well, had severely impaired proliferative capacity upon passage and further cultivation. Eventually all myoblasts from DMD muscle tissue exhibited defective growth potential. In contrast, the fibroblast yield and proliferative capacity from DMD samples did not differ from normal. Based on these findings, we propose a hypothesis for the etiology of DMD: Dividing myoblasts are required for muscle growth and maintenance, and the limited capacity of DMD myoblasts to grow is directly related to the progressive muscle degeneration characteristic of the disease.

    View details for Web of Science ID A1983RB47600054

    View details for PubMedID 6576361

Conference Proceedings


  • REGULATING THE MYOGENIC REGULATORS Blau, H. M. COMPANY BIOLOGISTS LTD. 1992: 9-18

    View details for Web of Science ID A1992BX82U00002

    View details for PubMedID 1341053

  • PURIFICATION AND PROLIFERATION OF HUMAN MYOBLASTS ISOLATED WITH FLUORESCENCE ACTIVATED CELL SORTING Blau, H. M., Webster, C., PAVLATH, G. K. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1990: 97-100

    View details for Web of Science ID A1990BR89S00012

    View details for PubMedID 2248160

  • LOCALIZATION OF MUSCLE GENE-PRODUCTS IN NUCLEAR DOMAINS - DOES THIS CONSTITUTE A PROBLEM FOR MYOBLAST THERAPY Blau, H. M., PAVLATH, G. K., Rich, K., Webster, S. G. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1990: 167-172

    View details for Web of Science ID A1990BR89S00019

    View details for PubMedID 2248136

  • RETROVIRAL LINEAGE MARKERS FOR ASSESSING MYOBLAST FATE INVIVO Blau, H. M., Hughes, S. M. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1990: 201-203

    View details for Web of Science ID A1990BR89S00022

    View details for PubMedID 2123371