Search

It remains challenging to generate reproducible, high-quality cDNA libraries from RNA derived from rare cell populations. Here, we describe a protocol for high-throughput RNA-seq library preparation, including isolation of 200 skeletal muscle stem cells from mouse tibialis anterior muscle by fluorescence-activated cell sorting and cDNA preparation. We also describe RNA extraction and cDNA preparation from differentiating mouse embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Juan et al. (2016) and Garcia-Prat et al. (2016)., Competing Interests: The authors declare no competing interests.

Muscle atrophy is the loss of skeletal muscle mass and strength in response to diverse catabolic stimuli. At present, no effective treatments except exercise have been shown to reduce muscle atrophy clinically. Here, we report that CRISPR/Cas9-mediated genome editing through local injection into gastrocnemius muscles or tibialis anterior muscle efficiently targets the biogenesis processing sites in pre-miR-29b. In vivo, this CRISPR-based treatment prevented the muscle atrophy induced by angiotensin II (AngII), immobilization, and denervation via activation of the AKT-FOXO3A-mTOR signaling pathway and protected against AngII-induced myocyte apoptosis in mice, leading to significantly increased exercise capacity. Our work establishes CRISPR/Cas9-based gene targeting on miRNA as a potential durable therapy for the treatment of muscle atrophy and expands the strategies available interrogating miRNA function in vivo., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers, and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs led to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The impact of glucose metabolism on muscle regeneration remains unresolved. We identify glucose metabolism as a crucial driver of histone acetylation and myogenic cell fate. We use single-cell mass cytometry (CyTOF) and flow cytometry to characterize the histone acetylation and metabolic states of quiescent, activated, and differentiating muscle stem cells (MuSCs). We find glucose is dispensable for mitochondrial respiration in proliferating MuSCs, so that glucose becomes available for maintaining high histone acetylation via acetyl-CoA. Conversely, quiescent and differentiating MuSCs increase glucose utilization for respiration and have consequently reduced acetylation. Pyruvate dehydrogenase (PDH) activity serves as a rheostat for histone acetylation and must be controlled for muscle regeneration. Increased PDH activity in proliferation increases histone acetylation and chromatin accessibility at genes that must be silenced for differentiation to proceed, and thus promotes self-renewal. These results highlight metabolism as a determinant of MuSC histone acetylation, fate, and function during muscle regeneration., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Skeletal stem cells (SSCs) are postulated to provide a continuous supply of osteoblasts throughout life. However, under certain conditions, the SSC population can become incorrectly specified or is not maintained, resulting in reduced osteoblast formation, decreased bone mass, and in severe cases, osteoporosis. Glutamine metabolism has emerged as a critical regulator of many cellular processes in diverse pathologies. The enzyme glutaminase (GLS) deaminates glutamine to form glutamate-the rate-limiting first step in glutamine metabolism. Using genetic and metabolic approaches, we demonstrate GLS and glutamine metabolism are required in SSCs to regulate osteoblast and adipocyte specification and bone formation. Mechanistically, transaminase-dependent α-ketoglutarate production is critical for the proliferation, specification, and differentiation of SSCs. Collectively, these data suggest stimulating GLS activity may provide a therapeutic approach to expand SSCs in aged individuals and enhance osteoblast differentiation and activity to increase bone mass., (Copyright © 2019 Elsevier Inc. All rights reserved.)

State of the art techniques have been developed to isolate and analyze cells from various tissues, aiming to capture their in vivo state. However, the majority of cell isolation protocols involve lengthy mechanical and enzymatic dissociation steps followed by flow cytometry, exposing cells to stress and disrupting their physiological niche. Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of isolation procedures and captures cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications while having negligible effects on DNA methylation. In addition to proposing a protocol to avoid isolation-induced artifacts, our study reveals previously undetected quiescence and early activation genes of potential biological interest., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7β1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD., (Copyright © 2017 The American Society of Gene and Cell Therapy. All rights reserved.)

Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Whether gene repositioning to the nuclear periphery during differentiation adds another layer of regulation to gene expression remains controversial. Here, we resolve this by manipulating gene positions through targeting the nuclear envelope transmembrane proteins (NETs) that direct their normal repositioning during myogenesis. Combining transcriptomics with high-resolution DamID mapping of nuclear envelope-genome contacts, we show that three muscle-specific NETs, NET39, Tmem38A, and WFS1, direct specific myogenic genes to the nuclear periphery to facilitate their repression. Retargeting a NET39 fragment to nucleoli correspondingly repositioned a target gene, indicating a direct tethering mechanism. Being able to manipulate gene position independently of other changes in differentiation revealed that repositioning contributes ⅓ to ⅔ of a gene's normal repression in myogenesis. Together, these NETs affect 37% of all genes changing expression during myogenesis, and their combined knockdown almost completely blocks myotube formation. This unequivocally demonstrates that NET-directed gene repositioning is critical for developmental gene regulation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Muscle stem cells (MuSCs) exhibit distinct behavior during successive phases of developmental myogenesis. However, how their transition to adulthood is regulated is poorly understood. Here, we show that fetal MuSCs resist progenitor specification and exhibit altered division dynamics, intrinsic features that are progressively lost postnatally. After transplantation, fetal MuSCs expand more efficiently and contribute to muscle repair. Conversely, niche colonization efficiency increases in adulthood, indicating a balance between muscle growth and stem cell pool repopulation. Gene expression profiling identified several extracellular matrix (ECM) molecules preferentially expressed in fetal MuSCs, including tenascin-C, fibronectin, and collagen VI. Loss-of-function experiments confirmed their essential and stage-specific role in regulating MuSC function. Finally, fetal-derived paracrine factors were able to enhance adult MuSC regenerative potential. Together, these findings demonstrate that MuSCs change the way in which they remodel their microenvironment to direct stem cell behavior and support the unique demands of muscle development or repair., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Repair of injured skeletal muscle by cell therapies has been limited by poor survival of injected cells. Use of a carrier scaffold delivering cells locally, may enhance in vivo cell survival, and promote skeletal muscle regeneration. Biomaterial scaffolds are often implanted into muscle tissue through invasive surgeries, which can result in trauma that delays healing. Minimally invasive approaches to scaffold implantation are thought to minimize these adverse effects. This hypothesis was addressed in the context of a severe mouse skeletal muscle injury model. A degradable, shape-memory alginate scaffold that was highly porous and compressible was delivered by minimally invasive surgical techniques to injured tibialis anterior muscle. The scaffold controlled was quickly rehydrated in situ with autologous myoblasts and growth factors (either insulin-like growth factor-1 (IGF-1) alone or IGF-1 with vascular endothelial growth factor (VEGF)). The implanted scaffolds delivering myoblasts and IGF-1 significantly reduced scar formation, enhanced cell engraftment, and improved muscle contractile function. The addition of VEGF to the scaffold further improved functional recovery likely through increased angiogenesis. Thus, the delivery of myoblasts and dual local release of VEGF and IGF-1 from degradable scaffolds implanted through a minimally invasive procedure effectively promoted the functional regeneration of injured skeletal muscle.

Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 acts to regulate gene function has remained enigmatic, despite the recent implication of its encoded miR-675 in limiting placental growth. We noted that vertebrate H19 harbors both canonical and noncanonical binding sites for the let-7 family of microRNAs, which plays important roles in development, cancer, and metabolism. Using H19 knockdown and overexpression, combined with in vivo crosslinking and genome-wide transcriptome analysis, we demonstrate that H19 modulates let-7 availability by acting as a molecular sponge. The physiological significance of this interaction is highlighted in cultures in which H19 depletion causes precocious muscle differentiation, a phenotype recapitulated by let-7 overexpression. Our results reveal an unexpected mode of action of H19 and identify this lncRNA as an important regulator of the major let-7 family of microRNAs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Regeneration requires both potential and instructions for tissue replacement. In planarians, pluripotent stem cells have the potential to produce all new tissue. The identities of the cells that provide regeneration instructions are unknown. Here, we report that position control genes (PCGs) that control regeneration and tissue turnover are expressed in a subepidermal layer of nonneoblast cells. These subepidermal cells coexpress many PCGs. We propose that these subepidermal cells provide a system of body coordinates and positional information for regeneration, and identify them to be muscle cells of the planarian body wall. Almost all planarian muscle cells express PCGs, suggesting a dual function: contraction and control of patterning. PCG expression is dynamic in muscle cells after injury, even in the absence of neoblasts, suggesting that muscle is instructive for regeneration. We conclude that planarian regeneration involves two highly flexible systems: pluripotent neoblasts that can generate any new cell type and muscle cells that provide positional instructions for the regeneration of any body region., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Pericytes are endothelial-associated cells that contribute to vessel wall. Here, we report that pericytes may derive from direct conversion of committed skeletal myoblasts. When exposed to Dll4 and PDGF-BB, but not Dll1, skeletal myoblasts downregulate myogenic genes, except Myf5, and upregulate pericyte markers, whereas inhibition of Notch signaling restores myogenesis. Moreover, when cocultured with endothelial cells, skeletal myoblasts, previously treated with Dll4 and PDGF-BB, adopt a perithelial position stabilizing newly formed vessel-like networks in vitro and in vivo. In a transgenic mouse model in which cells expressing MyoD activate Notch, skeletal myogenesis is abolished and pericyte genes are activated. Even if overexpressed, Myf5 does not trigger myogenesis because Notch induces Id3, partially sequestering Myf5 and inhibiting MEF2 expression. Myf5-expressing cells adopt a perithelial position, as occasionally also observed in wild-type (WT) embryos. These data indicate that endothelium, via Dll4 and PDGF-BB, induces a fate switch in adjacent skeletal myoblasts., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have an endless self-renewal capacity and can theoretically differentiate into all types of lineages. They thus represent an unlimited source of cells for therapies of regenerative diseases, such as Duchenne muscular dystrophy (DMD), and for tissue repair in specific medical fields. However, at the moment, the low number of efficient specific lineage differentiation protocols compromises their use in regenerative medicine. We developed a two-step procedure to differentiate hESCs and dystrophic hiPSCs in myogenic cells. The first step was a culture in a myogenic medium and the second step an infection with an adenovirus expressing the myogenic master gene MyoD. Following infection, the cells expressed several myogenic markers and formed abundant multinucleated myotubes in vitro. When transplanted in the muscle of Rag/mdx mice, these cells participated in muscle regeneration by fusing very well with existing muscle fibers. Our findings provide an effective method that will permit to use hESCs or hiPSCs for preclinical studies in muscle repair.

Exon skipping has been demonstrated to be a successful strategy for the gene therapy of Duchenne muscular dystrophy (DMD): the rational being to convert severe Duchenne forms into milder Becker ones. Here, we show the selection of U1 snRNA-antisense constructs able to confer effective rescue of dystrophin synthesis in a Δ44 Duchenne genetic background, through skipping of exon 45; moreover, we demonstrate that the resulting dystrophin is able to recover timing of myogenic marker expression, to relocalize neuronal nitric oxide synthase (nNOS) and to rescue expression of miRNAs previously shown to be sensitive to the Dystrophin-nNOS-HDAC2 pathway. Becker mutations display different phenotypes, likely depending on whether the shorter protein is able to reconstitute the wide range of wild-type functions. Among them, efficient assembly of the dystrophin-associated protein complex (DAPC) and nNOS localization are important. Comparing different Becker deletions we demonstrate the correlation between the ability of the mutant dystrophin to relocalize nNOS and the expression levels of two miRNAs, miR-1 and miR29c, known to be involved in muscle homeostasis and to be controlled by the Dys-nNOS-HDAC2 pathway.

Polycomb group (PcG) proteins exert essential functions in the most disparate biological processes. The contribution of PcG proteins to cell commitment and differentiation relates to their ability to repress transcription of developmental regulators in embryonic stem (ES) cells and in committed cell lineages, including skeletal muscle cells (SMC). PcG proteins are preferentially removed from transcribed regions, but the underlying mechanisms remain unclear. Here, PcG proteins are found to occupy and repress transcription from an intronic region containing the microRNA miR-214 in undifferentiated SMC. Differentiation coincides with PcG disengagement, recruitment of the developmental regulators MyoD and myogenin, and activation of miR-214 transcription. Once transcribed, miR-214 negatively feeds back on PcG by targeting the Ezh2 3'UTR, the catalytic subunit of the PRC2 complex. miR-214-mediated Ezh2 protein reduction accelerates SMC differentiation and promotes unscheduled transcription of developmental regulators in ES cells. Thus, miR-214 and Ezh2 establish a regulatory loop controlling PcG-dependent gene expression during differentiation.

Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts, miR-29 is repressed by NF-kappaB acting through YY1 and the Polycomb group. During myogenesis, NF-kappaB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-kappaB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its promyogenic function. Together, these results identify a NF-kappaB-YY1-miR-29 regulatory circuit whose disruption may contribute to RMS.

In a recent issue of Molecular Cell, Deato et al. (2008) elucidate the basis by which the muscle-specific activator MyoD recruits the core transcription machinery to the promoter of a key regulatory gene involved in myogenic differentiation.

The two metabolic sensors AMPK and SIRT1 take center stage as Fulco et al. reveal, in this issue of Developmental Cell, the signaling mechanism by which low glucose prevents the correct development of the myogenic program. These observations may hold some therapeutic promise against muscle wasting.

Duchenne muscular dystrophy (DMD) is caused by mutations in the X chromosome-linked DMD gene, which encodes the sarcolemma-stabilizing protein-dystrophin. Initial attempts at DMD therapy deployed muscle progenitor cells from healthy donors. The utilization of these cells is, however, hampered by their immunogenicity, while those from DMD patients are scarce and display limited ex vivo replication. Nonmuscle cells with myogenic capacity may offer valuable alternatives especially if, to allow autologous transplantation, they are amenable to genetic intervention. As a paradigm for therapeutic gene transfer by heterotypic cell fusion we are investigating whether human mesenchymal stem cells (hMSCs) can serve as donors of recombinant DMD genes for recipient human muscle cells. Here, we show that forced MyoD expression in hMSCs greatly increases their tendency to participate in human myotube formation turning them into improved DNA delivery vehicles. Efficient loading of hMSCs with recombinant DMD was achieved through a new tropism-modified high-capacity adenoviral (hcAd) vector directing striated muscle-specific synthesis of full-length dystrophin. This study introduces the principle of genetic complementation of gene-defective cells via directed cell fusion and provides an initial framework to test whether transient MyoD synthesis in autologous, gene-corrected hMSCs increases their potential for treating DMD and, possibly, other muscular dystrophies.

In skeletal muscle development, the myogenic regulatory factors myf5 and myoD play redundant roles in the specification and maintenance of myoblasts, whereas myf6 has a downstream role in differentiating myocytes and myofibers. It is not clear whether the redundancy between myf5 and myoD is within the same cell lineage or between distinct lineages. Using lineage tracing and conditional cell ablation in mice, we demonstrate the existence of two distinct lineages in myogenesis: a myf5 lineage and a myf5-independent lineage. Ablating the myf5 lineage is compatible with myogenesis sustained by myf5-independent, myoD-expressing myoblasts, whereas ablation of the myf6 lineage leads to an absence of all differentiated myofibers, although early myogenesis appears to be unaffected. We also demonstrate here the existence of a significant myf5 lineage within ribs that has an important role in rib development, suggested by severe rib defects upon ablating the myf5 lineage.

Cell therapy for Duchenne muscular dystrophy and other muscle diseases is limited by a massive early cell death following injections. In this study, we explored the potential benefit of heme oxygenase-1 (HO-1) expression in the survival of porcine myogenic precursor cells (MPCs) transplanted in pig skeletal muscle. Increased HO-1 expression was assessed either by transient hyperthermia or by HO-1 lentiviral infection. One day after the thermic shock, we observed a fourfold and a threefold increase in HSP70/72 and HO-1 levels, respectively. This treatment protected 30% of cells from staurosporine-induced apoptosis in vitro. When porcine MPC were heat-shocked prior to grafting, we improved cell survival by threefold at 5 days after autologous transplantation (26.3 +/- 5.5% surviving cells). After HO-1 lentiviral transduction, almost 60% of cells expressed the transgene and kept their myogenic properties to proliferate and fuse in vitro. Apoptosis of HO-1 transduced cells was reduced by 50% in vitro after staurosporine induction. Finally, a fivefold enhancement in cell survival was observed after transplantation of HO-1-group (47.5 +/- 9.1% surviving cells) as compared to the nls-LacZ-group or control group. These results identify HO-1 as a protective gene against early MPC death post-transplantation.

Pax3 and Pax7 play distinct but overlapping roles in developmental and postnatal myogenesis. The mechanisms involved in the differential regulation of these highly homologous proteins are unknown. We present evidence that Pax3, but not Pax7, is regulated by ubiquitination and proteasomal degradation during adult muscle stem cell activation. Intriguingly, only monoubiquitinated forms of Pax3 could be detected. Mutation of two specific lysine residues in the C-terminal region of Pax3 reduced the extent of its monoubiquitination and susceptibility to proteasomal degradation, whereas introduction of a key lysine into the C-terminal region of Pax7 rendered that protein susceptible to monoubiquitination and proteasomal degradation. Monoubiquitinated Pax3 was shuttled to the intrinsic proteasomal protein S5a by interacting specifically with the ubiquitin-binding protein Rad23B. Functionally, sustained expression of Pax3 proteins inhibited myogenic differentiation, demonstrating that Pax3 degradation is an essential step for the progression of the myogenic program. These results reveal an important mechanism of Pax3 regulation in muscle progenitors and an unrecognized role of protein monoubiquitination in mediating proteasomal degradation.

Epigenetic regulation of gene expression is a source of genetic variation, which can mimic recessive mutations by creating transcriptional haploinsufficiency. Germline epimutations and genomic imprinting are typical examples, although their existence can be difficult to reveal. Genomic imprinting can be tissue specific, with biallelic expression in some tissues and monoallelic expression in others or with polymorphic expression in the general population. Mutations in the skeletal-muscle ryanodine-receptor gene (RYR1) are associated with malignant hyperthermia susceptibility and the congenital myopathies central core disease and multiminicore disease. RYR1 has never been thought to be affected by epigenetic regulation. However, during the RYR1-mutation analysis of a cohort of patients with recessive core myopathies, we discovered that 6 (55%) of 11 patients had monoallelic RYR1 transcription in skeletal muscle, despite being heterozygous at the genomic level. In families for which parental DNA was available, segregation studies showed that the nonexpressed allele was maternally inherited. Transcription analysis in patients' fibroblasts and lymphoblastoid cell lines indicated biallelic expression, which suggests tissue-specific silencing. Transcription analysis of normal human fetal tissues showed that RYR1 was monoallelically expressed in skeletal and smooth muscles, brain, and eye in 10% of cases. In contrast, 25 normal adult human skeletal-muscle samples displayed only biallelic expression. Finally, the administration of the DNA methyltransferase inhibitor 5-aza-deoxycytidine to cultured patient skeletal-muscle myoblasts reactivated the transcription of the silenced allele, which suggests hypermethylation as a mechanism for RYR1 silencing. Our data indicate that RYR1 undergoes polymorphic, tissue-specific, and developmentally regulated allele silencing and that this unveils recessive mutations in patients with core myopathies. Furthermore, our data suggest that imprinting is a likely mechanism for this phenomenon and that similar mechanisms could play a role in human phenotypic heterogeneity.

Human muscle progenitor cells transduced with lentiviral vectors secreted high levels of blood clotting factor IX (FIX). When bioengineered into postmitotic myofibers as human bioartificial muscles (HBAMs) and subcutaneously implanted into immunodeficient mice, they secreted FIX into the circulation for >3 months. The HBAM-derived FIX was biologically active, consistent with the cells' ability to conduct the necessary posttranslational modifications. These bioengineered muscle implants are retrievable, an inherent safety feature that distinguishes this "reversible" gene therapy approach from most other gene therapy strategies. When myofibers were bioengineered from human myoblasts expressing FIX and vascular endothelial growth factor, circulating FIX levels were increased and maintained long term within the therapeutic range, consistent with the generation of a vascular network around the HBAM. The present study implicates an important role for angiogenesis in the efficient delivery of therapeutic proteins using tissue engineered stem cell-based gene therapies.

In this issue of Cell, G. Pavlath and coworkers demonstrate a novel role for Interleukin-4 (IL-4) in regulating the fusion of myoblasts with differentiated myotubes. The authors demonstrate that NFATc2 signaling in newly formed myotubes induces IL-4 expression and secretion which promotes myoblast fusion with pre-existing myotubes.

In a recent paper, the concept of a genotoxic stress-induced differentiation checkpoint has been proposed. The suggested function of this checkpoint is to preserve the integrity of the genome in terminally differentiated cells.