Repair and regeneration of adult skeletal muscle are mediated by satellite cells. In healthy muscle these rare mononucleate muscle precursor cells are mitotically quiescent. Upon muscle injury or degeneration, members of this self-renewing pool are activated to proliferate and then differentiate. Here we analyzed in single satellite cells the expression of a set of regulatory genes that are candidates for causal roles in satellite cell activation, maturation, and differentiation. Individual cells were identified as satellite cells and selected for analysis based on their physical association with single explanted myofibers or their position beneath the basal lamina in unperturbed muscle tissue. Using a multiplex single-cell RT-PCR assay we simultaneously monitored expression of all four MyoD family regulators of muscle determination and differentiation (MRFs) together with two candidate markers of satellite cell identity, c-met and m-cadherin. By making these measurements on large numbers of individual cells during the time course of satellite cell activation, we were able to define which expression states (possible combinations of the six genes) were represented and to specify how the representation of each state changed with time. Activated satellite cells began to express either MyoD or myf5 first among the MRFs; most cells then expressed both myf-5 and MyoD simultaneously; myogenin came on later in cells expressing both MyoD and myf5; and many cells ultimately expressed all four MRFs simultaneously. The results for fiber-associated satellite cells from either predominantly fast or slow muscles were indistinguishable from each other. The c-met receptor tyrosine kinase was also monitored because it is a candidate for mediating activation of quiescent satellite cells (Allen et al., 1995) and because it might also be a candidate molecular marker for satellite cells. A significant difficulty in studying mouse satellite cells has been the absence of molecular markers that could identify them in the quiescent state before expression of MRFs or desmin and distinguish them from fibroblasts. We show here that c-met receptor is present beneath the basal lamina on presumptive satellite cells in intact muscle and that c-met mRNA and protein are expressed by all myofiber-associated satellite cells from the time of explant through the course of activation, proliferation, and differentiation. c-met was not detected in muscle-derived fibroblasts or in other mononucleate cells from healthy muscle explants. When compared directly with m-cadherin, which has previously been suggested as a marker for quiescent satellite cells, m-cadherin mRNA was detected only in a small subset of satellite cells at early times after myofiber explant. However, at late times following activation (by 96 hr in this fiber culture system), c-met and m-cadherin were uniformly coexpressed. From the individual satellite cell expression types observed, a model of the satellite cell population at rest and during the time course of activation was generated.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.

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MyoD-deficient mice are without obvious deleterious muscle phenotype during embryogenesis and fetal development, and adults in the laboratory have grossly normal skeletal muscle and life span. However, a previous study showed that in the context of muscle degeneration on a mdx (dystrophin null) genetic background, animals lacking MyoD have a greatly intensified disease phenotype leading to lethality not otherwise seen in mdx mice. Here we have examined MyoD(-/-) adult muscle fibers and their associated satellite cells in single myofiber cultures and describe major phenotypic differences found at the tissue, cellular, and molecular levels. The steady-state number of satellite cells on freshly isolated MyoD(-/-) fibers was elevated and abnormal branched fiber morphologies were observed, the latter suggesting chronic muscle regeneration in vivo. Single-cell RNA coexpression analyses were performed for c-met, m-cadherin, and the four myogenic regulatory factors (MRFs.) Most mutant satellite cells entered the cell cycle and upregulated expression of myf5, both characteristic early steps in satellite cell maturation. However, they later failed to normally upregulate MRF4, displayed a major deficit in m-cadherin expression, and showed a significant diminution in myogenin-positive status compared with wildtype. MyoD(-/-) satellite cells formed unusual aggregate structures, failed to fuse efficiently, and showed greater than 90% reduction in differentiation efficiency relative to wildtype. A further survey of RNAs encoding regulators of growth and differentiation, cell cycle progression, and cell signaling revealed similar or identical expression profiles for most genes as well as several noteworthy differences. Among these, GDF8 and Msx1 were identified as potentially important regulators of the quiescent state whose expression profile differs between mutant and wildtype. Considered together, these data suggest that activated MyoD(-/-) satellite cells assume a phenotype that resembles in some ways a developmentally "stalled" cell compared to wildtype. However, the MyoD(-/-) cells are not merely developmentally immature, as they also display novel molecular and cellular characteristics that differ from any observed in wild-type muscle precursor counterparts of any stage.

Somatic stem cells cycle slowly or remain quiescent until required for tissue repair and maintenance. Upon muscle injury, stem cells that lie between the muscle fiber and basal lamina (satellite cells) are activated, proliferate, and eventually differentiate to repair the damaged muscle. Satellite cells in healthy muscle are quiescent, do not express MyoD family transcription factors or cell cycle regulatory genes and are insulated from the surrounding environment. Here, we report that the p38α/β family of mitogen-activated protein kinases (MAPKs) reversibly regulates the quiescent state of the skeletal muscle satellite cell. Inhibition of p38α/β MAPKs (a) promotes exit from the cell cycle, (b) prevents differentiation, and (c) insulates the cell from most external stimuli allowing the satellite cell to maintain a quiescent state. Activation of satellite cells and p38α/β MAPKs occurs concomitantly, providing further support that these MAPKs function as a molecular switch for satellite cell activation.

Summary Skeletal muscle satellite cells, located between the basal lamina and plasma membrane of myofibers, are required for skeletal muscle regeneration. The capacity of satellite cells as well as other cell lineages including mesoangioblasts, mesenchymal stem cells and side population (SP) cells to contribute to muscle regeneration has complicated the identification of a satellite stem cell. We have characterized a rare subset of the muscle SP that efficiently engraft into the host satellite cell niche when transplanted into regenerating muscle, providing 75% of the satellite cell population and 30% of the myonuclear population, respectively. These cells are found in the satellite cell position, adhere to isolated myofibers, and spontaneously undergo myogenesis in culture. We propose that this subset of SP cells (satellite-SP cells) characterized by ABCG2, Syndecan-4 and Pax7 expression, constitutes a self-renewing muscle stem cell capable of generating both satellite cells and their myonuclear progeny in vivo.

Skeletal muscle repair and regeneration requires the activity of satellite cells, a population of myogenic stem cells scattered throughout the tissue and activated to proliferate and differentiate in response to myotrauma or disease. While it seems likely that satellite cells would need to navigate local muscle tissue to reach damaged areas, relatively little data on such motility exist, and most studies have been with immortalized cell lines. We find that primary satellite cells are significantly more motile than myoblast cell lines, and that adhesion to laminin promotes primary cell motility more than fourfold over other substrates. Using timelapse videomicroscopy to assess satellite cell motility on single living myofibers, we have identified a requirement for the laminin-binding integrin α7β1 in satellite cell motility, as well as a role for hepatocyte growth factor in promoting directional persistence. The extensive migratory behavior of satellite cells resident on muscle fibers suggests caution when determining, based on fixed specimens, whether adjacent cells are daughters from the same mother cell. We also observed more persistent long-term contact between individual satellite cells than has been previously supposed, potential cell-cell attractive and repulsive interactions, and migration between host myofibers. Based on such activity, we assayed for expression of “pathfinding” cues, and found that satellite cells express multiple guidance ligands and receptors. Together, these data suggest that satellite cell migration in vivo may be more extensive than currently thought, and could be regulated by combinations of signals, including adhesive haptotaxis, soluble factors, and guidance cues. Stem Cells 2009;27:2527–2538

In addition to stimulating skeletal muscle growth and repair, Wnt7a/Fzd7 signaling increases the polarity and directional migration of myogenic progenitors and improves the efficacy of muscle stem cell therapy.