Molecular regulation of satellite cell function

Seminars in Cell & Developmental Biology 16 (2005) 575–584

Review

Molecular regulation of satellite cell function Chet E. Holterman a,b , Michael A. Rudnicki a,b,∗ b

a Ottawa Health Research Institute, Molecular Medicine Program, Ottawa, Ont., Canada K1H 8L6 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ont., Canada K1H 8M5

Available online 10 August 2005

Abstract Quiescent satellite cells are responsible for the repair of post-natal skeletal muscle. These cells are easily identified by their unique morphology within skeletal muscle as well as by several recently elucidated molecular markers. Careful examination of the function of these markers has provided insight into the early events surrounding satellite cell specification and activation. However, the origin of these cells, as well as the mechanisms by which this population is maintained within the adult remain elusive. Furthermore, the ability of non-muscle derived stem cells and the potential multipotency of satellite cells have altered the traditional views of skeletal muscle regeneration. © 2005 Published by Elsevier Ltd. Keywords: Skeletal muscle; Satellite cell; Pax-7; Myogenic regulatory factor; Self-renewal

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology and distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Self-renewal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of Pax7 in satellite cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multipotency of muscle satellite cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Under normal biological conditions, adult skeletal muscle is an extremely stable tissue. However, when damaged by extreme physical activity, trauma, or specific disease states, skeletal muscle possesses a remarkable ability for self-repair. The majority of this regeneration is carried out by the activa∗

Corresponding author. E-mail address: [email protected] (M.A. Rudnicki).

1084-9521/$ – see front matter © 2005 Published by Elsevier Ltd. doi:10.1016/j.semcdb.2005.07.004

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tion, proliferation and differentiation of a resident population of myogenic stem cells called satellite cells. Under normal conditions, satellite cells are quiescent but become activated in response to trauma giving rise to proliferating myogenic precursor cells that eventually differentiate and fuse to form multinucleated myotubes. It has been well demonstrated that quiescent satellite cells and their descendant myogenic precursors are the key effectors of muscle regeneration. Indeed, it has been shown that satellite cells are present in the skeletal muscle of all vertebrates, and that these cells are responsible for the majority of post-natal skeletal muscle growth and

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adult skeletal muscle repair. The molecular events that occur following the activation of satellite cells and their subsequent differentiation and fusion to form multinucleated myotubes have been well established. Furthermore, recent work has led to the identification of several molecular markers of quiescent satellite cells, providing insight into the early events surrounding satellite cell activation and myogenic commitment. What is not clear is the origin of the quiescent satellite cell, or the mechanism by which the quiescent satellite cell population is maintained within adult skeletal muscle. Importantly, recent work has challenged the notion that satellite cells are committed myogenic precursors and the sole effectors of muscle regeneration in adult skeletal muscle. Not only is there evidence to support the multipotency of satellite cells, but recent work has also demonstrated the existence of stem cell populations within muscle, discreet from quiescent satellite cells, that possess the ability to contribute to skeletal muscle regeneration. This review will examine the potential origins of quiescent satellite cells, the mechanisms of satellite cell activation and self-renewal, and the role of Pax7 in myogenic specification. As well, it will examine the nonmyogenic potential of satellite cells and the contribution of cells other than satellite cells to muscle regeneration.

suggesting that a mechanism exists for the self-renewal or maintenance of the quiescent satellite cell population [10,11]. Quiescent satellite cells are present throughout skeletal muscle but show an unequal distribution between different muscle groups and fiber types. In general, individual slow twitch fibers have a higher number of associated satellite cells than fast twitch fibers. It is, therefore, not surprising that muscles composed mainly of slow twitch oxidative fibers tend to contain more satellite cells than fast twitch glycolytic muscles [7,9]. The unequal distribution of satellite cells is not only apparent between different fiber types and muscle groups but also manifests itself along individual fibers with higher numbers of satellite cells associating with the neuromuscular junction as well as adjacent capillaries [9]. It is possible that the high density of satellite cells surrounding these structures may be due to the release of factors from the neuromuscular junction or adjacent capillaries that function in the homing of satellite cells or in the maintainence satellite cell number. Alternatively, it may be that the surrounding capillaries provide a source of progenitor cells that contribute to the quiescent satellite cell compartment of adult skeletal muscle.

3. Molecular markers 2. Morphology and distribution Originally described in the skeletal muscles of frogs, satellite cells have since been identified in mammalian, avian, and reptilian skeletal muscle [1–4]. These cells are classically defined by their unique position adjacent to the skeletal muscle fiber. Occupying depressions in the muscle fiber, satellite cells are situated between the plasma membrane and the basal lamina of the muscle fiber such that the basal lamina is continuous over the entire length of the fiber. These cells are further identifiable by their relatively minute amount of cytoplasm, sparse organelles, and high ratio of heterochromatin to euchromatin, indicative of the inactive state of these cells [5]. Upon activation, the morphology of the cell changes dramatically. Cytoplasmic extensions become apparent, accompanied by an increase in the cytoplasmic volume of the activated cell. As well, the amount of heterochromatin decreases and organelles, such as golgi, endoplasmic reticulum, ribosomes, and mitochondria become apparent [6,7]. These morphological criteria separate quiescent satellite cells from their descendent activated myogenic precursor cells (MPCs), the cells that contribute to muscle growth and repair. At birth, satellite cells account for 20–30% of sublaminar nuclei associated with mouse skeletal muscle. As mice grow and mature this number rapidly declines to approximately 5% at two months of age [8]. After sexual maturity, the number of satellite cells continues to decline, albeit at a greatly reduced rate, such that approximately 2% of sublaminar nuclei in senile mice are quiescent satellite cells [7,9]. Interestingly, the satellite cell compartment is maintained in adult muscle following multiple rounds of degeneration and regeneration

While the characteristic ultrastructure of quiescent satellite cells allows for easy identification by electron microscopy, it is virtually impossible to distinguish satellite cells from myonuclei by light microscopy using these criteria. Fortunately, several molecular markers for quiescent satellite cells have been discovered that allow for their identification by immunological methods. These include c-met, [12,13], VCAM1 [14], Syndecan 3/4 [15], M-cadherin [12,16], CD34 [17], MNF [18,19], and Pax7 [20]. Expression of the receptor tyrosine kinase c-met in quiescent satellite cells was first demonstrated by single-cell multiplex RT-PCR [12]. Importantly, it was demonstrated that c-met is expressed in all quiescent satellite cells regardless of muscle-type or the appendicular versus axial origin of the muscle [12]. Furthermore, c-met protein is detectable in both resting and regenerating muscle in vivo [13]. One day following crush injury a large proportion of mononuclear cells surrounding necrotic fibers are positive for c-met, suggesting that it also marks MPCs. As well, c-met can be detected in fiber-derived MPCs in vitro as well as in cultured C2C12 myoblasts, demonstrating that c-met expression is not restricted to quiescent satellite cells [12,21]. Vascular cell adhesion molecule 1 (VCAM1) is widely expressed in the mouse embryo during development but becomes limited to endothelial cells stimulated by inflammation as well as skeletal muscle in the adult. Skeletal muscle expression of VCAM1 appears to be limited to quiescent satellite cells and activated MPCs in vivo [14,22]. Interestingly, it has been observed that infiltrating lymphocytes show close association with VCAM1-positive cells in regenerating

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muscle, and that VCAM1 is capable of mediating cell–cell interactions by binding to ␣4␤1 integrin in vitro [14]. Interaction between VCAM1-expressing satellite cells or MPCs and infiltrating lymphocytes may result in local accumulation of cytokines required for satellite cell activation and MPC proliferation and differentiation [14]. Syndecans are cell-surface trans-membrane heparin sulfate proteoglycans that have been shown to function in FGF signaling [23]. Interestingly, a thorough examination of syndecan expression in skeletal muscle of both the developing and adult mouse revealed highly localized expression consistent with the position of satellite cells [15]. Furthermore, syndecan3 and syndecan4 expression in skeletal muscle completely overlaps with expression of c-met and is localized between the basal lamina and myofiber membrane, verifying that syndecan3 and syndecan4 are expressed by quiescent satellite cells [15]. As with c-met, syndecan expression is also detected in proliferating MPCs in culture. Homozygous deletion of either syndecan3 or syndecan4 results in severe yet unique defects in satellite cell activation and muscle regeneration [24]. Stimulation of cultured MPCs, isolated from syndecan3 −/− and syndecan4−/− animals, with HGF or FGF suggest that the syndecans play unique roles in signaling through the ERK1/2 MAP kinase pathway [24]. Clearly, further examination of the role of syndecan3 and syndecan4 in satellite cell biology will provide important insight into the signaling pathways that are integral to satellite cell activation and proliferation. The calcium-dependent cell adhesion molecule mcadherin has been identified as a marker of quiescent satellite cells and activated myogenic precursors, but is not detectable in differentiated myotubes [16,25]. M-cadherin mRNA is detectable in a subset of quiescent satellite cells by RTPCR [12], and immunostaining of isolated fibers verifies that some, but not all, quiescent satellite cells express mcadherin [17]. Those cells that express m-cadherin were also found to express the hematopoietic stem cell marker CD34, while those that did not express m-cadherin were likewise negative for CD34 [17]. Furthermore, it was found that mcadherin/CD34 expressing cells were also positive for Myf-5 based on expression of ␤-galactosidase targeted to the Myf5 locus [17]. However, previous studies have failed to demonstrate detectable Myf-5 expression in quiescent satellite cells and the question of whether or not quiescent satellite cells express Myf-5 remains controversial [12,26]. Importantly, the fact that most, but not all, satellite cells express mcadherin and CD34 suggests that there is heterogeneity within the satellite cell compartment, an idea previously proposed based on the observed differences in proliferation and differentiation of cultured MPCs [27]. The winged helix transcription factor myocyte nuclear factor (MNF) is expressed in quiescent satellite cells. The number of MNF positive cells increases when muscle is induced to regenerate, [18] suggesting that it is also expressed in MPCs. MNF can be detected in central nuclei of regenerated myotubes, but is down-regulated during the late stages

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of regeneration to the point where MNF is only detectable in the remaining quiescent satellite cells [18]. Interestingly, two isoforms of MNF, termed MNF-␣ and MNF-␤, are detected in skeletal muscle. These two isoforms display unique expression patterns, with MNF-␣ expressed in proliferating MPCs whereas MNF-␤ expression is limited to quiescent satellite cells and is down regulated upon activation [7,19]. Disruption of the MNF locus results in decreased numbers of satellite cells and significant impairment of skeletal muscle regeneration [7,19]. Indeed, it has been demonstrated that MNF-␤ is capable of forming a transcriptionally repressive complex with mSin3 family members [28]. It may be that MNF-␤ represses transcription of specific target genes in quiescent satellite cells and upon activation isoform switching disrupts this repression allowing for activation of the previously repressed genes. A closer examination of the functional differences between the two isoforms of MNF may provide critical insight into the early events of satellite cell activation. The paired box transcription factor Pax7 is a wellestablished marker of quiescent satellite cells. By in situ hybridization, Pax7 localizes to nuclei situated in discreet peripheral locations within resting adult skeletal muscle [20]. The number of Pax7 expressing cells corresponds well with the expected number of satellite cells [20]. Pax7 is also expressed in proliferating MPCs in vivo and myoblasts in vitro, but is downregulated following serum withdrawal and subsequent differentiation [20]. Interestingly, Pax7 is not detectable in a wide variety of non-myogenic cell lineages. Careful examination of skeletal muscle from adult Pax7 mutant mice revealed a striking lack of quiescent satellite cells [20]. Furthermore, attempts to culture primary myoblasts from adult Pax7 mutant animals were unsuccessful, supporting the notion that Pax7 is crucial for satellite cell function [20]. Whether or not Pax7 is required for the initial establishment of the satellite cell lineage or functions in survival and self-renewal of the satellite cell compartment remains to be clearly established (see Section 7). As is apparent, several markers for quiescent satellite cells have been established in recent years, however, the majority of these are also expressed in proliferating MPCs as well as other non-myogenic lineages. It is not surprising, therefore, that the quest for markers specific to quiescent satellite cells continues. To this end, Seale et al. have recently identified several potential satellite cell markers including IgSF4, HoxC10, Neuritin, and Klra18 as well as several novel genes that appear to be functional in skeletal muscle satellite cells [29]. Closer examination of these genes will determine whether or not the will be added to the list of satellite cell markers. It is possible that no one marker alone will prove to be unique to quiescent satellite cells. It may, therefore, prove useful to consider alternative methods for distinguishing between quiescent satellite cells and their activated descendents. For example, it may prove useful to employ combinations of markers to identify quiescent satellite cells

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immunologically. Combining a marker for satellite cells, such as Pax7 or c-met, with a marker that distinguishes between proliferating and quiescent cells, such as chromatin assembly factor (CAF)-1, may prove to be a more successful method for identifying and examining quiescent satellite cells [30].

4. Activation The cues that lead to satellite cell activation following muscle trauma remain to be fully identified. It has been clearly demonstrated that HGF/SF, the ligand for the c-met receptor tyrosine kinase, plays a crucial role in satellite cell activation. Early experiments designed to identify the factor or factors responsible for activating satellite cells demonstrated that several well characterized mitogens, such as EGF, FGF, IGF, and PDGF enhanced proliferation of MPCs but failed to activate quiescent satellite cells [31–34]. Bischoff demonstrated that extract from gently crushed muscle was capable of activating quiescent satellite cells in vitro, but did not identify the specific factor in the extract that possessed activating activity [32]. Eventually, HGF was identified as the critical activating factor in crushed muscle extract. Furthermore, it was shown that HGF/SF was present in the basal lamina of skeletal muscle fibers, providing a reservoir of HGF within skeletal muscle [13,35]. Interestingly, injection of HGF directly into the tibialis anterior of adult mice resulted in activation of quiescent satellite cells in the absence of trauma, verifying the ability of HGF to activate quiescent satellite cells in vivo. In addition, incubation of crushed muscle extract with anti-HGF abolished the ability of crushed muscle extract to activate quiescent satellite cells, implicating HGF as the critical factor for activation [13,36]. Recently, it has been suggested that nitrous oxide may play a role in release of HGF/SF from the extracellular matrix, implicating NO in satellite cell activation. Inhibition of nitric-oxide synthase I (NOS-I) reduces satellite cell activation in response to trauma in vivo [37]. NO is produced in response to fiber stretching in vitro, and inhibition of nitric-oxide synthase reduces the amount of HGF released following fiber stretching [38]. However, it has yet to be demonstrated whether this is a direct or indirect effect of NO in HGF release. Following activation, quiescent satellite cells upregulate members of the myogenic regulatory factor (MRF) family. It has been demonstrated that by 24 h following activation, the majority of satellite cells upregulate either MyoD or Myf5, followed by co-expression of these two factors by 48 h [12,26,39]. The ability to upregulate either MyoD or Myf-5 may be a function of the heterogeneous population of quiescent satellite cells or may represent responses to distinct cues following trauma. Interestingly, in MyoD−/− animals, muscle regeneration is severely impaired with an increase in the number of mononuclear cells in the damaged area. These mononuclear cells fail to fuse and differentiate and persist for extended periods of time [40]. Furthermore, MyoD−/−

MPCs in vitro continue to proliferate following serum withdrawal, suggesting that in the absence of MyoD, activated MPC have a propensity for proliferation and self-renewal [41]. A requirement for MyoD in myoblasts differentiation is further supported by the observation that differentiating MPCs express MyoD alone or Myf-5 and MyoD, but differentiating MPCs positive for Myf-5 alone are never observed [26]. These results have led to a model in which Myf-5 is required for the proliferation and possible self-renewal of satellite cells while MyoD is required for the differentiation of activated myogenic precursors. Analysis of muscle regeneration in viable Myf-5 mutant mice should further increase our understanding of the roles of Myf-5 and MyoD following satellite cell activation.

5. Origins Satellite cells are apparent in limb muscles of the mouse during the late stages of pre-natal development, arising at approximately embryonic day 17 [8,9]. During development, activated satellite cells can be distinguished from migrating embryonic and fetal myoblasts based on their sensitivity to the phorbol ester TPA, their expression of acetylcholine receptors in culture, and their expression of MHC isoforms upon differentiation in culture [42]. While the temporal appearance of satellite cells has been established in most vertebrates, the developmental origin of these cells remains unclear. Early experiments using quail/chick chimeras suggested that satellite cells share a common somitic origin with the developing musculature [43]. During migration, the myoblasts that populate the limb during development do not express MyoD or Myf-5 but do express c-met, an established marker of quiescent satellite cells. In the absence of c-met, myogenic precursors fail to migrate into the limb buds, resulting in an absence of limb musculature [44]. The migrating myoblasts that give rise to the limb musculature are in a nonproliferative state during their migration, raising the possibility that satellite cells are derived from migrating myogenic precursors which remain quiescent rather than proliferating and differentiating to contribute to muscle development. More recently, myogenic cells resembling satellite cellderived MPCs have been isolated from explants of the embryonic dorsal aorta of mice [45]. These cells express myogenic markers, such as MyoD, Myf5, c-met, desmin, and mcadherin as well as a variety of vascular-endothelial markers. Interestingly, it was observed that MPCs isolated from adult muscle expressed many of the vascular-endothelial markers observed in the myogenic cells derived from the dorsal aorta, including VE-cadherin, VEGF-R2, and PECAM [45]. These results suggest an endothelial origin for quiescent adult satellite cells. Further support for this hypothesis comes from the fact that cells with myogenic capability can be isolated from the developing limbs of Pax3 null and c-met null mouse embryos [45]. The developing limbs of these animals lack the somitically derived migrating myogenic precursors; however,

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Fig. 1. Two possible origins for quiescent satellite cells have been proposed. The first potential origin is the somite (S). During development, migrating muscle precursors arise from the somite and populate the skeletal muscle compartment. Satellite cells may find their origin in these migrating precursors or alternatively may migrate individually from the somite. More recent work suggests that the dorsal aorta (DA) may give rise to quiescent satellite cells. During development, cells from the somite (red) migrate into and populate the roof and walls of the dorsal aorta. Satellite cell precursors may find their origin in cells of the dorsal aorta (green) or in the somatically derived cells of the dorsal aorta (yellow). Importantly, these origins are not mutually exclusive and may explain the heterogeneity observed within the satellite cell compartment.

the vasculature remains undisturbed suggesting a non-somitic origin for MPCs. Importantly, transplantation experiments show that myogenic cells derived from the dorsal aorta are capable of contributing to muscle growth and regeneration in

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vivo, but whether or not these cells contribute to the quiescent satellite cell compartment remains to be established [45]. One must bare in mind that during development the dorsal aorta is colonized by somitic angioblasts [46]. These cells contribute to the endothelial lining of vessels as well as the roof and walls of the aorta [46,47]. This raises the possibility that the cells responsible for the appearance of myogenic clones from dorsal aorta are indeed somitic in origin (Figs. 1–3). These results led to a hypothesis in which there exists a multipotent endothelial progenitor that responds to tissue specific signals and adopts an appropriate identity based on these cues [45,48]. Support for this hypothesis comes from experiments in which either quail or mouse dorsal aorta was transplanted into the limb bud of developing chicks. It was observed that cells of donor origin were able to contribute not only to the developing vascular and hematopoietic compartments as would be expected, but could also contribute to a wide variety of mesodermal tissues, including cartilage, bone and smooth and skeletal muscle [49]. Importantly, it is possible to expand cells from the dorsal aorta in vitro for extended periods of time with individual clones maintaining the capacity to differentiate into most mesodermal tissues [49]. These results verify the existence of a multipotent stem cell associated with the embryonic vasculature, dubbed mesoangioblasts [49]. An endothelial origin for satellite cells is not mutually exclusive of the more traditional belief that satellite cells are of a somitic origin. Several lines of evidence point to a heterogeneous satellite cell population. Quiescent satellite cells show differences in molecular markers, with some expressing CD34 and m-cadherin while others do not [12,17]. Examination of the kinetics of proliferation reveal that while the majority of satellite cells enter a proliferative phase and eventually fuse to form multinucleated myotubes, a small proportion proliferate at a much slower rate and, rather than

Fig. 2. (a) Assymetric cell division may be mediated by Numb localization. Assymetric localization of Numb results in one cell with high levels of Numb which undergoes terminal differentiation and a second daughter cell with low levels of numb which undergoes continued proliferation and may be capable of re-populating the quiescent satellite cell compartment. (b) Differential expression of the myogenic regulatory factors may be responsible for maintenance of the quiescent satellite cell compartment. Alternatively, Pax7 expression may be required for downregualtion of MyoD and the subsequent return to quiescence of myogenic precursors.

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Fig. 3. Multipotent progenitor cells isolated as part of the muscle SP population are capable of contributing to both hematopoetic and myogenic lineages. Under normal circumstances CD45+/Sca1+ cells isolated from the SP population of injured skeletal muscle contribute to both lineages. However, in the absence of Pax7, these cells are unable to undergo myogenic differentiation. Furthermore, infection of CD45+/Sca1+ cells from the SP population of uninjured skeletal muscle undergo myogenic conversion following infection with Pax7, implicating Pax7 in lineage restriction of this multipotent progenitor population.

differentiating, return to a quiescent state [50]. The possibility that satellite cells arise from two or more distinct developmental origins is an attractive explanation for the observed heterogeneity within the satellite cell compartment. Clearly, further work is required to unequivocally establish the developmental origins of satellite cells and whether or not different developmental origins result in heterogeneity within the satellite cell compartment.

6. Self-renewal Several models have been proposed to explain the maintenance of the quiescent satellite cell compartment in adult skeletal muscle. These include asymmetric cell division, contribution of other resident stem cells to the satellite cell compartment, and a return of proliferating MPCs to the quiescent state. Importantly none of these models are mutually exclusive. Asymmetric cell division where activated satellite cells divide to produce two daughter cells, one committed myogenic precursor and a second quiescent progenitor is an attractive explanation for the maintenance of the quiescent satellite cell compartment. Indeed, it has been demonstrated by radiolabeling experiments that activated satellite cells contribute

not only to regenerating fibers but also to the satellite cell compartment [9]. The mechanism by which asymmetric cell division occurs in activated satellite cells may be explained by the observation that the plasma-membrane associated protein Numb is segregated asymmetrically in activated MPCs [51]. This study demonstrated that, following injury, an increase in the level of activated Notch occurs in activated MPCs, and that this is paralleled by an increase in the Notch ligand, Delta. Conversely, the Notch inhibitor, Numb, is initially decreased following activation and is subsequently increased during regeneration [51]. Further investigation revealed that a proportion of mononuclear MPCs asymmetrically localized Numb, giving rise to daughter cells with either high levels of Numb or low/absent Numb expression [51]. Interestingly, cells expressing high levels of Numb were found to undergo differentiation, while low/absent expression of Numb, as well as constitutively active Notch, resulted in continued proliferation and inhibition of differentiation. These results suggest that asymmetric cell division, with respect to Numb localization, may play a pivotal role in the decision of activated cells to continue proliferation or undergo terminal differentiation (Fig. 2a). The role of Notch signaling in muscle regeneration is further supported by experiments comparing regenerative potential and Notch activation in young versus aged muscle in vivo.

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While no difference in satellite cell numbers were observed between young and old muscle, a dramatic decrease in myoblast generation and regenerative potential was observed in aged muscle as compared to young muscle [52]. The decreased regenerative potential of aged muscle was accompanied by a failure to upregulate Delta-1 and decreased levels of activated Notch. Inhibition of Notch activation by soluble Jagged-Fc in young muscle resulted in inhibition of regeneration, while forced activation of Notch in aged muscle enhanced regeneration, supporting a role for Notch signaling in the proliferation and differentiation of activated satellite cells [52]. While these results highlight the importance of asymmetric cell division with respect to Numb localization and Notch activation in fate determination of activated MPCs, it does not provide direct evidence for a role in satellite cell self-renewal. One might hypothesize that, following activation, satellite cells must be maintained in a proliferative state in order to give rise to the daughter cells required to repopulate the satellite cell compartment. Alternatively, asymmetric cell division following activation may result in a proliferative daughter cell that utilizes the Notch signaling pathway for fate determination, as well as a quiescent progenitor that repopulates the satellite cell compartment independent of Notch signaling. Differential expression of the myogenic regulatory factors following activation supports a model whereby activated satellite cells can return to a quiescent state and repopulate the satellite cell compartment. As previously outlined, activation of satellite cells results in upregulation of Myf5 or MyoD followed by co-expression of these factors in MPCs. Interestingly, in the absence of MyoD, activated MPCs show enhanced proliferation and fail to differentiate appropriately, suggesting that these cells represent an intermediate stage between activated MPCs and quiescent satellite cells [40,41]. In vitro it has been demonstrated that a small proportion of C2C12 myoblasts express Myf-5 but not MyoD. Importantly, following serum withdrawal these Myf5+/MyoD− cells fail to differentiate [53,54] Furthermore, these cells retain the ability to proliferate and give rise to differentiation-competent progeny. A recent study by Olguin et al demonstrated that forced expression of Pax7 results in downregulation of MyoD, inhibition of differentiation, and cell cycle withdrawal in myogenic cells [55]. As well, Pax7 is never detected in cells expressing myogenin, a marker of commitment to terminal differentiation. The ability of Pax7+/MyoD+ MPCs to downregulate MyoD and exit the cell cycle in vitro has also been reported by Zammit et al. [56]. Importantly, these cells, much like the previously described C2C12 reserve cells, can be restimulated to proliferate. These results support a model whereby activated satellite cells are able to return to a quiescent state and re-populate the satellite cell compartment. In this model, activated satellite cells enter a proliferative stage by expressing Myf-5 and/or MyoD. Symmetric division gives rise to a population of daughter cells, some of which down regulate MyoD and re-populate the quiescent satellite cell compartment whereas cells that maintain

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MyoD expression terminally differentiate and contribute to muscle repair (Fig. 2b). The biological relevance of Pax7 in the process remains to be clearly demonstrated, although in vitro studies suggest that Pax7 may function in MyoD downregulation and the subsequent return of activated MPCs to a quiescent state. The ability of stem cells from sources other than muscle to contribute to muscle regeneration has been well established [57,58]. However, the ability of these cells to contribute to the quiescent satellite cell compartment has only recently been clearly demonstrated. An elegant study by LaBarge and Blau [59] demonstrated the ability of bone marrow-derived stem cells to contribute to muscle regeneration as well as the quiescent satellite cell pool [59]. These results firmly establish that non-muscle stem cells are capable of contributing to the satellite cell compartment; however, the extent to which this occurs under normal in vivo conditions remains unclear. A muscle-derived source of stem cells known as SP cells can be isolated from adult skeletal muscle using fluorescence activated cell sorting, based on the exclusion of Hoechst 33342 dye. This population of cells has been shown to possess hematopeoitic potential as well as the ability to contribute to muscle regeneration. Based on the absence of satellite cell markers, Pax7 and Myf5-nlacZ, as well as the fact that muscle SP cells can be isolated from Pax7−/− muscles that lack quiescent satellite cells, muscle SP and quiescent satellite cells are believed to represent distinct cell populations [9]. When co-cultured with myoblasts in vitro, muscle SP cells have the ability to give rise to myogenic colonies. However, muscle SP derived from Pax7−/− animals fail to undergo myogenic conversion and show an increase in hematopoeitic conversion. Following intramuscular transplantation, wildtype muscle SP cells are capable of contributing to the quiescent satellite cell compartment [60]. Taken together these results suggest that muscle SP cells may represent satellite cell progenitors. It should be noted that none of these methods of selfrenewal/maintenance are mutually exclusive. Indeed, it is possible that all of these mechanisms play some role in maintaining the satellite cell compartment. Under normal physiological conditions asymmetric division or symmetric division followed by return to quiescence may be the main mechanism by which the satellite cell compartment is maintained. However, under extreme conditions, these mechanisms may not be sufficient for satellite cell maintenance. The ability of multi-potent stem cells to contribute to the satellite cell compartment may be crucial under conditions of extreme challenge. Clearly, further work is needed to establish which of these methods is/are functioning in the maintenance of quiescent satellite cells following injury.

7. Function of Pax7 in satellite cells The first evidence of a role for Pax7 in adult skeletal muscle came from representational difference analysis which

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demonstrated that Pax7 was uniquely expressed in primary myoblasts [20]. Further examination revealed expression of Pax7 in quiescent satellite cells as well as activated MPCs in regenerating muscle. While the majority of Pax7−/− mice fail to survive past two weeks of age, analysis of musculature of the few mice that survived to adulthood revealed a striking absence of quiescent satellite cells, suggesting that Pax7 is required for satellite cell specification. Interestingly, while muscle SP cells from wildtype animals are able to convert to myogenic cells when cultured with primary myoblasts, muscle SP from Pax7−/− mice are unable to undergo such a conversion. Furthermore, muscle SP cells from Pax7−/− show a much greater ability to form hematopoietic colonies, suggesting that Pax7 may play a role in lineage restriction. In support of this view, it has been demonstrated that the CD45+/Sca1+ myogenic precursor population within the muscle SP upregulates Pax7 in response to muscle trauma [61,62]. Furthermore, these cells express the myogenic markers MyoD and Desmin. When the same cells are isolated from damaged muscle of Pax7−/− animals, MyoD+ and Desmin+ cells are never observed, suggesting that Pax7 is required for the myogenic specification of these cells. Importantly, infection of CD45+/Sca1+ cells from uninjured muscle with Pax7 retrovirus results in myogenic specification of these cells. Furthermore, injection of infected cells into the tibialis anterior muscle of mdx mice demonstrated the ability of these cells to contribute to regeneration in vivo. Taken together, these results support a role for Pax7 in myogenic specification (Fig. 3). This view has recently been challenged by the observation that immediately post-natal muscle from Pax7−/− animals contains quiescent satellite cells as identified by electron microscopy [63]. However, the number of satellite cells is greatly reduced in mutant animals as compared to wildtype littermates. Electron microscopy data for the existence of quiescent satellite cells in P60 mice is less than convincing and expression of b-galactosidase from the Pax7 locus may represent myogenic cells from sources other than quiescent satellite cells as would be consistent with the activation of Pax7 in mSP cells. The dramatic decrease in satellite cell number in post-natal mice and the severe regeneration deficit observed in adult mice verifies that Pax7 is crucial for adult skeletal muscle regeneration. The ability of a small subset of Pax7−/− mice (<10%) to survive to adulthood and respond to muscle trauma may be due to the ability of stem cells other than quiescent satellite cells to contribute to muscle growth and regeneration. Alternatively, given the heterogeneity within the satellite cell compartment, it may be that Pax7 is required for the specification of a specific subset of quiescent satellite cells and that in the absence of Pax7, only a small subset of quiescent satellite cells remains. Regardless, Pax7 plays a critical role in satellite cell biology. The work by Olguin and Zammit outlined in Section 6 suggests that Pax7 may regulate satellite cell propagation and self-renewal [55,56]. This fits well with the dramatic reduction of satellite cells in Pax7−/− muscle [63]. Importantly,

a role for Pax7 in the self-renewal of activated satellite cells does not preclude the possibility that Pax7 is also required for the myogenic specification of multipotent progenitors required for the maintenance of the quiescent satellite cell compartment. This view is supported by the ability of forced expression of Pax7 to convert CD34+/Sca1+ SP cells from uninjured muscle into myogenic cells. Until the developmental origins and mechanism of self-renewal of satellite cells are clearly established, it may prove difficult to clearly establish whether Pax7 is required for satellite cell specification, selfrenewal, or both.

8. Multipotency of muscle satellite cells It has long been believed that quiescent satellite cells are the sole population responsible for skeletal muscle regeneration in adults. Furthermore, these cells were thought to be committed myogenic progenitors capable only of contributing to myogenesis. In recent years, these views have been challenged by the discovery that muscle SP and stem cells from other tissues are capable of contributing to muscle regeneration. More recently, the commitment of quiescent satellite cells to the myogenic lineage has been questioned. The ability of C2C12 myoblasts to convert from myogenic to osteogenic or adipogenic cells following treatment with BMP2 or thiazolidinediones respectively is well established [64,65]. However, it has only recently been demonstrated that adult muscle satellite cells are capable of converting to non-myogenic lineages. Treatment of satellite cell derived primary myoblasts with BMPs or adipogenic inducers converts these cells into osteogenic or adipogenic cells [66]. Furthermore, myoblasts derived from single-fiber cultures are also capable of converting to osteogenic or adipogenic cells following BMP or adipogenic induction or culture in Matrigel [66,67]. Importantly, clonal analysis of single-fiber derived myogenic cells confirms that single clones are capable of giving rise to both myogenic, osteogenic, and adipogenic cells, verifying that satellite cells in vitro are multipotent [67,68]. While the multipotency of satellite cells has yet to be demonstrated in vivo, the accumulation of adipose tissue and ectopic bone formation within skeletal muscle in some human diseases suggests that satellite cells may be capable of non-myogenic commitment in vivo [9].

9. Concluding remarks Recent advancements in stem cell biology have demonstrated the ability of a variety of “stem cells” to convert to the myogenic lineage in vitro. Furthermore, the ability of these cells to contribute to muscle regeneration, under extreme in vivo conditions, has also been shown. However, their biological relevance and contribution to normal physiological damage remains to be established. The ability of cells other than quiescent satellite cells to contribute to muscle

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regeneration challenges the long held notion that quiescent satellite cells are the sole contributor to muscle regeneration in the adult. While satellite cells are still believed to be the major contributor to muscle regeneration, the ability of other stem cells to convert to the myogenic lineage and contribute to muscle regeneration opens several new avenues of investigation. Recent work also challenges the long held belief that satellite cells are committed myogenic precursors. It has demonstrated that satellite cells possess the ability to give rise to adipogenic and osteogenic cells in vitro. While there is no direct evidence for such conversion in vivo, ectopic bone formation and accumulation of adipose tissue within skeletal muscle under certain disease states indirectly suggests that similar events may occur in vivo. The ability of multiple cell types to contribute to muscle repair challenges the classical model of muscle regeneration and provides several new avenues of investigation. The potential to harvest multipotent stem cells and convert them to the myogenic lineage via Pax7 expression provides attractive clinical possibilities. A further understanding of the mechanisms of satellite cell self-renewal and stem cell commitment will further aid in our understanding of muscle repair and potentially provide promising cell based therapies for degenerative muscle disease.

Acknowledgments We would like to thank Dr. Iain McKinnell and Jeff Ishibashi for their comments regarding the manuscript.

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