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Ottawa Health Research Institute, Ottawa, Canada
ABSTRACT I. INTRODUCTION A. Skeletal Muscle Development: An Overview B. Adult Skeletal Muscle Characteristics C. Morphological Characteristics of Skeletal Muscle Regeneration D. Animal Models of Muscle Injury II. ADULT MUSCLE SATELLITE CELLS A. Identification of Muscle Satellite Cells B. Dynamics of Muscle Satellite Cells C. Embryonic Origin of Muscle Satellite Cells: Somitic Versus Endothelial D. Specification/Expansion of Muscle Satellite Cells: Role of Pax7 III. MUSCLE SATELLITE CELLS IN MUSCLE REPAIR A. Activation of Muscle Satellite Cell Upon Injury: Role of MRFs B. Fusion of Muscle Precursor Cells C. Self-Renewal of Muscle Satellite Cells D. Multipotentiality of Muscle Satellite Cells IV. ROLE OF SECRETED FACTORS IN THE REGULATION OF MUSCLE REGENERATION A. HGF B. FGFs C. IGFs D. TGF-{beta} Family E. IL-6 Family of Cytokines V. CONTRIBUTION OF OTHER STEM CELLS TO THE MUSCLE REPAIR PROCESS A. Nonmuscle Resident Stem Cells B. Muscle Resident Stem Cells VI. CONTRIBUTION OF DEGENERATING FIBER NUCLEI TO NEW MYOFIBER FORMATION A. The Amphibian Versus Mammalian Regenerative Process B. In Vitro Mammalian Cell Dedifferentiation C. Role of Msx Genes in Mammalian Muscle Regeneration D. Conclusion VII. PERSPECTIVES
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ABSTRACT |
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I. INTRODUCTION |
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In this review, the current understanding of the cellular and molecular processes of skeletal muscle regeneration is presented. First, the embryonic development as well as the structure and function of normal skeletal muscle are briefly presented. Indeed, muscle regeneration appears to recapitulate to some extent the embryonic developmental process. Second, the morphological characteristics of injured muscle and the various experimental models used to study skeletal muscle regeneration are introduced. Third, the role of adult muscle satellite cells in muscle repair is discussed, focusing on the morphological and molecular characterization of these cells and their activation after damage. Fourth, the current knowledge on the role for nonmuscle and muscle resident adult stem cells in muscle regeneration is reviewed. Finally, the Urodele dedifferentiation process is discussed in the context of mammalian muscle regeneration.
A. Skeletal Muscle Development: An Overview
All vertebrate skeletal muscles (apart from head muscles) are derived from mesodermal precursor cells originating from the somites (epithelial spheres of paraxial mesoderm) (for review, see Ref. 15). During embryonic development, specification of mesodermal precursor cells to the myogenic lineage is regulated by positive and negative signals from surrounding tissues (summarized in Fig. 1A). Specification to the myogenic lineage requires the upregulation of MyoD and Myf5, basic helix-loop-helix transcriptional activators of the myogenic regulatory factor family (MRF) (Fig. 1). This is demonstrated by the total loss of skeletal muscle in MyoD:Myf5 double knockout mice and the observation that putative muscle progenitor cells remain multipotential and contribute to nonmuscle tissues in the trunk and limbs of these mice (155, 232, 259; for review, see Ref. 15). Proliferative MyoD and/or Myf5 positive myogenic cells are termed myoblasts. Proliferating myoblasts withdraw from the cell cycle to become terminally differentiated myocytes that express the "late" MRFs, Myogenin and MRF4, and subsequently muscle-specific genes such as myosin heavy chain (MHC) and muscle creatine kinase (MCK) (Fig. 1B). Myogenin-deficient embryos die perinatally due to a deficit in myoblast differentiation as evidenced by an almost total absence of myofibers in these mutants (141, 223). Similarly, MRF4-deficient mice display a range of phenotypes consistent with a late role for MRF4 in the myogenic pathway (236, 249, 357, 348). Finally, mononucleated myocytes specifically fuse to each other to form multinucleated syncytium, which eventually mature into contracting muscle fibers (Fig. 1B). During the course of muscle development, a distinct subpopulation of myoblasts fails to differentiate, but remains associated with the surface of the developing myofiber as quiescent muscle satellite cells (Fig. 1B and discussed below). After sexual maturity, skeletal muscle is a stable tissue characterized by multinucleated postmitotic muscle fibers (85, 266).
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B. Adult Skeletal Muscle Characteristics
The muscle fibers are the basic contractile units of skeletal muscles. They are individually surrounded by a connective tissue layer and grouped into bundles to form a skeletal muscle (Fig. 2). As well as being rich in connective tissue, skeletal muscles are highly vascularized to provide essential nutrients for muscle function (Fig. 2A, arrowhead). As the myofiber matures, it is contacted by a single motor neuron and expresses characteristic molecules for contractile function, principally different MHC isoforms and metabolic enzymes (Fig. 2B). Both the motor neuron and the myoblast origin have been implicated to play a role in specifying the myofiber contractile properties, although the precise mechanisms remain to be defined (reviewed by Ref. 335). Nevertheless, individual adult skeletal muscles are composed of a mixture of myofibers with different physiological properties, ranging from a slow-contracting/fatigue-resistant type to a fast-contracting/non-fatigue-resistant type. The proportion of each fiber type within a muscle determines its overall contractile property (Fig. 2B). Despite having different physiological properties, the basic mechanism of muscle contraction is similar in all myofiber types and is the result of a "sliding mechanism" of the myosin-rich thick filament over the actin-rich thin filament after neuronal activation (for review, see Ref. 147). The connective tissue framework in skeletal muscle combines the contractile myofibers into a functional unit, in which the contraction of myofibers is transformed into movement via myotendinous junctions at their ends, where myofibers attach to the skeleton by tendons. Thus the functional properties of skeletal muscle depend on the maintenance of a complex framework of myofibers, motor neurons, blood vessels, and extracellular connective tissue matrix. Although this review focuses on the regeneration process of the myofibers, it is understood that revascularization, reinnervation, and reconstitution of the extracellular matrix are also essential aspects of the muscle regeneration process.
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C. Morphological Characteristics of Skeletal Muscle Regeneration
Adult mammalian skeletal muscle is a stable tissue with little turnover of nuclei (85, 266). Minor lesions inflicted by day-to-day wear and tear elicit only a slow turnover of its constituent multinucleated muscle fibers. It is estimated that in a normal adult rat muscle, no more than 1–2% of myonuclei are replaced every week (266). Nonetheless, mammalian skeletal muscle has the ability to complete a rapid and extensive regeneration in response to severe damage. Whether the muscle injury is inflicted by a direct trauma (i.e., extensive physical activity and especially resistance training) or innate genetic defects, muscle regeneration is characterized by two phases: a degenerative phase and a regenerative phase (Fig. 3A).
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The initial event of muscle degeneration is necrosis of the muscle fibers. This event is generally triggered by disruption of the myofiber sarcolemma resulting in increased myofiber permeability. Disruption of the myofiber integrity is reflected by increased serum levels of muscle proteins, such as creatine kinase, which are usually restricted to the myofiber cytosol. In human and animal models, increased serum creatine kinase is observed after mechanical stress (e.g., extensive physical exercises) and in the course of muscle degenerative diseases such as muscular dystrophies, all of which are characterized by the induction of a muscle regeneration process (79, 226, 238; reviewed in Refs. 13, 184, 292, 355). Reciprocally, the uptake of low-molecular-weight dyes, such as Evans blue or procion orange, by the myofiber is a reliable indication of sarcolemmal damage and is also associated with strenuous exercise and muscle degenerative diseases (46, 135, 201, 231, 297, 298). It has been hypothesized that increased calcium influx after sarcolemmal or sarcoplasmic reticulum damage results in a loss of calcium homeostasis and increased calcium-dependent proteolysis that drives tissue degeneration (reviewed in Refs. 2, 12, 30). Calpains are calcium-activated proteases that can cleave myofibrillar and cytoskeletal proteins and hence are implicated in the process (180; reviewed in Refs. 30, 90). Thus disrupted myofibers undergo focal or total autolysis depending on the extent of the injury.
The early phase of muscle injury is usually accompanied by the activation of mononucleated cells, principally inflammatory cells and myogenic cells. Present reports suggest that factors released by the injured muscle activate inflammatory cells residing within the muscle, which in turn provide the chemotactic signals to circulating inflammatory cells (reviewed in Refs. 247, 315). Neutrophils are the first inflammatory cells to invade the injured muscle, with a significant increase in their number being observed as early as 1–6 h after myotoxin or exercise-induced muscle damage (104, 230). After neutrophil infiltration and 
48 h postinjury, macrophages become the predominant inflammatory cell type within the site of injury (230, 315). Macrophages infiltrate the injured site to phagocytose cellular debris and may affect other aspects of muscle regeneration by activating myogenic cells (7, 191, 212, 256). Moreover, studies demonstrating the stimulation of peritoneal macrophages after intensive physical exercise suggest that a systemic factor capable of inducing an inflammatory response throughout the body is released following muscle damage (99, 196, 339). Although several mediators involved in the activation of the inflammatory response have been characterized, further studies are necessary to demonstrate their potential role in the muscle regeneration process in vivo (reviewed in Ref. 315). Thus muscle fiber necrosis and increased number of nonmuscle mononucleate cells within the damaged site are the main histopathological characteristics of the early event following muscle injury (Fig. 3B).
Muscle degeneration is followed by the activation of a muscle repair process. Cellular proliferation is an important event necessary for muscle regeneration as demonstrated by the reduced muscle regenerative capacity after exposure to colchicine (an inhibitor of mitotic division) or after irradiation (241, 244, 325, 331). Notably, the expansion of myogenic cells provides a sufficient source of new myonuclei for muscle repair (reviewed in Refs. 48, 129, 142). Numerous nuclear radiolabeling experiments have demonstrated the contribution of dividing myogenic cells to regenerating myofibers, and it is well accepted that following the myogenic proliferation phase, new muscle fibers are formed much as during bona fide embryonic myogenesis; myogenic cells differentiate and fuse to existing damaged fibers for repair or to one another for new myofiber formation (82, 289, 290, 326). Long-standing histological characteristics are still used to identify the mammalian skeletal muscle regeneration process. On muscle cross-sections, these fundamental morphological characteristics are newly formed myofibers of small caliber and with centrally located myonuclei (Fig. 3C). Newly formed myofibers are often basophilic (reflecting high protein synthesis) and express embryonic/developmental forms of MHC (reflecting de novo fiber formation) (134, 333). On muscle longitudinal sections and in isolated single muscle fibers, central myonuclei are observed in discrete portions of regenerating fibers or along the entire new fiber, suggesting that cell fusion is not diffuse during regeneration but rather focal to the site of injury (41). Fiber splitting or branching is also a characteristic feature of muscle regeneration and is probably due to the incomplete fusion of fibers regenerating within the same basal lamina (38, 41, 43). Fiber splitting is commonly observed in muscles from patients suffering neuromuscular diseases, in hypertrophied muscles, and in aging mouse muscles, all of which are associated with abnormal regenerative capacity (42, 57, 61, 264). Once fusion of myogenic cells is completed, newly formed myofibers increase in size, and myonuclei move to the periphery of the muscle fiber. Under normal conditions, the regenerated muscle is morphologically and functionally indistinguishable from undamaged muscle.
D. Animal Models of Muscle Injury
Although the degenerative phase and the regenerative phase of the muscle regeneration process are similar among different muscle types and after varying causes of injuries, the kinetics and amplitude of each phase may vary depending on the extent of the injury, the muscle injured, or the animal model (149, 187, 217, 237, 255). To study the process of muscle regeneration in a controlled and reproducible way, it has therefore been necessary to develop animal models of muscle injury.
The use of myotoxins such as bupivacaine (Marcaine), cardiotoxin (CTX), and notexin (NTX) is perhaps the easiest and most reproducible way to induce muscle regeneration (81, 133, 138, 139). These toxins have a wide range of biological activities, which are not entirely understood. For example, NTX is a phospholipase A2 neurotoxin peptide extracted from snake venoms that block neuromuscular transmission by inhibition of acetylcholine release. CTX is also a peptide isolated from snake venoms, but it is a protein kinase C-specific inhibitor that appears to induce the depolarization and contraction of muscular cells, to disrupt membrane organization, and to lyse various cell types. In our laboratory, 25 µl of 10 µM CTX (from Naja nigricollis snake venom) injected in adult mouse tibialis anterior muscle induces muscle degeneration leading to a wound coagulum with mononuclear cell infiltration within 1 day of injection. Inflammatory response and mononuclear cell proliferation is most active within 1–4 days of injection (Fig. 3B). Myogenic cell differentiation and new myotube formation is observed 5–6 days postinjection. By 10 days postinjection, the overall architecture of the muscle is restored, although most regenerated myofibers are smaller and display central myonuclei (Fig. 3C). The return to a morphologically and histochemically normal mature muscle is seen at 3–4 wk postinjection. Although injection of CTX is a highly reproducible way of inducing muscle regeneration, the potentially unknown effects of this toxin on various muscle cell types including satellite cells is a potential "caveat" to this protocol.
Alternative methods to myotoxin injection, which are possibly more physiologically relevant, are available. For example, the direct infliction of a wound by crushing and/or freezing the muscle or the denervation-devascularization by transplantation of a single muscle will trigger the process of muscle regeneration (187, 272). Transplantation of the extensor digitorum longus in the rat leads to rapid degeneration (within 2–3 days) of the transplanted fibers followed by the rapid appearance of regenerating fibers (within 5 days) and leading to a normal muscle by 60 days (51, 136, 137). Muscle regeneration can also be induced by repeated bouts of intensive exercise, and in fact, eccentric exercise (lengthening contraction) is particularly potent at inducing muscle damage (149; reviewed in Refs. 13, 98). Thus appropriate procedures that promote muscle damage will induce a controlled regeneration process.
Laboratory mouse models with abnormal degeneration due to the spontaneous or artificial deregulation of specific genes are also of interest (Table 1). For example, the mdx mouse is commonly used as an animal model of Duchenne muscular dystrophy (DMD) and as an alternative degeneration model for studying muscle repair (55; reviewed in Refs. 39, 328). The mdx mouse is a spontaneously occurring mouse line deficient for dystrophin because of a point mutation in exon 23 of the dystrophin gene, which forms a premature stop codon (283). Dystrophin is a major component of the dystrophin-glycoprotein complex (DGC), which links the myofiber cytoskeleton to the extracellular matrix. Disruption of this complex leads to increased susceptibility to contraction-induced injury and sarcolemmal damage leading to myofiber necrosis. Although mdx mice are normal at birth, skeletal muscles show extensive signs of muscle degeneration by 3–5wkof age (79, 235, 304). This acute muscle degeneration phase is accompanied by an effective regeneration process leading to a transient muscle hypertrophy (79, 235). After this period, the degeneration/regeneration activity continues at lower and relatively constant levels throughout the life span of the animal. However, for reasons that remain unclear, in the older animals (15 mo), muscle regeneration process is defective and the mice become extremely weak and die before wild-type littermates (68, 234, 235). To date there are various animal models in which the DGC has been disrupted by knocking out dystrophin-associated proteins such as dystroglycans and sarcoglycans (Table 1). These also provide useful insight in the degeneration/regeneration regulatory pathways (68, 297).
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A large variety of animal models are available for studying muscle regeneration; however, the mouse strain influences muscle regeneration (125, 128, 149, 205, 217, 255). For example, the regeneration process in Swiss SJL/J mice is more efficient than in Balb/c, C57BL6J, and B6AF-1, while A/J mice appear the least efficient. The underlying mechanisms for such strain differences are unclear, but the correlation between regeneration capacity and genetic background suggests the involvement of modifier genes. Differential expressions of Pax7 isoforms and MyoD have been correlated with efficiency of repair (165–167, 197). Furthermore, expression levels of basic fibroblast growth factor (FGF) have been correlated with the efficiency of repair within Balb/c and SJL/J muscles (10). Another experimental consideration is the observation that the muscle regeneration process follows a centripetal gradient (from the outer regions to the inner regions), which results in the formation of different zones within a regenerating muscle, each zone being in a different phase of degeneration or regeneration (51). The use of laboratory rodents has been fundamental in the understanding of mammalian skeletal muscle regeneration; through the use of such models the primary cellular component for muscle regeneration has been established to be the adult muscle satellite cell.
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II. ADULT MUSCLE SATELLITE CELLS |
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Muscle satellite cells are a population of undifferentiated mononuclear myogenic cells found in mammalian (49, 113, 202), avian (140), reptilian (157), and amphibian (202, 242) skeletal muscles including muscle spindles. When cultured in vitro, satellite cells display specific characteristics allowing their distinction from embryonic and fetal myoblasts (75, 77, 140). Muscle satellite cells are apparent as a distinct population of myoblasts during the midfetal (E12–13) to late stage (E18) of avian development (100, 140), during the 10th to 14th week of human limb development (75) and from around E17.5 in limbs of mouse embryos (reviewed in Ref. 76). Thus, in all organisms analyzed to date, the muscle satellite cell population appears distinct from the embryonic and fetal myoblasts populations. Furthermore, the temporal appearance of satellite cells follows the appearance of both embryonic and fetal myoblasts.
Since their first description by Mauro (202), muscle satellite cells have been primarily identified in situ by their morphological characteristics. Indeed, muscle satellite cells can unequivocally be identified by electron microscopy due to their distinct location within the basal lamina surrounding individual myofibers, juxtaposed between the plasma membrane of the muscle fiber and the basement membrane, hence their name (Fig. 2C, white arrow). Other important morphological features of satellite cells are an increased nuclear-to-cytoplasmic ratio, a reduced organelle content, and a smaller nucleus size displaying increased amounts of heterochromatin compared with fiber myonuclei (Fig. 2C, white arrow). These characteristics reflect the finding that satellite cells are mitotically quiescent and transcriptionally less active than myonuclei (269, 291). The identification of satellite cells by light microscopy is more ambiguous, although the use of markers such as laminin and dystrophin to respectively identify the basal lamina and the myofiber sarcolemma facilitate their identification. Moreover, the development of techniques to isolate and study single muscle fibers with their resident satellite cells in vitro has allowed great advances in understanding this cell population (Fig. 2D) (33, 258). Nevertheless, the difficult in vivo identification of satellite cells has hindered the study of this cell population and in effect the understanding of skeletal muscle regeneration. To circumvent such difficulties, scientists are focusing on identifying molecular markers specific to this cell population (summarized in Table 2 and discussed herein) (reviewed in Ref. 142).
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B. Dynamics of Muscle Satellite Cells
Satellite cells are present in all skeletal muscles and are associated with all muscle fiber types, albeit with unequal distribution. For instance, the percentage of satellite cells in adult slow soleus muscle is two- to threefold higher than in adult fast tibialis anterior or extensor digitorum longus muscles (Table 3) (117, 258, 265, 291). Similarly, high numbers of satellite cells are found associated with slow muscle fibers compared with fast fibers within the same muscle (117). Although these differences in satellite cell density between fiber types are well established, the regulatory mechanisms behind this phenomenon are less well understood. Increased density of satellite cells have been observed at the motor neuron junctions (312, 337) and adjacent to capillaries (265), suggesting that some factors emanating from these structures may play a role in homing satellite cells to specific muscle locations or in regulating the satellite cell pool by other means. The regulation of satellite cell density at the single fiber level is also suggestive of a role for the muscle fiber in regulating the satellite cell pool.
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The satellite cell population varies also with age (Table 3). Compelling evidence suggests a decrease in satellite cell density over time. During postnatal muscle growth, there is a dramatic decrease in the proportion of satellite cell nuclei. This decrease is mainly due to the dramatic increase in myonuclei number following satellite cell fusion. However, in some glycolytic muscles, it is combined with a net decrease in total satellite cell number (118). At birth, satellite cells account for 30% of sublaminar muscle nuclei in mice followed by a decrease to <5% in a 2-mo-old adult (35). After sexual maturity, satellite cell number continues to decrease, albeit not as dramatically (42, 61, 118, 273). At 1–4 mo of age, most murine single myofibers in culture yield satellite cells, whereas from 9 to 12 mo of age, up to 50% of extensor digitorum longus fibers fail to yield any satellite cells under similar culture conditions (Table 3) (42, 61). Thus the overall satellite cell number appears to decrease as a function of age.
C. Embryonic Origin of Muscle Satellite Cells: Somitic Versus Endothelial
Satellite cells are believed to constitute a myogenic cell lineage distinct from embryonic and fetal myoblast lineages (Fig. 1B). However, although the origin of embryonic and fetal myoblasts has been extensively studied and researchers have unanimously concluded that these myogenic precursors originate from the multipotential mesodermal cells of the somites (reviewed in Refs. 15, 229), the origin of satellite cells has been the subject of fewer studies and remains unclear with, to date, two standing hypotheses: a somitic versus an endothelial origin.
The somitic origin hypothesis emanates from traditional transplantation studies performed in avian models (11). In this fate-mapping analysis, embryonic somites from donor quail embryos were introduced into host chick embryos. After embryonic development, the contribution of quail cells to the host satellite cell compartment is determined using ultrastructural characteristics specific to quail nuclei to identify the donor cells. In this study, donor somitic cells are found to migrate from the somites into the developing chick limb contributing to both terminally differentiated muscle fibers and the host satellite cell population. Although the identification of quail nuclei was never definite and the somitic domain generating satellite cells was never characterized, this study suggests a common somitic origin for all myogenic cell lineages, including muscle satellite cells.
Several recent observations have challenged the view of a somitic origin for satellite cells. For example, bone marrow-derived myogenic cells are able to participate in skeletal muscle regeneration, although at low frequency, when injected intravenously, suggesting that some myogenic cells with similar functional characteristics as satellite cells originate from bone marrow-derived stem cells (discussed in sect. V) (37, 103, 132, 181). Subsequently, more studies have demonstrated the ability of nonmuscle resident cells to follow the myogenic lineage (reviewed in Ref. 119). However, more definitive conclusions come from the detailed clonal analysis of different mouse tissues at various developmental stages by De Angelis et al. (83). In this elegant study, the authors demonstrate the presence of clonal myogenic precursors within the embryonic dorsal aorta. When cultured in vitro, these clones display similar morphological characteristics and express myogenic and endothelial markers similar to that of adult muscle satellite cells. Furthermore, the same study shows that myogenic precursor clones can be derived from limbs of c-Met–/– and Pax3–/– mutants, which lack appendicular musculature due to the absence of migratory myoblasts of somitic origin. Thus myogenic precursors derived from these mutant limbs may be of endothelial origin. When directly injected into regenerating host muscles, these cells are incorporated into newly regenerating fibers, a hallmark of bona fide satellite cells. When embryonic aortas are transplanted into muscles of newborn immunodeficient mice, they can also give rise to many myogenic cells within the treated muscles and also within collateral untreated muscles. Moreover, when fetal limbs are transplanted under the skin of host animals and become vascularized by the host, myogenic cells of host origin are observed within the transplant. Taken together, these results suggest the presence of a multipotential cell population within the embryonic vasculature, which may differentiate as a function of the tissue it perfuses (83, 216). In the case of the skeletal muscle, the progenitors have the capacity to adopt a myogenic fate (83). However, although it is clear that these endothelial cells can contribute to new muscle fiber formation during muscle development and regeneration, it remains to be determined whether these cells can contribute to the quiescent sublaminar cell population historically defined as a satellite cell population (202). Indeed, the endothelial progenitors could represent an alternative cell population to satellite cells capable of muscle growth and repair. It is noteworthy, however, that adult satellite cells, in contrast to embryonic and fetal myoblasts, express both endothelial and myogenic markers, such as CD34 and MRFs (28).
The recent view of an endothelial origin for satellite cells is not mutually exclusive with the more traditional view of a somitic origin. In fact, during early embryogenesis the aortic endothelium and the somites are adjacent, suggesting a close proximity in origin of these two lineages (reviewed in Refs. 228, 233). Thus the presence of myogenic cells within the embryonic dorsal aorta does not rule out the possibility of an indirect somitic origin of the satellite cells. Moreover, emerging evidence that satellite cells may represent a heterogeneous population may be a reflection of this dual origin (reviewed in Ref. 213). Moreover, in the course of differing physiological or pathological states, myogenic cells of different origin may contribute differently to the myogenic repair. Thus myogenic repair may involve the activation of various myogenic cells depending on the extent of the injury or the local environment, and in particular, in response to damaged vasculature. Taken together, these studies highlight the need for further experiments designed to unequivocally identify the embryonic origin(s) of muscle satellite cells. Such studies will require the use of classical chimeric studies combined with lineage analysis using retroviral or genetic labeling.
D. Specification/Expansion of Muscle Satellite Cells: Role of Pax7
Although the embryonic origin of satellite cells remains to be determined, the gene responsible for the specification of progenitor cells to the satellite cell lineage has been recently identified (275). Using representational difference analysis (RDA) of cDNAs, our group has isolated Pax7 as a gene specifically expressed in cultured satellite cells and demonstrated its expression in quiescent and activated satellite cells in vivo (146, 275) (Fig. 2D). The Pax7 gene is a member of the paired box containing gene family of transcription factors implicated in development of the skeletal muscle of the trunk and limbs, as well as elements of the central nervous system (reviewed in Refs. 65, 198). Pax7 is closely related to Pax3, based on highly similar protein structures and partially overlapping expression patterns during embryonic development (121, 154). Interestingly, Pax3 is a key regulator of somitic myogenesis (200, 303). Detailed analysis of the distribution of Pax7 mRNA using Northern blot analysis by Seale et al. (275) demonstrated the expression of Pax7 in proliferating satellite cell-derived myoblasts and a rapid downregulation of Pax7 transcripts upon myogenic differentiation. Low levels of Pax7 expression were also detected in proliferating C2C12 mouse myoblasts, which is an established cell line originally derived from satellite cells (40, 344). However, Pax7 was not expressed at detectable levels in a variety of nonmuscle cell lines. In addition, analysis of poly(A)+ RNA from selected mouse tissues revealed expression of Pax7 at low levels in adult skeletal muscles only. Specific expression of Pax7 within muscle satellite cells in vivo was confirmed by in situ hybridization and immunocytochemistry analyses on fresh frozen muscle sections. Pax7 mRNA and protein were found in a subset of nuclei (5%) in discrete peripheral locations within undamaged wild-type skeletal muscle. Furthermore, the number of Pax7-positive cells increased in muscles undergoing regeneration such as in MyoD–/–, mdx, and mdx:MyoD–/– skeletal muscles. Centrally located nuclei within newly regenerated muscle fibers were also associated with Pax7 expression, suggesting that recently activated and fusing satellite cells express Pax7. Together, these data demonstrate the specific expression of Pax7 in quiescent and activated muscle satellite cells (275).
The analysis of Pax7–/– skeletal muscles demonstrated the important role for Pax7 in satellite cell development. Indeed, Pax7–/– mice appear normal at birth but fail to grow postnatally, leading to a 50% decrease in body weight by 7 days of age compared with wild-type littermates (199, 275). Pax7 mutant animals fail to thrive and usually die within 2 wk after birth (199, 275). This runted phenotype is characterized by a decreased skeletal muscle mass resulting from a fiber size decrease rather than a decrease in fiber number (S. Chargé, unpublished observation; Ref. 275). Pax7–/– skeletal muscles have a striking absence of satellite cells (275). Under standard derivation and growth conditions, primary cell cultures from mutant skeletal muscles failed to generate myoblasts; instead, mutant cultures were uniformly composed of fibroblasts and adipocytes. Furthermore, morphological analysis of mutant skeletal muscles by transmission electron microscopy confirmed the lack of satellite cells in Pax7-deficient musculature. Overall, the data to date suggest a key role for Pax7 in lineage determination, especially in the specification of myogenic progenitors to the satellite cell lineage. Recent studies have highlighted the multiple functions of the Pax genes, implicating Pax proteins in regulating organogenesis and maintaining proliferating, pluripotent stem cell populations (reviewed in Refs. 65, 198). Pax7 is unequivocally required for satellite cell development. However, whether Pax7 has a role in the specification or the survival of the satellite cell progenitor pool remains unclear. Understanding the molecular pathways regulated by Pax7 should prove useful in understanding the early event of satellite cell development.
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III. MUSCLE SATELLITE CELLS IN MUSCLE REPAIR |
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A. Activation of Muscle Satellite Cell Upon Injury: Role of MRFs
In the course of muscle regeneration, satellite cells first exit their normal quiescent state to start proliferating. After several rounds of proliferation, the majority of the satellite cells differentiate and fuse to form new myofibers or to repair damaged one. Satellite cell activation is not restricted to the damaged site. Indeed, damage at one end of a muscle fiber will activate satellite cells all along this fiber leading to the proliferation and migration of the satellite cells to the regeneration site (272). However, recruitment of satellite cells from adjacent muscles is seldom observed and requires damage to the connective tissue separating the two muscles (271, 272). After proliferation, quiescent satellite cells are restored underneath the basal lamina for subsequent rounds of regeneration (272). The process of satellite cell activation and differentiation during muscle regeneration is reminiscent of embryonic muscle development. In particular, the critical role of the MRFs is observed in both processes (Figs. 1 and 4).
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Upon exposure to signals from the damaged environment, quiescent satellite cells are activated and start proliferating at which stage they are often referred to as myogenic precursor cells (mpc) or adult myoblasts (Fig. 4). At the molecular level, activation of mpc is characterized by the rapid upregulation of two MRFs, Myf5 and MyoD (71, 73, 74, 110, 127, 197, 246, 285, 341, 353). In general, quiescent satellite cells do not have any detectable levels of MRFs. Although a low level of Myf5-lacZ expression has been reported in a subset of quiescent satellite cells using knock-in mice, this observation is likely an allele-specific phenomenon (28). Upon satellite cell activation, MyoD upregulation appears the earliest within 12 h of activation and is detectable before any sign of cellular division such as proliferative cell antigen nuclear (PCNA) expression (71, 73, 74, 79, 285, 341). A sensitive multiplex expression analysis at the single-cell level suggested that some satellite cells enter the MRF-positive compartment by expressing either Myf5 or MyoD; however, this state is rapidly followed by coexpression of the two (74). Activation of MyoD and Myf5 expression following muscle injury has also been observed in various in vivo models for muscle regeneration and in varying muscle types (32, 71, 110, 127, 246). Of particular interest is the study by Cooper et al. (71), which confirmed the initial upregulation of Myf5 and/or MyoD, followed by the coexpression of these MRFs by using CTX to induce muscle regeneration in Myf5-nlacZ mice combined with MyoD immunostaining. These data suggest an important role for MyoD in satellite cell differentiation. Supporting this view is the observation by Megeney et al. (210) that MyoD–/– mice have a reduced regenerative capacity characterized by an increase in mpc population and a decrease in regenerated myotubes. Furthermore, MyoD–/– muscles display an increased occurrence of branched myofibers suggestive of chronic or inefficient muscle regeneration in vivo (73). By electron microscopy, MyoD–/– satellite cells are morphologically normal (210). However, in vitro cultures of MyoD–/– satellite cells demonstrate a myogenic cell population with abnormal morphology characterized by a stellate, flattened appearance compared with the compact rounded appearance displayed by normal mouse myoblasts (260). Under low serum conditions, which usually are favorable for myogenic differentiation, MyoD–/– cells continue to proliferate and eventually yield a reduced number of mononucleated differentiated myocytes (73, 260, 342). The increased level of insulin-like growth factor I (IGF-I) expression in MyoD–/– cells is in accordance with the previously reported role for IGF-I in promoting myoblast proliferation (discussed in sect. IVC) (107, 260). In normal satellite cells, MyoD may downregulate IGF-I expression to promote myogenic differentiation. In addition, expression of M-cadherin is decreased in MyoD–/– cells (73, 260), and a requirement for M-cadherin has been reported for myoblast differentiation and fusion (150, 356). A recent study has isolated Slug, a zinc-finger protein of the snail family, as a direct downstream target of MyoD (358). Endogenous MyoD was shown to bind to the Slug promoter, and in vitro reporter analysis demonstrated the direct activation of this promoter by MyoD (358). Slug expression is dramatically increased in the late phase (4–10 days post-CTX injection) of muscle regeneration. Moreover, induction of muscle regeneration by CTX injection in Slug–/– muscle reveals a defective regenerative capacity with rare centrally nucleated fibers (i.e., rare regenerating myofibers) and smaller regenerated muscle area. Even though the data are suggestive of a role for Slug in muscle regeneration, the authors are rightly cautious in concluding that a number of developmental abnormalities could give rise to such defect. Indeed, Slug is a transcription factor with a broad expression pattern and is likely to play regulatory roles in multiple processes (67, 276). Nevertheless, one attractive hypothesis is that the muscle regeneration defect observed in MyoD–/– mice stems from the inappropriate expression of Slug. Understanding the downstream targets of Slug may shed some light on the molecular pathways regulating mpc activation. Overall, these data suggest an important role for MyoD in the process of satellite cell differentiation during muscle regeneration. However, further studies are required to determine the downstream targets of MyoD important in the regenerative process. Moreover, it remains unclear whether lack of MyoD during embryonic development or in mature fibers has an effect on muscle regeneration independently of satellite cell activity. A recent study argues against this possibility, wherein antisense nucleotides to inhibit MyoD expression during adult muscle regeneration were used successfully and muscle regeneration appeared somewhat delayed (352). In the future, this technique should prove useful in inhibiting individual factors during muscle regeneration in mice that have a similar developmental background.
Identifying a role for Myf5 in muscle regeneration has been more problematic. Indeed, until recently no viable Myf5–/– mice were available (164). Although Myf5-deficient mice display a delayed epaxial (deep back muscle) embryonic myogenesis and a normal hypaxial (trunk and limb muscles) embryonic myogenesis, no apparent phenotype in the adult muscle has been reported to date (reviewed in Ref. 156). These data combined with the reciprocal delay in hypaxial myogenesis in MyoD-deficient mice and the mutually exclusive expression of Myf5 and MyoD in early stages of embryonic muscle precursor cells have led to the hypothesis that Myf5 and MyoD support distinct myogenic lineages during embryonic muscle development (reviewed in Ref. 156). Similarly, circumstantial evidence suggests that Myf5 and MyoD may play distinct roles during muscle regeneration: Myf5 promotes satellite cell self-renewal (discussed below), whereas MyoD promotes satellite cell progression to terminal differentiation (discussed above). Analysis of viable Myf5-deficient mice under regeneration conditions and of Myf5-deficient myoblasts in culture should shed some light into these mechanisms.
After the mpc proliferation phase, expression of Myogenin and MRF4 (MRF members) is upregulated in cells beginning their terminal differentiation program (Fig. 4) (73, 74, 110, 127, 197, 285, 341). This is followed by the activation of the cell cycle arrest protein p21 and permanent exit from the cell cycle. The differentiation program is then completed with the activation of muscle-specific proteins, such as MHC, and the fusion of mpc to repair damaged muscle. The observation that MRF4 expression is in myonuclei of newly regenerated myofibers or young myotubes at a time after fusion suggests a distinct role from Myf5, MyoD, and Myogenin, possibly in myofiber maturation (359). Gross defects in embryonic muscle development of mutant mice for Myogenin and MRF4 have impeded further study of these genes in muscle regeneration.
Other factors important in regulating myogenic differentiation, possibly by acting upstreams of the MRFs, have been identified. Mice deficient in myocyte nuclear factor (MNF), a winged helix transcription factor important in regulating myoblasts cell cycle progression, are impaired in their ability to regenerate skeletal muscle (114, 115, and reviewed in 142). Recently, Fernando et al. (102) have demonstrated the requirement for caspase-3/MST1 signaling in initiating myoblasts differentiation in vitro. However, the requirement for caspase-3 activity for normal muscle regeneration remains to be demonstrated. Thus the activation of satellite cells following muscle injury results in the activation of the myogenic program, which allows expansion of the myogenic cell pool necessary for new myonuclei fusion and myofiber formation (Fig. 4).
B. Fusion of Muscle Precursor Cells
During the course of muscle regeneration, akin to during muscle development, mpc are required to specifically fuse to each other to form syncytial muscle fibers. Semi-stable intercellular junction structures that mediate cell-cell adhesion and regulate intracellular cytoskeleton architecture are important in the course of such complex tissue organization. Classical cadherins, which are transmembrane proteins mediating cell-cell interactions in a calcium-dependent manner, are thought to play important roles in these processes (reviewed in Refs. 116, 163). M-cadherin, in particular, has been postulated as an essential molecule for the specific fusion of myoblasts with each other during embryonic myogenesis and muscle regeneration (163, 219, 356). First, the preferential expression of M-cadherin in developing and regenerating skeletal muscles, as well as in skeletal muscle cell lines, is suggestive of a role for this molecule in myoblast fusion. More specifically, although M-cadherin mRNA can be detected in only a small subset of quiescent satellite cells, its protein remains constant in most of these cells. Moreover, M-cadherin expression within satellite cells is markedly induced upon muscle injury, suggesting a possible role for this protein in the muscle repair process (150, 219). Second, in vitro experiments using antagonistic peptides and antisense RNA strategies have demonstrated the essential role played by M-cadherin during myoblast fusion process without affecting the biochemical differentiation of myoblasts, as seen by normal upregulation of muscle-specific genes (175, 356). Finally, MyoD–/– satellite cells, which fail to fuse upon injury-induced activation, display a marked decreased in M-cadherin expression (73, 260). However, the essential role for M-cadherin in myoblast fusion has recently been placed in question by the analysis of M-cadherin–/– mice (144). In this careful analysis, M-cadherin-deficient mice did not show any gross developmental defect. In particular, the mutant mice developed normal skeletal musculature and demonstrated normal kinetics of muscle regeneration after CTX injection (144). The authors rightly suggest that such observation may be the result of a compensatory mechanism by other cadherins, such as N-cadherin and R-cadherin, which are present in skeletal muscle and may substitute for M-cadherin function. N-cadherin expression is upregulated in activated satellite cells following injury, but its function in myoblasts fusion remains to be determined (62). Thus, although M-cadherin may have an important role in fusion of myoblasts during muscle regeneration, it is not essential and other cadherins may also play a role during this process.
The role of m-calpain has also been suggested in cytoskeletal reorganization during myoblast fusion. M-calpain belongs to a family of calcium-dependent intracellular nonlysosomal cysteine proteases of relatively unknown functions (reviewed in Ref. 293). M-calpain activity is dramatically increased during fusion of embryonic primary myoblasts (78, 86, 180). In vitro fusion of myoblasts is prevented by calpastatin (a specific inhibitor of m- and µ-calpains) or by decreasing levels of m-calpain using an antisense strategy (20, 311). Conversely, myoblasts fuse earlier and faster after m-calpain injection or artificial decrease of endogenous calpastatin levels using antisense RNA (20, 311). The biological role for m-calpain in myoblast fusion is unclear because substrates for this proteinase during this process are unknown. A potential target of m-calpain is the intermediate filament desmin (90). Interestingly, cytoplasmic intermediate filament proteins such as vimentin, desmin, and nestin have been implicated in myoblast fusion during muscle regeneration (288, 319). Moreover, although the overall muscle formation in Desmin–/– mice appears normal, muscle regeneration appears impaired with delayed myoblast fusion (193, 288). Further analyses are required to determine the specific role for m-calpain and/or desmin in satellite cell fusion during regeneration. Recent advances uncovering the interacting extracellular molecules and the intracellular effectors that facilitate myoblast fusion in Drosophila should benefit the understanding of the mammalian myoblast fusion process (reviewed in Ref. 93).
C. Self-Renewal of Muscle Satellite Cells
Satellite cell self-renewal is a necessary process without which recurrent muscle regeneration would rapidly lead to the depletion of the satellite cell pool. Radiolabel-tracing experiments demonstrated that activated satellite cells contributed to both new myonuclei and satellite cell after muscle damage (124, 131, 143, 268, 272, 287, 347). Moreover, labeling experiments in the growing rat demonstrated the presence of two satellite cell populations (268). One population, representing 80% of the satellite cells, divided rapidly and was responsible for providing myonuclei to the growing rat. The other population, called "reserve cells," divided more slowly and was suggested to replenish the satellite cell pool (268). These observations are consistent with the idea that a small proportion of satellite cells that has undergone proliferation returns to the quiescent state, thereby replenishing the satellite cell pool (23, 24, 29, 268, 349). Satellite cell self-renewal may result from an asymmetric division generating two distinguishable daughter cells, one committed to myogenic differentiation and one stem cell "self." Alternatively, satellite cells may undergo symmetric division with one daughter cell being able to withdraw from the differentiation program and return to quiescence. Neither hypothesis has been proven wrong. Determining the molecular process involved in this mechanism remains a challenge.
In favor of satellite cell asymmetric division is the recent observation by Conboy and Rando (70) that Numb, a plasma membrane-associated cytoplasmic protein, is asymmetrically segregated within dividing satellite cells in vitro. Segregation of Numb proteins has been associated with cell fate determination in both invertebrate and vertebrate developmental processes including during Drosophila myogenesis (54, 58, 169, 252). Numb may influence cell fate by repressing Notch signaling, a pathway which is known to regulate cellular differentiation in different systems and species (88). In cultured satellite cells, activation of Notch-1 appears to promote the proliferation of "primitive" satellite cells (Numb–/Pax3+/Desmin–/Myf5–/MyoD–), whereas its inhibition leads to the commitment of the progenitor cells to the myoblast cell fate (Numb+/Pax3–/Desmin+/Myf5+) and their myogenic differentiation (70). Overall, the data suggest that asymmetric satellite cell division as marked by asymmetric inheritance of Numb may lead to satellite cell self-renewal by causing different patterns of gene expression. However, the specific role for Pax genes and Myf5 in this process remains to be substantiated.
Several lines of evidence suggest a role for Myf5 in facilitating satellite cell self-renewal. The increased proliferation and decreased differentiation phenotype of MyoD–/– cells is consistent with the notion that MyoD-deficient cells represent an intermediate stage between quiescent satellite cells and mpc (73, 260, 342). That observation and the demonstration that upon activation satellite cells express either Myf5 alone or MyoD alone, prior to coexpressing both MRFs and initiating terminal differentiation, have led to the hypothesis that Myf5+/MyoD–cells represent a developmental stage during which satellite cells undergo self-renewal. Interestingly, when human satellite cells or C2C12 murine cell line are induced to differentiate, a small population of undifferentiated Myf5+/MyoD–cells persists (24, 349). Moreover, these cells retain the capacity to self-renew and to give rise to differentiation-competent progeny (24, 349). However, expression of Myf5 in quiescent satellite cells is controversial (28, 74), suggesting that return to the quiescent state may require downregulation of Myf5. Thus satellite cell symmetric division followed by dedifferentiation of one of the daughter cells remains a possibility, especially in light of recent findings that mammalian myotubes are capable of dedifferentiation in vitro. This process may involve the activation of the transcriptional inhibitor Msx1 (discussed in sect. VI).
Furthermore, renewal of the satellite cell pool may not rely exclusively on the satellite cell compartment. Indeed, adult stem cells, other than satellite cells, capable of myogenic differentiation and of contributing to the satellite cell pool following transplantation have been described (discussed in sect. V). The observation that in the absence of Pax7 the number of hematopoietic precursors in muscle-derived stem cells is increased at the expense of myogenic precursors suggests a role for Pax7 in this process, by Pax7 promoting the determination of satellite cells and restricting alternative developmental pathways in multipotent stem cells (275).
Whatever the cellular mechanism(s) for satellite cell self-renewal, this does not appear to compensate for the chronic loss of myonuclei throughout a lifetime as reflected by the reduction in satellite cell number with aging (see sect. IIB and Table 3), nor does it compensate for the depletion of the satellite cell pool resulting from continuous activation of muscle repair in dystrophic muscles (42, 235, 329, 340). Exhaustion of the mitotic potential of satellite cells, or replicative senescence, may be responsible for the decrease in the satellite cell pool with age (85, 273, 329). For example, in DMD patients, where satellite cell proliferation is accentuated, telomere lengths (an indicator of the cell replicative age) are prematurely reduced compared with normal human senescence (84). Moreover, proliferative potential of satellite cells is reduced with age or after repeated rounds of regeneration in animal models (240, 270, 273, 340). Alternatively, the inability to sustain a constant satellite cell number may reflect an alteration in the aging environment rather than intrinsic defect in cellular capacity (26, 42, 50, 59, 60). Furthermore, the observation that multiple rounds of acute muscle regeneration do not deplete the satellite cell pool suggests that satellite cell self-renewal after a short but acute loss of myonuclei (i.e., muscle injury) is more efficient than after chronic covert myonuclei turnover (i.e., life time of day-to-day wear and tear). Thus different mechanisms for satellite cell self-renewal may be at play during varying muscle injuries and at varying ages.
D. Multipotentiality of Muscle Satellite Cells
It has been established for several years now that the commitment of skeletal myocytes is reversible under appropriate tissue culture conditions. Primary myoblasts from newborn mice and C2C12 can differentiate into osteogenic or adipogenic cells after in vitro treatment with bone morphogenetic proteins (BMP2) or adipogenic inducers (thiazollidinedione or fatty acids), respectively (162, 310). However, it is only recently that similar multipotential properties have been demonstrated for the adult muscle satellite cell (14, 323). Until these reports, the adult muscle satellite cell was generally considered a stem cell committed to the myogenic lineage. Work from our laboratory and others demonstrated the BMP induced osteogenic and adipogenic conversions of isolated adult murine satellite cells (14, 323). The osteogenic differentiation of primary myoblasts is characterized by a transient coexpression of myogenic markers (such as MyoD, Myf5, and Pax7) and osteogenic markers (such as alkaline phosphatase), suggesting a direct transdifferentiation from the myogenic lineage to the osteogenic lineage, rather than the passage through a common noncommitted progenitor. In vitro culture of single myofibers suggests the spontaneous conversion of satellite cells to the osteogenic and adipogenic lineages is a rare phenomenon. Satellite cells on freshly isolated muscle fibers do not express the myogenic markers Myf5 and MyoD, suggesting that quiescent satellite cells are more plastic and may enter nonmyogenic pathways more readily (74, 197, 285, 341). Supporting this view is the finding that Msx1, a homeobox protein involved in C2C12 dedifferentiation (see sect. VI), is expressed in quiescent satellite cell but downregulated upon satellite cell activation (73). Myoblasts from MyoD–/– mice do not display increased propensity to osteogenic and/or adipogenic differentiations, suggesting that MyoD alone may not suppress differentiation of myoblasts to these lineages (14). Overall, these data demonstrate the in vitro ability of satellite cells to differentiate into osteogenic or adipogenic lineages. However, satellite cell differentiation potential appears to be restricted to the mesenchymal range of cell lineages as demonstrated by their inability to undergo hematopoietic conversion (16).
Several in vivo observations have suggested the existence of mesenchymal progenitors within skeletal muscles. For example, ectopic bone formation within skeletal muscle has been described in some human diseases (reviewed in Ref. 282). Furthermore, accumulation of adipose tissue has been widely reported in skeletal muscle undergoing degeneration such as in DMD patients or the related mdx mice model and in other models of muscle regeneration (91, 235, 261). Overall, the data support the hypothesis that muscle satellite cells may be involved in the formation of adipogenic and osteogenic tissues under certain in vivo circumstances. Aberrant activation of satellite cells during muscle regeneration may lead to such reversal of lineage commitment at the expense of effective muscle regeneration. However, the hypothesis remains to be proven in vivo.
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IV. ROLE OF SECRETED FACTORS IN THE REGULATION OF MUSCLE REGENERATION |
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(TGF-) families, IGF, hepatocyte growth factor (HGF), tumor necrosis factor-
(TNF-), interleukin-6 (IL-6) family of cytokines, neural-derived factors, nitric oxide, and ATP, in maintaining a balance between growth and differentiation of satellite cells to restore a normal muscle architecture (reviewed in Refs. 142, 296). These studies have contributed to our knowledge on the effect of trophic factors, singly or in combination, on the proliferative and differentiative capacity of satellite cells in vitro. Nevertheless, the physiological functions in skeletal muscle regeneration in vivo have been shown for relatively few of these factors. Scatter factor/HGF was originally isolated from sera of partially hepatectomized rats and found to have mit
