On the non-equivalence of skeletal muscle satellite cells and embryonic myoblasts

Postnatal satellite cells, isolated from normal or previously denervated skeletal muscles of juvenile quails, were tested as to their capacity to participate in embryonic muscle ontogeny. They were grafted into 2-day chick embryo hosts, in place of a piece of brachial somitic mesoderm. Satellite cell implants were prepared from pellets either of freshly isolated cells or of cells precultured in vitro under proliferative conditions. Myogenic capacity of the implanted cells was attested by their ability to fuse into myotubes when cultured under differentiation conditions. In no case did the implanted satellite cells invade the adjacent wing bud or participate in wing muscle morphogenesis. They did not either give rise to myotubes at the site of implantation, nor did they even survive longer than 3 days in the embryonic environment. These negative results indicate that postnatal satellite cells, unlike embryonic myoblasts, are unable to take part in muscle embryogenesis. Although they derive from the same somitic myogenic cell line as the embryonic myoblasts, they therefore represent a differentiated non-totipotent type of myogenic cell.     

Fusion between myoblasts and adult muscle fibers promotes remodeling of fibers into myotubes in vitro

Muscle satellite cells are residual embryonic myoblast precursors responsible for muscle growth and regeneration. In order to examine the role of satellite cells in the initial events of muscle regeneration, we placed individual mature rat muscle fibers in vitro along with their satellite cells. When the satellite cells were allowed to proliferate, they produced populations of myoblasts that fused together to form myotubes on the laminin substrate. These myoblasts and myotubes also fused with the adult fibers. When they did so, the fibers lost their adult morphology, and by 8 days in vitro, essentially all of them were remodeled into structures resembling embryonic myotubes. However, when proliferating satellite cells were eliminated by exposure to cytosine arabinoside (araC), the vast majority of fibers retained their adult shape. Addition of C2C12 cells (a myoblast line derived from adult mouse satellite cells) to araC-treated fiber cultures resulted in their fusion with the rat muscle fibers and restored the ability of the fibers to remodel, whereas addition of either a fibroblast cell line or a transformed, non-fusing variant of C2C12 cells, or addition of conditioned medium from C2C12 cells, failed to do so. These results imply that myoblast fusion is responsible for triggering adult fiber remodeling in vitro.     

Divergent differentiation during embryonal histogenesis of skeletal muscle tissue

Morphometric analysis of the developmental processes of the satellite cells and myosimplasts has been performed in embryonal histogenesis of the skeletal muscle tissue in 17 human fetuses 8-27 weeks of the intrauterine development. The sequence of death of some myoblasts in embryonal histogenesis is described in details. Basing on the data obtained, a conception on existance of muscular-proliferative units (MPU) in composition of the skeletal muscles is put forward. The amount of the MPU determines the whole number of muscle fibers in the muscle. The anlage of the MPU occurs as a result of divergent differentiation of the stem myogenic cells at early stages of myogenesis (myosimplasts and myotubes) from the cells commited to mutual fusion. The fund of these cells is determined by the number of myogenic elements that are at the state of the proliferative rest. One of the mechanisms regulating the number of the resting cells is the growth rate of the simplast lengthwise. The resting cells, appearing at late stages of myogenesis (of the muscle fibers), are the sources for development of the myosatellites in mature muscle fibers. In dying myotubes there is a sharp disturbance in growth processes lengthwise, in biosynthesis of contractile proteins, in correlation between the number of nuclei in the satellite cells and those of simplasts.     

Context matters: in vivo and in vitro influences on muscle satellite cell activity

Skeletal muscle is formed during development by the progressive specification, proliferation, migration, and fusion of myoblasts to form terminally differentiated, contractile, highly patterned myofibers. Skeletal muscle is repaired or replaced postnatally by a similar process, involving a resident myogenic stem cell population referred to as satellite cells. In both cases, the activity of the myogenic precursor cells in question is regulated by local signals from the environment, frequently involving other, non-muscle cell types. However, while the majority of studies on muscle development were done in the context of the whole embryo, much of the current work on muscle satellite cells has been done in vitro, or on satellite cell-derived cell lines. While significant practical reasons for these approaches exist, it is almost certain that important influences from the context of the injured and regenerating muscle are lost, while potential tissue culture artifacts are introduced. This review will briefly address extracellular influences on satellite cells in vivo and in vitro that would be expected to impinge on their activity.     

Myotube Formation on Micro-patterned Glass: Intracellular Organization and Protein Distribution in C2C12 Skeletal Muscle Cells

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. Studies of myocyte differentiation, cell fusion, and muscle repair are limited by an appropriate in vitro muscle cell culture system. We developed a novel cell culture technique [two-dimensional muscle syncytia (2DMS) technique] that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. This technique is based on UV lithography–produced micro-patterned glass on which conventionally cultured C2C12 myoblasts proliferate, align, and fuse to neatly arranged contractile myotubes in parallel arrays. Combining this technique with fluorescent microscopy, we observed alignment of actin filament bundles and a perinuclear distribution of glucose transporter 4 after myotube formation. Newly formed myotubes contained adjacently located MyoD-positive and MyoD-negative nuclei, suggesting fusion of MyoD-positive and MyoD-negative cells. In comparison, the closely related myogenic factor Myf5 did not exhibit this pattern of distribution. Furthermore, cytoplasmic patches of MyoD colocalized with bundles of filamentous actin near myotube nuclei. At later stages of differentiation, all nuclei in the myotubes were MyoD negative. The 2DMS system is thus a useful tool for studies on muscle alignment, differentiation, fusion, and subcellular protein localization. (J Histochem Cytochem 56:881–892, 2008)     

Specific features of satellite cells and myoblasts at different stages of rat postnatal development

A cell culture consisting mainly of satellite cells and mononuclear myoblasts was derived from femoral muscles of infant (aged 3–7 days) and adult rats. Satellite cells identified by expression of the specific marker Pax7 accounted for approximately 80% of the isolated cell fraction. Mononuclear myoblasts represented by proliferating and postmitotic cell pools were identified immunocytochemically by the expression of markers Ki67 and desmin. Differentiation of satellite cells and myoblasts in the culture depended on the concentration of Ca²⁺ in the culture medium (F12 with different Ca²⁺ concentrations or DMEM). Differentiation of myogenic cells manifested in myoblasts fusion, formation of myotubes, and expression of myosin in myofibrils was observed only in the medium with a high Ca²⁺ concentration (2mM). Satellite cells and myoblasts from the muscles of newborn and adult rats did not differ noticeably in their capacity for differentiation.     

Sca-1 negatively regulates proliferation and differentiation of muscle cells

Satellite cells are tissue-specific stem cells critical for skeletal muscle growth and regeneration. Upon exposure to appropriate stimuli, satellite cells produce progeny myoblasts. Heterogeneity within a population of myoblasts ensures that a subset of myoblasts readily differentiate to form myotubes, whereas other myoblasts remain undifferentiated and thus available for future muscle growth. The mechanisms that contribute to this heterogeneity in myoblasts are largely unknown. We show that satellite cells are Sca-1(neg) but give rise to myoblasts that are heterogeneous for sca-1 expression. The majority of myoblasts are sca-1(neg), rapidly divide, and are capable of undergoing myogenic differentiation to form myotubes. In contrast, a minority population is sca-1(pos), divides slower, and does not readily form myotubes. Sca-1 expression is not static but rather dynamically modulated by the microenvironment. Gain-of-function and loss-of-function experiments demonstrate that sca-1 has a functional role in regulating proliferation and differentiation of myoblasts. Myofiber size of sca-1 null muscles is altered in an age-dependent manner, with increased size observed in younger mice and decreased size in older mice. These studies reveal a novel system that reversibly modulates the myogenic behavior of myoblasts. These studies provide evidence that, rather than being a fixed property, myoblast heterogeneity can be modulated by the microenvironment.     

Decorin expression in quiescent myogenic cells

Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells.     

Proliferation of muscle satellite cells on intact myofibers in culture

Muscle satellite cells are quiescent myogenic stem cells situated between the basal lamina and plasmalemma of mature skeletal muscle fibers. Injury to the fiber triggers the activation and proliferation of satellite cells whose progeny subsequently fuse to form new myotubes during regeneration. In this paper we report the proliferation of satellite cells on single muscle fibers isolated from adult rats and placed in culture. Viable fibers were liberated from muscle with collagenase and purified from non-muscle cells. The fibers were covered with a basal lamina and retained normal morphological characteristics. Each fiber contained two to three satellite cells per 100 myonuclei. Satellite cells showed little proliferative activity in medium with 10% serum but could be induced to enter the cell cycle by chick embryo extract or fibroblast growth factor. Other polypeptide mitogens such as epidermal growth factor, multiplication stimulating activity, and platelet-derived growth factor were ineffective. Mitogen-stimulated satellite cells fused to form new myotubes after 4-5 days in culture. These results imply that satellite cells are under positive growth control since they proliferate in contact with viable mature fibers when stimulated with mitogen. The mature fibers remained viable in culture but did not give rise to mononucleated cells. After several days, however, the fibers began to extend sarcoplasmic sprouts and underwent dedifferentiative changes that led to the formation of multinucleated cells resembling myotubes. These cells reexpressed embryonic isozymes of creatine kinase not made by the mature fibers.     

Satellite cells, myoblasts and other occasional myogenic progenitors: possible origin, phenotypic features and role in muscle regeneration

In the vertebrate embryo, skeletal muscle originates from somites and is formed in discrete steps by different classes of progenitor cells. After myotome formation, embryonic myoblasts give rise to primary fibers in the embryo, while fetal myoblasts give rise to secondary fibers, initially smaller and surrounding primary fibers. Satellite cells appear underneath the newly formed basal lamina that develops around each fiber, and contribute to post-natal growth and regeneration of muscle fibers. Recently, different types of non somitic stem-progenitor cells have been shown to contribute to muscle regeneration. The origin of these different cell types and their possible lineage relationships with other myogenic cells as well as their possible role in muscle regeneration will be discussed. Finally, possible use of different myogenic cells in experimental protocols of cell therapy will be briefly outlined.     

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a "myosheet," was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.     

c-Myb Inhibits Myoblast Fusion

Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3′ untranslated region (3′ UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3′ UTR of c-Myb was also important because the expression of c-Myb coding region with its 3′ UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3′ UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.     

Muscle satellite cell-specific genes identified by genetic profiling of MyoD-deficient myogenic cell

Satellite cells are committed myogenic progenitors that give rise to proliferating myoblasts during postnatal growth and repair of skeletal muscle. To identify genes expressed at different developmental stages in the satellite cell myogenic program, representational difference analysis of cDNAs was employed to identify more than 50 unique mRNAs expressed in wild-type myoblasts and MyoD-/- myogenic cells. Novel expression patterns for several genes, such as Pax7, Asb5, IgSF4, and Hoxc10, were identified that were expressed in both quiescent and activated satellite cells. Several previously uncharacterized genes that represent putative MyoD target genes were also identified, including Pw1, Dapk2, Sytl2, and NLRR1. Importantly, many genes such as IgSF4, Neuritin, and Klra18 that were expressed exclusively in MyoD-/- myoblasts were also expressed by satellite cells in undamaged muscle in vivo but were not expressed by primary myoblasts. These data are consistent with a biological role for activated satellite cells that induce Myf5 but not MyoD. Lastly, additional endothelial and hematopoietic markers were identified supporting a nonsomitic developmental origin of the satellite cell myogenic lineage.     

Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation

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

Characterization of myogenesis from adult satellite cells cultured in vitro

We describe several characteristics of in vitro myogenesis from adult skeletal muscle satellite cells from the rat and several amphibian species. The timing of cell proliferation and fusion into myotubes was determined, and in urodeles, myogenesis from satellite cells was clearly demonstrated for the first time. Growth factors are known to stimulate satellite cell proliferation. Acidic FGF mRNA was present in rat satellite cells during proliferation but it was not detected in myotubes. Fibronectin was synthesized in satellite cells during proliferation and expelled into the extracellular medium when the myotubes differentiated. We suggest that fibronectin plays a part in the formation of myotubes, as this process was inhibited by anti-fibronectin IgG. Adult satellite cells might differ from fetal myoblasts since they were observed to exhibit the opposite response to a phorbol ester (TPA) to that of the myoblasts. We therefore examined the possibility that the different levels of protein kinase C activity and different phorbol ester binding characteristics in the two cell types account for these opposite responses. Our results suggest that the difference is not connected with the phorbol ester receptor but might be caused by events subsequent to protein kinase C activation. Localized extracellular proteolytic activity might have a role in cell mobilization and/or fusion when satellite cells are activated. We showed that the content of plasminogen activators, chiefly urokinase, was larger in tissues from slow twitch muscles which regenerate more rapidly than fast muscles. The urokinase level rose sharply in cultures when cells fused into myotubes, and was twice as high in slow muscle cells as in fast ones. We also found that, in vitro, slow muscle satellite cells displayed greater myogenicity, but that phorbol ester inhibited their mitosis and myogenicity. We conclude that satellite cells acquire characteristics which differentiate them from myoblasts and correspond to the fast and slow muscles from which they originate.     

Acetylcholinesterase activity in developing skeletal muscle cells

Acetylcholinesterase activity has been demonstrated biochemically and cytochemically in developing chick embryo skeletal muscle cells growing in culture. The enzyme shows the same pattern of drug sensitivity as that of adult skeletal muscle acetylcholinesterase and in present in cultured myogenic cells before the time of cell fusion, the formation of myotubes, and the subsequent increase in rate of myosin synthesis. Myogenic cell fusion is accompanied, however, by a large increase in activity of acetylcholinesterase. The enzyme activity is restricted in these cultures to myogenic cells. Neighboring fibroblasts show no cytochemical responses when challenged with techniques showing intense activity in myoblasts and myotubes. In addition, evidence is presented which strongly suggests that acetylcholinesterase activity in dividing myogenic cells is not constant over the cell cycle.