Spontaneous and induced myogenesis in cell cultures from rat fetal liver

Fetal liver stroma consists of different cell populations. We found that the liver of 17- and 20-day rat fetuses contained skeletal muscle precursors that expressed MyoD. In primary cultures of liver cells from 15-, 17- and 20-day fetuses, spontaneous myotube formation was observed. The antigenic profile of these myogenic elements assayed by immunocytochemistry and PCR unambiguously indicated their skeletal muscle nature. Examination of major myogenic gene expression demonstrated that myogenic potencies cells from liver depended on the stage of fetal development cell cultivation. It was shown that fetal liver MSCs were capable of myotube formation in induction medium with 5-azacytidine. The results of our study show that 15- to 20-day prenatal rat liver contains mainly preexisting skeletal muscle precursors expressing MyoD and, probably, inducible muscle precursors.     

A laminin substrate promotes myogenesis in rat skeletal muscle cultures: analysis of replication and development using antidesmin and anti-BrdUrd monoclonal antibodies

Cells from newborn rat hindlimb show greatly enhanced myogenicity when grown on surfaces coated with poly-L-lysine followed by laminin (PLL/Lam) instead of the collagens routinely used. Coating with poly-L-lysine (PLL) alone or with PLL followed by collagen does not enhance myogenicity. Both myogenic and nonmyogenic cells, as distinguished by a monoclonal antibody specific for desmin, attach equally well to collagen- and laminin-coated surfaces, but there is a two- to five-fold increase in the number of myogenic cells on PLL/Lam by 72 hr, followed by increased myotube formation. To determine whether this increase in myogenic cells was a consequence of a selective increase in proliferation on PLL/Lam, incorporation of 5-bromodeoxyuridine into DNA followed by labeling with anti-BrdUrd antibody was used as an index of cell proliferation. The results indicate that desmin is expressed in replicating rat myoblasts, and that replication of myogenic cells is greatly enhanced on laminin compared to collagen. The rate of replication of nonmyogenic cells is the same on both substrates. Addition of 10 micrograms/ml laminin to the medium of cells seeded on PLL or collagen has no effect on myogenicity. We conclude that a laminin substrate enhances skeletal myogenesis in vitro by promoting selectively the replication of myoblasts. Cultures prepared from fetuses at 17 and 19 days gestation also show enhanced myogenicity when grown on PLL/Lam, while those from 15-day fetuses do not. Growth and development of fetal myoblasts on collagen were very poor, whereas myoblasts from the newborn rat do proliferate and differentiate on this substrate. Thus myogenic cells at different stages of fetal and neonatal development may require and respond to different extracellular environments. Myotube formation in the E63 clone of L8 rat myoblasts is inhibited by PLL/Lam.     

Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders

Interleukin-15 (IL-15) is a novel anabolic factor for skeletal muscle which inhibits muscle wasting associated with cancer (cachexia) in a rat model. To develop a cell culture system in which the mechanism of the anabolic action of IL-15 on skeletal muscle could be examined, the mouse C2 skeletal myogenic cell line was transduced with a retroviral expression vector for IL-15 and compared to sister cells transduced with a control vector. Overexpression of IL-15 induced fivefold higher levels of sarcomeric myosin heavy chain and alpha-actin accumulation in differentiated myotubes. Secreted factors from IL-15-overexpressing myogenic cells, but not from control cells, induced increased myofibrillar protein accumulation in cocultured control myotubes. IL-15 overexpression induced a hypertrophic myotube morphology similar to that described for cultured myotubes which overexpressed the well-characterized anabolic factor insulin-like growth factor-I (IGF-I). However, in contrast to IGF-I, the hypertrophic action of IL-15 on skeletal myogenic cells did not involve stimulation of skeletal myoblast proliferation or differentiation. IL-15 induced myotube hypertrophy at both low and high IGF-I concentrations. Furthermore, in contrast to IGF-I, which stimulated only protein synthesis under these culture conditions, IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured skeletal myotubes. These findings indicate that IL-15 action on skeletal myogenic cells is distinct from that of IGF-I. Due to the ability of IGF-I to stimulate cell division and its association with several forms of cancer, controversy exists concerning the advisability of treating cachexia or age-associated muscle wasting with IGF-I. Administration of IL-15 or modulation of the IL-15 signaling pathway may represent an alternative strategy for maintaining skeletal muscle mass under these conditions.     

Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation

Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent inhibition of differentiation was associated with MyoD degradation by the ubiquitin-proteasome pathway. MyoD overexpression in C2C12 myoblasts overrode the differentiation block imposed by hypoxic conditions. Thus, hypoxia by inducing MyoD degradation blocked accumulation of early myogenic differentiation markers such as myogenin and p21 and pRb, preventing both permanent cell cycle withdraw and terminal differentiation. Our study revealed a novel anti-differentiation effect exerted by hypoxia in myogenic cells and identified MyoD degradation as a relevant target of hypoxia.     

Distinct myogenic programs of embryonic and fetal mouse muscle cells: expression of the perinatal myosin heavy chain isoform in vitro.

Early embryonic and late fetal mouse myogenic cells showed distinct patterns of perinatal myosin heavy chain (MHC) isoform expression upon differentiation in vitro. In cultures of somite or limb muscle cells isolated from Day 9 to Day 12 embryos, differentiated cells that expressed perinatal MHC were rare and perinatal MHC was not detectable by immunoblotting. In cultures of limb muscle cells isolated from Day 13 to Day 18 fetuses, in contrast, the perinatal MHC isoform was easily detected and was expressed in a substantial percentage of myocytes and myotubes. Analyses of clonally derived muscle colonies and cytosine arabinoside-treated fetal muscle cell cultures suggested that different fetal muscle cell nuclei initiated perinatal MHC expression at different times. In both embryonic and fetal cell cultures, the embryonic MHC isoform was expressed by all differentiated cells examined. A small number of myotubes in fetal muscle cell cultures showed a mosaic distribution of MHC isoform accumulation in which the perinatal MHC isoform accumulated in a restricted region of the myotube near particular nuclei, whereas the embryonic MHC isoform accumulated throughout the myotube. Thus, the myogenic program of fetal, but not embryonic, mouse myogenic cells includes expression of the perinatal MHC isoform upon differentiation in culture.     

Myf-5 and myoD genes are activated in distinct mesenchymal stem cells and determine different skeletal muscle cell lineages.

Targeted inactivation of the myogenic determination genes myf-5 and myoD in mice resulted in moderate (Myf-5) or no muscle phenotypes (MyoD) and double knock-out mutants lacking both genes failed to develop any skeletal muscle. In order to determine the mechanism of this apparent genetic redundancy we investigated the basis of functional overlap between the two genes. Here we demonstrate that Myf-5 and MyoD are not expressed within the same muscle precursor cell, but rather determine different muscle cell lineages arising from independently committed stem cell populations. Selective ablation of Myf-5-expressing muscle precursors from differentiating ES cells does not prevent Myo-D-dependent muscle differentiation. The early muscle progenitor cells which normally express Myf-5 do not develop into later appearing MyoD cells, even when the myf-5 gene has been inactivated. Thus skeletal musculature in vertebrates develops from two separate cell lineages and complementation may occur at the cellular level, but not between different myogenic factor genes within one cell.     

Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.     

cis-4-Hydroxy-L-proline and ethyl-3,4-dihydroxybenzoate prevent myogenesis of C2C12 muscle cells and block MyoD1 and myogenin expression.

cis-4-Hydroxy-L-proline (cis-OH-Pro) and ethyl-3,4-dihydroxybenzoate (EDHB), two distinct inhibitors of collagen synthesis, prevented myogenesis in C2C12 mouse skeletal muscle cells. Both inhibitors blocked myotube formation and the expression of sarcomeric myosin heavy chain. Northern blot analysis showed that cis-OH-Pro- and EDHB-treated C2C12 muscle cells did not express the myogenic regulatory genes, MyoD1 and myogenin, but continued to express non-muscle isoforms of actin (beta and gamma) and alpha-tropomyosin. 10TFL2-3B cells, a C3H10T1/2 cell line permanently transfected with myogenin cDNA, constitutively expressed exogenous myogenin in the presence of cis-OH-Pro but failed to activate endogenous myogenin and to undergo myogenesis. These results demonstrate that commitment to terminal differentiation and activation of myogenic regulatory genes requires active synthesis of the extracellular matrix component collagen.     

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)     

The homeodomain protein Barx2 promotes myogenic differentiation and is regulated by myogenic regulatory factors

The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Here we use antisense-oligonucleotide inhibition of Barx2 expression in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, we isolated the Barx2 gene promoter and identified DNA regulatory elements that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contained binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors.     

Myf5 and MyoD activation define independent myogenic compartments during embryonic development

Gene targeting has indicated that Myf5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are missing in mouse embryos lacking both Myf5 and MyoD. To investigate the fate of Myf5:MyoD-deficient myogenic precursor cells during embryogenesis, we examined the sites of epaxial, hypaxial, and cephalic myogenesis at different developmental stages. In newborn mice, excessive amounts of adipose tissue were found in the place of muscles whose progenitor cells have undergone long-range migrations as mesenchymal cells. Analysis of the expression pattern of Myogenin-lacZ transgene and muscle proteins revealed that myogenic precursor cells were not able to acquire a myogenic fate in the trunk (myotome) nor at sites of MyoD induction in the limb buds. Importantly, the Myf5-dependent precursors, as defined by Myf5(nlacZ)-expression, deficient for both Myf5 and MyoD, were observed early in development to assume nonmuscle fates (e.g., cartilage) and, later in development, to extensively proliferate without cell death. Their fate appeared to significantly differ from the fate of MyoD-dependent precursors, as defined by 258/-2.5lacZ-expression (-20 kb enhancer of MyoD), of which a significant proportion failed to proliferate and underwent apoptosis. Taken together, these data strongly suggest that Myf5 and MyoD regulatory elements respond differentially in different compartments.     

Possible involvement of hic-5, a focal adhesion protein, in the differentiation of C2C12 myoblasts

Hic-5, a focal adhesion protein, has been implicated in cellular senescence and differentiation. In this study, we examined its involvement in myogenic differentiation. The hic-5 expression level in growing C2C12 myoblasts increased slightly on the first day and then gradually decreased until no hic-5 was detectable after 7 days of differentiation. In vivo, its expression level declined in the thigh and the calf skeletal muscle of mouse embryos after birth. The introduction of an antisense expression vector of hic-5 into C2C12 cells decreased the number of clones expressing the myosin heavy chain (MHC) upon exposure to the differentiation medium. In the cloned cells with low levels of hic-5, the efficiency of myotube formation was significantly reduced. The expression levels of MyoD, myogenin, MHC and p21 were also reduced in these clones. The results suggested that hic-5 plays a role in the initial stage of myogenic differentiation.     

Differential effects of cyclic uniaxial stretch on human mesenchymal stem cell into skeletal muscle cell

Both fetal and adult skeletal muscle cells are continually being subjected to biomechanical forces. Biomechanical stimulation during cell growth affects proliferation, differentiation and maturation of skeletal muscle cells. Bone marrow-derived hMSCs [human MSCs (mesenchymal stem cells)] can differentiate into a variety of cell types, including skeletal muscle cells that are potentially a source for muscle regeneration. Our investigations involved a 10% cyclic uniaxial strain at 1 Hz being applied to hMSCs grown on collagen-coated silicon membranes with or without IGF-I (insulin-like growth factor-I) for 24 h. Results obtained from morphological studies confirmed the rearrangement of cells after loading. Comparison of MyoD and MyoG mRNA levels between test groups showed that mechanical loading alone can initiate myogenic differentiation. Furthermore, comparison of Myf5, MyoD, MyoG and Myf6 mRNA levels between test groups showed that a combination of mechanical loading and growth factor results in the highest expression of myogenic genes. These results indicate that cyclic strain may be useful in myogenic differentiation of stem cells, and can accelerate the differentiation of hMSCs into MSCs in the presence of growth factor.     

Transdifferentiation of esophageal smooth to skeletal muscle is myogenic bHLH factor-dependent

Previously, coexpression of smooth and skeletal differentiation markers, but not myogenic regulatory factors (MRFs), was observed from E16.5 mouse fetuses in a small percentage of diaphragm level esophageal muscle cells, suggesting that MRFs are not involved in the process of initiation of developmentally programmed transdifferentiation in the esophagus. To investigate smooth-to-skeletal esophageal muscle transition, we analyzed Myf5nlacZ knock-in mice, MyoD-lacZ and myogenin-lacZ transgenic embryos with a panel of the antibodies reactive with myogenic regulatory factors (MRFs) and smooth and skeletal muscle markers. We observed that lacZ-expressing myogenic precursors were not detected in the esophagus before E15.5, arguing against the hypothesis that muscle precursor cells populate the esophagus at an earlier stage of development. Rather, the expression of the MRFs initiated in smooth muscle cells in the upper esophagus of E15.5 mouse embryos and was immediately followed by the expression of skeletal muscle markers. Moreover, transdifferentiation was markedly delayed or absent only in the absence of Myf5, suggesting that appropriate initiation and progression of smooth-to-skeletal muscle transdifferentiation is Myf5-dependent. Accordingly, the esophagus of Myf5(-/-):MyoD(-/-)embryos completely failed to undergo skeletal myogenesis and consisted entirely of smooth muscle. Lastly, extensive proliferation of muscularis precursor cells, without programmed cell death, occurred concomitantly with esophageal smooth-to-skeletal muscle transdifferentiation. Taken together, these results indicate that transdifferentiation is the fate of all smooth muscle cells in the upper esophagus and is normally initiated by Myf5.     

Myogenic differentiation of mesenchymal stem cells co‐cultured with primary myoblasts

TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co‐cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)‐transduced rat MSC co‐cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC—myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α‐sarcomeric actin, and MEF2 expression was up‐regulated upon co‐cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co‐cultures. In summary, we have determined optimal co‐culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell‐based therapies.