Regulation of skeletal muscle differentiation in fibroblasts by exogenous MyoD gene in vitro and in vivo

MyoD of the myogenic regulatory factors (MRFs) family regulates the skeletal muscle differentiation program. In this study, stably transfected NIH3T3-derived cell lines were established, in which exogenous MyoD was expressed at high levels. Transcriptional activation of endogenous muscle regulatory gene and induction towards the skeletal muscle lineages were observed with phase-contrast microscopy when continuously cultured in vitro. Moreover, to determine their ability of myogenic formation in vivo, the transfected cells were implanted in nude mice subcutaneously for up to 10 weeks. The morphological characterization of inductive cells was observed using transmission electron microscope and histological staining. Myogenesis of fibroblasts incubated in the medium was activated by overexpression of MyoD, and the cells were accumulated and fused into multinucleated myotubes. Correlatively, RT-PCR and immunohistochemistry confirmed the increased expression of characteristic downstream molecule myogenin and mysion heavy chains during myogenic differentiation. Ecoptic myogenesis was found and remained stable phenotype when the transfected cells were seeded in vivo. Our results suggest that MyoD can be considered to be a determining factor of myogenic lineages, and it may play an important role in the cell therapy and cell-mediated gene therapy of the skeletal muscle.