380. Dedifferentiation of Primary Murine Myoblasts Induced by Ectopic Expression of Msx1

Abstract

Top of pageAbstract Ectopic expression of msx1 was shown to induce dedifferentiation of multinucleated myotubes derived from myogenic cell line (C2C12) to mononucleated cells and these dedifferentiated cells displayed stem cell like pluripotency (Odelberg et al, Cell 103: 1099, 2000). We hypothesize that if the same phenomenon exists in primary cells, using this method, we can generate pluripotent cells from myoblasts or fibroblasts which are easily accessible and can be propagated in culture. In the present study, we transduced purified primary myoblasts isolated from muscles of adult mice with a retroviral vector that expresses msx1 gene regulated by a Tat off system and a neomyocin resistant gene expressing from 5′ LTR of retrovirus or a control vector that contains the same elements except the msx1 sequence reversed. In the presence of 6μg/ml doxycycline (an analog of Tetracycline, transduced myoblasts were selected with 400μg/ml G418 for 14 days. RT- PCR confirmed the expression of msx1 in clonal myoblasts of experimental group after withdrawal of doxycycline. To assess whether or not ectopic expression of msx1 can induce dedifferentiation of these primary myoblasts, first the myoblasts from experimental and control groups were forced differentiation by serum withdrawal and then the formed myotubes were placed into 96 well culture plates with one myotube per well. One to five days after withdrawal of doxycycline, 7.5% of multinucleated myotubes in experimental group cleaved into mononucleated cells or smaller myotubes. In contrast, none of myotubes in the control group cleaved. These mononucleated cells are capable of proliferating and the cells cleaved from single myotube were capable of propagating into multimillions of cells without signs of senescence. Two clones of these cells were characterized for their stem cell like properties. RT-PCR revealed that these cells expressed Oct 4, a marker of embryonic and mesenchymal stem cells, and NST, a marker of mesenchymal stem cells. In contrast, there was no detectable level of both markers in control group and uninfected primary myoblasts. When adding LIF into the culture, the growth rate of these dedifferentiated cells was increased but not the rate of control group and uninfected primary myoblasts. Immunochemistry showed that a fraction of these dedifferentiated cells were MyoD (a myogenic marker) negative while the whole population of myoblasts are MyoD positive. Most of these dedifferentiated cells had typical myoblast morphology but some of the cells appeared to have tendency to form ES cell-like clumps. After suppression of msx1 expression, these cells were cultured in adipogenic, osteogenic, chondrogenic or myogenic media to assess their pluripotency. In the osteogenic culture condition, some of the cells expressed alkaline phosphatase (a marker for osteogenesis). In the myogenic culture condition, all of the cells differentiated into myotubes. But we haven't detected the signals of adipogenesis and chondrogenesis in the present culture conditions. Thus, our data demonstrated that ectopic expression of msx1 could induce a subpopulation of primary mouse myoblast dedifferentiation. The further characterization of these dedifferentiated cells and development of methods to isolate them from main population are in the process.