Resident stem cells in the adult skeletal muscle are assumed to participate in normal tissue growth and in repair after injury by differentiating into committed myogenic precursors, presumably satellite cells. We have shown that androgens stimulate in vitro the myogenic differentiation of a mouse mesenchymal multipotent cell line (C3H-10T(1/2)) and inhibit its adipogenic differentiation and postulated that they would similarly affect the in vitro lineage commitment of mouse muscle-derived stem cells (MDSCs). To test this hypothesis, we isolated by the preplating procedure from the mouse hind limb muscles a MDSC cell population (pP6). This cell fraction comprised about 1% of the total muscle mononucleated cells and expressed the stem cell markers CD34 and Sca1+, but it was CD45-, as shown by immunocytochemistry and Western blot. The Sca1+ cells were further enriched by magnetic beads selection. Both populations of pP6 cells expressed androgen receptors and a low level of the early and intermediate myogenic markers, MyoD and myogenin, but not the late marker, myosin heavy chain II (MHC-II), as detected by Western blot. MyoD was more expressed in the other cell fractions, pP2 (mainly fibroblasts) and pP3-pP5 (rich in satellite cells). pP6 cells in adipogenic medium for 1 week differentiated into adipocytes, and in osteogenic medium for 2 and 4 weeks formed myofibroblasts and osteoblasts, as detected with Oil Red O, α-smooth muscle actin (ASMA), and alkaline phosphatase staining, respectively, and by Western blot for CBPα, ASMA, and alkaline phosphatase. In myogenic medium for 2 weeks, pP6 failed to form myotubes, intensify the MyoD and myogenin signals, or express MHC-II. However, when incubated with murine C2C12 myotubes on the top compartment of dual culture chambers the cells underwent intense myogenesis and led to a clear MHC-II signal. As predicted, testosterone (100 nM) and DHT (30 nM) increased myotube formation in dual cultures and reduced adipocyte number and stimulated osteogenesis in monocultures but did not affect fibrogenesis (myofibroblasts). Casodex and flutamide abolished these effects. In conclusion, we found that androgens do stimulate paracrinely induced myogenesis and inhibit adipogenesis in MDSC cultures, thus suggesting that a similar process may occur in vivo in the myofibers.
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