Myoblast differentiation from MPCs.
MPCs plated at 2 × 104/cm2 were treated with 3 μM 5-azacytidine in MPC expansion medium with 10 ng/mL EGF and 10 ng/mL PDGF-BB for 24 hours and maintained in the same expansion medium with 2% FCS, 10 ng/mL EGF, and 10 ng/mL PDGF-BB, but without 5-azacytidine for up to 15 days. After 5 and 14 days, cultures were analyzed by FACS for MYO-D, myogenin, fast-twitch myosin, and sarcomeric actin (A; specific antibody, thick line; control IgG, thin line) and on day 15 by immunohistochemistry and immunofluorescence for fast-twitch myosin (B, MPCs stained with anti–fast-twitch myosin antibody; 5-azacytidine–induced cells stained with control IgG, with anti–fast-twitch myosin antibody; arrow indicates multinucleated cell; 10 × and 40 × enlargement). Presence of fast-twitch myosin was confirmed by Western blot (C, clones nos. 1, 2, and 3; see also Figure12; β-actin serves as loading control). A representative example of more than 5 experiments is shown.
