Myogenic Differentiation of MyoD-hiPSCs

MyoD-hiPSCs were seeded onto CollagenI (Iwaki) or Matrigel (BD Biosciences) coated dishes without feeder cells. Matrigel was diluted 1∶50 with primate ES medium. MyoD-hiPSCs were trypsinized and dissociated into single cells. The cell number plated ranged from 2.0×10⁵ to 1.0×10⁶ per 10cm². Culture medium was changed to human iPS medium without bFGF and with 10 µM Y-27632 (Nacalai Tesque). After 24 h, 1 µg/mL doxycycline (LKT Laboratories) was added to the culture medium. After an additional 24 h, culture medium was changed to differentiation medium composed of alpha Minimal Essential Medium (αMEM; Nacalai Tesque) with 5% KSR (Invitrogen), 50 mU/L Penicillin/50 µg/L Streptomycin (Invitrogen), and 100 µM 2-Mercaptoethanol (2-ME). After an additional 5 days, culture medium was changed to DMEM with 5% horse serum (Sigma), 50 mU/L Penicillin/50 µg/L Streptomycin, 10 ng/mL recombinant human insulin-like growth factor 1 (Peprotech), 2 mM L-Glutamine and 100 µM 2-ME. Approximately 2 days later, myogenic properties were assessed.