Myogenic Differentiation of iPSCs

Myogenic differentiation was performed according to a previous protocol,³⁴ (link) with some modifications. MyoD-iPSCs were dissociated into single cells by incubation with 0.5× TrypLE Select, and single cells were seeded on growth factor reduced Matrigel (Corning)-coated dishes without feeder cells. Matrigel was diluted 1:100 with primate ESC medium (ReproCELL), and the culture medium was changed to primate ESC medium with 10 μM Y-27632 but without bFGF. After 24 h, Dox (1 μg/mL; LKT Laboratories) was added to the culture medium. After an additional 24 h, the culture medium was changed to differentiation medium, composed of alpha minimum essential medium (α-MEM) (Nacalai Tesque) with 5% KnockOut Serum Replacement (KSR) (Gibco), a 0.5% antibiotic-antimycotic solution (FUJIFILM Wako), and 200 μM 2-mercaptoethanol (Nacalai Tesque). After an additional 5 days, the culture medium was changed to DMEM (high glucose; Nacalai Tesque) with 2% horse serum (Sigma), a 0.5% antibiotic-antimycotic solution, 2 mM GlutaMAX (Gibco), and 200 μM 2-mercaptoethanol.