Myotube Differentiation of Human iPSCs

Myotube differentiation of human iPS cells was performed as previously described [8 (link),11 (link)]. On day 0, N-iPS cells, D-iPS and R-iPS cells were seeded at densities of 3.0 × 10⁵, 5.0 × 10⁵, and 1.0 × 10⁵ cells/well, respectively, onto 6-well plates coated with Matrigel (354230; Corning, New York, NY, USA). Since the growth rate differed depending on the type of iPS cells, the seeding cell density was changed so that cells reach semi-confluency during the same period. Cells were cultured in StemFit AK02N medium supplemented with 10 µM Y-27632, 0.1, 1.0 or 10 µM retinoic acid (RA) (Fujifilm Wako Pure Chemical, Osaka, Japan), and 0.5 µg/mL puromycin or 0.1 mg/mL neomycin. The next day, cells were cultured in ReproStem medium (ReproCELL, Yokohama, Japan) supplemented with the corresponding concentrations of RA and antibiotics. From day 2, doxycycline (Dox) (Sigma-Aldrich) was added to the medium at a concentration of 1.0 µg/mL. On day 3, the culture medium was changed to α-MEM (Invitrogen, Carlsbad, CA, USA) containing 5% knock-out serum replacement (Invitrogen), supplemented with Dox and RA. Cells were cultured for 14 days and the medium was changed with fresh medium every day.