Stemfit ak02n medium

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.

RPChiPS771 human induced pluripotent stem cells (iPSCs; REPROCELL) were maintained in StemFit AK02N medium (Ajinomoto) on 100 mm CellBIND cell culture dishes (Corning, 3296) precoated with vitronectin (ThermoFisher Scientific, A31804). Cell passages were performed as follows. hiPSCs were washed, dissociated with TrypLE^TM Select (Gibco), and resuspended in StemFit AK02N medium supplemented with 10 µM ROCK inhibitor (Y-27632; Wako) and replated at 1-2×10⁶ cells per dish.