Collagen type 1 coated 10 cm dishes

201B7, 409B2, and 454E2 iPSCs were provided by S. Yamanaka at Kyoto University. 201B7 was generated from a healthy donor using retroviral vectors [20 (link)]. 409B2 and 454E2 were integration-free hiPS clones generated from healthy donors using episomal vectors [21 (link)]. hiPSCs were cultured on iMatrix-511 (Nippi)-coated 6-well plates in StemFit AK02N medium (Ajinomoto) supplemented with penicillin/streptomycin/amphotericin B (1% v/v) (FujiFilm) as described previously [37 (link), 38 (link)]. Myogenic cells were induced from hiPSCs as described [37 (link), 38 (link)]. After floating culture, cells were induced to differentiate into myogenic cells on collagen type I-coated 10-cm dishes (Iwaki) in 10% FBS (Gibco)/DMEM (FujiFilm) in the presence of 1 μM SB431542 (Wako) for one week. The induced cells were then collected and incubated with ERBB3-APC (1B4C3, BioLegend), CD57(HNK-1)-PE (clone TB03, Miltenyi Biotec), and CD271-BB515 (C40-1457, BD Pharmingen). Myogenic cells were sorted as the CD57(-) CD271(+) ERBB3(+) fraction [37 (link), 38 (link)].

The human myoblast cell line Hu5/E18 was cultured in collagen type I-coated 10-cm dishes (Iwaki, Japan) or Cellmatrix type I-P (Nitta gelatin, Japan) -coated dishes in myoblast medium containing DMEM, 20 % FBS, 2 mM L-glutamine, 0.5 % penicillin/streptomycin and 2 % Ultroser G serum substitute (Pall, France). For in vitro neuro-muscular co-culture, Hu5/E18 cells were plated at a density of 1×10³ cells/ml on a Cellmatrix type I-P-coated micro cover glass (Matsunami, Japan) and cultured until the cells reached approximately 50 % confluence. Then, the medium was changed to MNM, and the cells were cultured for 4 additional days, until the formation of myotubes. Partially dissociated EBs derived from KhES1 cells were plated onto Hu5/E18-derived myotubes at a density of 2.5×10²–5×10³ cells/cm² and co-cultured with the myotubes for 3–4 days in MNM.