Establishment and Maintenance of Human Induced Pluripotent Stem Cells

Two hiPSC lines (201B7 and FF-PB-3AB4) were used in the experiments in this study. The validated hiPSC line 201B7 was purchased from Riken Cell Bank (Tsukuba, Japan) and transferred from on-feeder to feeder-free conditions in our laboratory. The hiPSC line FF-PB-3AB4 was established in our laboratory (Suzuki et al., 2019 (link)). These hiPSC lines were cultured according to a previously described method (Nakagawa et al., 2015 (link)) with slight modifications. In brief, the hiPSCs were maintained in StemFit AK02N medium (Ajinomoto, Tokyo, Japan) with penicillin (50 units/mL) and streptomycin (50 μg/mL) (#1514022; Gibco, Carlsbad, CA, USA) at 37°C with 5% CO₂. The medium was changed every other day and passaged every 7 days using 0.5x TrypLE Select (1x TrypLE Select [#A12859-01; Gibco] diluted 1:1 with 0.5 mM EDTA [#06894-14; Nacalai Tesque, Kyoto, Japan]/PBS [-]) and Rho-associated kinase (Rock) inhibitor (Y-27632; WAKO, Osaka, Japan). iMatrix-511 silk (#892021; Nippi, Tokyo, Japan) was used for precoating.