Culture of undifferentiated hiPSCs

The TkDN4-M (4M) hiPSCs line was a kind gift from Dr. M Ohtsu at The Institute of Medical Science, The University of Tokyo. 4M cells were cultured as previously described with minor modifications [23]. They were maintained on mitomycin C (MMC; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)-treated SNL feeder cells in hiPSCs medium (DMEM/Ham`s F12 (FUJIFILM Wako) supplemented with 20% Knockout Serum Replacement (KSR; GIBCO BRL, Palo Alto, CA, USA), 1x MEM nonessential amino acids (FUJIFILM Wako), 0.5x penicillin streptomycin(PS; FUJIFILM Wako), 55μM 2-melcaptoethanol (2ME Gibco) and 7.5μg/ml basic fibroblast growth factor (FGF2; Peprotech, Rocky Hill, NJ, USA). For passage, 4M colonies were detached using CTK solution, chipped by pipetting, and seeded onto MMC-treated SNL feeder (ECACC, Salisbury, UK) in hiPSCs medium once a week. The 15M63 hiPSCs line was kindly provided from the Center for iPS Cell Research and Application of Kyoto University (Kyoto, Japan) and maintained on vitronectin (Invitrogen, CA, USA) coated dishes in StemFit. For passage, 15M63 was dissociated with accutase (Innovative Cell Technologies, San Diego, USA) by pipetting and seeded on vitronectin coated dishes in StemFit (Ajinomoto, Tokyo, Japan) supplemented with 10μM Y27632 (Cayman Chemical, Ann Arbor, MI, USA).