Bone morphologic protein 4

Human-induced pluripotent stem cells (hiPSCs; 253G1; Riken, Ibaraki, Japan) were used in this study. Undifferentiated hiPSCs were cultured and maintained in primate embryonic stem cell medium (ReproCELL, Kanagawa, Japan) with 5 ng/mL basic fibroblast growth factor (bFGF; ReproCELL) on mitomycin C-treated mouse embryonic fibroblast cells (ReproCELL). Cardiomyogenic differentiation was induced as previously described with specific modifications (Matsuura et al., 2012 (link)). Briefly, cardiac differentiation was induced in StemPro 34 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 2 mM L-glutamine (Thermo Fisher Scientific), 50 μg/mL ascorbic acid (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 400 μM 1-thioglycerol (Sigma-Aldrich, St. Louis, MO, USA). The medium was also supplemented with several human recombinant proteins, including bone morphologic protein 4, activin A, bFGF (R&D Systems, Minneapolis, MN, USA), and VEGF (FUJIFILM Wako Pure Chemical Corporation), and small molecules, including IWR-1 and IWP-2 (Sigma-Aldrich). hiPSCs were dissociated using Accumax (Nacalai Tesque, Kyoto, Japan) and transferred to a bioreactor (ABLE Corporation & Biott, Tokyo, Japan). hiPSC-CMs were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich).