Mitomycin c treated mouse embryonic fibroblasts

Human-induced pluripotent stem cells (hiPSCs) (253G1; Riken, Tsukuba, Japan) were cultured in a primate embryonic stem cell medium (ReproCELL, Kanagawa, Japan) supplemented with the basic fibroblast growth factor (bFGF; ReproCELL, Kanagawa, Japan) at 37 °C. Mitomycin Ctreated mouse embryonic fibroblasts (ReproCELL, Kanagawa, Japan) were used as feeder cells. Cardiomyogenic induction was performed in the StemPro 34 medium (Thermo Fisher Scientific, Waltham, MA) containing 2 mM l-glutamine (Thermo Fisher Scientific, Waltham, MA), 50 mg/mL ascorbic acid (Wako, Pure Chemical Industries, Tokyo, Japan), and 400 mM 1-thioglycerol (Sigma-Aldrich, St. Louis, MO), as previously described [18 (link)].
To obtain hiPSC-CMs, hiPSCs were dissociated using Accmax (Nacalai Tesque, Kyoto, Japan), and induced in a bioreactor (ABLE Corporation & Biott Co., Tokyo, Japan). Human recombinant bone morphogenetic protein 4 (BMP4), activin A, bFGF, and vascular endothelial growth factor (VEGF) (R&D Systems, Minneapolis, MN), along with the small-molecule compounds IWR-1 and IWP-2 (Sigma-Aldrich, St. Louis, MO) were used for induction as follows: BMP4 from day 0–1; activin A, BMP4, and bFGF from days 1–4; IWR-1 and IWP-2 from days 4–6; and VEGF and bFGF after day 6. Flow cytometry analysis was performed on day 14 using a FACS Canto II (BD, Franklin Lakes, NJ) as previously reported [18 (link)].