Human iPSC Culture and Cardiomyocyte Differentiation

Undifferentiated hiPSCs used in this study were obtained from Beijing Cellapy Biotechnology. hiPSCs were cultured in Matrigel (Corning, United States)-coated plates in feeder-free culture conditions with fresh PGM1 PSC culture medium (Cellapy, China), and the cells were passaged at 80–90% confluence using a dissociation solution (Cellapy, China). The schematic shown in Supplementary Figure S1A illustrates the cell culture, differentiation, and dissociation timeline. The undifferentiated hiPSCs were induced to differentiate into cardiomyocytes by transient activation/inhibition of the Wnt signaling pathway. Cardiac differentiation methods were adapted from previously published articles (Lian et al., 2012; Sharma et al., 2015). Cells at 90% confluence were used for differentiation (day 0), and the medium was replaced with RPMI 1640 (Sigma, United States) with B27 supplement minus insulin (Thermo Fisher Scientific, United States), which was considered basal medium. Day 0 cells were incubated with 6 μmol of CHIR99021 (GSK-3 inhibitor) (Selleck, United States) for 48 h (day 2). Day 2 cells were cultured in the basal medium for 24 h (day 3). Day 3 cells were treated with 5 μmol of IWP2 (WNT inhibitor) (Selleck, United States) for 48 h (day 5). Then, day 5 differentiated cells were cultured in the basal medium for 48 h (until day 7). On day 7, the medium was replaced by RPMI 1640 with B27 supplement with insulin (Thermo Fisher Scientific, United States). Approximately 8 days after differentiation, some cells began to beat (Supplementary Video 1). Subsequently, cells were maintained in the medium containing RPMI 1640 plus B27 supplement with insulin for 7 days followed by maintenance in the cardiac enrichment medium [RPMI 1640 medium (Thermo Fisher Scientific, United States) supplemented with 4 mM sodium L-lactate (Sigma-Aldrich, United States)]. Cells were maintained in the cardiac enrichment medium for 3 days. After this enrichment phase, the medium was changed to RPMI 1640 with knockout serum replacement (KSR) (Thermo Fisher Scientific, United States). On day 20, spontaneously beating cardiomyocytes were dissociated with TrypLE Express enzyme (Thermo Fisher Scientific, United States), centrifuged, resuspended, and replated onto Matrigel-coated plates for follow-up experiments. hiPSC-derived cardiomyocytes were treated with 0.5 mM AICAR (Selleck, United States) or vehicle (DMSO) for 7 days from day 23 to day 30 (Supplementary Video 2). All cultures were grown at 37°C in 5% O₂ and 5% CO₂.