Monolayer-directed Cardiac Differentiation of hiPSCs

Monolayer-directed differentiation of hiPSC into CMs was performed as described previously with slight modifications [20 (link)]. Four days before initiation of cardiac differentiation, hiPSCs were split in a 1:8 ratio. On day 0 of cardiac differentiation, hiPSCs with 90% confluence were incubated with RPMI-B27 without insulin (Life Technologies, CA, USA) supplemented with 6 μM CHIR99021 (LC Laboratories, MA, USA) for 48 h. After withdrawn of CHIR99021, cells were kept in RPMI-B27 without insulin for another 48 h, and followed by IWR1 (LC Laboratories) treatment in the same basal medium for 48 h. Media were then switched to RPMI-B27 supplemented with insulin for further culturing. Beating hiPSC-CMs were expected to be observed from day 9 post initiation of cardiac differentiation. On day 11, derived hiPSC-CMs were metabolically purified by 4-day culture in RPMI-B27 without d-glucose, and then maintained in RPMI-B27 supplemented with insulin.