Cardiac Differentiation from Human iPSCs

Human iPSCs were maintained on Matrigel (BD Biosciences) coated plates in E8 medium (Life Technologies), which was changed daily33 (link). Prior to cardiac differentiation, hiPSCs were passaged once every four days with 0.5 mM EDTA at 37 °C for 5 minutes. After hiPSCs had achieved 95% confluence, cardiac differentiation was initiated by treating cells with 6 μM CHIR99021 (Selleck Chemicals) in RPMI + B27 without insulin for 48 hours (day 0 to day 2) according to a previously-published protocol23 (link). The medium was then changed to RPMI + B27 without insulin for 1 day, and 5 μM IWR1 (Selleck Chemicals) was added between days 3 to 5 of differentiation. From day 5 to 7, cells were returned to RPMI + B27 without insulin and without small molecules. Cells were then maintained on RPMI + B27 with insulin from day 7 onwards. Once spontaneous beating could be observed (usually between days 10 to 12 of differentiation), the medium was changed to RPMI without glucose + B27 with insulin for glucose starvation for 3 days to eliminate non-CMs in culture. This step typically resulted in greater than 90% CM purity. The surviving CMs were then passaged with trypLE^TM (Life Technologies) and replated for further studies.