Differentiation of hiPSC-CMs Using Small Molecules

Pluripotent stem cells were generated by reprogramming skin fibroblasts (ID MRIi004-A) of a healthy human donor according to established methods 19 (link). The study participant provided written informed consent and the investigation conformed to the principles outlined in the Declaration of Helsinki. For maintenance culture, hiPSCs were expanded in matrix-coated dishes (Geltrex LDEV-Free, Gibco), using Essential 8 medium (Gibco), which was changed on a daily basis. Cells were split at 85% confluency, using EDTA for dissociation (Versene solution; Gibco). A state-of-the-art differentiation protocol for hiPSC-CMs using small molecules to induce differentiation was adapted from Chen and colleagues 20 (link). Briefly, cardiac differentiation was initiated at about 85% confluence by applying chemically defined factors for 24 h (medium A) and subsequent 48 h (medium B, PSC Cardiomyocyte Differentiation Kit; Gibco). After 72 hours, differentiation of hiPSC-CMs was continued for 12 days with Cardiomyocyte Maintenance Medium (PSC Cardiomyocyte Differentiation Kit; Gibco) that was changed every other day. The workflow is displayed in Figure 1A.