Directed Cardiac Differentiation of hiPSCs

Protocol was adapted from Lian et al. [40 (link)] with some modifications as we previously reported [32 (link)]. Briefly, hiPSCs were grown until 70–90% confluence in six-well plates. Culture media was replaced with the differentiation medium RPMI 1640 + B-27™ Supplement (without insulin) (Thermo Fisher Scientific, USA) + 0.6 mM L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA), designated as RPMI/B27^-IN, and GSK3β inhibitor CHIR99021 (Selleckchem, USA) 4-12 μM was added [32 (link)]. The differentiation medium was changed after 24 h in order to remove/reduce CHIR concentration to less than 1.5 μM. Wnt inhibitor IWR-1 (Selleckchem, USA) 2.5 μM was added on day 2 [32 (link)] and was removed during the medium change on day 5. Cultures were maintained in the differentiation medium with the medium changed every 24 h. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. All small molecules were solubilized in DMSO (Sigma-Aldrich, USA). Cardiac markers: NK2 Homeobox 5 (NKX2–5), Troponin T and Myosin light chain 2a (MLC2a); endodermal marker hepatocyte nuclear factor 4 alpha (HNF4a; and mesodermal marker (CD44) were measured by flow cytometry on day 14 differentiation.