Cardiomyocyte Differentiation from Human Pluripotent Stem Cells

H1, H9, and PGP1 hPSCs were cultured on growth factor-reduced Matrigel (Corning, 354230)-coated well plates with mTeSR1 media (STEMCELL Technologies, 85850). For CM differentiations, hPSCs were passaged with Accutase (Innovative Cell Technologies, AT104) and treated with small molecular inhibitors as previously described.³² (link) Briefly, hPSCs were grown in mTeSR1 media (STEMCELL Technologies, 85850) until 90% confluence. Cells were then treated with 12 μM CHIR99021 (Tocris Bioscience, 4423) in RPMI 1640 media (Gibco, 11875) containing B27 supplement (Thermo, 17504), defined as day 0 of the differentiation. On day 1, CHIR99021 was removed and cells were cultured in RPMI 1640 media containing B27 minus insulin supplement (Thermo, A18956). On day 3, combined media consisting of 1:1 ratio of used media and fresh RPMI 1640 containing B27 minus insulin media and 5 μM IWP2 (Tocris Bioscience, 3533) were added to cells. Media were changed to RPMI 1640 containing B27 minus insulin on day 5, followed by media changes with RPMI media containing B27 supplement on days 7, 9, and 11. Cardiomyocytes were used for experiments as described on day 12.