Directed Cardiomyocyte Differentiation from Human Pluripotent Stem Cells

Human PSCs (WTC-11 or H7, Coriell) were maintained with mTeSR-1 media (StemCell Technologies) on Matrigel (BD Biosciences). Directed differentiation to cardiomyocytes was achieved through modulation of the Wnt pathway using small molecules^{28 (link)}. Briefly, on day 0 hPSCs were changed to mTesR with 1 μM Chiron 99021 (Tocris). After 24 h, media was changed to Roswell Park Memorial Institute (RPMI) 1640 media supplemented with B27 (minus insulin) containing 1 μM Chiron 99021. 48 h later, media was replaced with RPMI/B27(minus insulin) with 2 μM Wnt-C59 (Fisher). After 48 h, media was changed to the final cardio maintenance media, comprised of RPMI with B27 supplement containing insulin. Thereafter, media was replaced every 48 h. Between day 14 and 21, lactate selection with DMEM (no glucose) supplemented with 4 mM sodium lactate was performed for 72 h, after which cells were returned to cardio maintenance media (RPMI/B27)^{29 (link)}. Cardiomyocytes were used for experiments either directly following differentiation or were frozen and thawed for later use. Frozen cardiomyocytes were thawed into RPMI with B27 supplement containing insulin, 5% FBS, and 10 μM ROCK inhibitor Y-27632 (StemCell Technologies) for 24 h and then returned to cardio maintenance media prior to use.