Cardiomyocyte Differentiation of Pluripotent Stem Cells

All experiments were approved by the University of Washington Embryonic Stem Cell Oversight Committee (ESCRO) and conducted using the mT/mG 2APuro RuES-2 hESC line [14 (link)] and the IMR90 hiPSC line. PSCs were maintained and differentiated as previously described [6 (link)]. Briefly, PSCs were cultured in Matrigel-coated plates (BD Biosciences) and fed with MEF-conditioned media supplemented with 4 ng/mL bFGF (for RuES2) or mTeSR (for IMR90). For cardiomyocytes differentiation, PSC were passaged using versene–EDTA and replated at a density of 150,000–200,000 cells/cm². To start differentiation, we replaced the mTeSR with RPMI-B27 (Gibco) supplemented with L-glutamine, Matrigel, and 100 ng/mL recombinant human activin A (R&D Systems) [2 (link)]. After 24 h, the medium was switched to RPMI-B27 supplemented with L-glutamine and 10 ng/mL recombinant human bone morphogenetic protein-4 (BMP-4; R&D Systems). Four days later, the medium was aspirated, and the cells were subsequently fed every other day with RPMI-B27 containing L-glutamine. Cells typically began beating spontaneously on approximately day 14 after differentiation.