Detailed methods are provided in the Online Data Supplement . In brief, cardiomyocytes were generated from H7 hESCs using our recently reported directed differentiation protocol, which involves serum-free monolayer culture and serial treatment with activin A and bone morphogenetic protein-4 (BMP4)1 (link). Where indicated, this differentiation protocol was modified by supplementation with ErbB agonists or antagonists. At two weeks following induction of differentiation with activin, cultures were enzymatically dispersed to single cells and re-plated on glass coverslips at low density. Unless otherwise stated, phenotyping studies were performed on days 20–25 post-induction, using cell preparations comprised of ~60% cardiomyocytes (Online Figure I ).
For selected experiments, hESC-CM cultures were transduced with a lentiviral vector in which the proximal promoter-enhancer region of the chicken GATA6 (cGATA6) gene drives expression of enhanced green fluorescent protein (EGFP)29 (link), 30 (link). Parallel control experiments indicated ~50% transduction efficiency. Transduced cells were employed in electrophysiological or immunocytochemical studies at 3–4 days post-transduction, which corresponds to 22–25 days post-induction with activin.
For selected experiments, hESC-CM cultures were transduced with a lentiviral vector in which the proximal promoter-enhancer region of the chicken GATA6 (cGATA6) gene drives expression of enhanced green fluorescent protein (EGFP)29 (link), 30 (link). Parallel control experiments indicated ~50% transduction efficiency. Transduced cells were employed in electrophysiological or immunocytochemical studies at 3–4 days post-transduction, which corresponds to 22–25 days post-induction with activin.
