(A) Schematic of GiWi protocol for fully defined, growth factor-free differentiation of hPSCs to cardiomyocytes via treatment with small molecule modulators of Wnt signaling. (B–C) Cardiomyocytes were generated from 19-9-11 cells using the cardiac directed differentiation protocol described in (A), with exposure to 12 μM CHIR99021 at day 0 and 5 μM IWP4 at day 3. (B) At day 0, the indicated concentration of insulin was added into the culture medium. 15 days after differentiation, cTnT expression was determined by flow cytometry. Error bars represent the s.e.m. of three independent experiments. No statistically significant difference between samples were identified using one-way ANOVA and Tukey post tests (p>0.05). (C) 10 μg/ml insulin was added to the culture medium at different time points of differentiation. 15 days after initiation of differentiation, cells were analyzed for cTnT expression by flow cytometry. Error bars represent the s.e.m. of three independent experiments. * p<0.05, data were compared using one-way ANOVA and Tukey post tests, with the asterisk indicating day 1 is significantly different from other samples.