Activin-A levels during Day 0–1 modulate CM vs. DE differentiation.

<p>Pluripotent H1 ESCs were induced by changing medium to RPMI/B27 (no insulin), including CHIR (7.5 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5. Activin-A was included at the indicated levels during Day 0–1. Insulin (4,000 ng/ml) was included after Day 7. <b>Panel A</b>, a-e shows cells double-immunostained on Day 5 for Oct4 (red) and Sox17 (green); <b>e</b> is a positive control wherein cells were induced to DE with Activin-A (50 ng/ml) and Bmp4 (10 ng/ml) during Days 0–5. <b>Panel A f-i</b> shows cells immunostained with MF20 mAb on Day 14 to detect cardiomyocytes. <b>Panel B</b> depicts the effect of Activin-A levels during Day 0–1 on cardiomyocyte differentiation at Day 14, determined by flow cytometry using anti-cTnT. Cultures treated with 10 ng/ml Activin-A began to rhythmically contract at Day 6. Cells treated with 50 or 100 ng/ml Activin-A did not beat at any time. Bars indicate the average values combined from multiple experiments. Vertical lines = ±SEM. P-values were calculated by Student’s t-test. The p-value over the bar denoting 10 ng/ml Activin-A is relative to cells treated with CHIR only (0 ng/ml Activin-A), whereas the p-values over the bars denoting 50 and 100 ng/ml Activin-A are relative to cells treated with 10 ng/ml Activin-A. The size bar in Aa, which pertains to panels a-i, = 200 μm.