Wnt-modulated cardiomyogenesis is enhanced by Matrigel overlay.

<p>Pluripotent H1 ESCs expanded on Matrigel in mTeSR1 medium were treated with Matrigel overlays on Day -3 or -1. Differentiation was induced by changing medium to RPMI/B27 (no insulin) containing the small MW inhibitors CHIR (12 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5; insulin (4,000 ng/ml) was included after Day 7. <b>A</b>, scheme of cardiomyogenic induction using Matrigel overlay and small MW inhibitors; arrows denote days when medium was changed, before (green) and after (red) induction. <b>B</b>, typical flow cytometry results showing percentages of cardiac troponin-T (cTnT)-positive cells at Day 14. <b>C</b>, αMHC (MF20) immunostaining at Day 14. <b>D</b>, bar graph showing averaged flow cytometry results obtained in three independent determinations. Vertical lines depict ± SEM. Size bars in C = 200 μm. The p-value in D was calculated using Student’s t-test.</p

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.</p

RNA-seq heat map revealing endogenous gene expression patterns during cardiomyogenesis induced with CHIR alone.

<p>Pluripotent H1 ESCs expanded on Matrigel in mTeSR1 medium were induced to differentiate by changing the medium to RPMI/B27 (without insulin) including the small MW inhibitors CHIR (12 μmol/L) during Day 0–1 and IWP (5 μmol/L) during Days 3–5; insulin (4,000 ng/ml) was included after Day 7. RNA was purified from duplicate cultures on the indicated days, converted to cDNA, and processed to RNA-seq libraries as described in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118670#pone.0118670.s011" target="_blank">S1 Methods</a></b>. Colors indicate the range of each gene’s expression during the 14 day period, with least expression shown in green and highest expression shown in red (see inset). The number in each panel indicates the expression of each gene in transcripts per million (TPM) within each culture dish.</p