Early manipulation of the WNT and BMP pathways partially rescues the CM defects in GATA6 loss-of-function hESCs.
(A) Schematic for treatment with DOX (days 1-4), CHIR (3μM, days 0-2), and/or reduced BMP4 (5ng/mL, days 0-2, indicated as “LB”) during CM directed differentiation. (B) Day 5 flow cytometry quantification for %K+P+ double-positive cells, %KDR+ single-positive cells, or %PDGFRα+ single-positive cells in GATA6-/- hESCs transduced with iLGR5 or empty vector (EV) (n=5). Significance indicated by *p<0.05 according to a two-tailed paired Student’s t-test. (C) %cTnT+ CMs from days 13-18 of cardiac differentiation quantified by flow cytometry in GATA6-/- cells treated with CHIR LB or vehicle (DMSO) with normal BMP4 concentration treated control (n≥6). (D) Flow cytometry at day 5 of cardiac differentiation to quantifiy %K+P+ double-positive, %KDR+ single-positive, or %PDGFRα+ single-positive cells comparing GATA6-/- hESCs treated with CHIR LB with GATA6+/+ and GATA6-/- hESCs controls treated with vehicle and normal BMP4 concentration (n≥8). (E) %cTnT+ CMs from days 13-18 of cardiac differentiation quantified by flow cytometry in GATA6+/- or WT hESCs treated with CHIR (3μM) or DMSO (n≥7). Data represents the mean ± SEM, significance indicated by **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Student’s t-test (C) and one-way ANOVA (D) or two-way ANOVA (E) with Tukey’s multiple comparisons test.