FIGURE 1

Differentiation of Mature-Like Epicardial Cell Cultures from hiPSCs. (A) Schematic of protocol used for hEpiC differentiation. (B) Brightfield images of hEpiCs through four passages showing the gradual accumulation of the classic epithelial cell cobblestone morphology; scale bar: 500 µm. (C) qRT-PCR data for epicardial markers WT1 and TCF21 in hiPSCs and hEpiCs; n = 4-6 per cell line. (D) Confocal immunofluorescent images for TCF21 (green) and DAPI (blue) showing nearly 100% epicardial differentiation efficiency from hiPSCs; scale bar: 100 µm. (E,F) Time course qRT-PCR data for WT1 (E) and CDH1 (F) throughout hEpiC differentiation, suggesting an increase in epicardial nature over time; n = 3 per time point. (G) Confocal immunofluorescent images showing robust expression of epithelial markers after removal of Vitamin C and SB431542 from the culture media. Epicardial cells are labeled with WT1 (green), epithelial membranes with TJP1 (magenta), nuclei with DAPI (blue); scale bar: 50 µm. (H) qRT-PCR data for hEpiCs in the presence or absence of SB431542, showing no change in smooth muscle or fibroblast differentiation; n = 3 per condition. (I) Absolute counts of DAPI-labeled nuclei after exposure to different combinations of Vitamin C and SB431542; n > 20 per condition. (J) qRT-PCR data for hEpiCs in the presence or absence of SB431542 (SB), showing an increase in maturity when this compound is removed from the media; n = 3 per condition.