Fig. 1: Generation of cardiac organoids with ventricular cardiomyocytes and epicardial cells.

A Protocol to generate epicardial cells from human pluripotent stem cells. B Left; Representative flow cytometry analyses of Aldeflour (ALDH) in day15 retinol (ROH)-treated (ROH(+)) and untreated (ROH(-)) epicardial cells. Right; Quantification of the proportion of ALDH positive cells and total number of cells in ROH-treated and untreated epicardial population (N = 8 biologically independent samples). Statistical analysis was performed by two-sided unpaired t-test. C RT-qPCR expression analyses of ALDH1A2, TCF21, and GATA5 in the indicated populations (N = 4 biologically independent samples). Statistical analysis was performed by two-sided unpaired t-test. D Quantification of the proportion of ALDH positive cells and total number of cells in ventricular and atrial mesoderm-derived epicardial cells (N = 8 biologically independent samples). Statistical analysis was performed by two-sided unpaired t-test. E RT-qPCR expression analyses of epicardial marker genes in the indicated populations (N = 3 biologically independent samples). Statistical analysis was performed by two-sided unpaired t-test. F RT-qPCR expression analyses of epicardial marker genes in the indicated time points (N = 3 biologically independent samples). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons. G Schema of the coculture of GFP-positive epicardial cells and ventricular cardiomyocyte derived from hPSCs. H Representative immunostaining of day 1, 4, and 14 cardiac organoids. Scale bar; 200 μm. (N = 5 biologically independent samples). I Representative immunostaining of Ki67 in ventricular cardiomyocyte aggregates, cardiac organoids, and cardiac organoids treated with ROH. Scale bar; 20 μm. J Quantification of the relative number of cardiomyocytes in the cardiac organoids and cardiomyocyte aggregates (V-CM Agg) treated as indicated (N = 6 biologically independent samples). The number of cardiomyocytes in V-CM only without ROH (ROH(-)) was defined as 1.0 and the relative number of cardiomyocytes was calculated in each condition. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons. All error bars represent SEM. All values shown for the PCR analyses are relative to the housekeeping gene TBP. V-CM ventricular cardiomyocytes. Epi epicardial cells. Source data are provided as a Source Data file. Created with BioRender.com.