Fig. 2: Characterization epicardial-derived cells (EPDCs) in the cardiac organoids.

A Schema of the generation of the cardiac organoids using the non-labeled epicardial cells and RFP-positive ventricular cardiomyocytes derived from hPSCs, and the strategy to purify the EPDCs and the cardiomyocytes from the cardiac organoids by FACS. B Representative flow cytometry analyses of Aldefluor (ALDH) and CD90 expression in the cardiac organoids in the indicated time points. C Left; Representative flow cytometry analyses of 3 subpopulations in  the organoids. Right; RT-qPCR expression analyses of epicardial (ALDH1A2, BNC1), transcription factor (TCF21, GATA4, GATA6, HAND2), extracellular matrix (FN1, POSTN, COL1A1), and smooth muscle (ACTA2, MYH11, MYLK) genes in the indicated populations (N = 5 biologically independent samples). Adult and fetal cardiac fibroblasts (CF) and skin BJ fibroblasts were included as a reference. Epi: day15 epicardium prior to coculture, CF: adult and fetal cardiac fibroblasts (N = biologically independent samples each). BJF: skin BJ fibroblasts (N = 1). D Representative immunostaining of WT1 and GFP (EPDCs) in the untreated and SB431542 (SB)-treated cardiac organoids. Scale bar; 20um. (N = 5 biologically independent samples). E Upper; Representative flow cytometry analyses of ALDH and CD90 expression in the SB-treated cardiac organoids. Lower; Quantification of the proportion of ALDH+ cells in each population from the flow cytometry analyses (N = 6 biologically independent samples). F RT-qPCR expression analyses of FN1 and COL1 in CD90-positive populations in the SB-treated and untreated organoids (N = 5 biologically independent samples) and of ACTA2 and MYH11 in ALDH-negative / CD90-negative populations isolated (FACS) from the SB-treated and untreated organoids (N = 5 biologically independent samples). G Left; Representative flow cytometry analyses of ALDH and CD90 expression in the retinol (ROH)-treated cardiac organoids. Right; Quantification of the proportion of each population in the flow cytometry analyses (N = 6 biologically independent samples). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons in (C) and by two-sided unpaired t-test in (E)–(G). All error bars represent SEM. V-CM ventricular cardiomyocytes. Source data are provided as a Source Data file. Created with BioRender.com.