Feeder-free generation and characterization of endocardial and cardiac valve cells from human pluripotent stem cells

Figure 1. Feeder-free generation of endocardial and endothelial populations from iPSCs (A) Schematic of differentiation timeline. (B) Flow cytometry plots assessing mesoderm generation on day 4 of their respective differentiation strategies. (C) Quantification of CD13+/CD56+ mesoderm generation (n = 4, ∗p < 0.05, two-way ANOVA with Sidak’s multiple comparisons test, error bars ±SEM). (D) Flow cytometry plots assessing endocardial/endothelial differentiation efficiency on day 14 (also see Figure S1). (E) Timeline of CD31+ endocardial differentiation on days 10, 12, and 14 (n = 4, ∗p < 0.05 for WTC11, ##p < 0.01 for MSN02-4, two-way ANOVA with Dunnett’s multiple comparisons test, error bars ±SEM). (F) Quantification of CD31+ endocardial efficiency on day 14 (n = 5, ∗p < 0.05, two-way ANOVA with Sidak’s multiple comparisons test, error bars ±SEM).

Figure 2. BMP10-derived endocardial cells are distinct from VEGF-derived endothelial cells (A) RT-qPCR analysis of cardiac and endocardial gene expression on day 14 endocardial and endothelial cells (n = 4, ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA, error bars ±SEM). (B) Representative confocal microscopy of MSN02-4 endocardial and endothelial cells, stained for CD31 (GFP) and NKX2-5 (RFP). Scale bar, 50 μm. (C) Representative confocal microscopy of MSN02-4 endocardial and endothelial cells, stained for NFATc1 (GFP) and CDH5 (RFP) (also see Figure S2). White arrowheads = diffuse NFATc1 in the cytoplasm of endothelial cells. Scale bar, 50 μm.

Figure 3. EndMT of endocardial cells generates VIC and VEC populations (A) Flow cytometry plots following EndMT (also see Figure S1). (B) Quantification of CD31− VIC and CD31+ VEC populations following EndMT (n = 4, error bars ±SEM). (C) RT-qPCR analysis of mesenchymal and endothelial gene expression on day 14 endocardial cells and day 20/21 VICs/VECs (n = 4, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test, error bars ±SEM).

Figure 4. Immunofluorescence analysis of VICs and VECs (A) Representative confocal microscopy of WTC11 VICs stained for PERIOSTIN (GFP) and VIMENTIN (RFP). (B) Representative confocal microscopy of MSN02-4 VICs stained for αSMA (GFP) and SOX9 (RFP). (C) Representative confocal microscopy of WTC11 VECs stained for CDH5 (GFP) and CD31 (RFP).

Figure 5. Bulk RNA-seq characterization of iPSC-derived endocardial and endothelial cells (also see Figure S3 and File S1) (A) Heatmap of the top 50 DEGs ranked by variance between endocardial and endothelial populations. (B) Volcano plot of DEGs between endocardial vs. endothelial populations. Cutoff for log2 fold change was set to 1 and -1; padj < 10−3. (C) Top 10 biological process GO terms for upregulated DEGs in endocardial cells, ranked by p value. (D) Top 10 biological process GO terms for upregulated DEGs in endothelial cells, ranked by p value.

Figure 6. Bulk RNA-seq characterization of iPSC-derived VIC and VECs (also see Figure S4 and File S1) (A) Heatmap of top 50 DEGs ranked by variance between VIC and endocardial populations. (B) Volcano plot of DEGs between VIC vs. endocardial populations. Cutoffs for log2 fold change was set to 1 and -1; padj < 10−3. (C) Top 10 biological process GO terms for upregulated DEGs in VICs, ranked by p value. (D) Heatmap of top 50 DEGs ranked by variance between VEC, endocardial, and endothelial populations. (E) Volcano plot of DEGs between VECs vs. endocardial populations. Cutoff for log2 fold change was set to 1 and -1; padj < 10−3. (F) Top 10 biological process GO terms for upregulated DEGs in VECs, ranked by p value.

Figure 7. Comparison of iPSC-derived VICs and VECs to human fetal heart valve cells (A) PCA plot of iPSC-derived VICs, VECs, and human fetal heart valve cells from a normal 15-week-old fetus (labeled Liu_W15). (B) Pearson correlation matrix comparing iPSC-derived VICs and VECs to their respective human fetal valve populations. (C) Heatmap with unsupervised clustering on the top quartile of genes sorted by variance, showing human fetal valve VICs and VECs clustering with their respective iPSC-derived populations.