Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells

Abstract

Rationale: Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology.

Objective: Here, we performed droplet-based single-cell RNA sequencing (scRNA-seq) of the human iPSCs after iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity.

Methods and results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of NO. Nonendothelial cell populations resulting from the differentiation protocol were identified, which included immature cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with 4 major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes, respectively.

Conclusions: Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of nonendothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.

Keywords: computational biology; endothelial cells; induced pluripotent stem cells; myocytes, cardiac; stem cells.

Figure 1.. Monolayer-based differentiation of human iPSCs to endothelial cells.

(A) Schematic representation of endothelial cell differentiation from human iPSCs. At day 12 of differentiation, bona fide iPSC-ECs are purified by MACS sorting with bead-conjugated CD144 antibody and cultured for up to five passages. t-SNE plots of scRNA-seq at (B) day 8 and (C) day 12 of human iPSCs subjected to EC differentiation. Heatmaps of enriched gene expression for each cluster of cells in (D) day 8 and (E) day 12 of differentiation.

Figure 2.. Identification of bona fide iPSC-EC cluster.

(A) Number of cells counted in each of ten clusters of day 8 (top) and of eight clusters of day 12 (bottom) of iPSC-EC differentiation. (B) Expression of early endothelial cell signatures (CD34, KLF2, ECSCR), (C) mature endothelial cell signatures (CDH5, ERG, FLT1), and (D) non-endothelial cell signatures (mesenchymal marker ACTA2, cardiac marker TNNT2, hematopoietic marker KIT) visualized by ViolinPlots (left) and FeaturePlots (right).

Figure 3.. Canonical correlation analysis of day 8 and day 12 iPSC-EC differentiation.

(A) Canonical correlation analysis (CCA) of day 8 and day 12 samples combines the two datasets. (B) t-SNE plot of CCA shows cells from two independent samples correlating based on transcriptional similarity. (C) Heatmap (left) and t-SNE plot (right) of single integrated analysis reveal seven canonically-correlated (CC) clusters (CC0-CC6). (D) Hierarchical analysis of seven CC clusters. (E) Percentage of cell number is determined for each CC cluster. (F) Pathway enrichment analysis shows statistically significant gene ontologies. (G) Intercellular communication analysis amongst CC clusters. Line color indicates ligands broadcast by the cell population of the same color (labeled). Lines connect to cell populations where cognate receptors are expressed. Line thickness is proportional to the number of ligands where cognate receptors are present in the recipient cell population. Loops indicate autocrine circuits. Map quantifies potential communication but does not account for anatomic position or boundaries of cell populations. (H) Biological function and identity of each CC cluster are identified and are labeled accordingly.

Figure 4.. Novel molecular signatures that define heterogeneous populations of human iPSC-ECs.

(A) Bona fide iPSC-EC cluster on the final day of differentiation is further analyzed by sub-clustering. The iPSC-ECs are divided into four distinct sub-clusters, marked by expression of CLDN5, APLNR, GJA5, and ESM1. (B) All iPSC-EC sub-clusters show high expression of endothelial markers KDR and CDH5 while no expression of cardiac markers TNNT2 or NKX2–5. (C) Heatmap shows enriched genes of four iPSC-EC sub-clusters. (D) Representative ViolinPlots and FeaturePlots of enriched genes in sub-clusters. (E) Pathway enrichment analysis reveals statistically significant gene ontologies for each iPSC-EC sub-cluster. (F) GJA5+ cluster represents arterial-like endothelial cells (left). ESM1+ cluster represents activated endothelial cells with enriched expression of genes related to angiogenesis, regulation of cell death, cell adhesion molecule binding, and response to stress and chemical stimulus.