Flow-Induced Transcriptomic Remodeling of Endothelial Cells Derived From Human Induced Pluripotent Stem Cells

Abstract

The vascular system is essential for the development and function of all organs and tissues in our body. The molecular signature and phenotype of endothelial cells (EC) are greatly affected by blood flow-induced shear stress, which is a vital component of vascular development and homeostasis. Recent advances in differentiation of ECs from human induced pluripotent stem cells (hiPSC) have enabled development of in vitro experimental models of the vasculature containing cells from healthy individuals or from patients harboring genetic variants or diseases of interest. Here we have used hiPSC-derived ECs and bulk- and single-cell RNA sequencing to study the effect of flow on the transcriptomic landscape of hiPSC-ECs and their heterogeneity. We demonstrate that hiPS-ECs are plastic and they adapt to flow by expressing known flow-induced genes. Single-cell RNA sequencing showed that flow induced a more homogenous and homeostatically more stable EC population compared to static cultures, as genes related to cell polarization, barrier formation and glucose and fatty acid transport were induced. The hiPS-ECs increased both arterial and venous markers when exposed to flow. Interestingly, while in general there was a greater increase in the venous markers, one cluster with more arterial-like hiPS-ECs was detected. Single-cell RNA sequencing revealed that not all hiPS-ECs are similar even after sorting, but exposing them to flow increases their homogeneity. Since hiPS-ECs resemble immature ECs and demonstrate high plasticity in response to flow, they provide an excellent model to study vascular development.

Keywords: RNA sequencing; endothelial cells; flow; induced pluripotent stem cells; shear stress; single-cell RNA sequencing.

FIGURE 1

Characterization of hiPS-ECs. (A), PECAM1 and VE-cadherin IF staining of hiPS-ECs (white) and uptake of oxLDL by hiPS-ECs (red). Nuclei are shown in blue. Scale bar 50 μm. (B), Matrigel tube assay of hiPS-ECs and HUVECs (scale bar 50 μm). The cells were plated on Matrigel and imaged every day for 72 h. (C), Phase-contrast images of hiPS-ECs after 24 h exposure to flow-induced shear stress and under static conditions. The cells in both conditions are from the same differentiation batch and images have been taken at the same time point just before processing them for scRNASeq. Scale bar 50 μm.

FIGURE 2

Characterization of single-cell RNA sequencing clusters (HEL47.2 cells). (A), UMAP (Uniform Manifold Approximation and Projection) plot of flow and static cells. (B), UMAP plot of the delineated clusters. (C), Dot plot for marker genes of the delineated clusters. (D), Feature plot of blood EC (PECAM1, CDH5) and lymphatic EC (PROX1, PDPN) EC marker gene expression. (E), Violin plot of EC marker genes (KDR, CD34) in all identified clusters.

FIGURE 3

The expression of venous and arterial genes in hiPSC-ECs flow and static clusters (HEL47.2). (A), Venous genes. (B), Arterial genes. (C), Volcano plot on the differential expression of flow arterial-like cluster 3 genes compared to all the other flow clusters.

FIGURE 4

Gene Ontology (GO) analysis of the scRNASeq and bulk RNASeq data (DAVID Bioinformatics Resources). (A), Venn diagram of upregulated and downregulated genes shared between scRNASeq and bulk RNASeq. (B), Upregulated and downregulated GO terms in bulk RNASeq data (C), Upregulated and downregulated GO terms in scRNASeq data. (D), A list of genes included in the GO term categories that were both upregulated and downregulated.

FIGURE 5

The effect of flow on hiPS-EC gene expression (HEL47.2). (A), Violin plots for selected genes upregulated by flow. (B), Downregulated genes in response to flow.

FIGURE 6

Bulk RNASeq analysis of the effect of flow compared to static condition. (A), A volcano plot of differentially expressed genes in flow compared to static hiPS-ECs. (B), Heat map of the 50 most significantly changed genes by flow. Red denotes upregulation and blue downregulation.

FIGURE 7

NOTCH and VEGF pathway gene expressions in scRNASeq (HEL47.2). (A), NOTCH pathway gene expression in flow and static clusters. (B), VEGF pathway genes in flow and static clusters.