ETV2 Overexpression Promotes Efficient Differentiation of Pluripotent Stem Cells to Endothelial Cells

Differentiating endothelial cells (ECs) from human pluripotent stem cells (hPSCs) typically takes 2 weeks and requires parameter optimization. Overexpression of cell type-specific transcription factors in hPSCs has shown efficient differentiation into various cell types. ETV2, a crucial transcription factor for endothelial fate, can be overexpressed in hPSCs to induce rapid and facile EC differentiation (iETV2-ECs). We developed a two-stage strategy which involves differentiating inducible ETV2-overexpressing hPSCs in a basal induction medium during stage I and expanding them in an endothelial medium during stage II. By optimizing seeding density and medium composition, we achieved 99% pure CD31+ CD144+ iETV2-ECs without cell sorting in 5 days. iETV2-ECs demonstrated in vitro angiogenesis potential, LDL uptake, and cytokine response. Transcriptomic comparisons revealed similar gene expression profiles between iETV2-ECs and traditionally differentiated ECs. Additionally, iETV2-ECs responded to Wnt signaling agonist and TGFβ inhibitor to acquire brain EC phenotypes, making them a scalable EC source for applications including blood-brain barrier modeling.