ETV2 Overexpression Promotes Efficient Differentiation of Pluripotent Stem Cells to Endothelial Cells
Yunfeng Ding, Soniya Tamhankar, Feifan Du, Tessa Christopherson, Nate Schlueter, Jenna R. Cohen, Eric V. Shusta, Sean P. Palecek
Abstract
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.