Hyperoxia impairs induced pluripotent stem cell-derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype.

Background: Induced pluripotent stem cells (iPSCs) directed to endothelial identity (iPSC-ECs) are emerging as a potent tool for regenerative medicine in vascular disease. However, iPSC-ECs lose expression of key identity markers under standard in vitro conditions, limiting their clinical applications. Methods: To model physiological in vivo conditions, we examined the bioenergetics, presence of key cell markers, and proliferative and angiogenic capacity in iPSC-ECs at late and early passage under hyperoxic (21%) and physiological (4%) oxygen concentrations. Results: Physoxia resulted in relative preservation of mitochondrial bioenergetic activity, as well as CD144 expression in late passage iPSC-ECs, but not proliferative capacity or tube formation. Single cell RNA sequencing (scRNA-seq) revealed that late passage hyperoxic iPSC-ECs develop an endothelial-to-mesenchymal phenotype. Comparing scRNA-seq data from iPSC-ECs and from atherosclerotic ECs revealed overlap of their transcriptional phenotypes. Conclusions: Taken together, our studies demonstrate that physiological 4% oxygen culture conditions were sufficient to improve mitochondrial function in high passage cells, but alone was insufficient to preserve angiogenic capacity. Furthermore, late passage cells under typical conditions take on an endothelial-to-mesenchymal phenotype with similarities to ECs found in atherosclerosis.