Abstract 1146: Metabolic Regulation Of Induced Pluripotent Stem Cell Derived Endothelial Cells Regenerative Capacity

Introduction: Induced pluripotent stem cells (iPSCs) derived endothelial cells (ECs) have emerged as a novel treatment strategy to restore limb perfusion in a murine model of peripheral artery disease (PAD). However, our lab has demonstrated that iPSC-EC in culture experience a failure of mitophagy and loss of cellular identity, limiting therapeutic implementation. Metabolic regulation of this mechanism is unknown. Methods: y6 human iPSCs were differentiated as previously described (Patsch Nat Cell Bio 2015) to generate ECs. Resulting iPSC-ECs were cultured under varying oxygen conditions (standard 21% vs. physoxic 4%). Markers of cellular identity and metabolic profile were analyzed. Results: iPSC-ECs lost cellular identity markers VE-Cadherin (55% positive in physoxic vs. 29% positive in standard) and PECAM1 (46% positive in physoxic vs. 39% positive in standard) over a period of two weeks (p<0.01). Single-cell RNA sequencing demonstrated the loss of cellular identity coincided with upregulation of endothelial-to-mesenchymal transition (EndMT), including COL1A1/2 and TGF-β. Physoxia significantly altered key metabolic proteins compared with standard conditions. Neuropilin 1 (Nrp1), an angiogenic related protein, was significantly increased in standard conditions (40% increased, p<0.01). PFKFB3, pathological upregulation of which correlates with increased EMT, was favorably downregulated in physoxic (52% lower, p<0.05) compared to standard conditions. Conclusion: iPSC-ECs under standard culture conditions demonstrated loss of EC identity with EndMT transition by single-cell transcriptomic analysis and by Western blot analysis of key metabolic proteins. Physoxia favorably impacted retention of key iPSC-EC identity markers. While increased Nrp1, as seen under standard conditions, can be associated with angiogenesis, the concomitant increase in PFKFB3 suggests an unfavorable metabolic activation and shift towards EndMT. Overactivation of PFKFB3 can results in net loss of energy in iPSC-ECs which leads to uncontrolled EndMT, and impaired regenerative capacity. Our results demonstrate iPSC-ECs effectively maintain metabolic control when grown in physoxic conditions.