Selective Elimination of Genetic Variants of Human Embryonic Stem Cells from High Vulnerability to Ferroptosis

Despite the great promise of human pluripotent stem cell (hPSC)-based cell therapy, safety concerns arise from genetic aberrations during in vitro culture, due to their uncertain consequences. Notably, these genetic aberrations confer a survival trait known as "culture-adaptation", allowing aberrant hPSCs to evade apoptosis and outcompete normal cells. Thus, it is crucial to develop strategies for selectively eliminating aberrant hPSCs to ensure the safety of therapeutic applications. Herein, we discovered that hPSCs with genetic variations exhibited increased glycolysis and active fatty acid biosynthesis. Surprisingly, these variants, showing resistance to stress-induced apoptosis, were paradoxically susceptible to ferroptosis by the treatment of RAS-selective lethal 3 (RSL3), a glutathione peroxide 4 inhibitor. The selective sensitivity to RSL3 resulted from elevated levels of polyunsaturated fatty acids containing phospholipids, driven by the up-regulation of acyl-coenzyme A synthetase long-chain family member 4 through Yes1-associated protein 1 activity. Importantly, the distinct sensitivity of normal hPSCs and metabolic variants to ferroptosis enabled the targeted removal of genetically aberrant hPSCs through RSL3 treatment, while normal hPSCs transiently exposed to RSL3 maintained their pluripotency and normal differentiation capacity. These findings hold important promise for the maintenance of genetically normal hPSCs during extended in vitro culture, thereby ensuring the safety and efficacy of hPSC-based cell therapies.