IF1 restrains excessive elevation of the mitochondrial membrane potential and safeguards the epithelial state of human induced pluripotent stem cells

Human pluripotent stem cells (hPSCs) rely predominantly on glycolysis and exhibit relatively low mitochondrial respiration. Under these conditions, the mitochondrial F1Fo ATP synthase tends to operate in reverse mode, hydrolyzing ATP. ATP synthase inhibitory factor subunit 1 (IF1) inhibits this F1Fo ATP hydrolysis, but its role in hPSCs remains unclear. Here, we generated human induced pluripotent stem cells (hiPSCs) with stable IF1 knockdown (IF1-KD). IF1-KD enhanced F1Fo ATP hydrolysis and elevated the mitochondrial membrane potential (MMP). Although core pluripotency transcription factors were maintained, IF1-KD cells exhibited a partial epithelial-mesenchymal transition (EMT)-like state and biased trilineage differentiation. Mechanistically, the elevated MMP was accompanied by enhanced store-operated Ca²⁺ entry (SOCE) and nuclear translocation of NFATc3. Moreover, lowering the MMP attenuated SOCE, and NFATc3 overexpression reproduced the EMT-like gene expression. These results support a model in which IF1, by inhibiting F1Fo ATP hydrolysis, prevents excessive elevation of the MMP and thereby suppresses the transition to a partial EMT-like state via the MMP–SOCE–NFAT axis, contributing to the maintenance of the epithelial state associated with hiPSC pluripotency.