Mitochondrial Pyruvate Carrier Differentially Controls the Self‐Renewal and Differentiation of Human Pluripotent Stem Cells

Mitochondria are crucial for cell fate determination, yet their roles in human pluripotent stem cell (hPSC) fate changes have remained underexplored. Here, we designed a CRISPR library targeting 661 mitochondrial proteins and identified the MPC (mitochondrial pyruvate carrier) as a critical regulator of hPSC self‐renewal and pluripotency. Notably, MPC inhibition reduced hPSC self‐renewal and endoderm differentiation while promoting mesoderm differentiation, with no effect on ectoderm differentiation, all mediated by influencing glycolytic acetyl‐CoA production. Specifically, the decrease in acetyl‐CoA following MPC inhibition affected histone acetylation in hPSCs, compromising self‐renewal. In contrast, MPC inhibition did not impact histone acetylation in differentiated cells; instead, it reduced the acetylation of non‐histone proteins—EP300 and SMAD2—thereby enhancing mesoderm differentiation and repressing endoderm differentiation, respectively. These findings suggest that distinct effector proteins respond to variations in acetyl‐CoA levels at different developmental stages, leading to a context‐dependent regulation of cell fate determination by glycolytic acetyl‐CoA in hPSCs.