PRODH safeguards human naive pluripotency by limiting mitochondrial oxidative phosphorylation and reactive oxygen species production

Naive human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naive hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901, a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy, DNA damage, and apoptosis. Remarkably, MitoQ, a mitochondria-targeted antioxidant, effectively restored the pluripotency and proliferation of PRODH-knockdown naive hESCs, indicating that PRODH maintains naive pluripotency by preventing excessive ROS production. Concomitantly, PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus, we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production, and thereby safeguarding naive pluripotency of hESCs. Downregulation of PRODH promotes oxidative phosphorylation and ROS production, which in turn impair pluripotency and proliferation of naive but not primed hESCs, revealing a crucial role of PRODH in safeguarding human naive pluripotency.PRODH is expressed in naive hESCs at a higher level compared to their primed counterparts. MEK inhibitor present in naive culture media upregulates PRODH by suppressing MYC. PRODH depletion boosts mtOXPHOS and ROS production in naive hESCs. PRODH promotes proteolytic degradation of the ETC complex components. Downregulation of PRODH promotes oxidative phosphorylation and ROS production, which in turn impair pluripotency and proliferation of naive but not primed hESCs, revealing a crucial role of PRODH in safeguarding human naive pluripotency.