Mitochondrial NADH-redox inflexibility constrains genomic and epigenetic stability in pluripotent stem cells

The electron transport chain (ETC) is essential for NAD⁺ regeneration and proliferation. While many cell types tolerate ETC inhibition when pyruvate or aspartate is supplied, pluripotent stem cells (PSCs) enter a reversible paused state even at abundant pyruvate levels. Here, we show that ETC inhibition triggers severe NADH reductive stress in mouse embryonic stem cells (mESCs), driven mainly by threonine dehydrogenase (TDH). TDH-derived NADH establishes a metabolic environment that disfavors cells with compromised mitochondrial function, maintains inhibition of pyruvate dehydrogenase (PDH), and is associated with increased genomic and epigenetic stability at the cellular population level. ETC inhibition similarly induces pausing in early mouse embryos and in human pluripotent stem cells (hPSCs). In hPSCs, combined inhibition of the one-carbon metabolism enzymes serine hydroxymethyltransferase (SHMT1/2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) effectively reduced reductive stress and rescued the paused phenotype. Together, these findings support a model in which limited mitochondrial redox adaptability represents a conserved metabolic feature of pluripotent stem cells and in which NADH reductive stress is associated with genomic and epigenetic stability.