α‐Ketoglutarate inhibits the pluripotent‐to‐totipotent state transition in stem cells

In early mouse embryogenesis, the distinct enrichment of α-ketoglutarate (αKG) in blastocysts and L-2-hydroxyglutarate (L-2HG) in 2-cell (2C) embryos serves as a key metabolic signature. While elevated L-2HG levels inhibit the resolution of totipotency during the transition from the 2C stage to the blastocyst, the role of αKG remains elusive. Mouse embryonic stem cells (mESCs) cultured in vitro naturally harbor a subpopulation that transitions dynamically into a 2C-like totipotent state, providing a convenient model to investigate the role of αKG in totipotency reprogramming. This study demonstrates that αKG significantly inhibits the pluripotency to totipotency transition through upregulating ten-eleven translocation (TET) DNA hydroxylases. We further show that reducing endogenous αKG levels via glutamine withdrawal or inhibiting αKG-dependent dioxygenases by blocking succinate dehydrogenase (SDH) markedly enhances the induction of 2C-like cells (2CLCs). Finally, leveraging the potent SDH inhibitor dimethyl malonate (DMM), we have developed a highly efficient protocol for 2CLC induction, producing cells that transcriptionally resemble mid-to-late 2C embryos. Our findings deepen the understanding of the metabolic regulation of totipotency and provide a previously undescribed approach for capturing totipotent-like stem cells in vitro.