SP2 impedes the pluripotent-to-totipotent state transition in an RA-dependent manner

The pluripotency-to-totipotency transition represents a transient developmental reversal that remains not fully understood. Although chemical approaches can induce a 2-cell (2C)-like state in mouse embryonic stem cells (mESCs), how cells choose between totipotency acquisition and alternative developmental fates remains unclear. Here, we established a chemical induction system that efficiently drives mESCs toward the 2C-like state. We found that mESCs under induction treatment followed distinct trajectories, with a subset activating developmental programs rather than acquiring 2C-like features. SP2 was preferentially expressed along this developmental trajectory and progressively reduced during successful 2C-like induction. Loss of SP2 enhanced 2C-like cell formation, whereas SP2 overexpression impaired the transition. Transcriptomic analysis of SP2-deficient cells showed reduced expression of developmental genes, including Hox family genes, accompanied by increased expression of 2C-associated genes. Consistent with this finding, SP2 occupied the promoters of Hox family genes in the reprogrammed cells. Notably, the effect of SP2 loss was particularly evident upon activation of retinoic acid (RA) signaling, linking SP2-dependent developmental gene activation to the RA-responsive branch of the transition. Altogether, our findings identify SP2 function as a barrier that limits acquisition of the 2C-like state by promoting a competing developmental program, highlighting the importance of coordinated extracellular signaling and transcriptional regulation in directing cell fate transitions.