GSE175610
AMPK activation reverts mouse epiblast stem cells to naïve state
Reversion from primed to naïve pluripotent status has been achieved by various signaling manipulation, but it is still unclear what signaling is the actual driving force to get over the hurdle from primed to naïve pluripotency. We previously reported that activation of AMP kinase (AMPK) contributed to maintenance of naïve pluripotency. Here, we further show that AMPK activators, AICAR, A769662 or metformin, can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to naïve pluripotent state. Primed mEpiSCs in our naïve cell culture condition with leukemia inhibitory factor (LIF) and 2 kinase inhibitors (2i) (2iL) never gave rise to naïve state cells. Addition of AICAR alone even in the absence of 2iL or either of AMPK inhibitors with LIF induced appearance of naïve-like cells from primed mEpiSCs. Through maintenance and passages of these cells in 2iL condition, clear naïve-like morphology colonies were purely obtained. They showed core naïve protein expression, and global naïve gene expression profiles. These cells contributed to chimeric mice including germline transmission. Inhibition of p38 signaling abolished the AMPK-elicited reversion and forced activation of p38 in primed mEpiSCs partially reproduced the naïve cell induction, suggesting that p38 is one of the critical downstream in AMPK activation. Single cell RNA-seq analysis under AICAR stimulation successfully demonstrated the reversion process with appearance of intermediate naïve-like population. AMPK pathway should be a novel critical driving force in reversion of primed to naïve pluripotency. Overall design: We analyzed single cell RNA-seq profiles of naïve mESCs (500 cells), primed mEpiSCs (500 cells), AICRA+LIF d8 (9000 cells), AICAR+LIF d16 (9000 cells) and AICAR+LIF reverted d16+10p (1000 cells),we used combination of Biotin-CD9 and Biotin-CD63 (Biolegend) conjunction with TotalSeq™-A095(1-5) PE Streptavidin, respectively. naïve mESCs tagged with A951,primed mEpiSCs tagged with A952,AICRA+LIF d8 tagged with A953, AICAR+LIF d16 tagged with A954, AICAR+LIF reverted d16+10p tagged with A955. 5 samples: A951: Naïve mESCs (Rex1-GFP) A952: Primed mEpiSCs (Oct4GIP) A953: AICAR+LIF d8 A954: AICAR+LIF d16 A955: AICAR+LIF reverted d16+10p