Human Naive Pluripotency Attained by Transient Inhibition of mTor

Mouse embryonic stem cells (mESCs) are in naive pluripotency that represents the ground state of development, from which all cells in the mouse embryo are derived. In contrast, human embryonic stem cells (hESCs) are in a primed state of pluripotency with many different properties. Despite intense efforts to generate naive human pluripotent stem cells (hPSCs), it has not been possible to derive naive hPSCs without relying on transgene overexpression or chemicals. Here, we show that a transient treatment with Torin1, a selective inhibitor of mTOR, converted hPSCs from primed to naive pluripotency. The naive hPSCs were maintained in the same condition as mESCs in defined media with 2iLI (MEK inhibitor, GSK3b inhibitor, LIF and Insulin). Like mESCs, they exhibited high clonal efficiency, rapid cell proliferation, active mitochondrial respiration, X chromosome activation, DNA hypomethylation, and transcriptomes similar to those of human blastocysts than primed hESCs. Most importantly, the naive hPSCs significantly contributed to mouse embryos when transferred to mouse blastocysts. mTor inhibition induced nuclear translocation of TFE3, a critical transcription factor at the interplay of autophagy and pluripotency. TFE3 with mutated nuclear localization signal blocked the conversion from primed to naive pluripotency. It appears that by mimicking diapause at the cellular level, naive pluripotency in human can be readily attained from primed hPSCs, thus establishing the unified ground state of pluripotency in mammals. Overall design: We compared the differences in gene expression between primed state and naive state human pluripotent stem cells. RNAseq was performed on primed H9 (PH9), naive H9 (NH9), primed RUES2 (PRUES2), and naive RUES2 (NRUES2) with 3 biological replicates for each line. For each sample, colonies of hPSCs were manually picked and homologized in 1ml TRIzol reagent (Thermo Fisher Scientific). RNeasy Mini Kit (QIAGEN) was used for RNA extraction. Quality of purified RNA was monitored by agarose gel electrophoresis. PolyA+ RNA enrichment, cDNA library preparation, sequencing and RPKM calculation were performed at our core facility with standard procedure.