Alternative routes to induce naive pluripotency in human embryonic stem cells

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naive pluripotent state. Efforts have been made to trigger naive pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naive genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor1Ascorbic Acid1Forskolin) facilitating rapid induction of transgene-free naive pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naive hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naive pluripotency genes, and exhibited dependence on signaling pathways similar to naive mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naive pluripotency. Collectively, we demonstrate an alternate route to capture naive pluripotency in hESCs that is fast, reproducible, supports naive mESC derivation, and allows efficient differentiation