Reinforced STAT3 activity sustains self-renewal of human embryonic stem cells in a naive-like state

Only rodent embryonic stem (ES) cells can self-renew in the pristine state of pluripotency called the naive or ground state. Human ES (hES) cells self-renew in the so-called primed state of pluripotency, which is an obstacle to research, hindering cost-effective cultivation in media devoid of animal-derived products, genetic stability, and genome engineering. Here we show that forced expression of a hormone-dependent STAT3-ERT2, in combination with LIF and inhibitors of MEK and GSK3beta, allows hES cells to escape from the primed state, and enter a new state designated as TL2i, characterized by the activation of STAT3 target genes, regular passaging by single cell dissociation, and the expression of naive state-specific transcription factors. We used microarrays to analyse the gene expression changes occuring during the adaptation to the naive culture conditions. We then compared our dataset to previously published dataset of mouse ESc and EpiSc, human primed and naive ESc, and human embryos. Human ES cells were genetically modified to overexpress the STAT3-ERT2 transcription factor. The modified cells were then cultured, on MEFs, in different conditions : (i) F = bFGF, (ii) TL = LIF + Tamoxifen, (iii) TL2i = LIF + Tamoxifen + MEK inhibitor +GSK3b inhibitor. We overexpressed STAT3-ERT2 in two different human ES cell lines : the H9 cell line and a cell line newly derived in our lab, designated Oscar. We analysed two clones H9-STAT3T-ERT2 (cl2 and cl14) and one clone Oscar-STAT3-ERT2. The TLOscar wer reverted to bFGF culture condition. We included three biological replicates per condition.