SummaryDuring the transition from the inner cell mass (ICM) cells of blastocysts to pluripotent embryonic stem cells (ESCs) in vitro, a normal developmental program is replaced in cells that acquire a capacity for infinite self-renewal and pluripotency. We explored the underlying mechanism of this switch by using RNA-Seq transcriptome analysis at the resolution of single cells. We detected significant molecular transitions and major changes in transcript variants, which include genes for general metabolism. Furthermore, the expression of repressive epigenetic regulators increased with a concomitant decrease in gene activators that might be necessary to sustain the inherent plasticity of ESCs. Furthermore, we detected changes in microRNAs (miRNAs), with one set that targets early differentiation genes while another set targets pluripotency genes to maintain the unique ESC epigenotype. Such genetic and epigenetic events may contribute to a switch from a normal developmental program in adult cells during the formation of diseased tissues, including cancers.

The pluripotent state, which is first established in the primitive ectoderm cells (PE) of blastocysts, is lost progressively and irreversibly during subsequent development 1 . For example, development of postimplantation epiblast from PE involves significant transcriptional and epigenetic changes, including DNA methylation and X inactivation 2 , which creates a robust epigenetic barrier and prevents their reversion to a PE-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signaling, but they respond to Activin/bFGF to form self-renewing epiblast stem cells (EpiSC), which exhibit essential properties of epiblast cells 3,4 , that differ from embryonic stem cells (ESC) derived from PE 5 . Here we show reprogramming of advanced epiblast cells from E5.5 -E7.5 embryos with uniform expression of N-cadherin and inactive X chromosome, to ES-like cells (rESC) in response to LIF-STAT3 signaling. Cultured epiblast cells (cEpi) overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, involving DNA demethylation, X reactivation and expression of Ecadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Notably, using this new approach, we report reversion of established EpiSC to rESC. Furthermore, unlike epiblast and EpiSC, rESC contribute to somatic tissues and germ cells in chimeras. This is a tractable model to investigate signaling molecule induced epigenetic reprogramming that can promote reacquisition of the fundamental pluripotent state.Previous studies showed that epiblast cells, unlike PE, are refractory to LIF-STAT3 signaling 3,4 ; instead they respond to Activin/bFGF to generate self-renewing EpiSC. EpiSC differ epigenetically from ESC, as they have an inactive X-chromosome and they cannot form chimeras when introduced into blastocysts. However, we set out to re-examine if postimplantation epiblast cells could undergo reprogramming to ESC-like cells in response to LIF-STAT3 signaling. We isolated epiblast tissue on embryonic day (E) E5.5 -E7.5 from transgenic embryos with an Oct4-ΔPE-green fluorescent protein (GFP) reporter 6 . This reporter, with the distal enhancer and lacking the proximal enhancer for Oct4, shows Next, for the culture of epiblast, we used LIF and fetal calf serum (FCS) on mouse embryonic fibroblasts feeder cells (MEFs), which is the standard condition used for the derivation of ESC from PE, and for reprogramming of somatic cells to induced pluripotent stem cells (iPS) 5,[8][9][10][11] . The epiblast tissue was dissected to remove the most proximal region (the site of PGC and PGC precursors 2 ), and the outer visceral endoderm (Fig. 1a). All the epiblast cells uniformly showed an inactive X-chromosome, and were positive for Ncadherin (see below). Notably, we then trypsinised the epiblast tissue and used single cell suspension from individual epiblasts for culture, unlike previous studies where the epiblast tissue was left inta...