Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells

Abstract

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...

LacdiNAc (GalNAcβ1-4GlcNAc) Contributes to Self-Renewal of Mouse Embryonic Stem Cells by Regulating Leukemia Inhibitory Factor/STAT3 Signaling

“…In particular, both cell types are maintained by the same extrinsic signals, namely, activin/ nodal and FGF2 signaling. Recent studies have demonstrated that EpiSCs, which are more advanced developmentally than mESCs, can be induced to revert to mESC-like cells [41,42,[49][50][51]. FAB-SCs are generated from the preimplantation epiblast under defined culture conditions, including the presence of FGF2 and activin, and they can be induced to revert to the mESC-like state.…”

“…It has been reported that LIF/STAT3 signaling contributes to the reversion of primed state EpiSCs to naïve state mESCs [49,50]. Additionally, a recent study showed that weak transduction of the LIF/STAT3 signal is a possible barrier to this reversion [51].…”

“…A recent study reported that LIF/STAT3 signaling contributes to reversion from the primed state to the naïve state [49][50][51]. Therefore, we investigated whether the regulation of LIF/ STAT3 signaling by LacdiNAc expression contributed to reversion.…”

Concise Review: Culture Mediated Changes in Fate and/or Potency of Stem Cells

“…In a second series of studies, similar dedifferentiation from an EpiSC to an ICM-like state was accomplished by supplementing standard mESC medium, containing leukemia inhibitory factor (LIF), with small molecules targeting epigenetic modifiers and/or signaling pathways differentially used by the two pluripotent cell types [19,20]. Moreover, Bao et al [21] demonstrated that EpiSC derived from E5.5 to E7.5 embryos could spontaneously revert to an ICMlike ground state when cultured for 14-35 days on MEFs supplemented with LIF but without any other factors. The reverted ESC could generate germline-competent chimeras and progressively lost the histone 3 lysine 27 trimethylation (H3K27me3) mark on the inactive X chromosome, which was associated with loss of X-chromosome silencing.…”

Pluripotency of Male Germline Stem Cells

“…Gene expression analysis indicates shared expression of Oct4 and SSEA1 but differential expression of a group of genes, including Stella, Rex1, Pecam1, Gbx2, and Fgf5 (Bao et al, 2009;Brons et al, 2007;Tesar et al, 2007). Differential expression of GFP by Oct4 regulatory sequences can be used to distinguish the two different pluripotent states (Han et al, 2010a;2010b).…”