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

Abstract

The pluripotent state, which is first established in the primitive ectoderm cells of blastocysts, is lost progressively and irreversibly during subsequent development. For example, development of post-implantation epiblast cells from primitive ectoderm involves significant transcriptional and epigenetic changes, including DNA methylation and X chromosome inactivation, which create a robust epigenetic barrier and prevent their reversion to a primitive-ectoderm-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signalling, but they respond to activin/basic fibroblast growth factor to form self-renewing epiblast stem cells (EpiSCs), which exhibit essential properties of epiblast cells and that differ from embryonic stem (ES) cells derived from primitive ectoderm. Here we show reprogramming of advanced epiblast cells from embryonic day 5.5-7.5 mouse embryos with uniform expression of N-cadherin and inactive X chromosome to ES-cell-like cells (rESCs) in response to LIF-STAT3 signalling. Cultured epiblast cells overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, resulting in DNA demethylation, X reactivation and expression of E-cadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Using this approach, we report reversion of established EpiSCs to rESCs. Moreover, unlike epiblast and EpiSCs, rESCs contribute to somatic tissues and germ cells in chimaeras. Further studies may reveal how signalling-induced epigenetic reprogramming may promote reacquisition of pluripotency.

Figure 1

Reprogramming epiblast cells from E6.5 embryos to generate rESC. (a) Derivation of cEpi from E6.5 epiblast. Epiblast tissue was divested of the proximal region (white line, panel 1) and visceral endoderm; a single cell suspension (black arrow) was cultured, which formed cEpi colonies. Note AP positive cells in cEpi in the last panel. (b) Derivation of rESC from cEpi. Note the appearance of clusters of Oct4-ΔPE-GFP-positive cells in cEpi colonies (black arrowheads), and corresponding white arrowheads for GFP in the panel below. Note that the rESC are uniformly AP positive. Scale bar: 100μm. (c) Schematic representation of reprogramming of epiblast through cEpi, and finally rESC.

Figure 2

Changes in gene expression profile. (a) Reverse transcription real-time PCR of marker genes in EpiSC, cEpi, rESC at early (P4) and late (P24) passages, and ESC. Note progressive loss of markers of epiblast detected in cEpi and EpiSC (at the top) and enhancement of expression of genes in rESC that resemble ESC (b) Whole-genome cluster analysis of transcriptomes of cEpi, rESC at early (P4) and late (P24) passages, and ESC. The labeled numbers are the corresponded Pearson correlation coefficients between different cDNA samples. Note that rESC resemble ESC and not cEpi that are more like the original epiblast cells as described above. Note also the changes between the early (P4) and late (P24) passages of rESC.

Figure 3

Dynamic changes in cell surface adhesion molecules; both E-cadherin and N-cadherin are detected uniformly in E6.5 epiblast, which is undetectable in single cell suspension. During culture, N-cadherin expression is heterogeneous in cEpi and eventually disappears completely and replaced by E-cadherin in rESC. Scale bar: 20μm.

Figure 4

Epigenetics changes during reprogramming of epiblast cells. (a) Female cEpi exhibit uniform accumulation of H3K27me3 associated with the inactive X (white arrowhead), which is gradually lost from individual cells during culture and finally lost in rESC. Scale bar: 20 μm (b) Changes in DNA methylation of Stella and Rex1 during reprogramming of epiblast. Although Stella and Rex1 are repressed in epiblast cells, these loci are initially unmethylated; they undergo DNA methylation transiently in cEpi and stably in EpiSC. Reprogramming to form rESC results in the loss of DNA methylation.

Figure 5

Contribution of rESC to chimeras. (a) Progeny derived from chimera with rESC showing germline transmission indicated by black arrow. (b) E6.5 chimera with ROSA-lacZ rESC: Note contribution of early passage rESC both to the epiblast and to the extraembryonic ectoderm (ExE), a derivative of trophectoderm cells. (c) E6.5 chimera derived with ROSA-lacZ ESC; Note contribution predominantly to the epiblast. (d) Schematic representation of reprogramming of cEpi and EpiSC to rESC. Note the epigenetic and transcriptional changes during reprogramming of cEpi and EpiSC.