Tracking the embryonic stem cell transition from ground state pluripotency

Abstract

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.

Keywords: ES cells; Epiblast; Methylome; Pluripotency; Rex1; Transcriptome.

Fig. 1.

Expression of the RGd2 reporter before and after implantation. (A,B) Immunofluorescent staining for GFP (Rex1GFPd2) (red) and Gata4 (grey) at (A) E4.5 and (B) E5. Arrowheads show GATA4-positive nuclei. Scale bar: 20 μm. ExE, extra-embryonic ectoderm; Epi, epiblast.

Fig. 2.

Multilineage specification of ES cells upon release from 2i. (A) Protocol for monolayer differentiation of naïve ES cells in N2B27 by withdrawal of 2i. (B) Immunofluorescent staining for Oct4, Sox1 and brachyury (T). Lower panels (q-w) show enlarged insets from 48 h and 72 h, with respective inset number in parentheses. (C,D) RT-qPCR for selected (C) pluripotency and (D) early post-implantation epiblast markers. Expression levels are shown as fold change relative to naïve ES cells in C and to 48 h samples in D. GAPDH was used for normalisation. Error bars indicate s.d. from two biological replicates.

Fig. 3.

Expression of transcription factors during transition of ES cells. (A) GFP flow cytometry profile at indicated time points post-2i withdrawal (10,000 cells per time-point). Wild-type ES cells were used as the negative control (neg). (B) Immunofluorescent staining for GFP, Nanog and Otx2. (C) Otx2 versus Nanog fluorescence intensity per cell in arbitrary units (a.u.), as quantified by Volocity. X and Y intercepts of the red lines mark the cut-off for Nanog- and Otx2-negative cells, respectively. (D) Distribution of Otx2 expression in Nanog subpopulations. (E) GFP versus Nanog fluorescence intensity per cell. X and Y intercepts of the red lines mark the cut-off for Nanog- and GFP-negative cells, respectively. (F) Distribution of Nanog expression in Rex1 subpopulations at 25 h. (G) Immunofluorescent staining for Oct4 and Sox2.

Fig. 4.

Downregulation of Rex1 tracks exit from the naïve state. (A) Protocol for sorting and clonogenicity assays. (B) Clonogenicity of cells from 2i and differentiating subpopulations sorted at indicated time points, plated in serum/LIF (Serum/L) or 2i supplemented with LIF (2i/L). Data are mean±s.d. from two technical replicates (500 cells were plated). (C) Sorting of 25 h-cultures into four subpopulations based on GFP levels by flow cytometry. Lower plot shows the GFP profiles of post-sort subpopulations. (D) Clonogenicity of four subpopulations shown in C. Data are mean±s.d. from two technical replicates. (E) Diagram summarising phases of transition from the naïve state. (F) Clonogenicity of the indicated subpopulations. Data are mean±s.d. from three biological replicates each with two technical replicates (800 cells were plated). (G) Immunoblot of total cell lysates from sorted subpopulations. β-Tubulin and Gapdh are loading controls. (H) Expression of selected general (red) and naïve (orange) pluripotency and early post-implantation epiblast (blue) markers in single cells measured using the Fluidigm system. Scale bar represents log2 transformed expression value.

Fig. 5.

Transcriptional changes in ES cells during progression from naïve pluripotency. (A) Expression of MEK/ERK and Wnt/β-catenin transcriptional effectors and targets from three independent replicates measured by microarray profiling. Scale represents log2 transformed expression value. (B) Enriched KEGG pathway categories in the differentially expressed gene sets ranked according to P value (P<0.05). (C) OCR and ECAR levels of 2i versus 25 h populations (left) and sorted 25h-H and 25h-L subpopulations with unsorted whole population (right) (data are mean± s.d. from six technical replicates). (D) Expression of general (red) and naïve (orange) pluripotency, early post-implantation (blue) and lineage-priming factors (black) detected by microarray. Scale represents log2 transformed expression value.

Fig. 6.

Comparison of transcriptional changes during pluripotency progression in ES cells and in the embryo. (A) Functional grouping of genes that show similar regulation in ES cells and in the embryo. (B) Expression of genes from selected pathways.

Fig. 7.

Acquisition of DNA methylation during transition from naïve pluripotency. (A) mRNA expression of factors that modulate DNA methylation. (B) Global genomic methylation in CG context (mCG) in 2 kb tiles. ns, non significant, *P<0.05 (one-way multiple comparisons ANOVA corrected with Tukey's test). (C,D) Percentage of mCG (C) in the promoters (−1000 bp to +500 bp of TSS) of expressed genes (RPKM≥10), (D) in genome-wide naïve and super enhancers and (E) in the promoters of selected pluripotency-associated genes.