Isolation of epiblast stem cells from preimplantation mouse embryos

Abstract

Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The preimplantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.

Figure 1. Intrinsic and extrinsic factors contribute to the acquisition of distinct pluripotent states from pre-implantation mouse embryos

To derive pluripotent stem cells, pre-implantation blastocyst stage embryos at E3.5 (shown as a 3D reconstruction immunostained for Oct3/4 (red) and Cdx2 (green)) were (A) explanted and grown for 6 days under specific conditions (see methods) at which point few cells remained positive for the Oct3/4. (B) Using passaging protocols described in this manuscript, both ‘flat’ and ‘domed’ colony morphologies were evident by day 16. (C) ‘Domed’ colonies gave rise to Oct3/4+ mouse ES cell lines while (D) ‘flat’ colonies produced Oct3/4+ EpiSCs. (E) The derivation efficiency for blastocyst-derived mouse ES cells and EpiSCs was 28% and 26% respectively in strain 129 but intrinsic genetic elements present in other strains as well as extrinsic modulation of signaling pathways altered the efficiencies.

Figure 2. Pre- and post-implantation-derived EpiSCs share gene expression and DNA methylation patterns that are distinct from other pluripotent states

(A) Quantitative RT-PCR of pre-implantation-derived EpiSCs (E3.5) showed gene expression patterns consistent with post-implantation derived EpiSCs (E5.5) and not pre-implantation derived mouse ES cells (E3.5). (B) Correlations of whole genome microarray data showed a strong correlation between pre- and post-implantation derived EpiSCs and their clear distinction from other pluripotent cells such as FAB-SCs and mouse ES cells. Bisulfite pyrosequencing of pre-implantation-derived EpiSCs (E3.5) reveled hypermethylation of CpGs in the (C) Dppa3 promoter region and hypomethylation in (D) Oct3/4 promoter region. The location of analyzed CpGs is indicated in red.

Figure 3. Pre- and post-implantation-derived EpiSCs utilize similar & developmentally-relevant mechanisms to regulate pluripotency and differentiation

(A) Pre-implantation-derived EpiSCs are pluripotent as evidenced by haematoxylin and eosin stained histological sections of teratomas. Examples of specific cell types are denoted with an arrow. Additional images are found in Figure S1. Both pre- (E3.5) and post- (E5.5) implantation derived EpiSCs exited pluripotency and differentiated into neural rosettes in response to Alk-4/5/7 inhibition while mouse ES cells maintained their pluripotency as evidenced by (B) morphology and (C) hierarchical cluster analysis of whole genome microarray data of treated and untreated samples.

Figure 4. Acquisition of distinct pluripotent states

Summary diagram depicting the multiple avenues which have provided access to the two pluripotent states (mouse ES-like and epiblast-like) and highlighting the contribution of this manuscript to the field. See also Figure S2.