Two temporally distinct pluripotent stem cells (PSCs) have been derived in mouse: “naïve” embryonic stem cells (ESCs)1,2 and “primed” epiblast stem cells (EpiSCs)3,4. They differ from each other in molecular signatures and phenotypic properties5. As embryo development is a dynamic process not only in time but also in space, it remains unknown whether pluripotent states with distinct regional properties may exist. Through optimization of culture parameters using mouse epiblast explants, we derived with high efficiency a novel pluripotent cell type sharing features of the posterior-proximal (PP) epiblasts of post-implantation mouse embryos. We designated this new cell type as region-selective EpiSCs, or rsEpiSCs. Mouse rsEpiSCs have high cloning efficiency and faster growth kinetics and differ from EpiSCs at global transcriptomic, epigenomic and metabolic levels.
Conventional human ESCs exist in a primed pluripotent state similar to mouse EpiSCs due to their similar colony morphology, signaling dependency, poor cloning efficiency and epigenetic features. We tested mouse rsEpiSCs-based culture on human ESCs and found human ESCs could self-renew while maintaining karyotypic stability after long-term culture (the cells were thus named human rsESCs after treatment). Moreover, human rsESCs share similar molecular characteristics with mouse rsEpiSCs.
We analyzed the grafting properties of distinct primed pluripotent cells in isolated non-intact and non-viable mouse E7.5 epiblasts. mEpiSCs were previously reported to efficiently incorporate and differentiate in anterior, posterior as well as distal part of the E7.5 mouse epiblasts6-8. In contrast, mouse rsEpiSCs only selectively colonize and give rise to derivatives of the three embryonic germ layers after being grafted to the posterior pole of isolated non-intact and non-viable E7.5 mouse embryos. Surprisingly, unlike mouse EpiSCs, we found that after grafting conventional human ESCs could hardly survive inside E7.5 mouse epiblasts regardless of the location. Interestingly, just like mouse rsEpiSCs, human rsESCs could efficiently integrate into the posterior region of isolated non-intact and non-viable mouse E7.5 epiblast and differentiated into three embryonic germ lineages after 36 h in vitro embryo culture. These results indicate that we have captured a spatially distinct primed pluripotent state that confers ex-vivo intra- and inter-species chimeric competency.
This protocol describes in detail of isolation and preparation of non-intact and non-viable mouse E7.5 embryos for grafting. The epiblast grafting and in vitro embryo culture procedures will also be elaborated.