Context: In ungulates, a unique feature of early development is the delayed progression of the blastocyst prior to implantation. During this period, the epiblast proliferates and retains its pluripotency for several days before initiating gastrulation. During this time, extraembryonic tissues show increased growth and elongate to several tens of centimetres in length.

Objective: Our aim was to elucidate how these developmental processes occur and are synchronized, and to understand their implications for the phenotype of pluripotent embryonic stem cells derived from these embryos.

Methods: We performed integrated single-cell transcriptomic and epigenomic analyses of cells from both early and late-stage porcine blastocysts, as well as from derived pluripotent epiblast stem cells (EpiSCs).

Results: We observed dynamic changes in the activity of gene regulatory modules in extraembryonic tissue, at the onset of blastocyst elongation, while the activity of gene regulatory modules remains relatively constant in the epiblast. When comparing embryonic epiblast cells to EpiSCs, we detected only subtle global differences in transcriptomic and epigenomic profiles. However, we identified two distinct subpopulations of primed pluripotent cells within EpiSCs. One is characterized by the expression of transcription factors GRHL2 and TFAP2A and the other by ZNF521 and ZEB1. We confirmed that these two populations coexist in our lines and in those published by other laboratories. Using targeted cellular enrichment techniques, we demonstrated that these states are interconvertible, highlighting their plasticity and the dynamic equilibrium between them.

Conclusion: We confirmed the presence of these pluripotent subpopulations in human, bovine, ovine, and porcine stem cell lines, but not in rodent cells. Our preliminary data suggest that EpiSCs from non-rodent species may undergo early precommitment toward either the amnion or the late epiblast, regulated by specific signalling pathways.