Molecular and cellular mechanisms underlying embryo colonization by pluripotent stem cells in primates

Unlike rodents, generating interspecies chimeras in primates remains a challenge. During the early stages of embryonic development, naïve pluripotent stem cells (PSCs) possess a unique ability to integrate into the pre-implantation embryo and differentiate into any cell type found in the adult organism. Recent findings have established that, primarily due to a defective cell cycle, human naïve PSCs do not survive or proliferate after being injected into a host pre-implantation embryo. Similarly, it has been shown that naïve PSCs from non-human primates are less efficient at colonizing pre-implantation embryos compared to rodent PSCs. Therefore, a deeper understanding of the mechanisms governing embryo colonization could lead to the creation of highly competent primates PSCs. The pre-implantation embryo presents a hostile and challenging environment for foreign cells, which are likely eliminated by active cell death mechanisms. Cell competition serves as a quality-control measure during pre-implantation development and throughout adult life. In this study, we examined the behavior of primate PSCs in an in vitro cell competition assay and their ability to colonize rabbit and cynomolgus monkey embryos. To this end, we reprogrammed human, chimpanzee, marmoset and rhesus monkey PSCs to a naïve state of pluripotency using a new culture regime designated ALGöX. Our in vitro competition assays showed that human, rhesus monkey, and marmoset PSCs were progressively outcompeted by chimpanzee PSCs. Consequently, we qualified the chimpanzee PSCs as the "winners", in contrast to the PSCs from the other primates, which acted as the "losers" in the competition assays. Additionally, our experiments demonstrated that chimpanzee and marmoset PSC lines have a greater ability to colonize than those from human and rhesus monkey PSCs after 5-10 cells were injected into rabbit and cynomolgus morulae. While human and rhesus monkey PSCs did not survive post-injection, an average of 10 to 50 chimpanzee PSCs were found in 95% of the injected morulae. Furthermore, singlecell transcriptomic analysis of chimpanzee PSCs, conducted before and after injection, revealed adaptative transcriptomic changes after 48 hours of embryo culture. Crucially, we identified the MEK-ERK and PI3K-AKT signaling pathways that may dictate the chimpanzee PSCs' colonization competency and success in cell competition. Data supporting these findings will be presented, underscoring the robust correlation between the outcome of cell competition in vitro and the capability of primate PSCs to colonize host embryo.