In human and non-human primates, naïve pluripotent stem cells (nPSCs) lack the ability to generate intra- and interspecies chimeras using mammal embryos. Although the reasons remain unclear, the pre-implantation embryo being a competitive environment may account for poor colonization capability. Understanding the mechanisms governing embryo colonization could establish highly competent human nPSC lines.

In this study, we reprogrammed human, chimpanzee, marmoset and rhesus monkey nPSCs towards a naïve state of pluripotency using ALGöX culture regime. We next examined the competitiveness of primates nPSCs in vitro and their ability to colonize pre-implantation embryos from rabbit, cynomolgus macaque, pig and bovine. Our in vitro competition assays showed that human, rhesus, and marmoset nPSCs were always progressively yet actively outcompeted by the chimpanzee nPSCs. Further experiments demonstrated that only the chimpanzee nPSC line colonized 100% of injected morulae.

Single-cell transcriptomic analysis of the unique chimpanzee nPSC line revealed signaling pathways such as MEK/ERK and PI3K/AKT may dictate the chimpanzee nPSCs behavior during colonization. We found that the inhibition of ERK and AKT negatively altered colonization efficiency in chimpanzee PSCs, specifically through upregulation of pro-apoptotic factors BIM and BAX.

Interestingly, we found that human PSCs display lower levels of phosphorylated ERK and AKT compared to the chimpanzee nPSCs. Therefore, increasing the activity of both pathway in human naïve PSCs may improve their competitiveness and colonization capability. For this, inducible transgenic human nPSC lines expressing constitutively active forms of phosphorylated ERK or AKT were generated. We determined that the sole reactivation of either ERK or AKT was sufficient to improve colonization capability in human nPSCs, as an average of 10-15 cells were found in 100% of embryos post-injection. Importantly, the synergy between genetic and chemical activation of both ERK and AKT using small agonists considerably enhanced the colonization capability in human nPSCs, as an average of 10-40 cells were found in 100% of injected rabbit and cynomolgus morulae. Lastly, we determined that AKT reactivation in human nPSCs also improved their competitiveness as they outcompeted wild-type human nPSCs after 48h of in vitro competition assays. These findings highlight the importance of both pathways for better competitive assets and high embryo colonization.