Apoptosis, G1 phase stall and premature differentiation account for low chimeric competence of Human and rhesus monkey naïve pluripotent stem cells

Abstract

After reprogramming to naïve pluripotency, human pluripotent stem cells (PSCs) still exhibit very low ability to make interspecies chimeras in mice and pigs. Whether this is because they are inherently devoid of the attributes of chimeric competency or because naïve PSCs in general cannot colonize embryos from distant species remains to be elucidated. Here, we have used different types of mouse, human and rhesus monkey naïve PSCs and we have analyzed their ability to colonize both distant (rabbit) and close (cynomolgus monkey) embryos. Mouse embryonic stem cells (ESCs) remained mitotically active after injection into rabbit and cynomolgus embryos and contributed to the formation of the neural tube in postimplantation rabbit embryos. In contrast, primate naive PSCs colonized rabbit and cynomolgus embryos with much lower efficiency, regardless of the reprogramming protocols. Unlike mouse ESCs, they slowed DNA replication following colony dissociation and, after injection into host embryos, they stalled in the G1 phase of the cell cycle and differentiated prematurely, regardless of host species, distant or close. These results show that, unlike mouse ESCs, primate naive PSCs do not make chimeras because they are inherently unfit to stay mitotically active during embryo colonization.

Running the full human developmental clock in interspecies chimeras using alternative human stem cells with expanded embryonic potential

“…These data, at least in part, may account for why naïve hPSCs have met limited success in generating human interspecific organogenesis within animal embryos 73 , 74 , 81 . At a minimum, the limited proficiency for generating chimeras with primed rhesus PSC in these and other studies aligned with the principle that primed EpiSC are incompetent in chimera formation in pre-implantation murine embryos 86 , 100 , 101 .…”

“…1 ) 10 . Nevertheless, despite using highly sensitive mitochondrial DNA PCR detection assays, these studies collectively revealed that injection of naïve-reverted hPSC into a mouse, rabbit, or ungulate blastocysts or morulae, or alternatively naïve-reverted non-human primate (NHP) rhesus PSC injected into a mouse, rabbit, or monkey embryos, resulted in limited human or monkey fetal chimerism (Table 1 ) 73 , 81 – 86 .…”

“… Yes N.A. Fu et al 85 Naïve NHP-PSC Pig – 0.001–0.0001% Yes Yes Yes No No Aksoy et al 86 Naïve hPSC, Naïve NHP-PSC Mouse, Rabbit – undetectable N.A. N.A.…”

In Vivo Generation of Organs by Blastocyst Complementation: Advances and Challenges

“…Over the last few years, different culture conditions have been assessed and different pluripotent states (naive, primed and intermediate) have been achieved for hESCs in order to maximize interspecies chimera formation efficiency (51)(52)(53)(54)(55)(56)(57)(58)(59). However a recent report suggests that even naive human PSCs are inherently unfit for chimera formation as, upon dissociation and injection into host embryos, they stop dividing and undergo premature differentiation (60). On the other hand, it has been observed that pluripotent stem cells grafted heterochronically in embryos undergo apoptosis (61)(62)(63).…”

Nuclear transfer and the development of genetically modified/gene edited livestock

“…The causes of the very limited chimerism are unclear, but they could be due to differences in the developmental stages represented by the stem cells and the host embryo (Mascetti and Pedersen 2016). The type of stem cell and the culture conditions can also determine the viability of the cells in interspecies chimeras (Aksoy, et al 2021, Fu, et al 2020. It is also noteworthy that the greater evolutionary divergence between pigs and human (>90 million years) compared to that between mice and rats (< 20 million years) renders this approach incompatible under these experimental conditions.…”