Cross-species single-cell transcriptomic analysis reveals pre-gastrulation developmental differences among pigs, monkeys, and humans

Abstract

Interspecies blastocyst complementation enables organ-specific enrichment of xenogeneic pluripotent stem cell (PSC) derivatives, which raises an intriguing possibility to generate functional human tissues/organs in an animal host. However, differences in embryo development between human and host species may constitute the barrier for efficient chimera formation. Here, to understand these differences we constructed a complete single-cell landscape of early embryonic development of pig, which is considered one of the best host species for human organ generation, and systematically compared its epiblast development with that of human and monkey. Our results identified a developmental coordinate of pluripotency spectrum among pigs, humans and monkeys, and revealed species-specific differences in: (1) pluripotency progression; (2) metabolic transition; (3) epigenetic and transcriptional regulations of pluripotency; (4) cell surface proteins; and (5) trophectoderm development. These differences may prevent proper recognition and communication between donor human cells and host pig embryos, resulting in low integration and survival of human cells. These results offer new insights into evolutionary conserved and divergent processes during mammalian development and may be helpful for developing effective strategies to overcome low human–pig chimerism, thereby enabling the generation of functional human organs in pigs in the future.

Conserved features of non-primate bilaminar disc embryos and the germline

“…Similarly, during this period Nodal expression becomes restricted to the posterior epiblast in rabbit and pig ( Ploger and Viebahn, 2018 ; Yoshida et al., 2016 ), the region of the formation of the primitive streak, which has also been determined in 3D cultures of human embryos, and more recently in gastruloids ( Moris et al., 2020b ; Xiang et al., 2020 ). Although the temporal pattern may be skewed due to the in vitro culture of the human embryo, cross-species comparison of RNA-sequencing datasets show highest correlation of E10 and E12 pig epiblast cells with E8 and E10 human epiblasts, respectively ( Liu et al., 2021 ). Conserved features of left-right patterning and axial elongation have also been studied in rabbit, pig, and cow embryos, which highlight some remarkable species-specific mechanisms ( Ploger and Viebahn, 2018 ; Schroder et al., 2016 ).…”

Fumarylacetoacetate hydrolase gene as a knockout target for hepatic chimerism and donor liver production

“…The leap from integrating naive hiPSCs into mice to integrating human/primate cells into porcine embryos has been challenging, partially due to the lack of data about embryonic stages in these organisms. Understanding of stage matching in primate and porcine embryos was recently bolstered by the identification of transcriptome evolution patterns during embryological development in pigs, humans, and cynomolgus monkeys ( Liu et al., 2021 ). Based on the progression of pluripotency transcriptomes, r2iLGoY-hiPSCs ( Liu et al., 2017 ) were correlated with the earliest pig ICM cells (E5–6), NHSM-hiPSCs fell between the spherical and filamentous stages (E11–12), and primed hiPSCs corresponded to filamentous stages (E12–13).…”

“…All porcine hosts were E4–6 embryos that were complemented with either primed or naive donor hiPSCs. Based on the work of Liu et al. (2021) , at least some of these hiPSCs represented an inappropriately late developmental trajectory compared with the pig embryo.…”

“…(2021) , at least some of these hiPSCs represented an inappropriately late developmental trajectory compared with the pig embryo. The most successful attempt was the combination of naive hEPSCs and cynomolgus monkey recipient embryos, which have a closer developmental trajectory and closer evolutionary distance to humans than pigs do ( Liu et al., 2021 ; Tan et al., 2021 ). Further attempts to more closely correlate the transcriptomic evolution of donors and recipients will be needed, perhaps using cells such as r2iLGoY-hiPSCs, which correspond to an early embryonic stage in pigs.…”

“…From a technical standpoint, the engraftment and proliferation of primate embryonic stem cells (ESCs) within pig blastocysts has been persistently low ( Das et al., 2020 ; Fu et al., 2020 ; Nowak-Imialek et al., 2020 ; Wu et al., 2017 ). We discuss the role of naive and primed human pluripotent cells in blastocyst complementation and how human cell choice can be optimized based on differences between human, pig, and monkey embryonic transcriptomes during development ( Liu et al., 2021 ). From an ethical standpoint, human-derived cells in the blastocyst must be prevented from differentiating into germline cells and neural tissue to maintain the recipient porcine phenotype.…”