Major transcriptomic, epigenetic and metabolic changes underlie the pluripotency continuum in rabbit preimplantation embryos.

Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit preimplantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis, and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic and metabolic map of the pluripotency continuum in rabbit preimplantation embryos, and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with those of humans and non-human primates.

Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells.

After reprogramming to naive pluripotency, human pluripotent stem cells (PSCs) still exhibit very low ability to make interspecies chimeras. Whether this is because they are inherently devoid of the attributes of chimeric competency or because naive PSCs cannot colonize embryos from distant species remains to be elucidated. Here, we have used different types of mouse, human, and rhesus monkey naive PSCs and analyzed their ability to colonize rabbit and cynomolgus monkey embryos. Mouse embryonic stem cells (ESCs) remained mitotically active and efficiently colonized host embryos. In contrast, primate naive PSCs colonized host embryos with much lower efficiency. Unlike mouse ESCs, they slowed DNA replication after dissociation and, after injection into host embryos, they stalled in the G1 phase and differentiated prematurely, regardless of host species. We conclude that human and non-human primate naive PSCs do not efficiently make chimeras because they are inherently unfit to remain mitotically active during colonization.

Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors.

Rabbit induced pluripotent stem cells (rbiPSCs) possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs) 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA) were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5), epiblast in 10% mid-blastocysts (E4.5), and embryonic disk in 1.4% pre-gastrulae (E6). Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency.

A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits.

Conventional rabbit embryonic stem cell (ESC) lines are derived from the inner cell mass (ICM) of pre-implantation embryos using methods and culture conditions that are established for primate ESCs. In this study, we explored the capacity of the rabbit ICM to give rise to ESC lines using conditions similar to those utilized to generate naive ESCs in mice. On single-cell dissociation and culture in fibroblast growth factor 2 (FGF2)-free, serum-supplemented medium, rabbit ICMs gave rise to ESC lines lacking the DNA-damage checkpoint in the G1 phase like mouse ESCs, and with a pluripotency gene expression profile closer to the rabbit ICM/epiblast profiles. These cell lines can be converted to FGF2-dependent ESCs after culture in conventional conditions. They can also colonize the rabbit pre-implantation embryo. These results indicate that rabbit epiblast cells can be coaxed toward different types of pluripotent stem cells and reveal the dynamics of pluripotent states in rabbit ESCs.