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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.

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 pre-implantation 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 that of humans and non-human primates.Summary StatementRabbit preimplantation embryos share characteristics with human and monkey embryos with respect to timing of early lineage segregation and expression of marker genes for naive and primed pluripotency.

Induced pluripotent stem cells (iPSCs) are obtained by genetically reprogramming adult somatic cells via the overexpression of specific pluripotent genes. The resulting cells possess the same differentiation properties as blastocyst-stage embryonic stem cells (ESCs) and can be used to produce new individuals by embryonic complementation, nuclear transfer cloning, or in vitro fertilization after differentiation into male or female gametes. Therefore, iPSCs are highly valuable for preserving biodiversity and, together with somatic cells, can enlarge the pool of reproductive samples for cryobanking. In this study, we subjected rabbit iPSCs (rbiPSCs) and rabbit ear tissues to several cryopreservation conditions with the aim of defining safe and non-toxic slow-freezing protocols. We compared a commercial synthetic medium (STEM ALPHA.CRYO3) with a biological medium based on fetal bovine serum (FBS) together with low (0&ndash, 5%) and high (10%) concentrations of dimethyl sulfoxide (DMSO). Our data demonstrated the efficacy of a CRYO3-based medium containing 4% DMSO for the cryopreservation of skin tissues and rbiPSCs. Specifically, this medium provided similar or even better biological results than the commonly used freezing medium composed of FBS and 10% DMSO. The results of this study therefore represent an encouraging first step towards the use of iPSCs for species preservation.

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Summary 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., Graphical Abstract, Highlights • Mouse ESCs are highly effective in colonizing rabbit and non-human primate embryos • Rhesus monkey and human naive PSCs ineffectively colonize rabbit and monkey embryos • Most rhesus/human naive PSCs differentiate prematurely upon injection into embryos • Rhesus monkey PSCs stall in the G1 phase after transfer into rabbit embryos, Aksoy and colleagues report that mouse embryonic stem cells (mESCs) remained mitotically active after injection into rabbit and cynomolgus monkey embryos. In contrast, primate naive PSCs colonized rabbit and cynomolgus embryos with much lower efficiency. Unlike mouse ESCs, they died of apoptosis. The surviving cells slowed DNA replication, stalled in the G1 phase of the cell cycle, and differentiated prematurely, thus questioning their very nature.

International audience; Pluripotency describes the ability of a stem cell to give rise to all cell types in mature organisms. Two main types of pluripotent stem cells (PSCs) have been described: naïve and primed. In rodents, naïve-state pluripotency characterises mouse embryonic stem cells (mESCs) that have been derived from the pre-implantation epiblast. In primates (human and monkey) and lagomorphs (rabbits), ESCs only exist in the primed state of pluripotency. Primed-state pluripotency characterises the epiblast of developmentally more advanced embryos, closer to the commitment to differentiation. Contrary to naïve PSCs, PSCs in the primed state of pluripotency fail to make chimeras when introduced into host blastocysts. Several methods have been developed to reprogram human and monkey ESCs to naïve-like pluripotency using various cocktails of growth factors and small molecules. However, none of them works in rabbits, suggesting that the molecular network of naïve pluripotency differ between primates and lagomorphs. This study aimed to decipher the molecular signature of naïve pluripotency in the rabbit. Using single-cell 10x Chromium RNA-sequencing of rabbit embryos, we analysed the expression of genes regulating pluripotency, as well as genes involved in other processes that shape pluripotency (cell-cycle machinery, chromatin-related regulators, metabolism). From those data, we observed strong similarities between rabbits, primates, and rodents. However, we also observed differences in the expression of well-known marker of naïve pluripotency in mice, such as Zinc-Finger Protein 42 (ZFP42, also known as REX1) or Teratocarcinoma-Derived Growth Factor 1 (TDGF1, also known as CRIPTO). We also identified novel molecular markers useful to characterize naïve-state pluripotency in rabbits that could then be used to capture naïve-state pluripotency in rabbit PSCs.

International audience; Both embryo-derived (ESC) and induced pluripotent stem cell (iPSC) lines have been established in rabbit. They exhibit the essential characteristics of primed pluripotency. In this review, we described their characteristic features at both molecular and functional levels. We also described the attempts to reprogram rabbit pluripotent stem cells (rbPSCs) toward the naive state of pluripotency using methods established previously to capture this state in rodents and primates. In the last section, we described and discussed our current knowledge of rabbit embryo development pertaining to the mechanisms of early lineage segregation. We argued that the molecular signature of naive-state pluripotency differs between mice and rabbits. We finally discussed some of the key issues to be addressed for capturing the naive state in rbPSCs, including the generation of embryo/PSC chimeras.

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 post-implantation 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.

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International audience; Epigenetic modifications and their regulations participate in the phenotype establishment of each cell type. Accordingly, cell differentiation is accompanied by heritable changes of epigenetic signatures, while cellular reprogramming requires resetting of these epigenetic modifications. However, in many protocols, when differentiated cells are reprogrammed to the induced pluripotent stem cells (iPSCs) state, somatic epigenetic signature is only partially reprogrammed. We believe understanding the underlying mechanisms of those epigenetic changes would provide a more precise and efficient way of reprogramming cell fate. One of the main feature of epigenetic reprogramming is chromatin structure remodeling by covalent modifications on DNA and histones. This remodeling often involves changes in transcription as a result of chromatin opening and increased binding of transcription factors. Another consequence is a higher chromatin mobility that could participate in the acceleration of cell cycle and cell proliferation. In view of this, we used rabbit iPSCs to interrogate the effect of chemical inhibitors of DNA methyltransferases (SGI1027), histone deacetylases (BRD4354 and UF010) and methyltransferases (A366 and UNC06) on chromatin structure and subsequent cell cycle features. For this purpose, cells were cultured with the inhibitors for 24, 48, 96 or 192 hours. The cell cycle was then analyzed thanks to an improved FUCCI cell cycle reporter system and TimeLapse analysis. We also performed immunostainings to confirm the efficiency of inhibitors on their epigenetic targets. Results show that cell cycle lengths are fairly similar between controls and iPSC cultured with inhibitors. However, a significant decrease is observed, with a G1-phase

International audience

International audience; Pluripotency describes the ability of a stem cell to give rise to all cell types in mature organisms. Two main types of pluripotent stem cells (PSCs) have been described: naïve and primed. In rodents, naïve-state pluripotency characterises mouse embryonic stem cells (mESCs) that have been derived from the pre-implantation epiblast. In primates (human and monkey) and lagomorphs (rabbits), ESCs only exist in the primed state of pluripotency. Primed-state pluripotency characterises the epiblast of developmentally more advanced embryos, with a higher concentration of heterochromatin and an inactivated X chromosome in female cells. The study aimed to decipher the chromatin context of naïve and primed pluripotency in the rabbit embryo, from the compacted morula stage until the onset of the gastrulation in the epiblast. Using immuno-fluorescence, we analysed the distribution of DNA-methylation and hydroxymethylation, Histone 3 lysine 9 and 27 methylation, as well as Histone 2A lysine 119 ubiquitination. In parallel, we analysed the expression of genes encoding the erasers and writers of those epigenetic marks using single-cell Biomark qPCR. From these experiments, we observed a progressive loss of heterochromatin marks in the pluripotent cells of early blastocysts (Inner Cell Mass or ICM), followed by gain of heterochromatin in more advanced stages (in the epiblast). Those variations are associated with the transient downregulation of genes promoting heterochromatin formation in the ICM, while genes promoting euchromatin formation are downregulated in the epiblast. This study helped us to identify the chromatin landscape and its regulators associated to the naïve state of pluripotency in rabbits.

Since the derivation of the first pluripotent embryonic stem cell lines in mice in the early 1980s, a plethora of lines has been obtained from various mammalian species including rodents, lagomorphs and primates. These lines are distinguished by their molecular and functional characteristics and correspond to the different pluripotency states observed in the developing embryo between the "blastocyst" and "gastrula" stages. These cell lines are positioned along a gradient, or continuum of pluripotency, the ends of which are epitomized by the naïve and primed states, respectively. Conventional human pluripotent stem cells self-renew in the primed state of pluripotency (ie, at the bottom of the gradient), a position that is undoubtedly the cause of their natural instability. Recent studies aim to generate naive human pluripotent stem cells (at the top of the gradient). The importance of this research in the perspective of medical applications will be discussed.

International audience; In the rabbit, many attempts have been made to reprogram pluripotent stem cells (PSC) to the naïve state, but none of the conditions tested could lead the naïve state as defined in the mouse. These previous studies also highlighted differences in the molecular signature of the naïve and primed pluripotency states between rabbit vs. mouse. In order to address these differences and identify new markers of naïve and primed pluripotency in rabbit, it is necessary to characterize in more details the transcriptome of the pluripotent cells in vivo, i.e. in the rabbit embryo. We, therefore, performed a Biomark qPCR single-cell analysis of rabbit embryos around the time of lineage segregation, from the morula stage to the early gastrulating stage. We identified a transcriptomic continuum of pluripotency in vivo specific to the rabbit, although we observed many similarities to the mouse. These results will allow us to judge the state of pluripotency of rabbit PSC we will derive in vitro, as compared to the embryo, and to find new targets to reprogram them towards the naïve state.

International audience; Depuis la dérivation des premières lignées de cellules souches embryonnaires pluripotentes chez la souris au début des années 1980, une pléthore de lignées a été obtenue chez diverses espèces de mammifères, dont les rongeurs, les lagomorphes1 et les primates. Ces lignées se distinguent par leurs caractéristiques moléculaires et fonctionnelles et correspondent aux différents états de pluripotence observés chez l’embryon, entre les stades blastocyste et gastrula. Ces lignées se répartissent le long d’un gradient, ou continuum de pluripotence, dont les deux extrémités sont symbolisées par les états appelés naïf et amorcé. Les cellules souches pluripotentes humaines sont dans un état de pluripotence amorcé (au bas du gradient), une position qui est sans doute la cause de leur instabilité naturelle. Les recherches récentes visent à obtenir des cellules souches pluripotentes humaines à l’état naïf (en haut du gradient). L’importance de ces recherches dans la perspective d’applications médicales est discutée dans cette revue.

Le travail présenté dans ce mémoire vise à définir les conditions de dérivation et de culture de cellules souches pluripotentes ou PSC (Pluripotent Stem Cells) de lapin aptes à produire des chimères germinales. Les PSC sont des cellules capables de se différencier dans les trois lignages embryonnaires (ectoderme, mésoderme, et endoderme) et de s’auto-renouveler, c’est-à-dire de se multiplier indéfiniment en culture sans perdre leur caractère pluripotent. Chez la souris, ces cellules sont à la base des techniques de transgénèse permettant des modifications génétiques ciblées. Il existe trois types de PSC murines: (i) les cellules souches embryonnaires ou ESC (Embryonic Stem Cells), qui dérivent du bouton embryonnaire d’embryons précoces; (ii) les cellules souches de l’épiblaste ou EpiSC (Epiblast-derived Stem Cells) qui dérivent de l’épiblaste d’embryons post-implantatoires ; et (iii) les cellules souches pluripotentes induites ou iPSC (induced Pluripotent Stem Cells) obtenues après reprogrammation de cellules somatiques. On peut classer ces cellules en deux catégories en fonction des voies de signalisation qui activent les gènes responsables de leur pluripotence : (i) les cellules à l’état naïf de pluripotence, dépendantes de la cytokine LIF et de la voie de signalisation gp130/Stat3, comme les ESC et iPSC murines. Les mPSC sont capables de coloniser un blastocyste receveur pour former des chimères germinales et présentent les caractéristiques transcriptomiques et épigénétiques des cellules pluripotentes de l’épiblaste précoce de l’embryon pré-implantatoire ; (ii) les cellules à l’état amorcé de pluripotence, dépendantes du facteur FGF2 et de la voie de signalisation TGFβ/Activine/Nodal, comme les EpiSC murines et les PSC des autres mammifères. Les EpiSC sont incapables de former des chimères après injection dans un blastocyste receveur. Elles sont donc engagées vers la différenciation et proches des cellules de l’épiblaste tardif de l’embryon post-implantatoire. Le lapin est un animal modèle intéressant car, d’une part, il est physiologiquement et génétiquement plus proche des primates que ne l’est la souris et, d’autre part, il présente un développement embryonnaire et un système de placentation identiques à ceux des primates. Les PSC de lapin sont dans un état amorcé de pluripotence, incapables de produire des chimères. La fabrication de PSC à l’état naïf faciliterait le développement des technologies de la transgènèse, et permettrait à terme la création de lapins modèles de maladies humaines, ou de créer des réacteurs biologiques. Mon travail vise donc à déterminer les conditions permettant de dériver et de cultiver de telles cellules chez cette espèce. Dans ce but, je développe différents projets qui visent : (i) à caractériser la pluripotence in vivo chez le lapin aux niveaux transcriptomique, épigénétique et métabolique ; et (ii) à capturer in vitro un état de pluripotence proche de celui de l’épiblaste de lapin au stade E4-E5.5, capable de produire des chimères germinales. Ce travail me permettra d’obtenir des PSC de lapin à l’état naïf de pluripotence, mais également de produire des données sur les mécanismes soutenant la pluripotence chez un mammifère non-murin, autre que les primates. En effet, les comparaisons multi-espèces sont nécessaires à la compréhension et à la généralisation des mécanismes biologiques et développementaux. Ce mémoire se présente donc en trois parties qui développent respectivement l’état de l’art sur les PSC, l’analyse des travaux que nous avons réalisés sur les PSC de lapin et une discussion amenant les projets en cours. Il regroupe également mes publications sur le sujet.

The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians.

Master

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