Jean Lépine

Keywords: Pluripotency, Stem cells, Primates, Human, Embryology, Embryogenesis Je souhaite dans

Pluripotency is defined by the ability of a cell to self-renew and to differentiate in to the three primary germ layers, mesoderm, ectoderm and endoderm. In the mouse, two types of pluripotency have been defined: naïve and primed, the former originating from the naïve epiblast of the pre-implantation embryo, and the latter from the post-implantation epiblast.

These two states, despite sharing the core characteristics of pluripotency, differ in the molecular pathways and transcriptional networks underpinning their regulation. In primates, naïve pluripotency as defined in mice cannot be captured in vitro. In spite of the many protocols established to reprogram primed human cells to a "naïve-like" state, the issue remains unsolved, suggesting that other regulators of primates' naïve pluripotency exist and remain to be identified. In the mouse, it has recently been shown that Netrin-1, a protein belonging to the Laminins superfamily, is a regulator of naïve pluripotency. In this work, we thus undertook to characterize Netrins family function in primates' pluripotency regulation. We demonstrated that, unlike for the mouse and macaque, NTN1 expression is not associated with pluripotency in human, but rather triggers differentiation of naïve-like human pluripotent stem cells (PSCs) to mesodermal lineages. NTN1 antagonist DRAXIN, on the contrary, is enriched in the human pre-implantation epiblast and shields PSCs against NTN1-induced differentiating, therefore constituting a new potential regulator of pluripotency in human.

Rôle de la signalisation Nétrine dans la régulation de la pluripotence du primate humain et non-humain

Résumé

La pluripotence se définit comme la capacité d'une cellule à s'auto-renouveler et à se différencier dans les trois lignages primaires que sont le mésoderme, l'ectoderme et l'endoderme. Chez la souris, deux types de pluripotence ont été définis : la pluripotence naïve, trouvant son origine dans l'épiblaste pré-implantatoire, et la pluripotence amorcée, issue de l'épiblaste post-implantatoire. Ces deux types de pluripotence, bien que partageant les caractéristiques centrales précédemment citées, divergent sur les signaux moléculaires et les réseaux transcriptionnels qui les régissent. Chez les primates, la pluripotence naïve telle que définie chez la souris ne peut être capturée in vitro. En dépit de protocoles mis au point pour reprogrammer les cellules humaines amorcées vers un état « pseudo-naïf », cette problématique reste non résolue à ce jour, et suggère que la pluripotence chez le primate est régie par d'autres voies moléculaires encore non identifiées. Chez la souris, il a été récemment découvert qu'une molécule de la superfamille des laminines, Nétrine-1, est un régulateur de la pluripotence naïve, nous avons donc entreprit dans le présent projet de caractériser le rôle des Nétrines dans la pluripotence chez le primate. Nous avons démontré que l'expression de NTN1, contrairement au macaque et à la souris, n'est pas associée à la pluripotence chez l'homme, mais déclenche au contraire la différentiation des cellules souches pluripotentes humaines (CSPs) en lignages mésodermiques. L'antagoniste de NTN1, DRAXIN, est au contraire enrichi dans l'épiblaste pré-implantatoire humain et protège les CSPs de la différenciation induite par NTN1, constituant de ce fait un nouveau régulateur potentiel de la pluripotence chez l'homme.

Stem Cell and Brain Research Institute (SBRI), Inserm 1208; 18 Avenue du Doyen Jean Lépine, Bron.

Mots-clés : Pluripotence, Cellules souches, Primates, Humain, Embryologie, Embryogénèse n'auraient pas eu la même saveur, et merci à mes stagiaires, Alex, Julien et Nawel qui ont tous participé avec assiduité à l'acquisition des résultats de ce projet.

Merci à ma famille et à mes amis, Nico (les deux), Béa, Sémi, Mehdy, et tous ceux que j'oublie et qui m'en voudront (mais que j'aime bien quand même), d'avoir supporté des monologues sur des problèmes de labo auxquels, pour la plupart d'entre vous, vous ne compreniez rien. Merci aussi de tolérer mon esprit scientifique toujours prêt à saccager un débat sur les ovnis, les fantômes ou l'astrologie. Hélas pour vous, ce dernier point ne va pas prendre fin avec cette thèse… Enfin, j'aimerais adresser des remerciements spéciaux aux personnes qui ont étroitement travaillé avec moi sur ce projet, et sans lesquelles il ne serait probablement pas arrivé à terme. Premièrement, à Guillaume Marcy, qui m'a enseigné à peu près tout ce que je sais en bioinformatique, avec lequel j'ai eu de longues et nombreuses discussions indispensables à ma réflexion, et sans qui ce manuscrit ne serait tout simplement que l'ombre de ce qu'il est.

Ensuite, à Cloé Rognard, que j'ai un jour qualifiée d'ange gardien de l'équipe, et qui l'a en tout cas certainement été pour moi. Les milliers d'heures passées au P2 auraient été ressenties au centuple sans elle, elle fut une aide indispensable de tous les instants en culture comme en biomol, une oreille patiente et attentive à toutes les difficultés ainsi qu'à toutes les plaisanteries (d'une qualité parfois franchement discutable) ; une merveilleuse collègue et une amie.

Enfin, j'adresse mes infinis remerciements à Irène Aksoy, qui m'a encadré de la plus excellente des manières durant mon stage de master (stage auquel, soit dit en passant, je n'aurais strictement rien changé…), et a continué de le faire en thèse. Tous les étudiants ne bénéficient pas d'un encadrement si étroit, tous ne sont pas défendus et soutenus comme je l'ai été par Irène. Je souhaitais initialement dédier ce manuscrit à ceux de mes professeurs d'école, de collège et même de lycée, qui disaient de moi que je n'étais bon qu'à récolter des heures de colle, mais c'est finalement à toi que je le dédie, Irène. Merci pour tout. Embryogenesis is characterized by the progressive specialization of stem cells, that commit as a matter of time into tissues of restricted function, giving the adult organism. This process of specialization is also called differentiation, and the potential of stem cells to a given extent of specialization is referred to as potency. Because mouse embryos are small sized, low cost and easy to obtain, mouse has become over the past decades the golden standard to study early stages of mammals' embryogenesis [START_REF] Taft | Virtues and limitations of the preimplantation mouse embryo as a model system[END_REF]. It is in mouse that the fundamental concepts of pre-implantation development, and pluripotency, have been first and the most thoroughly described.

Table of Contents

Figures and Tables Index

Pluripotency in vivo

Pre-implantation stages of eutherian mammals' embryogenesis are articulated around the segregation of embryonic and extra-embryonic tissues. In the mouse model, before E2.5, the embryo is constituted of blastomeres undergoing regular rounds of division. These blastomeres are totipotent, meaning they hold the capacity to give both embryonic and extraembryonic lineages. At E3.0, the first differentiation event occurs, and these blastomeres segregate into two tissues: the trophectoderm (TE), contributing to the fetal portion of the placenta, and the Inner Cell Mass (ICM). The ICM undergoes the second round of differentiation of the embryo at E3.5, timepoint marking the beginning of the blastocyst stage.

Two populations of cells arise then, the primitive endoderm (PE) and the epiblast (Epi). The former gives rise to the parietal and visceral endoderm of the yolk sac and to a subpopulation of cells in the early gut tube [START_REF] Schrode | Anatomy of a blastocyst: Cell behaviors driving cell fate choice and morphogenesis in the early mouse embryo[END_REF]. The latter is composed of Pluripotent Stem Cells (PSCs), entirely devoted to the formation of the embryo strictly speaking and holding the capacity to form any cell type in an adult organism (Figure 1).

a) Molecular regulation of the ICM onset

Between the 8-cell and 32-cell stages of mouse development, the combination of symmetrical and asymmetrical divisions yields two populations of blastomeres: outer and inner [START_REF] Humiêcka | Mouse blastomeres acquire ability to divide asymmetrically before compaction[END_REF]. Though identical in morphology and potency, these two populations start to diverge, i) in their polarity status, ii) in the differential expression of a subset of genes [START_REF] Guo | Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression Analysis from Zygote to Blastocyst[END_REF]. Among those genes, three in particular have been shown to be critical in the ICM vs TE specification: Caudal-related homeobox 2 (Cdx2), Nanog and Oct4.

Cdx2 is detectable in a subset of blastomeres as soon as the 8-cell stage of development and is then significantly more expressed in outer blastomeres, until being fully restricted to the TE at the blastocyst stage (Ralston and Rossant, 2008). Cdx2 -/-mutant embryos, although they form morphologically normal blastocysts, fail to implant. This is due to a complete lack of TE specification in said embryos (Strumpf et al., 2005). Cdx2 is therefore necessary to trigger TE allocation.

Oct4 (also known as Pou5f1), a POU family protein, is expressed in the oocyte and in all blastomeres of the cleavage stage embryo, and is then restricted to the ICM and Epiblast (Scholer et al., 1989). Oct4 -/-mutant embryos, although they form blastocysts, do not develop an ICM and instead yield cells all committed to TE [START_REF] Nichols | Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[END_REF]. In complete opposition to Cdx2, Oct4 Is therefore necessary to ICM commitment, and its absence favors TE differentiation.

Nanog, a variant homeodomain protein, is expressed at the 8-cell stage and, as for Oct4, is subsequently maintained in the ICM and Epiblast. Nanog null mutants form an ICM, but when derived in vitro (surgical removal of TE), cells appear to not persist in undifferentiated masses, but instead entirely commit to a PE-like fate in 4 days [START_REF] Mitsui | The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[END_REF][START_REF] Silva | Nanog Is the Gateway to the Pluripotent Ground State[END_REF]. This demonstrates that Nanog, unlike Oct4, is not necessary to ICM formation, but is rather critical to its maintenance.

Altogether, Cdx2 and Oct4/Nanog therefore work in an ambivalent fashion, restricting each other's expression to promote respectively TE and ICM commitment [START_REF] Okumura-Nakanishi | Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells[END_REF]Strumpf et al., 2005;Wang et al., 2010) (Figure 2). This process is also the founding step to the epiblast segregation, and therefore pluripotency establishment in vivo.

b) Emergence of the epiblast

After ICM establishment at E3.0, the cavitation process occurs, leading to the appearance of the blastocoelic cavity. This event marks the entrance in the blastocyst stage of development, at E3.5. In the meantime, ICM cells begin to differ in the expression of several factors, in a salt-and-pepper manner. Two subsets of cells can be distinguished then: those expressing Nanog, and those expressing Gata6 (Chazaud et al., 2006;Gerbe et al., 2008;Kurimoto et al., 2006;[START_REF] Plusa | Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst[END_REF]Rossant et al. 2003). The latter migrate toward the blastocoelic cavity and form an epithelialized layer, the PE, and the former stay in between the TE and newly formed PE epithelium, yielding the pluripotent epiblast. Before they are fully segregated into either Epi or PE, these cells are referred to as "biased", meaning their engagement in one or either way is not complete. Epi-biased cells, in addition to the expression of Nanog, upregulate the Fibroblast Growth Factor 4 (Fgf4), while PE-biased cells upregulate its receptor, Fgfr2 [START_REF] Guo | Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression Analysis from Zygote to Blastocyst[END_REF]. Conversely, both proteins are respectively downregulated in PE-and Epi-biased cells. Fgf4 binding to its receptor activates the MEK/ERK pathway, and leads to the upregulation of Gata6. Once expressed, Gata6 in turns inhibits Nanog expression, promoting PE differentiation (Figure 2). Removal of Fgf4, Fgfr2 or any downstream actor of the cascade leads to a failure of commitment into PE (Arman al., 1998;Chazaud et al., 2006;Feldman et al., 1995;Kang et al. 2013).

As stated earlier, Oct4 is critical to the ICM establishment and maintenance, and its expression is maintained and just as mandatory in the epiblast. Nanog is paramount to the epiblast vs PE establishment. However, alongside Nanog and Oct4, a third factor is critical to the epiblast emergence: the SRY (Sex-determining Region Y) box 2 transcription factor, also known as Sox2. Sox2 overlaps Oct4 in terms of kinetics and patterning of expression in the early mouse embryo, but unlike Oct4, null mutants develop to the blastocyst stage and exhibit an ICM. This ICM though, fails to allocate an epiblast, while both PE and TE are present in the subsequent hours [START_REF] Avilion | Multipotent cell lineages in early mouse development depend on SOX2 function[END_REF]. Oct4, Sox2 and Nanog are therefore altogether necessary to the epiblast emergence, and thus pluripotency in vivo, reason why they are often referred to as the triumvirate of pluripotency. Those three factors also appear to crosstalk and regulate one another [START_REF] Liang | Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells[END_REF]. Oct4 and Sox2 form a transcriptional complex that regulates both Oct4 and Sox2 expression [START_REF] Chew | Reciprocal Transcriptional Regulation of[END_REF][START_REF] Okumura-Nakanishi | Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells[END_REF], and promotes Nanog upregulation [START_REF] Kashyap | Regulation of Stem cell pluripotency and differentiation involves a mutual regulatory circuit of the Nanog, OCT4, and SOX2 pluripotency transcription factors with polycomb Repressive Complexes and Stem Cell microRNAs[END_REF][START_REF] Rodda | Transcriptional regulation of Nanog by OCT4 and SOX2[END_REF].

Pluripotency in vitro -Pluripotent Stem Cells derivation a) Conventional Embryonic Stem Cells (ESCs)

In 1981, a procedure to expand epiblast cells in vitro was developed [START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF][START_REF] Martin | Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[END_REF]. These cells, initially cultured on a layer of mitotically inactivated fibroblasts in a medium containing calf serum, were called Embryonic Stem (ES) cells. ES cells have a high nuclear/cytoplasmic ratio, form tightly packed, dome-shaped colonies and can be dissociated in single cells able to expand and form colonies de novo. This property is referred to as clonogenicity [START_REF] Hackett | Regulatory principles of pluripotency: From the ground state up[END_REF]. As for the epiblast, ES cells can differentiate into any cell type from the three primary germ layers (ectoderm, mesoderm and endoderm) [START_REF] Smith | Embyronic-Derived Stem Cells: Of Mice and Men[END_REF], and can be propagated in vitro indefinitely. This property of self-replication without genetic impairment is called "self-renewal" and defines, along with differentiation into the three germ layers, pluripotency.

In addition to their in vitro potential, ES cells also hold the remarkable attribute to reenter embryogenesis. Indeed, when injected in a host blastocyst, they are capable of participating in the development of the embryo, even if they have been precedingly manipulated and propagated in vitro [START_REF] Bradley | Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines[END_REF]. Exogenous ESCs yield progeny in all tissues and organs of the host with no bias, proving that their potential remains intact. This unique capacity of embryo colonization is called chimeric competency, because the resulting embryo is a chimera between two distinct individuals.

In 1988, it was found that feeder cells used to co-culture ESCs could be replaced by a single cytokine, Leukemia Inhibitory Factor (LIF) [START_REF] Smith | Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides[END_REF][START_REF] Williams | Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells[END_REF]. LIF is naturally secreted by the feeders in presence of ES cells, and its removal alone leads to their differentiation and the subsequent arrest of their propagation after a few days. It was shown that the molecular signal of LIF is transduced by the gp130 receptor [START_REF] Yoshida | Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gp130 signalling pathways[END_REF] and inhibits differentiation through activation of the JAK-STAT3 pathway [START_REF] Niwa | Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3[END_REF]. LIF triggers the cross phosphorylation of JAKs, that in turn recruits and phosphorylates STAT3, allowing it to enter the nucleus and activate the transcription of target genes (summarized on Figure 3 and further discussed in part I/2.d)).

ES cells cultured in serum/LIF exhibit heterogeneity in terms of morphology and expression of several genes, among which notably, Nanog [START_REF] Chambers | Nanog safeguards pluripotency and mediates germline development[END_REF][START_REF] Hayashi | Dynamic Equilibrium and Heterogeneity of Mouse Pluripotent Stem Cells with Distinct Functional and Epigenetic States[END_REF].

Serum batches need to be screened before use, and only a few mouse strains are permissive to ES cells derivation with such culture conditions. Therefore, replacing serum was critical to restrain variability and understand the extrinsic factors supporting pluripotency. It was observed that upon removal of serum and LIF, established ES cell lines preferentially differentiate into neurectodermal lineages (Ying et al., 2003b). In Xenopus embryo, the TGFβ superfamily growth factor BMP4 is known to suppress neuralization [START_REF] Sasai | Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus[END_REF].

Based on this observation, BMP4 was used in replacement of serum in ES culture media, and was shown to be sufficient, through inhibition of Id target genes, to suppress differentiation in the presence of LIF (Ying et al., 2003a)

b) 2i/LIF ESCs

As stated in part I/1.b), the ERK/MAPK pathway is activated in a non-cell autonomous manner through Fgf4-Fgfr2 binding in the mouse blastocyst, and promotes the second event of differentiation of ICM into PE. In line with this observation in vivo, chemically suppressing FGF/ERK in vitro with the MEK inhibitor PD098059 leads to an enhancement of self-renewal, and to an inhibition of differentiation in embryoid bodies formation assays [START_REF] Burdon | Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells[END_REF].

In opposition to FGF/ERK, it was shown that Wnt pathway activation was associated to pluripotency maintenance in ES cells [START_REF] Merrill | Wnt pathway regulation of embryonic stem cell self-renewal[END_REF]. Wnt ligands trigger detachment of GSK3 from β-catenin, preventing its phosphorylation and degradation. Accordingly, inhibition of GSK3 with a pharmacological inhibitor, 6-bromoindirubin-3'oxime, in addition to LIF, maintains the undifferentiated phenotype of ES cells, although reducing their viability [START_REF] Sato | Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor[END_REF].

On the basis of these findings, both inhibition of FGF/ERK and GSK3 was succeedingly attempted, with the combined use of PD098059 and CHIR99021 pharmacological inhibitors [START_REF] Ying | The ground state of embryonic stem cell self-renewal[END_REF]. This approach, called 2i, yields complete suppression of differentiation (Figure 3). When LIF is added, ES cells, contrary to their Serum/LIF counterparts, are largely homogeneous in morphology and gene expression [START_REF] Wray | Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation[END_REF]. 2i/LIF (also referred to as ground state condition, see part I/3.e)) is now widely adopted, and allows derivation of ES cell lines from otherwise restrictive mouse strains.

c) Epiblast-derived Stem Cells (EpiSCs)

After uterine implantation, the mouse embryo changes shape, and goes from blastocyst to a cup shaped structure called the egg cylinder [START_REF] Kaufman | The Atlas of Mouse Development[END_REF]. The epiblast does not cease to exist but is submitted to new molecular signals secreted by the trophoblast and the yolk sac.

These include Bone Morphogenic Proteins (BMPs), Fibroblasts Growth Factors (FGFs) and

Wnt family molecules [START_REF] Beddington | Axis development and early asymmetry in mammals[END_REF]. In XX embryos, post-implantation Epiblast cells also show random inactivation of one X, whereas both are maintained active during the pre-implantation period [START_REF] Kalantry | Recent advances in X-chromosome inactivation[END_REF]. These differences between pre-and postimplantation epiblast, however, do not appear to be associated with a loss of pluripotency after implantation, as both types of cells maintain the capacity to differentiate into any cell lineage from the primary germ layers.

In line with this observation, PSCs were derived from the post-implantation Epiblast (between E5.5 to 7.5) in 2007, and named post-implantation Epiblast-derived Stem Cells (EpiSCs). EpiSCs, just as ES cells, show the ability to self-renew and to differentiate into the three germ layers, and exhibit expression of the pluripotency triumvirate Oct4, Nanog and Sox2 [START_REF] Brons | Derivation of pluripotent epiblast stem cells from mammalian embryos[END_REF][START_REF] Tesar | New cell lines from mouse epiblast share defining features with human embryonic stem cells[END_REF].

d) Induced Pluripotent Stem Cells (iPSCs)

In 2006, Takahashi and Yamanaka identified 24 genes upregulated in either ESCs or tumors as potential candidates to induce pluripotency in somatic cells. By overexpressing each factor individually, no pluripotent characteristic could be observed, to the opposite of simultaneous expression of all 24 genes. Individual withdrawal of these factors allowed to break down the list to 4 critical factors whose absence did not allow to produce pluripotent colonies from somatic cells. These were Oct4, Sox2, Klf4 and c-Myc, known as the OSKM cocktail. Simultaneous overexpression of OSKM in mouse embryonic or somatic fibroblasts leads to effective conversion in pluripotent cells, named induced Pluripotent Stem cells (iPSCs). IPSCs can form teratomas, exhibit the growth properties and morphology of ESCs, and express gene markers specific of ESCs. Intriguingly, Nanog is dispensable to the reprogramming cocktail, as confirmed by double Knockout of Nanog in mouse embryonic fibroblasts (MEFs) submitted to OSKM ectopic expression. After induction, Nanog -/-are efficiently reprogrammed to induced pluripotency, demonstrating that endogenous Nanog expression is not necessary to the process [START_REF] Schwarz | Nanog is dispensable for the generation of induced pluripotent stem cells[END_REF]. Such cells, however, lack the capacity to produce adult chimeric mice, as wild-type first generation iPSCs, revealing a default in germline transmission. A proposed explanation is that Nanog is dispensable for the first steps of reprogramming, i.e. de-differentiation and acquisition of "pre-pluripotent characteristics", but paramount to germline-competency and acquisition of ground state pluripotency (further discussed in part I/3.e)) [START_REF] Silva | Nanog Is the Gateway to the Pluripotent Ground State[END_REF]. Accordingly, the second generation of iPSCs achieved germline transmission with addition of Nanog overexpression, or specific selection of Nanog-positive colonies [START_REF] Okita | Generation of germline-competent induced pluripotent stem cells[END_REF][START_REF] Wernig | In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state[END_REF].

From Naïve to Primed: The Continuum of Pluripotency a) Discrepancies of ESCs and EpiSCs

When they were first derived, it was observed that EpiSCs exhibit a number of differences with ES cells (summarized on Figure 4). First, they are derived and maintained in vitro with a medium containing Activin A and FGF2, contrary to the conventional Serum-BMP4/LIF ES cells medium, and no EpiSC line can be derived and/or maintained with such conventional conditions. In addition, application of the activin receptor inhibitor SB431542 leads to rapid differentiation of EpiSCs, showing that their pluripotency, unlike that of ESCs, depends strictly on the activin/nodal pathway. Second, they are morphologically distinct from ES cells, growing as flat monolayered colonies rather than tight dome-shaped ones. Third, when passaged by molecular dissociation with trypsin, EpiSCs undergo widespread cell death contrary to ESCs, and need to be mechanically dissociated in smaller clumps for amplification. Fourth, EpiSCs

show no expression of some pluripotency markers expressed in ESCs, such as Rex1 (Zfp42), and strongly reduced expression of others such as Gbx2; they also express early lineage markers such as Fgf5 (ectoderm), Eomes, Brachyury (mesoderm) and Gata6 (endoderm) [START_REF] Coronado | A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency[END_REF]. Fifth, Unlike ESCs, EpiSCs also appear to not express Germ line markers such as Blimp1 and Stella. Sixth, their transcriptomic signature, as shown by microarray studies, is similar to the late epiblast but distinct from ESCs and ICM/early-epiblast cells, consistent with their origin. Lastly, when micro-injected in pre-implantation mouse embryos, EpiSCs are uncapable of colonizing the host and to yield chimeras, as opposed to

ESCs [START_REF] Brons | Derivation of pluripotent epiblast stem cells from mammalian embryos[END_REF][START_REF] Tesar | New cell lines from mouse epiblast share defining features with human embryonic stem cells[END_REF].

b) Epigenetic landscape of ESCs and EpiSCs

Epigenome is defined by the accumulation of DNA and histones chemical modifications affecting the transcription of genes. These changes are of two main types: DNA methylation and histone modifications. DNA methylation plays a repressive role in gene transcription regulation, while histone modifications can be of two types, repressive or activating [START_REF] Thiagarajan | The epigenome in pluripotency and differentiation[END_REF]. During early development, global DNA demethylation occurs [START_REF] Smith | DNA methylation: Roles in mammalian development[END_REF], followed by selective de novo methylation. The early pluripotent epiblast is therefore characterized by a globally hypomethylated genome. Accordingly, ESCs exhibit low levels of DNA methylation, to the opposite of EpiSCs, derived from a later developmental stage [START_REF] Takahashi | Epigenetic differences between naïve and primed pluripotent stem cells[END_REF]. Histone modifications also differ sharply, particularly in enhancer sites, and also for genes that are expressed in both ESCs and EpiSCs. Oct4, for example, is controlled by its distal enhancer (DE) in ESCs, and by its proximal enhancer (PE)

in EpiSCs [START_REF] Tesar | New cell lines from mouse epiblast share defining features with human embryonic stem cells[END_REF]. In the case of female cell lines, EpiSCs exhibit one inactive X chromosome, as shown by the presence of the H3K27me3 mark, unlike ESCs that display two active chromosomes. This is consistent with in vivo observations (mentioned in part I/2.c)) that female peri-implantation epiblast cells only show one active X chromosome versus two in preimplantation epiblast.

Culturing ESCs in EpiSCs medium (i.e. with Activin and Fgf2) leads to their conversion in EpiSCs. The converse, however, is not possible without genetic modification or epigenetic reversion [START_REF] Guo | Klf4 reverts developmentally programmed restriction of ground state pluripotency[END_REF]. Taken together, these observations suggest that a one-way epigenetic barrier exists between ESCs and EpiSCs, consistently with the latter being derived from a more developmentally advanced and more restricted tissue [START_REF] Takahashi | A developmental framework for induced pluripotency[END_REF].

c) Cell Cycle of ESCs and EpiSCs

ES cells have unusual proliferative capacities. They do not suffer contact inhibition, are anchorage-independent and never undergo cell-cycle arrest or quiescence in conventional culture conditions, suggesting they hold a unique regulation of cell-cycle [START_REF] Burdon | Signalling, cell cycle and pluripotency in embryonic stem cells[END_REF].

In somatic cells, proliferation is mainly controlled by the regulation of the G1 to S phase transition. This process is monitored by the phosphorylation status of the retinoblastoma protein (RB). During the G1 phase, RB is hypophosphorylated and sequesters the E2F family transcription factors, blocking entry into S phase. As G1 phase progresses, RB is sequentially phosphorylated by Cyclin/Cyclin-dependent-kinases (CDKs) tandems, Cyclin D/Cdk4 or Cyclin E/Cdk6, leading to the release of E2F that in turns allows expression of Cyclin E. The Cyclin E/Cdk2 complex subsequently finalizes RB phosphorylation, leading to complete release of E2F and entry into S phase [START_REF] Harbour | The Rb/E2F pathway: Expanding roles and emerging paradigms[END_REF][START_REF] Harbour | Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1[END_REF]. In addition to this RB/E2F pathway, the c-Myc gene has also been demonstrated to control the G1/S phase transition.

It has been shown that ES cells have an unusually short G1 phase of approximately 1h, and at the same time do not exhibit hypophosphorylated RB [START_REF] Savatier | Analysis of the cell cycle in mouse embryonic stem cells[END_REF]. They are resistant to the Cyclin D/Cdk4-6 inhibitor p16ink4a, do not arrest in G1 phase at confluency (a process mediated by increasing hypophosphorylation of RB), and when triple knocked-out (TKO) for the RB family genes (p107-/-, p130-/-, Rb-/-), remain unaffected and carry on proliferating. These observations strongly suggest that ES cells escape from contact inhibition and G1 checkpoint is at least in part due to shielding against RB regulating activity of the G1/S transition. Moreover, ES cells show low levels of Cyclin D1 and D3, and no Cyclin D2 at all.

Cdk4 kinase activity is also undetectable. This reflects the state of pre-implantation epiblast cells, that also appear to have low amounts of D-type cyclins. Once gastrulation in vivo and differentiation in vitro start, however, D-type cyclins expression is strongly increased and Cdk4 kinase activity restored, showing that response to G1 regulatory control is recovered [START_REF] Savatier | Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells[END_REF].

When ESCs are converted in EpiSCs by treatment with FGF2 and Activin, the proportion of cells in G1 phase, monitored by FACS-sorting using a cell cycle phase-specific fluorescent system, drastically increases (from ~3% to ~25%) [START_REF] Coronado | A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency[END_REF]. On the contrary, when conventional ESCs are transferred to 2i/LIF condition, only rare cells are found left to be in G1 phase. On the other hand, ESCs in G1 phase are more prone to differentiation, as evidenced by their response to Retinoic Acid (RA). When isolated from their population, ESCs in G1 submitted to a RA treatment form more mixed and differentiated colonies than their S and G2/M counterparts. This is also true, though to a lesser extent, for RA-untreated G1 cells, demonstrating that the G1 phase of the cell cycle is favorable to differentiation.

Thus, G1 phase lengthens as ES cells transit from 2i/LIF to Serum-BMP/LIF and Activin/Fgf2, demonstrating that ESCs and EpiSCs differ, in addition to their precedingly mentioned discrepancies, in their cell cycle and proliferation rate. This divergence, as the entirety of the properties of EpiSCs, suggest that they are more prone to differentiate than their ESCs counterparts, because G1 is the privileged phase for differentiation. This is consistent with their in vivo provenance of peri-implantation epiblast, poised to initiate gastrulation.

d) The Metabolic Switch of PSCs

Because ESCs are rapidly proliferating, energy must be balanced accordingly. [START_REF] Hayashi | Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells[END_REF][START_REF] Ohinata | A Signaling Principle for the Specification of the Germ Cell Lineage in Mice[END_REF].

EpiLCs, though short-lived and heterogeneous, hold characteristics consistent with the existence of a conceptual phase of development and pluripotent state named "Formative" [START_REF] Smith | Formative pluripotency: The executive phase in a developmental continuum[END_REF]. This state corresponds to the period of time observed in vitro, where naïve cells subjected to differentiating cues are not immediately responsive and need to exit their naïve status first. During this time, a transient population appears able to differentiate into PGCLCs.

The formative state is thus defined by both the ability to give rise to PGCLCs, and a transcriptional signature in between ESCs and EpiSCs, where naïve markers are shut down but early differentiation markers are not yet expressed [START_REF] Kinoshita | Pluripotency Deconstructed[END_REF][START_REF] Kinoshita | Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency[END_REF]. In vivo, such a population exists transiently between E5.5 and E6.75 [START_REF] Ohinata | A Signaling Principle for the Specification of the Germ Cell Lineage in Mice[END_REF].

Another

f.2. WiEpiSCs

EpiSCs cultured in Activin/Fgf2 express high levels of germ layers markers, and have a high propensity to spontaneous differentiation. This differentiation, largely stochastic, is a source of heterogeneity in EpiSCs. While Activin and Fgf2 alone sustain self-renewal in the primed state, it was shown that combined with BMP and Wnt, they collaborate to promote epithelial-to-mesenchymal transition (EMT). Wnt, downstream of BMP, is also required for mesodermal differentiation [START_REF] Huelsken | Requirement for β-catenin in anterior-posterior axis formation in mice[END_REF][START_REF] Liu | Requirement for Wnt3 in vertebrate axis formation[END_REF]. Blocking the Wnt pathway with inhibitors (such as XAV939, IWP2 and IWR1), or by genetically knocking down β-catenin, consistently leads to a more homogenous primed state. Such cells, named Wnt-inhibited EpiSCs (WiEpiSCs), are more permissive to clonal expansion in single cells and easier to derive [START_REF] Kurek | Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells[END_REF]. They express reduced levels of mesodermal and endodermal markers, and increased levels of pluripotency markers compared to conventional EpiSCs.

Because of their enhanced quality and homogeneity, WiEpiSCs are sometimes considered as representative of the "ground state" of primed pluripotency [START_REF] Kim | Modulation of βcatenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal[END_REF][START_REF] Sumi | Epiblast Ground State Is Controlled by Canonical Wnt/β-Catenin Signaling in the Postimplantation Mouse Embryo and Epiblast Stem Cells[END_REF][START_REF] Tsakiridis | Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors[END_REF]Wu et al., 2015).

g) The Pluripotency Gene Network

Pluripotency is regulated at two levels: 1) Extrinsic signals, 2) intrinsic signals originating from a regulatory gene network. Those two layers of regulation are contextdependent, vary depending on the pluripotent state of the cell, and mainly rely on the action of transcription factors (TFs).

TFs are proteins that recognize and bind DNA sequences to either activate or repress their transcription. They can bind promoter-proximal DNA regions or more distal elements that can be from several tens of bases to hundreds of kilobases away. Enhancers, the elements involved in positive gene regulation, are bound by multiple TFs that can recruit both the transcription machinery, and chromatin regulators/ remodelers in order to allow access to promoter regions. In ESCs, as mentioned earlier, the OSN (Oct4, Sox2, Nanog) TFs are considered as the core triad of pluripotency, because their simultaneous expression is characteristic of pluripotency both in vivo and in vitro [START_REF] Avilion | Multipotent cell lineages in early mouse development depend on SOX2 function[END_REF][START_REF] Masui | Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells[END_REF][START_REF] Mitsui | The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[END_REF][START_REF] Nichols | Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[END_REF][START_REF] Silva | Nanog Is the Gateway to the Pluripotent Ground State[END_REF]. OSN are the centerpieces of a regulatory circuitry governing pluripotency and behave so in two manners: One, by cooperating to positively regulate their own promoters; Two, by co-occupying genes, simultaneously repressing expression of those involved in lineage commitment and activating those involved in the maintenance of self-renewal [START_REF] Young | Control of the embryonic stem cell state[END_REF].

At the top of the pluripotency regulation system are extrinsic signals, i.e. LIF, Bmp4, Wnt, FGF and Activin, as detailed precedingly. Stat3, β-catenin and Smad1, the downstream effectors of LIF, Wnt and Bmp4, overlap with most part of OSN binding sites, thus coupling extrinsic signals with the intrinsic regulatory gene circuitry. This is the first level of pluripotency regulation.

Once OSN expression is established and maintained, forming the core of the Pluripotency Gene Network (hereafter PGN), naïve ESCs then express an "ancillary" set of genes, such as Rex1, Esrrb, Klf4, Tbx3 and Tfcp2l1, that constitute the second layer of pluripotency regulation (Figure 6). Unlike OSN, these factors are individually dispensable for pluripotency establishment and maintenance, but they globally reinforce and stabilize the network to shield it against pro-differentiating cues. This extended PGN, much like OSN, is self-promoting through positive feedback loops, and its components stabilize each other's expression [START_REF] Festuccia | Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells[END_REF][START_REF] Hackett | Regulatory principles of pluripotency: From the ground state up[END_REF][START_REF] Dunn | Defining an essential transcription factor program for naïve pluripotency[END_REF]. In conventional ESCs populations, components of the extended PGN are heterogeneously expressed, stochastically on or off at the single-cell level. This is consistent with the heterogenous nature of such populations and the propensity of individual cells to metastably shift close to the primed state [START_REF] Chambers | Nanog safeguards pluripotency and mediates germline development[END_REF][START_REF] Martello | Esrrb is a pivotal target of the Gsk3/Tcf3 axis regulating embryonic stem cell self-renewal[END_REF][START_REF] Toyooka | Identification and characterization of subpopulations in undifferentiated ES cell culture[END_REF][START_REF] Yamaji | PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells[END_REF]. This shift is at least in part due to the fact that, without GSK3 inhibition by CHIRON, conventional ESCs express the TCF3 factor that antagonizes OSN and destabilizes the network. With addition of 2i in the ground state condition, TCF3 expression is inhibited and the network is stabilized, heterogeneity is thus suppressed and ancillary factors expressed at high and stable levels in most cells [START_REF] Wray | Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation[END_REF]. Though most members of the extended network are upregulated in 2i/LIF compared to Serum/LIF, some have a particularly higher expression in the former, such as Tfcp2l1 and Rex1, and seem to play a critical role in the maintenance of the naïve state. Tfcp2l1 alone, for instance, can drive EpiSCs to reprogram in the naïve state when overexpressed [START_REF] Grabole | Prdm14 promotes germline fate and naive pluripotency by repressing FGF signalling and DNA methylation[END_REF][START_REF] Martello | Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor[END_REF].

Accordingly with the rest of their characteristics, the regulatory gene circuitry of ESCs and EpiSCs largely differs. Though EpiSCs express Oct4 and Sox2 to a comparable level to

ESCs, Oct4 expression in EpiSCs is controlled by its proximal enhancer, as mentioned in part I/3.b). In the primed state, Nanog expression is also attenuated. In addition to these nuances in OSN expression, EpiSCs also and foremost do not express, or to very reduced levels, most of the extended pluripotency network. On the contrary, they do express lineage-associated factors and other TFs, such as Otx2, Zic2, Zfp281, Zscan10, and Oct6, not expressed in neither ground state nor conventional ESCs.

Interestingly, when ESCs are converted to EpiLCs (Fgf/Activin/Fibronectin/1% KOSR),

Oct4 occupancy is drastically changed from genes associated with naïve pluripotency (Klf4, Tbx3, Prdm14) to post implantation epiblast/primed pluripotency-associated genes (Fgf5, Oct6, Wnt8a). This phenomenon goes along with a global transformation of the chromatin landscape. Oct4 also changes TFs interactors, switching from Esrrb and Klf4/5 (in addition to Sox2 and Nanog) to Otx2 and Zic2/3. The newly formed Oct4/Otx2 tandem has been shown

to bind, in this context, previously inaccessible chromatin domains, triggering the expression of primed-associated genes [START_REF] Buecker | Reorganization of enhancer patterns in transition from naive to primed pluripotency[END_REF]. This suggests that, as for naïve cells, primed cells express their own ancillary factors forming an extended primed pluripotency network.

II/ Pluripotency in Primates.

Human Embryonic Stem Cells (hESCs)

In 1998, ES cell lines were derived for the first time from human blastocysts [START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF]. These cells (hereafter named hESCs), although derived from the ICM like mouse ES cells (hereafter mESCs), do not share the same characteristics, however [START_REF] Weinberger | Dynamic stem cell states: Naive to primed pluripotency in rodents and humans[END_REF]. First, they are not responsive to LIF, and need to be cultured in primed conditions instead (I.e. FGF/Activin A) to self-renew. In addition, hESCs lose pluripotency upon MEK-ERK inhibition in conventional conditions [START_REF] Dahéron | LIF/STAT3 Signaling Fails to Maintain Self-Renewal of Human Embryonic Stem Cells[END_REF]. Second, they have a primed morphology of flat, monolayered colonies and need to be passaged by mechanical dissociation. Third, most of the extended pluripotency network/naïve pluripotency network as defined in mouse is not expressed in hESCs [START_REF] Chia | A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity[END_REF]. Fourth, hESCs epigenome differs largely with that of mESCs, with H3K27 histone repressive marks being appended to developmental genes and hESCs exhibiting one inactivated X chromosome (Xi) in female cell lines, like EpiSCs but unlike mESCs [START_REF] Silva | X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells[END_REF].

Thus, hESCs differ greatly from mESCs; however, they do not have all the primed characteristics from a mouse standard. They do not, for instance, express some of the classical EpiSCs markers such as FGF5 or CDH2; but they do express some of the naïve markers such as PRDM14 and REX1 [START_REF] Chia | A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity[END_REF]. hESCs genome methylation is also closer to that of conventional mESCs (although distinct from ground state mESCs) than to EpiSCs [START_REF] Hackett | Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency[END_REF][START_REF] Shipony | Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells[END_REF], and hESCs exhibit both a nuclear and cytoplasmic localization of the TFE3 factor, located either in one or the other in mouse ground state and primed conditions, respectively [START_REF] Betschinger | Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3[END_REF].

The question then arises of why hESCs diverge so much in their characteristics from mESCs. Although some similarities of hESCs with EpiSCs are striking, hESCs have a somewhat different phenotype, revealing a potential intermediate state between primed and naïve. However, it is worth reminding that both of these states have been discovered and characterized in mouse. A critical question thus arises: does the naïve state as defined in mouse exist in humans, and more generally, in primates? Answering this question requires a stricter definition of naïve pluripotency.

In mouse, the naïve state can be defined on the basis of either the functional (i.e. chimeric competency), molecular (reliance on cytokines and growth factors) or genetic (expression of the extended pluripotency network) characteristics of ground state ESCs.

However, the primed and naïve pluripotent states are also and foremost defined by their tissue of origin in vivo, i.e. the post-and pre-implantation epiblast, respectively. Defining naïve pluripotency in primates can thus not be extrapolated from either functional, molecular or genetic characteristics of mouse cells, but reclaims to define the identity of the primates' preimplantation epiblast, and understand in what measure it diverges from that of the mouse.

The naïve state of primates' pluripotency in vivo a) Early embryonic development of the mouse and primates

In 1994, Denis Duboule suggested the model of the "Developmental hourglass", based on the observation that all vertebrate species diverge in the earliest and latest stages of their embryonic development, but share more common characteristics during the mid-embryonic period, also called phylotypic [START_REF] Duboule | Temporal colinearity and the phylotypic progression: A basis for the stability of a vertebrate Bauplan and the evolution of morphologies through heterochrony[END_REF][START_REF] Hanken | The shape of life: Genes, development, and the evolution of animal form[END_REF]. The phylotypic period starts when the common anatomical features of the basic body plan of a given phylum are established. In the particular case of mammals, this period starts after gastrulation, when neurulation, establishment of the antero-posterior axis and appearance of the mesodermal somites occur. In mouse, this period lies between E7.5 and E8.0, and is concomitant with the differentiation of the last pool of epiblast cells. In primates, and more specifically humans, it extends from E15 to E20, between the beginning of gastrulation and formation of the notochord (Figure 7) [START_REF] Irie | The developmental hourglass model: A predictor of the basic body plan[END_REF][START_REF] Shahbazi | Deconstructing and reconstructing the mouse and human early embryo[END_REF]. According to the developmental hourglass model, important divergence in primates' and mouse embryogenesis before gastrulation should thus exist. This is proven correct in several aspects of development.

The first, maybe most obvious difference is that of timing. Mouse embryonic development lasts for 19 days, while human's extends to 9 months. This difference of duration is not only reflected by the final number of cells composing the individual, but also by a difference in their proliferation rate. Indeed, mouse late blastocysts (E4.5) consist of ∼150 cells, which reflects a cell cycle comprised between 14 and 15h [START_REF] Plusa | Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst[END_REF] while at the same stage (E7), human blastocysts are formed of ∼250 cells, reflecting a cell cycle of ∼21h [START_REF] Niakan | Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse[END_REF]. In mouse, the rapid proliferation rate of ICM and early epiblast cells is due to a high expression of Cdk2, promoting a fast G1 to S-phase transition.

High CDK2 is not observed in human or marmoset ICM and early epiblast cells. Moreover, the key G2/M checkpoint kinase WEE1 is highly expressed, exemplifying a different behavior in cell cycle regulation [START_REF] Blakeley | Erratum to Defining the three cell lineages of the human blastocyst by single-cell RNAseq[END_REF][START_REF] Boroviak | Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis[END_REF][START_REF] Yan | Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells[END_REF].

Another critical difference is that of structural organization upon implantation. At E5, the mouse embryo attaches to the endometrium and epiblast cells form a rosette-like structure, embedded in between TE and PE. That rosette subsequently invaginates to form a cup-shaped structure called the egg-cylinder, conserved until the beginning of gastrulation at E7.5 [START_REF] Shahbazi | Deconstructing and reconstructing the mouse and human early embryo[END_REF]. In primates the blastocyst, once implanted, forms a bilaminar embryonic disc composed of an apical layer of epiblast cells and a basal layer of visceral endoderm. At this stage compared to the mouse embryo, one more extra-embryonic tissue appears, the amniotic epithelium, only initiated upon gastrulation in rodents (Figure 7) [START_REF] Boroviak | Primate embryogenesis predicts the hallmarks of human naïve pluripotency[END_REF].

The third important difference between primates and rodents early embryogenesis is embryonic diapause. This process, also known as discontinuous development, is characterized in mammals by a delayed implantation when environmental conditions are unfavorable to postnatal development [START_REF] Lopes | Embryonic diapause and its regulation[END_REF]. In eutherian mammals, it occurs at the blastocyst stage, where the embryo hatches from the zona pellucida and then remains in a so-called "dormant state", i.e., retains its full developmental potential but is under proliferation arrest. Embryonic diapause has been widely described in a number of placental species, including rodents, but has not yet been depicted in primates [START_REF] Ptak | Embryonic diapause is conserved across mammals[END_REF].

In mouse, delayed blastocysts have been shown to be able to stay dormant for several weeks, and keep the ability to resume development afterwards in response to estrogens.

Though mESCs can be derived from normal and diapaused embryos alike, the first mESCs lines derived were from diapaused embryos [START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF], and this appears to generally be a facilitating factor of derivation [START_REF] Brook | The origin and efficient derivation of embryonic stem cells in the mouse[END_REF]. Embryonic diapause has been linked in mouse to LIF/gp130 signaling, c-Myc and mTOR activity [START_REF] Bulut-Karslioglu | Inhibition of mTOR induces a paused pluripotent state[END_REF][START_REF] Nichols | Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines[END_REF][START_REF] Scognamiglio | Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause[END_REF]. Although the precise mechanisms underlying this phenomenon's regulation are still poorly understood, the correlation with mESCs derivation efficiency and pluripotency-associated pathways raises the question of a link between this process and the naïve state per se in mouse. Whether the molecular tooling of embryonic diapause remains in primates, despite it not being used in vivo, and whether this absence can be correlated with difficulties to capture the naïve state remains to be elucidated.

In addition to these three major differences, Zygotic Genome Activation (ZGA) also occurs at different timings in mouse and primates, at the 2-cell stage in the former, and between the 4-and 8-cell stage in the latter [START_REF] Blakeley | Erratum to Defining the three cell lineages of the human blastocyst by single-cell RNAseq[END_REF] and mechanisms of X chromosome dosage compensation, process by which male and female cells conserve an equivalent number of X chromosome transcripts, also appear to differ [START_REF] Petropoulos | Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos[END_REF]. Taken altogether, these differences could explain a divergence between mouse and primates pluripotency regulation, and thus, differences between hESCs and mESCs. However, the question of the proximity between hESCs and the pluripotent human pre-implantation epiblast remains. More generally, the question is: how is the primates' naïve state defined in vivo?

b) The hallmarks of the naïve primates' epiblast

In part I/3., we depicted the differences existing between mouse ESCs and EpiSCs, i.e.

the naïve and primed pluripotent states in vitro. These differences reflect the identity of preimplantation epiblast versus its post-implantation counterpart in vivo, and characteristics of the former define the hallmarks of naïve pluripotency in mouse. Such hallmarks are not yet entirely unraveled in primates, but are under active investigation and so far revealed at least eight features, based on the identity of human and non-human primates' pre-implantation epiblast.

These include ERK independence, apolarity, DNA hypomethylation and X chromosome activation, all of which are common with the mouse. Three features are specific to primates:

the expression of a primates' specific naïve network, a primates' specific naïve transposable elements (TEs) signature, and the ability to differentiate into amniotic epithelial cells (Figure 8) [START_REF] Boroviak | Primate embryogenesis predicts the hallmarks of human naïve pluripotency[END_REF].

b.1 ERK independence

In mouse, the FGF/ERK pathway is a critical inducer of differentiation and in the blastocyst, PE specification is mainly controlled by FGF paracrine secretion (cf. part [START_REF] Burdon | Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells[END_REF]) (Arman al., 1998;Chazaud et al., 2006;Feldman et al., 1995;Kang et al. 2013). When mouse cells shift from naïve to primed pluripotency, i.e. are converted into EpiSCs, they necessitate FGF for self-renewal. Similarly, hESCs, that closely resemble EpiSCs, rely on FGF signaling to self-renew. In primates' blastocyst, though additional signals are involved, FGF is as for mouse implied in PE specification. It was also shown for both human and marmoset that high NANOG expression could be maintained without FGF, showing independency to this pathway for self-renewal [START_REF] Boroviak | Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis[END_REF][START_REF] Roode | Human hypoblast formation is not dependent on FGF signalling[END_REF].

I/1.b))(

b.2 Apolarity

When grown in vitro, mESCs form small, tightly packed dome-shaped colonies. In conventional serum/LIF conditions, this morphology is heterogeneously distributed, but when cells are cultured in ground state 2i/LIF culture conditions, i.e. homogeneously naïve, this becomes the only morphology. Colonies of cells in primed conditions, on the other hand, adopt a flat, monolayered morphology. This difference between primed and naïve cells is attributed, at least in part, to polarity. In vivo, between the 16-and 32-cell stages of the morula, polarity is established in outer blastomeres and drives differentiation into TE. Inner cells on the contrary remain apolar, a characteristic critical to their adoption of a pluripotent identity (Anani et al. 2014, Boroviak and[START_REF] Takashima | Resetting transcription factor control circuitry toward ground-state pluripotency in human[END_REF]. At the blastocyst stage, the pluripotent epiblast conserves an apolar status, while both PE and TE are polarized epithelia (Bedzhov and Zernicka-Goetz, 2014;Enders et al., 1986;Plusa et al., 2005). This changes only upon implantation, when Epi cells arrange in a rosette-like structure, form extensive adherens junctions and concentrate their organelles at apical ends (Bedzhov and Zernicka-Goetz, 2014;Enders et al., 1986). The apolar and polar status of pre-and post-implantation epiblast, respectively, is conserved in primates. In vitro, EpiSCs and hESCs harbor ultrastructural characteristics of the postimplantation epiblast such as apical microvilli and tight junctions [START_REF] Brons | Derivation of pluripotent epiblast stem cells from mammalian embryos[END_REF]Krtolica et al., 2007;Sathananthan et al., 2002;[START_REF] Tesar | New cell lines from mouse epiblast share defining features with human embryonic stem cells[END_REF]. Therefore, naïve primates' cells, in accordance with in vivo observations, should be apolar and recapitulate in vitro the domeshaped morphology of 2i/LIF mESCs.

b.3 DNA Hypomethylation

During germline differentiation and pre-implantation development, the genome of both primates' and mouse undergo massive hypomethylation, allowing enhanced access to gene promoters for transcription (Seisenberger et al., 2013a,b). In the pre-implantation epiblast, genome-wide hypomethylation is observed, to the only exception of imprinted regions, and progressive methylation proceeds upon implantation until gastrulation [START_REF] Takashima | Resetting transcription factor control circuitry toward ground-state pluripotency in human[END_REF]Smallwood et al., 2011;Smith et al., 2014Smith et al., , 2012)). In mouse, this process is retrieved in vitro with mESCs in 2i/LIF, but not serum/LIF condition, while EpiSCs show higher levels of methylation, consistent with their tissue of origin. Accordingly, hESCs show high levels of methylation, in discordance with the hypomethylated human ICM and early epiblast. In both rodents and primates therefore, hypomethylation is a hallmark of naïve pluripotency in vivo.

b.4 X Chromosome Activation

In most mammalian species, including rodents and primates, random X chromosome inactivation (XCI) occurs in female cells to compensate for X-linked gene expression [START_REF] Escamilla-Del-Arenal | Evolutionary diversity and developmental regulation of X-chromosome inactivation[END_REF]. In mouse, the paternal X chromosome is silenced at the 4cell stage and transiently reactivated in the epiblast, concomitantly with naïve pluripotency establishment. Once the embryo implants, XCI occurs in the post-implantation epiblast as pluripotent cells transit from naïve to primed. Such a phenomenon is also observed in primates embryo [START_REF] Dupont | Different flavors of X-chromosome inactivation in mammals[END_REF][START_REF] Petropoulos | Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos[END_REF], making dual activation of X chromosomes a mark of pre-implantation epiblast cells and thus, naïve pluripotency in both primates and rodents.

b.5 Primate-specific naïve network

Numerous single-cell RNA sequencing analyses [START_REF] Blakeley | Erratum to Defining the three cell lineages of the human blastocyst by single-cell RNAseq[END_REF][START_REF] Boroviak | Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis[END_REF][START_REF] Liu | Single-cell RNAsequencing reveals the existence of naive and primed pluripotency in pre-implantation rhesus monkey embryos[END_REF][START_REF] Nakamura | A developmental coordinate of pluripotency among mice, monkeys and humans[END_REF][START_REF] Petropoulos | Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos[END_REF][START_REF] Xue | Global expression profiling reveals genetic programs underlying the developmental divergence between mouse and human embryogenesis[END_REF][START_REF] Yan | Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells[END_REF] showed that primates (cynomolgus monkey, rhesus monkey, marmoset and human) and rodents epiblasts share common components of the pluripotency network. In addition to the core OSN triumvirate, some members of the extended network such as KLF4, TFCP2L1, SALL4, TBX3 and TDGF1 are expressed in primates. However, critical components of the mouse network are absent, namely KLF2, ESRRB, BMP4, NR0B1 and FBXO15. It is becoming increasingly clear that primates indeed express their own extended, naïve-specific pluripotency network, though the exact nature and regulation of this network is still under investigation. Several members have been yet identified, notably KLF17 [START_REF] Guo | Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass[END_REF], the activator protein-2 (AP2) transcription factor C (TFAP2C) [START_REF] Pastor | TFAP2C regulates transcription in human naive pluripotency by opening enhancers[END_REF], ARGFX, and several members of the TGF-β signaling pathway, including NODAL, GDF3, TGFBR1, LEFTY1, SMAD2, SMAD4 and TDGF1 (see part II/2.c)) [START_REF] Blakeley | Erratum to Defining the three cell lineages of the human blastocyst by single-cell RNAseq[END_REF][START_REF] Boroviak | Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis[END_REF][START_REF] Nakamura | A developmental coordinate of pluripotency among mice, monkeys and humans[END_REF][START_REF] Petropoulos | Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos[END_REF].

b.6 OCT4 expression control

In mouse, differential enhancer control of Oct4 expression is a clear distinction between the primed and naïve states (cf part I/). Primed cells indeed rely on the proximal enhancer (PE)

to express Oct4 while naïve cells rely on the distal enhancer (DE) both of which are located upstream of the Oct4 gene. In primates, pre-implantation blastocysts, both PE and DE are, surprisingly, inaccessible to the transcription machinery. However, two enhancers are located downstream of the OCT4 transcription termination site. When these sequences are ablated in primed hESCs, normal OCT4 expression is maintained, but if so is done in naïve-like reprogrammed hESCs (See part II/3.b), OCT4 expression is dramatically decreased [START_REF] Pastor | TFAP2C regulates transcription in human naive pluripotency by opening enhancers[END_REF]. The naïve primate marker TFAP2C appears to be the main activator of these downstream enhancers, confirming that they are naïve-specific regulators to the naïve state.

Therefore, while upstream distal enhancer control of OCT4 expression is not, contrary to what is observed in mouse, a discriminating hallmark of naïve pluripotency in primates, OCT4 yet has a differential regulation in the primed and naïve states.

b.7 Primate-specific "transpocriptome"

Transposable elements (TEs) (or "jumping genes") are DNA sequences able to change location within the genome through duplication or excision processes. In mammalian species, TEs count for approximately half of the genome and play a critical role in several biological processes such as transcriptional modulation, coding and long non-coding RNA metabolism or germline and soma mutagenesis [START_REF] Bourque | Ten things you should know about transposable elements 06 Biological Sciences 0604 Genetics[END_REF]. In 2015, Göke and colleagues investigated the function of endogenous retroviruses (ERVs), a subclass of TEs, based on the observation that hESCs express genes from active ERVs, and that ERVs are, in mouse, indicators of totipotency. It was found that ERVs are systematically transcribed in the human pre-implantation embryo, and that the long terminal repeats (LTRs) flanking ERVs can be used as stage-specific markers. More specifically, the LTR7-class of HERVHs are only expressed in NANOG-positive, i.e. naïve pre-implantation epiblast cells, along with members of the HERVK class [START_REF] Göke | Dynamic transcription of distinct classes of endogenous retroviral elements marks specific populations of early human embryonic cells[END_REF]. Therefore, in vivo naïve pluripotency of primates is characterized by a specific "transposcriptomic" (TEs transcriptome) signature, which is likely, however, to be species-specific.

b.8 Extra-embryonic potential

As mentioned precedingly, primates epiblast undergoes differentiation into amniotic epithelium before gastrulation, a major difference to mouse development [START_REF] Boroviak | Primate embryogenesis predicts the hallmarks of human naïve pluripotency[END_REF]. This means that, theoretically, naïve primate cells should retain the capacity to differentiate into amniotic epithelial cells. However, the pathways governing this process are yet unknown, and the precise transcriptomic and epigenetic identity of the amniotic epithelium is still largely unraveled.

c.2 NODAL/TGF-β signaling

Among the primate-specific pre-implantation epiblast genes, several are members of the NODAL/TGF-β signaling pathway (NODAL, GDF3, TGFBR1, LEFTY1, SMAD2, SMAD4

and TDGF1), suggesting a critical role in naïve pluripotency identity. Interestingly, culture of human pre-implantation embryos with the NODAL/TGF-β inhibitor SB431542 leads to an increase in the number of NANOG-positive ICM cells [START_REF] Van Der Jeught | Treatment of human embryos with the TGFβ inhibitor SB431542 increases epiblast proliferation and permits successful human embryonic stem cell derivation[END_REF]. However, performing the same experiment with higher concentrations leads to an opposed result of dramatic reduction of NANOG expression [START_REF] Blakeley | Erratum to Defining the three cell lineages of the human blastocyst by single-cell RNAseq[END_REF], and using another inhibitor, A83-01, on marmoset embryos [START_REF] Boroviak | Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis[END_REF] does not seem to provide modulation of NANOG expression. The function of NODAL/TGF-β signaling in primates pre-implantation epiblast thus remains elusive, and cannot be seen so far as a hallmark of naïve pluripotency in vivo.

c.3 LIF signaling/ JAK dependency

One paramount element of mESCs self-renewal is the phosphorylation and nuclear translocation of Stat3 in response to LIF. In human embryos, members of the JAK/STAT3 pathway (IL6R, GP130/IL6ST, JAK1 and STAT3) are expressed at significantly higher levels in the pre-implantation epiblast than in the PE or TE [START_REF] Bourillot | GP130 signaling and the control of naïve pluripotency in humans, monkeys, and pigs[END_REF] and treatment of embryos cultured in vitro in serum-free conditions with LIF significantly improves blastocyst formation [START_REF] Dunglison | Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium[END_REF]. Consistently, LIFR is expressed in all pre-implantation human lineages, demonstrating that human embryos are responsive to LIF. In the cynomolgus monkey, a subset of pre-implantation epiblast cells express IL6R, GP130/IL6ST, STAT3, but not JAK1 at high levels. Interestingly, a drastic downregulation of those members occurs upon implantation, suggesting that LIF signaling is indeed a characteristic of the naïve primates preimplantation epiblast [START_REF] Bourillot | GP130 signaling and the control of naïve pluripotency in humans, monkeys, and pigs[END_REF][START_REF] Nakamura | A developmental coordinate of pluripotency among mice, monkeys and humans[END_REF]. However, a compelling observation is the low level of expression of LIF itself in all human pre-implantation lineages or, in the case of the cynomolgus monkey, undetectable. On the strict basis of in vivo observations, LIF/STAT3 activation cannot be considered so far as a hallmark of the naïve primates' epiblast, however, it appears mandatory to any reprogramming method yielding naïve-like cells from hESCs (See part II/3.b)).

The naïve state of primates' pluripotency in vitro a) Of the purpose of primates naïve cells

Capture of the naïve state in primates, and more specifically in humans, is desired for at least three reasons. The first is use of naïve hPSCs as a tool for regenerative medicine.

Contrary to hESCs, naïve cells should be, as mESCs, more genetically stable [START_REF] Fu | DNA repair mechanisms in embryonic stem cells[END_REF][START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF], more amenable to genetic editing [START_REF] Zwaka | Homologous recombination in human embryonic stem cells[END_REF] and free of any differentiation bias, allowing to efficiently derive any desired tissue with no epigenetic imprinting [START_REF] Ying | The ground state of embryonic stem cell self-renewal[END_REF]. Most hopes in using PSCs for regenerative medicine are based on iPSCs, however, current methods of hiPSCs generation yield cells with limited proficiency of lineage-specific differentiation, and epigenetic imprinting of donor cells [START_REF] Kim | Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells[END_REF].

The tabula rasa of naïve-state conversion is hoped to solve such problems.

Another potential application, more controversial, is that of organ culture. In 2008, after establishment of the 2i/LIF protocol, Buehr and colleagues derived for the first time naïve cells from a rat blastocyst [START_REF] Buehr | Capture of Authentic Embryonic Stem Cells from Rat Blastocysts[END_REF]. Two years later, naïve 2i/LIF rat cells were used by Kobayashi et al. in a major breakthrough experiment. Naïve rat cells were injected in a mouse

Pdx1-/-blastocyst (pancreatogenesis disabled) and efficiently complemented pancreatogenesis, generating an interspecific chimera, a mouse with a functional rat pancreas [START_REF] Kobayashi | Generation of Rat Pancreas in Mouse by Interspecific Blastocyst Injection of Pluripotent Stem Cells[END_REF]. More recently a similar experiment was performed in anephric Sall1-/-mutant rats, in which naïve mouse cells successfully complemented kidney formation [START_REF] Goto | Generation of pluripotent stem cell-derived mouse kidneys in Sall1-targeted anephric rats[END_REF]. These experiments showed that naïve cells are not only able to form chimeras, but to do so in other species in the proper conditions. Such approach, termed "blastocyst complementation", raised hopes of similar success with human naïve cells injected in host species such as pigs or non-human primates, allowing human organ culture in non-human hosts to address donations shortage [START_REF] Wu | Generation of human organs in pigs via interspecies blastocyst complementation[END_REF]. Recently, a first step was performed toward this direction by Wu and colleagues who injected naïve-like human iPSCs in pig embryos and obtained chimerism, though to a limited extent [START_REF] Wu | Interspecies Chimerism with Mammalian Pluripotent Stem Cells[END_REF].

Finally, the capture of the naïve state in primates is fundamental to the comprehension of primates early embryonic development, and would provide a valuable model to the investigation of the molecular and epigenetic mechanisms occurring before implantation. As a tool, human naïve cells would not only give insight in pre-implantation, but also in postimplantation development, as this period is, for obvious ethical reasons, challenging to investigate.

b) Reprogramming strategies to naïve-like pluripotency

In 2007, one year after the derivation of the first mouse iPS cells (hereafter miPSCs),

Takahashi and Yamanaka successfully repeated the experiment on human cells. After induction with the OSKM cocktail, cells exhibited pluripotent properties, but did not display a similar morphology to miPSCs or mESCs [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF]. Additionally, they expressed some early lineage markers and conserved imprinting from their tissue of origin [START_REF] Kim | Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells[END_REF]. Altogether, human iPSCs (hereafter hiPSCs) exhibited properties more similar to primed mouse cells or to hESCs than to naïve cells. This was not surprising, accounting to the fact that the first generation of miPSCs themselves displayed primed characteristics. The second generation, yielding cells resembling the naïve state, included addition of NANOG to the OSKM cocktail or subcloning of NANOG-positive clones among the reprogrammed population [START_REF] Okita | Generation of germline-competent induced pluripotent stem cells[END_REF][START_REF] Wernig | In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state[END_REF].

In 2010, starting from this postulate, Buecker and colleagues attempted reprogramming of human fibroblasts with a doxycycline-inducible combination of OSKM and NANOG. This yielded human iPSCs with naïve properties, named "hLR5 iPSCs". hLR5 displayed a domeshaped morphology, expressed some mouse naïve pluripotency markers, could be passaged by trypsin dissociation and were dependent on LIF to self-renew. However, they remained dependent on the constant activation of their five transgenes [START_REF] Buecker | A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells[END_REF]. The same year another approach was adopted by Hanna and colleagues, that combined overexpression of OCT4, KLF4 and KLF2, with LIF stimulation and inhibition of both GSK3b and ERK1/2 (2i/LIF conditions) [START_REF] Hanna | Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs[END_REF]. In both of these studies, despite the apparent naïve characteristics of the cells, constitutive activation of the transgenes was mandatory (although forskolin, a protein kinase A agonist, could transiently compensate for absence of OCT4, KLF4

and KLF2 in the study of Hanna and colleagues) leaving many questions unanswered regarding the extrinsic regulation of the human PGN.

In 2013, Gafni and colleagues reported the first transgene-free reprogramming method of hESCs to a naïve-like state, using exclusively small molecules and growth factors. This protocol, termed NHSM, yielded cells with a number of naïve characteristics including, notably, the ability to colonize a mouse blastocyst [START_REF] Gafni | Derivation of novel human ground state naive pluripotent stem cells[END_REF], although this last feature has been the subject of controversy, and was not retrieved in other hands afterwards.

These three experiments paved the way to the quest of human naïve pluripotency, and in the following years, a broad number of protocols blossomed, yielding variable proximity to the naïve human epiblast, and a diverse set of molecular and functional naïve characteristics.

All of these methods are based on the simultaneous LIF stimulation and inhibition of GSK3

and ERK/MEK, though this can be combined with other approaches, thus leading to different outcomes and shedding light on different aspects of pluripotency regulation in humans (Figure 9 and Table 1). In the subsequent paragraphs, we will detail these aspects, often overlapping between several protocols. It is worth noting that, although we mentioned the compelling role of LIF signaling in vivo, its necessity in vitro appears unquestionable and demonstrates that, as for the mouse, LIF/STAT3 signaling plays a critical role in primates naïve pluripotency regulation.

b.1 MEK/ERK reinforced inhibition

In addition to LIF stimulation and Wnt inhibition, reinforcement of FGF/ERK inhibition appears as a recurring way to induce naïve-like reprogramming. In their original protocol NHSM, Gafni and colleagues make use of a Protein Kinase C (PKC) inhibitor in addition to PD03 (classical MEKi), a strategy also used in the Reset [START_REF] Takashima | Resetting transcription factor control circuitry toward ground-state pluripotency in human[END_REF] and 5i/L/A protocols [START_REF] Theunissen | Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency[END_REF]. PKC is part of a regulatory loop promoting MEK/ERK activation. PKC activates Ras, leading to the phosphorylation cascade activating ERK that in turn phosphorylates PKC [START_REF] Krueger | Down-regulation of Cdx2 in colorectal carcinoma cells by the Raf-MEK-ERK 1/2 pathway[END_REF]. Use of a PKCi (Gö6983) on mESCs has been shown to suppress differentiation [START_REF] Dutta | Self-renewal versus lineage commitment of embryonic stem cells: Protein kinase C signaling shifts the balance[END_REF]. In the 5i/L/A method of Theunissen and colleagues, the serine/threonine B-Raf and tyrosine kinase c-Src are also targeted in addition to MEK and PKC [START_REF] Theunissen | Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency[END_REF]. B-Raf is a direct activator of MEK [START_REF] Leonardi | BRAF mutations in papillary thyroid carcinoma and emerging targeted therapies (Review)[END_REF], while c-Src is an activator of Ras, upstream kinase of the MAPK pathways [START_REF] Du | The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways[END_REF]. In the case of 5i/L/A, 4 simultaneous MEK/ERK actors are therefore inhibited. The last inhibitor is IM-12, an alternative to CHIR99021 GSK3 inhibition. This suggests that in human cells, MEK inhibition with PD03 alone is not sufficient and/or specific enough to promote activation of the PGN. Reinforced MEK/ERK inhibition through other inhibitors is one strategy to overcome this problem.

b.2 Activin/Nodal activation

Nodal is a member of the Transforming Growth factor beta (TGF-β) superfamily, able to bind heterodimeric Activin receptors. When bound, Nodal or Activin activate the phosphorylation and nuclear translocation of SMAD2 and SMAD3 that in turn regulate transcription of their target genes. In Xenopus, Zebrafish and rodents, Nodal has been shown

to regulate mesodermal and endodermal differentiation in vivo. In humans, it was shown in vitro that Nodal signaling inhibits neurectoderm differentiation, the default commitment path of hESCs in conventional culture media [START_REF] Vallier | Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway[END_REF], and that the Activin/Nodal and FGF pathways cooperate to maintain pluripotency [START_REF] Vallier | Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells[END_REF]. SMAD2/3, as STAT3, are indeed able to co-occupy enhancers with OSN and thus, promote pluripotency [START_REF] Hill | Transcriptional control by the SMADs[END_REF].

Consistently mouse EpiSCs, just as hESCs, reclaim both Activin and FGF to self-renew [START_REF] Brons | Derivation of pluripotent epiblast stem cells from mammalian embryos[END_REF][START_REF] Tesar | New cell lines from mouse epiblast share defining features with human embryonic stem cells[END_REF][START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF]. On the contrary, mESCs differentiate in response to Nodal/Activin but rely on BMP4, another TGF-β superfamily member, in combination with LIF to self-renew [START_REF] Fei | Smad2 mediates Activin/Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells[END_REF]. BMP4, in spite of being a TGF-β member, acts through SMAD1, 5 and 8 instead of SMAD2/3, thus having a different impact on transcription regulation [START_REF] Miyazono | Bone morphogenetic protein receptors and signal transduction[END_REF].

If mESCs are taken as a reference, primates naïve ESCs should therefore require BMP4 for self-renewal, and differentiate in response to Nodal/Activin, intriguingly however, the converse is observed [START_REF] Nemashkalo | Morphogen and community effects determine cell fates in response to BMP4 signaling in human embryonic stem cells[END_REF]. Most reprogramming methods require addition of a layer of mouse embryonic fibroblasts (MEFs), also called feeder cells (see part I/). In addition to Wnt molecules and FGF, MEFs secrete TGF-β-I, Activin A and antagonists of BMPs [START_REF] Villa-Diaz | Analysis of the factors that limit the ability of feeder cells to maintain the undifferentiated state of human embryonic stem cells[END_REF]. When MEFs are not used, naïve-like human culture conditions (so called feeder-free) require Activin A [START_REF] Theunissen | Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency[END_REF] or TGF-β-I [START_REF] Gafni | Derivation of novel human ground state naive pluripotent stem cells[END_REF]. This suggests that Activin/Nodal activation is mandatory to primed and naïve-like cells self-renewal alike, consistently with the observation that Nodal signaling members are upregulated in the primates naïve epiblast (see paragraph II/2.c.2).

b.3 JNK and p38 inhibition

The MAPK superfamily, in addition to the MEK/ERK pathway, includes the JNKs (c-Jun Nterminal kinases) and p38 pathways [START_REF] Neganova | A critical role for p38MAPK signalling pathway during reprogramming of human fibroblasts to iPSCs[END_REF]. In mouse, LIF withdrawal induces activation of JNK1 and 2 that in turn phosphorylate Klf4, inhibiting its transcription and transactivation activity [START_REF] Yao | JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity[END_REF]. P38/MAPK, on the other hand, has been shown to promote mESCs differentiation into either neuronal or cardiomyocytes lineages [START_REF] Aouadi | p38 Mitogen-Activated Protein Kinase Activity Commits Embryonic Stem Cells to Either Neurogenesis or Cardiomyogenesis[END_REF][START_REF] Wu | P38 Mitogen-activated protein kinase controls a switch between cardiomyocyte and neuronal commitment of murine embryonic stem cells by activating myocyte enhancer factor 2C-dependent bone morphogenetic protein 2 transcription[END_REF].

JNKs, p38, or their upstream activator AMPK [START_REF] Meisse | Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells[END_REF], are targeted in several reprogramming protocols, namely NHSM [START_REF] Gafni | Derivation of novel human ground state naive pluripotent stem cells[END_REF], 3iL [START_REF] Chan | Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast[END_REF], 4i (rhesus monkey-specific) [START_REF] Fang | Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts[END_REF], EPSCs [START_REF] Gao | Establishment of porcine and human expanded potential stem cells[END_REF] and, optionally, 5i/L/A (thus being 6i/L/A) [START_REF] Theunissen | Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency[END_REF]. Although it was shown that JUNs, downstream effectors of JNKs, can co-occupy enhancers of OCT4, NANOG, SMAD2 and SMAD3 in hESCs [START_REF] Li | Genome-scale screens identify JNK-JUN signaling as a barrier for pluripotency exit and endoderm differentiation[END_REF], the process by which JNK/JUN pathway inhibition and, even more so, that of p38 and AMPK promotes the human and rhesus monkey naïve state remains to be elucidated.

b.4 Transitory PGN reinforcement

In their Reset protocol, Takashima and colleagues use a doxycycline-inducible system to overexpress NANOG and KLF2 in primed hESCs, an approach shown to convert mouse EpiSCs to the ground state of pluripotency [START_REF] Hall | Oct4 and LIF/Stat3 Additively Induce Krüppel Factors to Sustain Embryonic Stem Cell Self-Renewal[END_REF]. After overexpression, cells are cultured in 2i/LIF with a reduced concentration of CHIR (GSK3i) compared to classical 2i/LIF conditions, and with a PKC inhibitor (Gö6983). Interestingly, reprogrammed hESCs would conserve naïve characteristics after dox withdrawal, suggesting that NANOG and KLF2 overexpression is sufficient to ignite the extended PGN, that subsequently self-maintains without ectopic expression of any factor.

A similar approach was adopted by Chen and colleagues in 2015, who demonstrated that a transient reinforcement of STAT3 signaling through a tamoxifen-inducible system successfully reprograms hESCs to a naïve-like state [START_REF] Chen | Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[END_REF]. Upon withdrawal of tamoxifen, reprogrammed hESCs can subsequently be cultured with 2i/LIF, in FGF2-and feeder-free conditions, suggesting, as for Reset cells, that transitory PGN reinforcement is sufficient to elicit naïve pluripotency.

b.5 Epigenome resetting

The epigenome of a cell is characterized by both its global DNA methylation level and the apposition pattern of repressive and activating histone marks [START_REF] Smith | DNA methylation: Roles in mammalian development[END_REF][START_REF] Thiagarajan | The epigenome in pluripotency and differentiation[END_REF]. DNA methylation is carried out by DNA methyltransferases (DNMTs) while demethylation is operated by ten-eleven translocation (TET) enzymes. In part I/, we mentioned that early development is characterized by global demethylation followed by progressive de novo methylation. As a consequence, pre-implantation (naïve) epiblast cells exhibit a hypomethylated genome compared to post-implantation (primed) epiblast cells, more developmentally advanced. The epigenome of primed and naïve cells is not only a consequence, but also an intrinsic determinant of their state. A dynamic interplay exists between chromatin state and transcription factors activity determining which genes are expressed, and thus what regulatory loops are triggered [START_REF] Thiagarajan | The epigenome in pluripotency and differentiation[END_REF].

Accordingly, reprogramming somatic cells to induced pluripotency triggers widespread epigenetic changes resembling that of epiblast cells [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF][START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF], but the converse is also true: By using chromatin remodelers, transcriptomic status of a cell can be changed, as exemplified by Esteban and colleagues who used L-Ascorbic acid (Vitamin C), a known TET activator, to enhance iPSC generation in both mouse and human [START_REF] Esteban | Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem Cells[END_REF].

Several naïve-reprogramming protocols consistently exploit chromatin remodelers to enhance reprogramming [START_REF] Duggal | Alternative Routes to Induce Na€ıve Pluripotency in Human Embryonic Stem Cells[END_REF][START_REF] Gao | Establishment of porcine and human expanded potential stem cells[END_REF], or as a direct catalyst of the process [START_REF] Guo | Erratum: Correction: Epigenetic resetting of human pluripotency[END_REF][START_REF] Ware | Histone Deacetylase Inhibition Elicits an Evolutionarily Conserved Self-Renewal Program in Embryonic Stem Cells[END_REF]. In their 2iL/Toggle protocol, Ware and colleagues first use a mixture of Histone deacetylase inhibitors (HDACi) to erase histone repressive marks of hESCs, and then culture them, successfully, in 2i/LIF conditions [START_REF] Ware | Histone Deacetylase Inhibition Elicits an Evolutionarily Conserved Self-Renewal Program in Embryonic Stem Cells[END_REF]. A similar approach was chosen by Guo and colleagues, who culture cells in three successive media, the first containing Valproic Acid (or VPA), a HDAC inhibitor [START_REF] Guo | Erratum: Correction: Epigenetic resetting of human pluripotency[END_REF].

Whether epigenome resetting/remodeling is used as a reprogramming driver or not, all naïve-reprogramming protocols to this day yield cells with altered epigenomes, resembling that of primates/human pre-implantation epiblast to different degrees. This demonstrates how critical the epigenetic status of a cell is in its identity, and in this case, in its route toward naïve pluripotency.

phosphorylate TFs, leading to their degradation [START_REF] Jeronimo | The Mediator Complex: At the Nexus of RNA Polymerase II Transcription[END_REF]. In the context of cancer cells, it was shown that CDK8/19 inhibition leads to global hyperactivation of enhancers and thus, hypertranscription [START_REF] Pelish | Mediator kinase inhibition further activates super-enhancer-associated genes in AML[END_REF]. suggesting that CDK8/19 might be the common effectors of these pathways in naïve pluripotency destabilization.

b.7 Hippo pathway inhibition/ YAP activation

Yes-associated proteins (YAP) are signaling effectors inhibited by the Hippo pathway.

They have been shown to be upregulated in several types of cancer (Mo et al., 2014), in mouse progenitor and stem cells, including mESCs [START_REF] Ramalho-Santos | Stemness": Transcriptional profiling of embryonic and adult stem cells[END_REF] and in vitro, the Hippo pathway is a barrier to iPSCs reprogramming [START_REF] Qin | Transcriptional analysis of pluripotency reveals the hippo pathway as a barrier to reprogramming[END_REF][START_REF] Qin | Systematic identification of barriers to human Ipsc generation[END_REF]. Consistently, Qin and colleagues demonstrated that YAP overexpression or Hippo inhibition with Lysophosphatidic acid (LPA) elicits naïve-like characteristics in hESCs, termed Yin-PSCs (YAP-induced naïve PSCs) [START_REF] Qin | YAP Induces Human Naive Pluripotency[END_REF]. However, transcriptomic analysis of Yin-PSCs suggests they might be more closely related to cleavage stages embryos than to the ICM/naïve epiblast.

b.8 Tankyrase inhibition

In 2016, Zimmerlin and colleagues generated naïve-like hESCs from conventional primed hESCs by culture in 2i/LIF supplemented with the Wnt pathway modulator XAV939 [START_REF] Zimmerlin | Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality[END_REF]. These culture conditions, termed LIF-3i, allowed direct reprogramming from Activin/FGF primed culture medium, while transfer to 2i/LIF conditions usually triggers immediate cell differentiation of hESCs [START_REF] Hanna | Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs[END_REF][START_REF] Theunissen | Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency[END_REF]. Though

XAV939 is an inhibitor of Tankyrase, known when used alone to favor B-catenin destabilization and destruction [START_REF] Huang | Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling[END_REF], it paradoxically acts in synergy with CHIR99021 (GSK3i)

to increase Axin expression and thus, Wnt signaling and B-catenin targets transcription in mouse EpiSCs [START_REF] Kim | Modulation of βcatenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal[END_REF]. In both LIF-3i culture and EPSCs culture conditions from Gao and colleagues [START_REF] Gao | Establishment of porcine and human expanded potential stem cells[END_REF], joint use of XAV939 and CHIR99021 is adopted.

Though the effect of this combination may be to strictly increase Wnt activation, it remains to be investigated if it acts through other molecular pathways.

b.9 MUC1* activation

In several types of cancer, the Mucin 1 receptor MUC1 is found to be strongly upregulated. It has been shown that the truncated form of MUC1, MUC1-C/MUC1* is capable of functioning as an independent growth factor receptor, binding the Nucleoside diphosphate kinase A (NME1/NM23) ligand [START_REF] Mahanta | A minimal fragments of MUC1 mediates growth of cancer cells[END_REF]. In primed hESCs, it was shown that undifferentiated colonies are NM23-negative and MUC1*-positive, while differentiated cells lose expression of NM23 and express only the full-length form of MUC1 [START_REF] Hikita | MUC1* mediates the growth of human pluripotent stem cells[END_REF].

Culturing hESCs with either a dimeric form of NM23 (NM23-H1) [START_REF] Smagghe | MUC1* Ligand, NM23-H1, Is a Novel Growth Factor That Maintains Human Stem Cells in a More Naïve State[END_REF] or a recombinant form of its paralog NME7 [START_REF] Carter | A Primitive Growth Factor, NME7AB, Is Sufficient to Induce Stable Na€ıve State Human Pluripotency; Reprogramming in This Novel Growth Factor Confers Superior Differentiation[END_REF] reportedly triggers a conversion to a pluripotent state exhibiting naïve properties without use of LIF, FGF or any inhibitor. The naïve-like cells thus produced need to be further characterized and, so far, the mechanisms that underpin the function of MUC1* remain poorly understood. However, it is worth noting that the full-length MUC1 is able to block the phosphorylation-mediated degradation of B-catenin by GSK3B [START_REF] Huang | MUC1 oncoprotein blocks glycogen synthase kinase 3β-mediated phosphorylation and degradation of β-catenin[END_REF] and microarray data available on all reprogrammed human cell lines of that time. This analysis revealed that the transcriptome of naïve-like hPSCs was a) Significantly different from primed hPSCs, and b) significantly different between cell lines reprogrammed with different protocols [START_REF] Takashima | Resetting transcription factor control circuitry toward ground-state pluripotency in human[END_REF]. Later the same year, Huang and colleagues compared transcriptomes of different reprogrammed hPSCs lines with that of human pre-implantation embryos and demonstrated that they displayed improved but variable similarity with the human epiblast compared to primed cells [START_REF] Huang | The naive state of human pluripotent stem cells: A synthesis of stem cell and preimplantation embryo transcriptome analyses[END_REF].

As new reprogramming methods and scRNA-seq analyses were published along the recent years, these observations only tended to be confirmed [START_REF] Aksoy | Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells[END_REF][START_REF] Gao | Establishment of porcine and human expanded potential stem cells[END_REF][START_REF] Guo | Erratum: Correction: Epigenetic resetting of human pluripotency[END_REF][START_REF] Messmer | Transcriptional Heterogeneity in Naive and Primed Human Pluripotent Stem Cells at Single-Cell Resolution[END_REF][START_REF] Yang | Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency[END_REF] and remain true to this day :

human naïve-like PSCs display variable transcriptomes, reflecting the diversity of methods used for their reprogramming, and some of them only [START_REF] Chen | Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[END_REF][START_REF] Guo | Erratum: Correction: Epigenetic resetting of human pluripotency[END_REF][START_REF] Theunissen | Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency[END_REF] closely resemble the human epiblast. In all the considered studies, however, transcriptomic comparison is obtained by either calculating the expression foldchange for a selected set of genes between two populations, or by graphical representations such as Principal Component Analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE) or Uniform manifold approximation and projection (UMAP). In PCA representations two or three dimensions are selected to be displayed, representing each a fraction of the differentially expressed genes of the dataset. T-SNE and UMAP, on the other hand, perform a 2-or 3D projection of all dimensions (genes) of the dataset. In both cases, the resulting visualizations are approximations of the transcriptomic distance between cells (single-cell RNA-seq) or populations of cells (Bulk RNA-seq). This gives a robust overview of coarse differences, but hardly suffice to highlight finer changes between close populations.

Thus, although such analyses have sometimes been relied on to claim the full efficiency of primates' PSCs naïve-reprogramming, they by no means constitute such evidence, and could even be considered to demonstrate the opposite.

c.2 Cell-surface markers

RNA-sequencing analyses, despite the diversity of information they provide, remain limited to the transcriptome and cannot thus reflect the entire biological reality. Another layer of analysis, taking into account the translated proteins is therefore necessary.

In 2005 Andrews and colleagues demonstrated that hESCs displayed heterogeneity in their self-renewal capacity, and that this was reflected at the population level by expression of surface antigens. The Specific Embryonic Antigen 3 (SSEA3) was found to be expressed only in the undifferentiated fraction of cells that accounted for most of the colony forming ability [START_REF] Enver | Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells[END_REF]. In another study two markers (tetraspannin CD9 and the pericellular matrix proteoglycan GCTM-2) were demonstrated to be co-expressed with OCT4 by hESCs at levels inversely correlated with the differentiation state of the cells [START_REF] Hough | A continuum of cell states spans pluripotency and lineage commitment in human embryonic stem cells[END_REF].

Since then, a broad number of surface markers have been identified, not only reflecting pluripotency in human, but also discriminating the primed and naïve-like states [START_REF] Bredenkamp | The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells[END_REF][START_REF] Collier | Identifying Human Naïve Pluripotent Stem Cells -Evaluating State-Specific Reporter Lines and Cell-Surface Markers[END_REF][START_REF] Collier | Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States[END_REF][START_REF] Pastor | Naive Human Pluripotent Cells Feature a Methylation Landscape Devoid of Blastocyst or Germline Memory[END_REF][START_REF] Shakiba | CD24 tracks divergent pluripotent states in mouse and human cells[END_REF]. The analysis of expression of these markers by sc-RNA seq and flow cytometry offers a new layer of characterization for reprogrammed hPSCs, and demonstrates further that they largely differ depending on the method used for their conversion. Interestingly, only a fraction of these cells express the Sushi Containing Domain 2 (SUSD2) marker, recently identified as a surface marker of the human pre-implantation epiblast [START_REF] Bredenkamp | The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells[END_REF].

c.3 Chimeric competency

As reviewed above, reprogramming methods to naïve primates' pluripotency are diverse and numerous. Cells obtained with these methods, despite displaying common features of putative naïve pluripotency, also exhibit many discrepancies with one another. This is in apparent contradiction with the definition of the naïve state, expected to be at the high end of the pluripotency spectrum, and thus mostly homogenous. Two reasons could explain these differences: 1) Naïve-like cells are incompletely reprogrammed, and on different positions in the pluripotency spectrum. 2) Reprogramming methods "force" the cells out of the primed state toward an artificial state adapted from the culture conditions. Although these cells would share some characteristics with the naïve primates' epiblast, they would be in a state of their own, only partially overlapping with the pluripotency spectrum (Figure 9B).

In part II/1., we mentioned that all the criteria defining the naïve state could fall into three categories: I) molecular, i.e., the activated and repressed pathways underpinning selfrenewal in the naïve-state, II) Genetic, the gene network expressed in response of such cues, and contributing to maintain them. III) Functional, the ability of naïve cells to re-enter embryonic development at the time-point they originate from. In the preceding sections II/3.c.1 and c.2, we showed that aspects I) and II) have been largely characterized in human naïve-like cells, and so far demonstrate their heterogeneity. While transcriptomic proximity of some hPSCs with the human epiblast exist [START_REF] Chen | Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[END_REF][START_REF] Guo | Erratum: Correction: Epigenetic resetting of human pluripotency[END_REF][START_REF] Theunissen | Molecular Criteria for Defining the Naive Human Pluripotent State[END_REF], it was until recently unknown if they retained the functional capacity to re-enter development. The ability to form chimeras, and even more so, interspecies chimeras, is the litmus test for ground state pluripotency in mouse and, in the current state of knowledge, should be so in primates.

Although colonization of naïve-like human and rhesus monkey cells in mouse blastocysts has been described in early studies [START_REF] Fang | Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts[END_REF][START_REF] Gafni | Derivation of novel human ground state naive pluripotent stem cells[END_REF], we mentioned that these results failed to be reproduced in subsequent attempts by other groups [START_REF] Masaki | Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells[END_REF][START_REF] Theunissen | Molecular Criteria for Defining the Naive Human Pluripotent State[END_REF]. Furthermore in a more recent work, naïve-like human iPSCs were injected in pig embryos, and although survival could sometimes be observed at pre-implantation stages, most cells were not able to survive past implantation, resulting in very low fetus colonization rates [START_REF] Wu | Interspecies Chimerism with Mammalian Pluripotent Stem Cells[END_REF].

These rather inconclusive data were put in perspective again this year by two studies.

The first, published by Tan and colleagues, described the fate of human extended PSCs (hEPSCs) injected in cynomolgus macaque early blastocysts [START_REF] Tan | Article Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo ll ll Article Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo[END_REF]. In this work, all blastocysts injected were reported to retain hEPSCs, although proliferation could not be demonstrated, as the number of human cells found in the host never exceeded the number of initially injected cells (25). Additionally, although the authors reported colonization of human cells at post-implantation stage d.p.f. (day post fertilization) 19, this observation was not supported by visual evidence.

In the second study, Aksoy and colleagues systematically assessed the capacity of naïve-like macaque monkey PSCs reprogrammed with a broad variety of methods (TL2i, NHSM-v, E-NHSM , 4i/L/b, t2iLGöY, 5iL/A and LCDM) and human iPSCs (TL2i and t2iLGöY)

to colonize rabbit blastocysts, phylogenetically as distant to primates as rodents, but exhibiting structural and timing features closer to primates [START_REF] Aksoy | Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells[END_REF]. These experiments revealed poor surviving and colonization capacity of both macaque monkey and human reprogrammed cells, with the best results being however obtained with t2iLGöY [START_REF] Guo | Erratum: Correction: Epigenetic resetting of human pluripotency[END_REF] and TL2i [START_REF] Chen | Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[END_REF] cells. This was in sharp contrast with what was obtained with mouse 2i/LIF, bona fide naïve cells, that did not only yield excellent survival rates but also actively proliferated. In the same work, colonization was assessed in macaque embryos, and did not provide better results than with more distantly related species. Further characterization demonstrated that injected cells, both human and macaque, either underwent apoptosis or prematurely differentiated and were unable to remain as mitotically active as their mouse counterparts.

These results reveal that reprogrammed primates' cells lack the naïve-defining capacity of colonizing a pre-implantation blastocyst. Unlike their mouse counterparts, they are unable to remain seamlessly proliferating after dissociation or when re-introduced in an unfavorable environment, and thus cannot participate in the development of the host. Taken collectively with all the characterization of primates naïve-like PSCs performed so far, this questions their very nature and suggests that no current reprogramming method allows self-renewal in a true naïve-state, i.e., a state faithfully mirroring the primates' naïve epiblast. This implies that gaps remain in our understanding of human and non-human primates' pluripotency regulation, and that potential new molecular and genetic regulators are to be yet identified.

III/ Netrin signaling 1. Netrin-1 discovery

Netrins, from the Sanskrit "Netr", one who guides, are a family of molecules belonging to the superfamily of laminins and sharing with them homology in the N-terminal domains. The first Netrin, unc-6, was discovered in the worm C. elegans, in a screen aiming at identifying key genes in neural development [START_REF] Hedgecock | The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans[END_REF]. In pioneer studies, the unc-6 gene was found to code for a 591 amino-acids secreted protein able to bind two transmembrane receptors, unc-5 and unc-40 [START_REF] Ishii | UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans[END_REF][START_REF] Leung-Hagesteijn | UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans[END_REF]. A gradient of unc-6 was shown, in the worm, to guide the newly formed axons along the epidermis toward their tissue of destination, depending on the type of receptor they express. A few years later, two unc-6 homologs were discovered in the chicken and were named Netrin-1 and -2 [START_REF] Serafini | The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6[END_REF]. These proteins were shown to share important homology (~50%) with their worm counterpart, but also to be involved in a very similar process of axon guidance during embryogenesis. At the end of neurulation, Netrin-1 is secreted by the floor plate cells and diffuses in a gradient toward the roof plate, reaching the newly differentiated commissural neurons. These detect the signal through structures called growth cones, and subsequently outgrow axons toward the floor plate and the ventral midline [START_REF] Kennedy | Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord[END_REF][START_REF] Serafini | Netrin-1 Is Required for Commissural Axon Guidance in the Developing Vertebrate Nervous System[END_REF]. Depending on a) the type of receptor expressed by the axon growth cone and b)

the type of ligand expressed by the neighboring cells and target tissues, the axon is either attracted or repelled by Netrin-1 and other chemoattractant signals until it reaches its destination and connects to the target area [START_REF] Barallobre | The Netrin family of guidance factors: Emphasis on Netrin-1 signalling[END_REF] (Figure 10).

Since the establishment of this model, Netrin-1 homologs have been identified in many other invertebrate and vertebrate species, including mammals [START_REF] Gan | Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets[END_REF][START_REF] Harris | Guidance cues at the Drosophila CNS midline: Identification and characterization of two Drosophila Netrin/UNC-6 homologs[END_REF][START_REF] Matus | Molecular evidence for deep evolutionary roots of bilaterality in animal development[END_REF]. As it turns out, both the amino-acid sequence and the function of the protein in neurogenesis are well conserved across bilateral species, though it has been found to be also involved in many other developmental and somatic processes such as pancreatic branching morphogenesis, angiogenesis and spatial control of lung-bud development [START_REF] Cirulli | Netrins: Beyond the brain[END_REF].

The Netrin family a) Netrin ligands

Since Netrin-1 discovery, other Netrins were identified. They all share homology in their N-terminal regions with the amino-terminal domains of laminins, classifying them as members of the laminin superfamily [START_REF] Rajasekharan | Protein family review The netrin protein family[END_REF]. In mammals, and more specifically primates, six Netrins have been identified so far: Netrin-1, -3, -4, -5, -G1 and -G2.

The Netrin-2 homolog identified in the chick, duplication of the Netrin-1 gene, does not appear to exist in mammalian species. Netrins can be divided in two categories, depending of the homology they share with laminins. Netrin-1, -3 and -5 share homology with the γ-chain of laminins and are thus also referred to as γ-Netrins. Netrin-4, -G1 and -G2 are closer related to the β-chain, and thus named β-Netrins (Figure 11A).

In addition to their structural relation to laminins, Netrins also diverge in their own specific structures. Netrin-1, -3, -4 and -5 are secreted proteins, producing gradients involved in neurogenesis and other developmental processes. Netrin-G1 and -G2 (also named G-Netrins), however, are evolutionary and structurally more distant and are attached to the cell membrane by glycophosphatidylinositol (GPI) anchors instead of being secreted [START_REF] Cirulli | Netrins: Beyond the brain[END_REF][START_REF] Rajasekharan | Protein family review The netrin protein family[END_REF]. In the current state of knowledge, G-Netrins are thus reported to act as receptors binding their own ligands, NGLs (Netrin-G Ligands), are not able to interact with the other Netrins receptors, and likely support different molecular pathways than the rest of the family [START_REF] Sun | Netrins: Versatile extracellular cues with diverse functions[END_REF].

b) Netrin receptors

Aside from G-Netrins, secreted Netrins are able to bind two types of receptors: the DCC/Neo1 (DCC and Neogenin) family, and the UNC5 (primates' UNC5A-D and mouse UNC5H1-4) family. Both groups are closely related members of the immunoglobulin superfamily and composed of an extracellular domain, a single-pass transmembrane region, and a cytoplasmic domain [START_REF] Bradford | Netrin-1: Diversity in development[END_REF][START_REF] Sun | Netrins: Versatile extracellular cues with diverse functions[END_REF] (Figure 11B). Although it does not appear to be their preferential receptor, Netrins have also been reported to bind the DS Cell Adhesion Molecule (DSCAM), which is expressed by commissural axons during neurogenesis, participating in their pathfinding to the floor plate [START_REF] Andrews | Dscam guides embryonic axons by Netrin-dependent and -independent functions[END_REF]Liu et al., 2009).

c) Draxin

In addition to all Netrin members and receptors, another protein associated to the family was identified in 2009. This molecule, named Draxin has been first described in vivo in the mouse embryo [START_REF] Islam | Draxin, a repulsive guidance protein for spinal cord and forebrain commissures[END_REF] and then reported in the chick, where it is secreted by the roof plate during later stages of neurulation. It was shown that Draxin is able to directly bind Netrin-1, forming an opposing gradient and preventing it to act on its receptors, regulating its guiding action (Figure 11A). Another ability of the protein is reportedly to bind at least two of said receptors, DCC and Neo1, thus also acting as a competitive inhibitor of Netrin-1 (Gao et al., 2015).

Interestingly, the DRAXIN gene was reported in humans, and DRAXIN protein was shown to retain the same capacity of interaction and antagonism with human NTN1, suggesting a conserved function in molecular regulation [START_REF] Gao | A floor-plate extracellular protein-protein interaction screen identifies Draxin as a secreted Netrin-1 antagonist[END_REF].

Netrins in cancer

Colorectal Carcinoma is often associated with a deletion in the chromosomal region 18q21. In 1990, it was found that when the deletion was present, suppression of a specific gene located in the area was responsible for the phenotype. This gene was named Deleted in Colorectal Carcinoma (DCC), and later found to be the receptor of Netrin-1, homolog of unc-40 in C. elegans [START_REF] Fearon | Identification of a chromosome 18q gene that is altered in colorectal cancers[END_REF][START_REF] Keino-Masu | Deleted in Colorectal Cancer (DCC) encodes a netrin receptor[END_REF].

Further studies demonstrated that DCC was not only active in response to Netrin-1

binding, but could also induce cell death in absence of it [START_REF] Mehlen | The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis[END_REF]. This type of behavior was previously described for other receptors that were termed "dependence receptors" (DRs) [START_REF] Bredesen | p75NTR and the concept of cellular dependence: seeing how the Other Half Die[END_REF][START_REF] Rabizadeh | Induction of Apoptosis by the Low-Affinity NGF Receptor[END_REF]. DRs, as their name suggests, rely on the presence of their ligand to trigger a broad variety of responses (survival, proliferation, differentiation) but instead of being inactive in absence of it, trigger pro-apoptotic signals. Thus, when correctly expressed, DRs regulate the balance between proliferation and cell death, participating in the maintenance of one tissue's homeostasis and leading to overgrowth when suppressed or downregulated. The finding that DCC belonged to the DRs category opened a new avenue in cancer research [START_REF] Bredesen | Apoptosis and Dependence Receptors: A Molecular Basis for Cellular Addiction[END_REF][START_REF] Mehlen | The dependence receptor hypothesis[END_REF], and numerous other receptors were subsequently found to behave in a similar manner. Among these, having the same dependence to Netrin-1 as DCC, are the Neogenin and UNC5 human family receptors [START_REF] Guenebeaud | The Dependence Receptor UNC5H2/B Triggers Apoptosis via PP2A-Mediated Dephosphorylation of DAP Kinase[END_REF][START_REF] Llambi | Netrin-1 acts as a survival factor via its receptors UNC5H and DCC[END_REF][START_REF] Tanikawa | p53RDL1 regulates p53dependent apoptosis[END_REF].

Mechanistically, it was shown that, in the absence or their ligand, both DCC/Neo and UNC5 receptors are accessible and cleaved in their intracellular domain by caspase 3 (Figure 12B). On the contrary, binding of Netrin-1 blocks access to the cleavage site of caspases, at least in part through a dimerization process changing the configuration of the intracellular domains. Caspase 3 is then still recruited, but has no proteolytic effect on DCC/Neo or UNC5H

receptors. Instead, it is the first link of several possible molecular chains leading to various effects [START_REF] Arakawa | Netrin-1 and its receptors in tumorigenesis[END_REF]. Though those are still probably not all identified, activated pathways reported so far include PI3k/Akt [START_REF] Lv | Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor[END_REF][START_REF] Yin | Netrin-1 promotes gastric cancer cell proliferation and invasion via the receptor neogenin through PI3K/AKT signaling pathway[END_REF], p53 [START_REF] Tanikawa | p53RDL1 regulates p53dependent apoptosis[END_REF], ERK1/ERK2, [START_REF] Forcet | Netrin-1mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation[END_REF], FAK [START_REF] Huyghe | Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling[END_REF][START_REF] Liu | Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction[END_REF][START_REF] Ren | Focal adhesion kinase in netrin-1 signaling[END_REF] and YAP/TAZ [START_REF] Yin | Netrin-1 promotes metastasis of gastric cancer by regulating YAP activity[END_REF] (Figure 12).

Involvement of Netrin-1 and its receptors in tumorigenesis is now broadly demonstrated [START_REF] Bernet | Netrin-1 and its receptors in tumour growth promotion[END_REF][START_REF] Boussouar | Netrin-1 and its receptor DCC are causally implicated in melanoma progression[END_REF][START_REF] Goldschneider | Dependence receptors: A new paradigm in cell signaling and cancer therapy[END_REF][START_REF] Grandin | Structural Decoding of the Netrin-1/UNC5 Interaction and its Therapeutical Implications in Cancers[END_REF][START_REF] Sung | Cancer-associated fibroblasts produce netrin-1 to control cancer cell plasticity[END_REF], but other members of the Netrin family such as Netrin-

Netrin-1 in mouse pluripotency

In 2015, starting from the postulate that Netrin-1, through the binding of its receptors, can control proliferation and cell death, Lavial and colleagues investigated the action of the protein on mouse iPSCs reprogramming [START_REF] Ozmadenci | Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance[END_REF]. By adding recombinant Netrin-1 to the OSKM cocktail, the authors observed a significant enhancement of the reprogramming yield, normally associated with widespread cell death. This improvement was found to be mainly allowed by the anti-apoptotic effect of Netrin-1, binding an increasingly expressed DCC along the conversion process.

In the same study, Ntn1 was overexpressed in mouse ESCs cultured in serum/LIF. By doing so, a significant improvement of self-renewal was observed in the dishes, with a higher fraction of cells being fully undifferentiated, compared to the classically heterogenous serum/LIF populations (Figure 13A). Although binding of UNC5B was shown to be partially responsible, this finding was compelling because it did not imply a simple inhibition of apoptosis. Indeed, commitment to differentiation and self-renewal were here affected and thus, pluripotency per se.

This intriguing observation led to a recently published study by the same group [START_REF] Huyghe | Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling[END_REF]. In this work, Serum/LIF (heterogenous naïve state) and 2i/LIF ("ground state" / homogenous naïve state) mESCs transcriptomes were compared, and the Ntn1-Unc5b-Neo1

axis was found to be a specific component of the latter, suggesting an involvement in naïve pluripotency regulation. In serum/LIF cells, Ntn1-Unc5b-Neo1 activation was shown to increase Nanog levels, as well as that of the naïve mouse pluripotency marker Esrrb. Ntn1 over-expression in mouse ES cell lines combined with LIF treatment was also found to supplement for 2i (MEK and GSK3 inhibitors) and to sustain self-renewal in a ground-like state for several passages, indicating that the Ntn1-Unc5b-Neo1 axis replaces, at least on the short run, MEK and GSK3 inhibition. In concluding mechanistic analyses, the authors demonstrated that the Ntn1-Unc5b-Neo1 promotes naïve pluripotency through FAK activation, leading to 1) Indirect ERK1/2 inhibition, a pathway promoting differentiation and 2) GSK3 destabilization, allowing stronger Wnt pathway activation (Figure 13B).

In regard of these recent findings, we thus decided to investigate the function of Netrin-1 and the Netrins family as a whole in primates pluripotency regulation. We investigated the expression of the family in both human and non-human primates single-cell RNA-seq data sets, and generated a human cell line, hSTAN1, conditionally overexpressing the NTN1 gene to address the effect of NTN1-signaling exogenous activation in human pluripotent stem cells.

Results

I. Netrins in the human and non-human primates' embryo

I.1. Netrin family genes in the mouse naïve compartment

To investigate the function of Netrins in primates' pluripotency regulation, we first analyzed publicly available single-cell RNA-sequencing datasets published in the recent years, to establish an overview of the netrins' family genes expression in the primates' preimplantation embryo. As a reference, we started by analyzing several datasets published in the mice early embryo and PSCs [START_REF] Cheng | Single-Cell RNA-Seq Reveals Cellular Heterogeneity of Pluripotency Transition and X Chromosome Dynamics during Early Mouse Development[END_REF][START_REF] Kolodziejczyk | Single Cell RNA-Sequencing of Pluripotent States Unlocks Modular Transcriptional Variation[END_REF][START_REF] Nowotschin | The emergent landscape of the mouse gut endoderm at single-cell resolution[END_REF]Wu and Belmonte, 2015b). We observed in the embryo that Ntn1, Unc5b and Neo1 are the only members of the family to be expressed in the epiblast, regardless of the stage (Figure 14A). While Unc5b and Neo1 are expressed from E4.5 (pre-implantation) to E7.5 (post-implantation / pre-gastrulation), Ntn1 expression is confined to the early (E4. (heterogenous naïve state) counterparts (Figure 14E). From a dataset generated from primed EpiSCs (Wu et al., 2015) we finally found that, although Neo1 and Unc5b are present at high levels, Ntn1 expression is replaced by Ntn3 and Ntn4 (Figure 14F). These results recapitulate what was previously demonstrated in vitro, and confirm that Ntn1-Unc5b-Neo1 form the axis of netrins family genes in the mouse naïve pluripotency context.

I.2. Netrin family genes in the non-human primate embryo

With this reference in mind, we then examined netrins family genes in the cynomolgus macaque embryo, by exploring a single-cell RNA-seq dataset published by Nakamura and colleagues in 2016 [START_REF] Nakamura | A developmental coordinate of pluripotency among mice, monkeys and humans[END_REF]. We found that, in addition to NTN1, UNC5B and NEO1, genes coding for two other Netrin family members are expressed in the primate's preimplantation embryo: the membrane-anchored NTNG1, and the NTN1 antagonist DRAXIN.

Both are enriched in PE, although DRAXIN was detected at lower levels in all other preimplantation tissues (Figure 15A). As for the mouse, we observed that NTN1 is strongly enriched in the early EPI and impoverished in other tissues, albeit expressed at low levels in mid-and late-EPI. While the UNC5B and NEO1 receptors were found to be expressed in all pre-implantation tissues, UNC5B is enriched in the early-, and subsequently late-EPI, while NEO1 shows only one peak in the late-EPI (Figure 15B). This suggests a pattern similar to the mouse, where NTN1 and UNC5B are the privileged actors of naïve pluripotency/early epiblast regulation [START_REF] Huyghe | Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling[END_REF]. To verify this hypothesis, we asked whether NTN1+ cells of the pre-implantation macaque embryo exhibit higher levels of expression of pluripotency markers, and observed that NANOG, OCT4 and SOX2 are all expressed at higher levels in NTN1+ cells compared to NTN1-(Figure 15C). Interestingly, NTN1-antagonist DRAXIN expression does not seem to affect pluripotency, as DRAXIN+ cells show similar expression of OSN compared to DRAXIN-. Upon implantation, we noticed that in addition to NTN1, NTNG1, DRAXIN, UNC5B and NEO1, NTN4 and UNC5D are expressed (Figure 15D and E). We observed that after its peak of expression at E6, NTN1 is swiftly downregulated and is not expressed again until E16-17, stages preceding the onset of gastrulation (Figure 15F). In sharp contrast, DRAXIN expression is strongly rising, along with UNC5B and NEO1, upon implantation until E20. NTN4 and UNC5D have a distinct pattern, both being expressed solely at the peri-gastrulation stages. Collectively, these data show that mouse and macaque monkey share expression of the NTN1-UNC5B-NEO1 axis at pre-implantation stages, with an enrichment of NTN1 in the naïve epiblast.

I.3. Netrins and DRAXIN in the human pre-implantation embryo

We then asked if Netrin family genes are expressed in a similar fashion in the human pre-implantation embryo, and inspected to this end other publicly available single-cell RNA sequencing data [START_REF] Blakeley | Erratum to Defining the three cell lineages of the human blastocyst by single-cell RNAseq[END_REF][START_REF] Stirparo | Erratum: Correction: Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human preimplantation epiblast[END_REF]. We first investigated expression of Netrin ligands (i.e., secreted NTN1, -3, -4 and -5), G-Netrins and DRAXIN. While NTN5 was very weakly detected, and NTN3 absent in all tissues, NTN1, -4 and -G2 were found to be strongly expressed in the zygote and rapidly downregulated at the 8-cell stage (Figure 16A-B). Surprisingly, unlike the mouse and macaque, NTN1 expression is completely extinguished from the compacted morula stage onwards. Only three genes were detected from this point:

NTN4, NTNG1 and DRAXIN (Figure 16C). Accordingly with its temporal pattern of expression, we found that NTN1 is associated with lower expression levels of core pluripotency markers NANOG, OCT4 and SOX2 (Figure 16D). On the contrary, the DRAXIN+ population was enriched for NANOG and OCT4 compared to DRAXIN-cells, although SOX2 exhibited a similar level between the two groups. Neither NTN4 or NTNG1, on the other hand, seem to have an influence on core pluripotency, as illustrated by strictly similar OSN patterns of expression between NTN4+ and -or NTNG1+ and -populations. Altogether, these observations indicate that despite their relative genetic proximity, macaque and human display critical differences of Netrins, and more specifically NTN1 expression during early embryogenesis.

I.4. DRAXIN-positive cells of the human pre-implantation embryo

Because DRAXIN+ cells of the human pre-implantation embryo are specifically enriched in the EPI and PE and display higher levels of OCT4, SOX2 and NANOG, we decided to further characterize this population. We generated volcano plots of the differentially expressed genes (DEGs) of the DRAXIN+ vsgroups and observed that aside from OSN, several other pluripotency genes are upregulated in the former, including the naïve markers SUSD2 and DPPA4 (Figure 17A andB). Interestingly, we also noticed that elements of NODAL signaling, including NODAL itself, GDF3 and LEFTY2 represent some of the most upregulated genes of the DRAXIN+ population. On the contrary, the PE marker GATA6 is the most downregulated gene of the group (p-value and Foldchange considered). Accordingly, DRAXIN and GATA6 show robust anti-correlation among the dataset (Figure 17C). To define more clearly the phenotype of DRAXIN+ cells, we interrogated the KEGG (Kyoto Encyclopedia of Genes and Genomes) database with the most up-and down-regulated genes of the population (Log2(FC)>0,25 or <-0,25) and found that DRAXIN+ cells are enriched for several terms associated with active amino acids biogenesis and metabolism (Figure 17D). Among the most enriched terms are "Signaling pathways regulating pluripotency of stem cells" and "Oxidative phosphorylation", regrouping 30 genes each. On the contrary, downregulated genes return only one term, "Cell cycle", suggesting that DRAXIN-cells proliferate faster than their DRAXIN+ counterparts. We performed a similar analysis with the Gene Ontology (GO) database using most upregulated genes of the DRAXIN+ population, and observed that numerous terms are consistent with rich amino-acid metabolism and active oxidative phosphorylation (Figure 17E). GO analysis also returned terms associated with planar cell polarity and epithelium establishment, including activation of the Wnt signaling pathway.

Collectively, these findings strongly suggest that DRAXIN+ cells epitomize the naïve pluripotent compartment of the human pre-implantation embryo, i.e., the early epiblast, exhibiting expression of naïve markers and genes associated with oxidative phosphorylation, the preferential metabolic mode of naïve cells in vitro.

I.5. Netrin receptors in the human pre-implantation embryo

We next explored expression of netrin receptors (UNC5A-D, DCC and NEO1) in the same dataset. We observed that all receptors, except UNC5B, exhibit zygotic expression followed by a rapid downregulation at the 8-cell stage (Figure 18A andB). Among these, only NEO1 sustains a high and steady level at the compacted morula, early ICM and blastocyst (both EPI and PE) stages, while others (UNC5A, -C, -D and DCC) are expressed at very low levels and/or by a negligible fraction of cells. UNC5B shows a sharply different pattern, mostly undetected until the blastocyst stage where it is strongly upregulated in both EPI and PE (Figure 18C). Because only NEO1 and UNC5B are expressed at this developmental point, we decided to further characterize the NEO1+ and UNC5B+ populations. We found that, while UNC5B+ cells show higher levels of NANOG and OCT4, the converse is true for NEO1+ cells, exhibiting lower NANOG and OCT4 than their negative counterparts. In both UNC5B+ and NEO1+ populations, SOX2 expression is unaffected (Figure 18D). In addition to these findings, we observed that NEO1 and UNC5B expression anti-correlate in the dataset, despite their overlap at the blastocyst stage. Furthermore, UNC5B was found to strongly correlate with NANOG and anti-correlate with the PE marker GATA6 (Figure 18E). Taken together, these observations indicate that, while NEO1 is ubiquitously expressed in the human preimplantation embryo, UNC5B expression is likely a marker of the early epiblast.

To further demonstrate this hypothesis, we studied UNC5B+ cells transcriptome by generating volcano plots of UNC5B+ vs UNC5B-DEGs. Similarly to the DRAXIN+ population, we found multiple pluripotency-associated genes to be upregulated in UNC5B+ cells, including the naïve marker DPPA4. Consistently with our preceding observations, SOX2 is the only pluripotency marker considered that exhibits a similar level of expression between UNC5B+ andcells. We observed that enrichment in pluripotency genes expression in UNC5B+ is accompanied by a strong enrichment for NODAL signaling members such as GDF3, LEFTY1, -2, TDGF1 and NODAL (Figure 19A andB). KEGG pathway and GO analyses with the most differentially expressed genes from the UNC5B+/-populations showed that UNC5B+, as DRAXIN+ cells, are enriched for terms associated with pluripotency, oxidative phosphorylation and active amino acids biogenesis and metabolism (Figure19C and D). Altogether, these results confirm that UNC5B expression in the human pre-implantation embryo is correlated to naïve pluripotency and likely epitomizes the early epiblast, thus phenotypically overlapping with DRAXIN+ cells.

To obtain a complete overview of Netrin family genes patterns in the human preimplantation embryo, we finally analyzed NEO1+ cells transcriptome. As previously mentioned, this population spans from the zygote to blastocyst stages with high enrichment in all tissues, including the EPI and PE. We found that, despite their representation in the pluripotent compartment, NEO1+ cells are strongly downregulated for NANOG, OCT4, and SUSD2. The NODAL signaling members found upregulated in the UNC5B+ population are also all downregulated in NEO1+ cells (Figure 20A andB). In line with these observations, we found through KEGG pathway analysis that NEO1+ cells are impoverished for genes associated with oxidative phosphorylation (Figure 20C), while GO databases returned numerous terms associated with meiosis and ciliogenesis. This suggests that NEO1, despite being expressed in the human epiblast, is not a marker of naïve pluripotency and is likely involved in different early embryogenesis mechanisms.

II. Netrin family genes in primates Pluripotent Stem Cells

II.1. Netrin family genes in non-human primate PSCs

In order to know if our observations in vivo could be verified in vitro, we then decided to explore netrin family genes expression in primates PSCs. We first performed bulk RNAsequencing on rhesus monkey PSCs (LyonES cell line, characterized by [START_REF] Wianny | Derivation and Cloning of a Novel Rhesus Embryonic Stem Cell Line Stably Expressing Tau-Green Fluorescent Protein[END_REF] in the primed state or reprogrammed to naïve-like pluripotency with three methods : ENHSM (modification of the original NHSM protocol from [START_REF] Gafni | Derivation of novel human ground state naive pluripotent stem cells[END_REF], 4i [START_REF] Fang | Generation of naive induced pluripotent stem cells from rhesus monkey fibroblasts[END_REF] and

TL2i [START_REF] Chen | Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[END_REF]. In primed cells, we noticed very low levels of NTN1, but high DRAXIN, NTN4, NEO1, UNC5B and UNC5D, consistently with our observations in the post-implantation macaque embryo (Figure 21A). Unlike the embryo however, NTN5 expression was here detected. After reprogramming with the 4i and TL2i method, we observed a slight increase of NTN1, a decrease of DRAXIN and UNC5D, and almost complete disruption of NTN4 and -5 expression. In these cells, the NTN1-UNC5B-NEO1 triad is thus expressed along with DRAXIN, similarly to what was found in the macaque early epiblast. Cells reprogrammed with the third method, ENHSM, however showed a pattern of netrin family genes expression almost exactly similar to the primed condition. This is consistent with previous reports showing weak or partial acquisition of naïve characteristics from NHSM cells [START_REF] Aksoy | Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells[END_REF][START_REF] Theunissen | Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency[END_REF]. We confirmed these results with quantitative PCR (qPCR) analysis on rhesus PSCs reprogrammed with the three protocols already mentioned and two more, LCDM [START_REF] Yang | Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency[END_REF] and TLCDK8i (Original protocol from Lynch et al., 2020 combined with STAT3ER activation). When comparing these cells with their primed counterparts, we observed an upregulation of NTN1, NEO1, a downregulation of NTN4 with all reprogramming methods, and an upregulation of UNC5B with TL2i, 4i and TLCDK8i (Figure 21B). This is in accordance with our findings in bulk RNA-seq, and consistent with observations made in the embryo. A compelling observation, however, is the co-increase of NTN3 and NTN1, observed neither in vivo nor in vitro through bulk RNA-seq.

II.2. Netrin family genes in human PSCs

We next explored netrin family genes expression in human PSCs, and for this first looked over raw microarray data from naïve-like 5i/L/A cells (Original data from [START_REF] Theunissen | Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency[END_REF]. We observed that Netrin ligands and DRAXIN are weakly expressed compared to NANOG, and that among the receptors, UNC5B, NEO1 and DCC exhibit the highest expression levels, two or three-fold higher than their ligands (Figure 21C). We additionally compared human cell lines reprogrammed with the 5i/L/A or t2iLGöY / Reset [START_REF] Takashima | Resetting transcription factor control circuitry toward ground-state pluripotency in human[END_REF] methods to their primed counterparts, and noticed important discrepancies in netrin family genes between the two protocols. While 5i/L/A cells upregulate all genes of the family upon reprogramming except the UNC5B and -D receptors, t2iLGöY only show weak upregulation of NTN3 and -4, and downregulation of DRAXIN, NEO1, DCC and UNC5D

(Figure 21D). These results suggest that, contrary to the macaque and mouse, culture of human PSCs in media sustaining self-renewal in a naïve-like state does not trigger a systematic upregulation of NTN1, and yield overall various outcomes in Netrin family genes expression.

III. NTN1 overexpression in primates Pluripotent Stem Cells.

III.1. NTN1 overexpression in rhesus PSCs -STAN1 cells

To investigate the effect of Netrin signaling in primates' pluripotency, we decided to overexpress NTN1 in primed LyonES rhesus cells. For this, we used a Tet-On 3G, doxycyclineinducible PiggyBac system called "XLone", for which robust transgene induction in 24h has been demonstrated in human PSCs [START_REF] Randolph | PiggyBac system for human pluripotent stem cells and derivatives[END_REF]. We replaced Xlone's GFP cassette by a NTN1-HA-tag fusion gene and electroporated LyonES rhesus cells with the XLone-NTN1-HA construction, resulting in the creation of the STAT3ER-NTN1 (STAN1) cell line (Figure 22A). After selection of the clones, we confirmed induction efficiency through western blot analysis, and selected two clones for further characterization (Figure 22B). To explore a potential dose-effect of NTN1 induction, we submitted STAN1 cells to a doxycycline (hereafter dox) scale comprising five different concentrations (0, 100, 500, 1000 and 2000 ng/mL) for 72h. For both clones, we observed discrete morphological changes at the highest (1000 and 2000 ng/mL) dox concentrations, with a light flattening and thinning of colonies (Figure 22C). After 72h, no cell death or marked change of proliferation could be observed in any +dox condition compared to the -dox control. We analyzed the transcription profile of induced STAN1 clones by qPCR, using as a reference non-induced STAN1 cells. We first confirmed gradual and efficient induction of NTN1-HA in response to the dox scale, and observed that other Netrin genes are either not detected (NTN3 and-5) or not affected (NTN4 and DRAXIN) by NTN1 induction (Figure 22D). Among the receptors, only NEO1 shows a light and gradual increase in response to NTN1 induction in both clones, while UNC5B and -D exhibit different behaviors from one clone to another with no clear increase. We then tested expression of several core, primed and naïve pluripotency, as well as early lineage differentiation markers, and did not observe significant downregulations in any case. Several genes from the pluripotency categories (KLF5, FGF5, PITX2, DPPA5 and ICAM1) are upregulated upon induction in both clones, albeit with foldchanges not exceeding 3 with the highest dox concentrations. Most of the differentiation genes tested however, are unaffected by NTN1 induction, with the exception of GATA6 that exhibits a slender increase in the 1000 and 2000 ng/mL dox conditions. Collectively, these data suggest that NTN1 overexpression only triggers discrete morphological and transcriptomic changes in macaque primed PSCs.

III.2. NTN1 overexpression in primed human PSCs -hSTAN1 cells

Because of the minor changes observed in rhesus PSCs, and because such cells show increased instability compared to human PSCs when cultured in media initially designed for the latter, we decided to carry on experiments with human PSCs. We used for this the male ES cell line F-OS3-10, expressing a STAT3-ER transgene (derived and characterized by [START_REF] Chen | Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[END_REF]. Using a similar protocol to STAN1 cells creation, we thus generated human STAN1 (hSTAN1) cells (Figure 23A). We verified hSTAN1 clones' induction by western blotting and immunostaining, and selected two clones (c10 and c15) for subsequent work (Figure 23B). After a 72h dox induction at 1µg/mL, we noticed that hSTAN1 colonies homogeneously express the NTN1-HA protein, although net-like aggregates form at the periphery (Figure 23C). We submitted hSTAN1 cells to a dox scale modified from our previous experiments, adding very low concentrations (10, 30, 100, 300 and 1000ng/mL) to explore a possible low-dose effect of induction. Upon treatment, we observed sharp morphological changes in both clones from the +100 to +1000 points (Figure 23D). Phenotype is stronger but comparable to STAN1 cells, with a flattening and thinning of colonies without size change.

After 72h of induction, we noticed no increase of cell death or proliferation in +dox dishes in comparison to the control. By qPCR, we analyzed the transcriptome of induced hSTAN1 cells compared to their non-induced counterparts. Increase of NTN1 transcripts in response to dox was confirmed but, contrary to what was observed in rhesus cells, we found an overall decrease of several pluripotency markers upon induction, including NANOG, OCT4, and the gene coding for a primed surface marker CD24 (Figure 24A). Three pluripotency genes:

SOX2, BMP4 and CDH2 are unaffected by dox treatment. Despite the overall downregulation of pluripotency, we however did not notice an equivalent upregulation of early lineage markers EOMES, GATA6 and NESTIN. Among the netrin genes family, only UNC5B and DRAXIN returned detectable levels of expression, both decreasing upon NTN1 induction. Interestingly, we observed that 11 (NANOG, OCT4, PITX2, C-MYC, KLF4, FGF5, DPPA5, DRAXIN, UNC5B, EOMES and GATA6) out of the 18 genes tested show a peak of expression at the 30 ng/mL dox point. By immunostaining, we confirmed that the NANOG, OCT4 and CD24 proteins are visibly downregulated at the +300 and +1000ng/mL dox points, but could not observe any peak of signal at +30ng/mL (Figure 24B). At the highest dox concentration, we noticed that NANOG and OCT4 expression is extinguished in most of the cells, but that both proteins remain co-expressed in patches in the central part of colonies. We observed a similar pattern for the primed cell-surface marker CD24. The SOX2 protein, on the other hand, is homogeneously expressed in all hSTAN1 colonies regardless of the dox treatment applied, consistently with our previous results. Altogether, these data show that primed hSTAN1 cells undergo marked morphological changes upon NTN1 induction and, unlike rhesus STAN1 cells, undergo pluripotency exit in a cluster fashion.

III.3. NTN1 overexpression in reprogrammed human PSCs -TL2i-hSTAN1 cells

To address if NTN1 overexpression has a similar effect on pluripotency in a naïve versus to primed context, we next reprogrammed our hSTAN1 cells with the TL2i method. After reprogramming, TL2i-hSTAN1 displayed the typical naïve-like morphology and characteristics reported in previous publications [START_REF] Aksoy | Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells[END_REF][START_REF] Chen | Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[END_REF]. Upon treatment with the dox scale applied on primed hSTAN1, we observed a change of morphology gradually accentuating with concentration (Figure 25A). Induced TL2i-hSTAN1 colonies are flattened with an overall increase of size, reaching a point close to confluency after 72h in the +1000 condition. By applying the same doxycycline treatment to a control TL2i-OS3 cell line, no morphological transformation could be seen at any concentration, confirming that changes observed with TL2i-hSTAN1 are caused by the XLone-NTN1 transgene activation. We next analyzed TL2i-hSTAN1 transcriptome through qPCR and found, similarly to primed hSTAN1, that NTN1 expression is efficiently induced in response to dox (Figure 25B). While we observed a slight increase of NTN3, all other netrin ligands, receptors and DRAXIN share a similar pattern, with a peak of expression at +10 and +30 ng/mL dox (NTNG1, UNC5A-C) or +30 ng/mL (DRAXIN, NTN4, NEO1 and UNC5D) followed by a gradual downregulation. We found a similar behavior for core, primed and naïve pluripotency markers with the exception of KLF5, the only pluripotency gene upregulated upon induction (Figure 25C). As reported for primed hSTAN1, commitment to early lineage differentiation could not be demonstrated, as no endodermal (GATA6, SOX17), ectodermal (FST, PAX6) or mesodermal (EOMES) marker is upregulated as a matter of induction. Instead, all rather exhibit a pattern similar to pluripotency markers, with a single peak of expression at +30 followed by a downregulation. To gain better insight on the contrasting low-vs high-dose effects, we performed immunostaining on cells treated with the lowest (+10, +30) and highest (+1000) dox concentrations. We found faint expression of NTN1-HA at the lowest doses, while strong signal is visible at the +1000 concentration (Figure 25D). As observed for primed hSTAN1, we noticed that NTN1 is thoroughly expressed in TL2i-hSTAN1 colonies, but enriched in a net-like structure at the periphery. OCT4 staining revealed that, as for primed hSTAN1, reprogrammed cells heterogeneously lose expression of the core pluripotency marker, and remaining signal is located in the central area of colonies. Unlike what was suggested by our qPCR analysis, we did not find an increase of OCT4 staining in the +30ng/mL condition compared to untreated cells. Collectively, these data show that naïve-like hSTAN1 cells, as their primed counterparts, change morphology and lose pluripotency in a dose-dependent fashion upon NTN1 induction.

In both cases, however, no commitment to early-lineage differentiation could be demonstrated, and transcriptomic analyses returned a peak of expression in discordance with protein observations at the lowest doxycycline doses.

IV.1. TL2i-hSTAN1 cells commit to mesodermal lineage after dox induction.

IV.1.a. TL2i-hSTAN1 cells undergo pluripotency exit upon induction

To characterize NTN1 overexpression effect, we first evaluated the induction efficiency of our system. In absence of treatment (-dox condition), we found that most cells (91.9%) express no detectable NTN1 (hereafter NTN1-zero population); however, 7.6% display low levels of the transcript (Figure 27A andB). We termed that NTN1 expression interval "endogenous" (NTN1-endo population). Upon dox treatment, we observed emergence of cells expressing NTN1 up to 10-fold higher than the NTN1-endo levels. Cells expressing NTN1 levels in that interval represent 46.6% of the population at the +10 ng/mL dox concentration, and increase to 79.2 and 84.5% at the +30 and +1000 doses, respectively (Figure 27B). As such levels of expression were not observed in the -dox condition, we considered this as product of the XLone-NTN1 system's induction, and refer to it as exogenous NTN1 (NTN1-exo population). Interestingly, these observations are in sharp contrast with the kinetics of induction initially reported by Randolph and colleagues, where plateau phase is reached at the +1000 dox dose, and transgene-positive cells do not exceed 40% of the population with doses under 200ng/mL [START_REF] Randolph | PiggyBac system for human pluripotent stem cells and derivatives[END_REF], versus 10ng/mL in our hands. Incidentally, we noticed that few cells (0.4%, or 9 cells) from the -dox condition express exogenous levels of NTN1. This could be indicative of a system's leak; however, we did not observe any differentially expressed genes in these cells compared to their NTN1-zero counterparts (data not shown).

Next, we sought to evaluate if TL2i-hSTAN1 cells, as suggested by our previous findings, lose pluripotency upon induction. For this, we performed Uniform manifold approximation and projection (UMAP) for each of the four conditions, and generated feature plots of NANOG expression. As expected, we found that the -dox population exhibits widespread NANOG expression, while NTN1 is limited to low levels in rare cells (Figure 28A).

Upon induction, a drastic change occurs, with a strong decrease of NANOG+ cells and converse increase of NTN1+. This phenotype gradually accentuates with dox concentration.

By generating violin plots of NANOG expression in NTN1-zero, NTN1-endo and NTN1-exo sub-populations for each condition, we noticed that NTN1-zero cells retain high NANOG, in contrast to NTN1-exo that show almost complete extinction of the gene in the +dox conditions (Figure 28B). NTN1-endo cells exhibit an intermediate behavior, with reduced but not entirely downregulated NANOG compared to NTN1-zero. This anti-correlation of NANOG and NTN1-HA is in accordance with our previous findings, and strongly suggests pluripotency exit of hSTAN1 cells in response to induction. To confirm this hypothesis, we investigated expression of additional markers of core, primed, formative and naïve pluripotency in the whole dataset.

With heatmap and dotplot representations, we observed that non-induced TL2i-hSTAN1 cells express strong core pluripotency markers OCT4, NANOG, SOX2 and the naïve marker TFAP2C (Figure 29A). Surprisingly, however, we found other naïve markers (TFCP2L1, KLF4, HHLA1) to be expressed by a relatively small fraction of cells. Conversely, primed and formative markers were both expressed at significant levels. This suggests, as outlined in introduction and in previous work [START_REF] Aksoy | Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells[END_REF], that naïve-like human cells are more heterogenous than their mouse counterparts and not fully recapitulate naïve characteristics.

Upon dox induction, we observed a gradual downregulation of all the pluripotency genes analyzed, to the one exception of SOX2, consistently with our previous data on TL2i-hSTAN1. With violin plot representations, we confirmed for NANOG a reduction both in the fraction of positive cells and average level of expression compared to -dox, but noticed a different pattern for OCT4, expressed by an equivalent number of cells in the +dox conditions versus -dox, but spanning out lower, more heterogenous levels in +dox (Figure 29B). SOX2, on the other hand, exhibits a slight increase upon induction, but remains stable and otherwise unaffected by dox concentration changes. Altogether, these results confirm our previous findings that hSTAN1 cells undergo pluripotency exit upon treatment, we thus next tried to determine if early lineage specification is occurring. We explored expression of mesoderm, endoderm and ectoderm markers and found that uninduced TL2i-hSTAN1 express both early mesodermal (MIXL1, TBXT, EOMES, LEFTY1, CER1) and ectodermal (VIM, NES, FST) genes in an heterogenous fashion (Figure 29C). Endodermal markers (GATA4, -6 and SOX17) are also expressed, though to a lesser extent. Upon treatment, we observed a sharp dose-dependent downregulation of all early lineage genes, with the exception of NESTIN that shows a slight increase following induction. As previously described by qPCR analysis, these results suggest that, although they exit pluripotency, induced TL2i-hSTAN1 cells do not commit to early-lineage differentiation.

IV.1.b. NTN1-exo cells commit to striated muscle differentiation

To gain better insight on NTN1 effect on TL2i-hSTAN1 cells, we undertook to characterize the NTN1-exo populations of the +dox conditions. For this we isolated NTN1-exo cells in all treated conditions and compared them to their null counterparts (Figure 30A). With the most differentially expressed genes (average Log2FC>0,25 or <-0,25) thus obtained, we interrogated GO databases, and found that NTN1-exo cells are enriched for terms associated with muscle system processes, including myofibril assembly, sarcomere and actomyosin structures organization and striated muscle differentiation (Figure 30B). We also found several terms associated with Golgi-vesicle organization, transport and budding, consistent with active NTN1-HA translation and secretion. We performed GSEA analysis with all DEGs from NTN1exo cells and confirmed enrichment for Golgi-transport, protein secretion, actin-based structure organization, but also cilium organization (~200 genes) (Figure 30C). We noticed that NTN1exo cells are downregulated for terms associated with ribosome biogenesis and ribosomal RNA processes, consensus transcriptomic terms for naïve-like human cells [START_REF] Huang | The naive state of human pluripotent stem cells: A synthesis of stem cell and preimplantation embryo transcriptome analyses[END_REF], which confirms their disengagement from naïve pluripotency.

We then asked if the NTN1-exo phenotype observed is varying depending on dox concentration. When generating GO and GSEA analyses on NTN1-exo DEGs from +10, +30 and +1000 conditions individually, we found similar terms enriched compared to the pooled +dox populations, with only slight differences of order (data not shown); we thus decided to evaluate the transcriptomic distance between our four experimental conditions by comparing the most differentially expressed genes (average Log2FC>0,25) of the +10, +30 and +1000 conditions compared to -dox. We found a significant overlap of 170 genes between the three treated conditions, and noticed that the +30 and +1000 points share the highest proximity index (Figure 30D). This was confirmed by hierarchical clustering representation, on which all +dox conditions cluster together, but +30 and +1000 segregate a separate branch. Altogether, this indicates that NTN1 triggers striated muscle differentiation of TL2i-hSTAN1 cells, but does not yield a different phenotype at low compared to high dox concentrations. More likely, commitment is gradual and dose-dependent.

IV.1.c. NTN1 overexpression has a paracrine differentiating effect

Because NTN1 is a secreted protein and has been extensively demonstrated to act, in different contexts, as a short-and long-range signal, we then asked if in addition to its autocrine effect on the NTN1-exo population, NTN1 has a paracrine effect on the NTN1-zero neighboring cells. To address this question, we compared the transcriptomes of NTN1-zero cells in induced context to the NTN1-zero population of the control (-dox) condition (Figure 31A). As precedingly, we performed successive GO analysis and GSEA, and found that, as NTN1-exo cells, NTN1-zero are enriched for muscle system processes, though to a lesser extent than NTN1-exo (Figure 31B). We also found enrichment for "ossification" and "osteoblast differentiation", a cell type belonging, as straited muscle cells, to late mesodermal lineages.

Through GSEA, we found for +dox NTN1-zero cells terms associated with a single family of enrichment, ciliogenesis, and a converse downregulation of ribosomal biogenesis and rRNA processes (Figure 31C). Collectively, these data suggest that NTN1 paracrine and autocrine signals trigger effects partially similar, but the latter has power to induce commitment in a different late mesodermal lineage.

IV.2. DRAXIN-positive

TL2i-hSTAN1 do not undergo autocrine or paracrine effects from NTN1.

IV.2.a. DRAXIN-positive TL2i-hSTAN1 cells remain pluripotent upon induction

As previously investigated in the embryo and other primates PSCs, we nest asked if secreted Netrins other than NTN1 are expressed in TL2i-hSTAN1, and if their expression is affected by induction. By dotplot and heatmap representations, we found that NTN3, -4 and -5 are expressed by infinitesimal fractions of cells regardless of dox treatment; however, we noticed strong expression of DRAXIN by a majority of cells in the -dox condition (Figure 32A).

Upon NTN1 induction, we observed a strong downregulation of DRAXIN in both the fraction of positive cells and average level of expression. On UMAP representations, it appears that DRAXIN-and NTN1-positive cells are mutually exclusive and cluster separately (Figure 32B).

We confirmed this observation by calculating the correlation coefficient between DRAXIN and NTN1 in each condition. While in -dox, no correlation could be established, we found a significant anti-correlation between the genes in all +dox conditions, consistently with our previous findings (Figure 32C).

Because DRAXIN-positive cells form a distinct cluster from their NTN1-exo counterparts upon induction, and because DRAXIN has been previously demonstrated to prevent NTN1 action in different contexts, we then asked if its expression could counteract NTN1 pro-differentiation effect in TL2i-hSTAN1. We isolated and pooled together the DRAXIN+ populations of the +dox conditions, and generated a volcano plot representation with all DEGs from DRAXIN+ vs DRAXIN-cells. Interestingly, we found several pluripotencyassociated genes, including SOX2, OCT4, ESRG and NANOG to be strongly enriched in DRAXIN+ cells (Figure 33A). Consistently, we found NANOG and DRAXIN to positively correlate in the whole dataset (Figure 33B), as previously suggested by their similar patterns of expression in UMAP representations. Using the most differentially expressed genes of the DRAXIN groups (Average Log2FC>0.25 or <-0,25), we then interrogated the KEGG database, and found DRAXIN+ cells to be enriched for pathways regulating pluripotency and TGFβ/NODAL signaling (Figure 33C). We also noticed enrichment for cell cycle, DNA replication and base excision repair. DRAXIN-, on the other hand, are enriched for focal adhesion and actin cytoskeleton regulation, but also apoptosis. Additionally, while terms indicative of DNA replication, proliferation and stem cell self-renewal were confirmed, we found by GO analysis that DRAXIN+ cells undergo chromatin remodeling and DNA reconfiguration compared to their negative counterparts (Figure 33D). Collectively, these observations strongly suggest that

IV.2.b. DRAXIN-positive cells constitute the most pluripotent compartment of the TL2i-hSTAN1 population

As previously showed, NTN1 and NANOG anti-correlate upon induction, and NTN1zero cells, despite undergoing paracrine effects from the NTN1-exo population, retain high NANOG and hallmarks from pluripotency. Because we demonstrated that DRAXIN+ cells remain pluripotent in treated conditions, we wondered if they epitomize entirely the pluripotent compartment of induced TL2i-hSTAN1, overlapping with NTN1-zero cells. For this, we identified and compared six subpopulations based on the co-expression of DRAXIN and NTN1 (endogenous or exogenous). In absence of induction, we noticed that in the majority NTN1zero fraction, more than half of the cells express DRAXIN, representing 56.8% of the whole population (Figure 34A). In induced conditions, despite an overall reduction compared to -dox, the NTN1-DRAXIN+ group systematically represents more than half of NTN1-zero cells. By generating violin plots of OCT4, SOX2, NANOG, DPPA4 and SALL4 expression, we found that NTN1-DRAXIN+ cells exhibit higher levels of all considered pluripotency markers than their DRAXIN-counterparts (Figure 34B). Interestingly, we also found higher OSN, DPPA4 and SALL4 in NTN1-exo DRAXIN+ compared to NTN1-exo DRAXIN-cells. By performing a similar analysis in the -dox population, we observed overall higher levels of said markers, albeit to a lesser extent, in DRAXIN+ versus DRAXIN-cells (Figure 34C). Altogether, these findings reveal that DRAXIN-positive cells represent the most pluripotent compartment of TL2i-hSTAN1 cells, regardless of NTN1 induction, and further suggest that DRAXIN has a shielding effect towards NTN1, alleviating both paracrine and autocrine effects.

IV.3. NTN1 effect is partially due to NEO1 activation.

IV.3.a. Netrin receptors expression in TL2i-hSTAN1

To characterize NTN1 mode of action, we then asked what known netrin receptors are expressed by TL2i-hSTAN1 cells. We found that, among the UNC5 and DCC/NEO1 families, only UNC5B and NEO1 are detected at significant levels (Figure 35A). We noticed that without induction, UNC5B is expressed by a majority of cells (56.1%), while NEO1+ only represent 40.2% of the population (Figure 35B andC). Upon induction, this pattern is inverted, NEO1 taking over at the expense of UNC5B to reach 66.6% versus 29.6% of positive cells at the highest dox concentration, respectively. Moreover, while no correlation of expression could be established between UNC5B and NEO1 in the whole dataset, we found a negative and positive one with NTN1, respectively (Figure 35D). This shows that, upon induction, TL2i-hSTAN1 switch expression from UNC5B to NEO1.

We next sorted out 8 subpopulations (lettered A to H) based on the expression of UNC5B, NEO1 and NTN1, and compared different configurations in the -dox condition (Figure 36A). We first collected DEGs of NTN1-zero UNC5B+ versus NEO1+ cells and generated volcano plots. As a result, we found that selected pluripotency markers are not significantly enriched in one or the other population, indicating that pluripotency does not rely on UNC5B or NEO1 expression in absence of induction (Figure 36B). We noticed, however, that NTN1endo expression leads to a change in enriched genes from both NEO1+ and UNC5B+ populations, suggesting that both receptors are responsive to a low-dose autocrine signal (Figure 36C). By comparing UNC5B and NEO1-positive cells to their negative counterparts, we observed that, in a non-induced context, UNC5B expression is associated with modest, but greater transcriptional changes that NEO1 expression (Figure 36D). Collectively, these results indicate TL2i-hSTAN1 pluripotency is not affected by UNC5B or NEO1 expression in a noninduced context, and that both receptors have an overall discrete effect on the cells transcriptome.

IV.3.b. NEO1-positive and NTN1-exo TL2i-hSTAN1 cells phenotype overlap

We then investigated NEO1 and UNC5B expression impact on cell identity in treated conditions. For this, we first compared NEO1+ cells with or without NTN1-exo expression (collectively referred to as NEO1-specific) to all NEO1-negative cells in all +dox conditions (Figure 37A). With the differentially expressed genes from these populations, we generated a volcano plot and observed important downregulation of NEO1-specific cells for several pluripotency genes, including OCT4, SOX2 and NANOG, along with NODAL and LEFTY2 (Figure 37B). We performed gene set enrichment analysis interrogating GO databases and found that, while NEO1-negative cells are enriched for ribosomal biogenesis, rRNA metabolism and mitochondrial genes expression, all consensus terms of naïve-like cells, NEO1-specific return terms associated with endocytosis, Golgi-vesicle transport, cilia assembly and mitosis (Figure 37C). Interrogation of KEGG databases retrieved terms confirming the naïve-pluripotent identity of NEO1-negative cells, and enrichment of endocytosis for NEO1-specific cells (Figure 37D). Interestingly, the NEO1-specific population additionally display with this method enrichment for terms associated with osteoclast differentiation and focal adhesion. These results collectively suggest that cells expressing NEO1 without UNC5B are engaged in differentiation processes, form cilia and undergo active proliferation, while NEO1-negative cells, regardless of UNC5B expression, retain a pluripotent identity.

We next analyzed UNC5B-specific cells with a similar approach (Figure 38A). By using DEGs of UNC5B+NTN1-and UNC5B+NTN1+ versus all UNC5B-negative subpopulations, we generated a volcano plot and found that, to the opposite of NEO1-specific cells, UNC5Bspecific are strongly enriched for OCT4, ESRG, NODAL and LEFTY2 (Figure 38B). We noticed, however, that unlike the NEO1-negative population, other pluripotency markers, including NANOG and SOX2, are not significantly enriched in UNC5B-specific cells. We also observed a distinctive upregulation of the NODAL signaling gene TDGF1 in the latter. Despite these differences, NEO1-negative and UNC5B-specific groups return partially similar GOterms upon gene set enrichment analysis (namely ribosomal biogenesis and rRNA metabolism), while UNC5B-negative cells share common terms with their NEO1-specific counterparts (Golgi-vesicle transport and cilia assembly) (Figure 38C). By KEGG-pathway analysis, we confirmed that UNC5B-specific cells are enriched for terms associated with pluripotency (Figure 38D). Altogether, these results suggest distinct effects from NEO1 and UNC5B expression in induced TL2i-hSTAN1. While the former is associated with actin-based morphological changes, cilia formation and differentiation, the latter is correlated to pluripotent characteristics.

IV.3.c. NEO1 and/or UNC5B activation in induced TL2i-hSTAN1is not the main cause of NTN1 effect.

To further understand these phenotypic differences, we then undertook to identify the genes expressed in response to one or the other receptor's activation. For this, we speculated that NEO1 or UNC5B co-expressed with autocrine NTN1 are strongly activated compared to a NTN1-negative context, where ligand is spatially distant and in limiting quantities. We thus compared NEO1+NTN1+ and UNC5B+NTN1+ (hereafter activated-NEO1 and -UNC5B,) to NEO1+NTN1-and UNC5B+NTN1-cells (hereafter silent-NEO1 and -UNC5B), respectively (Figure 39A). We generated volcano plots of these comparisons, in which the varying parameter is exogenous NTN1 expression, and found for both UNC5B+ and NEO1+ cells important transcriptomic changes depending on ligand expression (Figure 39B and Supplementary Figure S1). In NEO1+ cells, we noticed that NTN1 absence is associated with higher NANOG, SOX2, OCT4, ESRG, DPPA4, and NODAL signaling markers NODAL, TDGF1, LEFTY2, FST. NEO1+NTN1+ on the contrary, express high levels of NTN1-exo top markers such as (B2M, H19, COL1A1, LGALS1, ANXA1, ACTC1) (Supplementary Figure S2 and Table S2). For UNC5B+ cells, we observed similar impact of NTN1 expression to a reduced extent (overall lower p-values). We then compared NTN1-exo cells expressing or not NEO1 or UNC5B, to assess activated-receptors impact in a NTN1+ background. We found strikingly limited transcriptional effect of both NEO1 and UNC5B in this context, with 8 (NEO1, MALAT1, H19, COL1A1, TOP2A, TPX2, MKI67, UBE2C) and 6 (UNC5B, TDGF1, GAL, HIST1H1B, HIST1H4C, UBE2C) upregulated genes, respectively (average Log2FC>0,2 and p-value<10 -5 ) (Figure 39C). Moreover, OCT4 is the only pluripotency gene downregulated upon one receptor (NEO1) expression. This intriguingly suggests that most transcriptomic effects observed in NTN1-exo cells are caused by the sole effect of NTN1 rather by activation of its receptors, although activated NEO1 is associated with transcription of some top NTN1exo markers.

IV.3.d. UNC5B expression effect on TL2i-hSTAN1 cells is weaker than that of DRAXIN.

As precedingly observed, NEO1+ cells are associated in an induced context to differentiation of TL2i-hSTAN1, while UNC5B is correlated to maintenance of pluripotent characteristics. Because we previously suggested that DRAXIN acts as a shield against NTN1 pro-differentiation cues and is thus also associated with pluripotency, we asked which of UNC5B or DRAXIN has the strongest pro-pluripotency effect. For this, we identified 4 subpopulations based on combined UNC5B and DRAXIN expression, and first compared DRAXIN+UNC5B+ cells to DRAXIN-UNC5B+. By volcano plot representations, we found that DRAXIN expression yields important transcriptomic changes in a UNC5B+ context, with enrichment for both pluripotency and NODAL-signaling genes and concomitant downregulation of NTN1-exo markers (B2M, H19, COL1A1, LGALS1, ANXA1, ACTC1) (Figure 40A). Conversely, we observed sharply more discrete effects of UNC5B expression in a DRAXIN+ context, with a slight downregulation of NODAL-signaling members, and slight upregulation of ETV4 and OCT4 (average Log2Fc<0,2) (Figure 40B). We consistently found with violin plot representations that DRAXIN+ cells systematically exhibit higher levels of some selected pluripotency and NODAL-associated genes than their UNC5B+ counterparts (Figure 40C). Among said genes, we found that only ETV4 and, to a lesser extent, NANOG and GDF3, benefit from combined DRAXIN and UNC5B expression. Collectively, these data demonstrate that DRAXIN has a significantly stronger effect on TL2i-hSTAN1 pluripotency, and more generally transcriptome, than UNC5B expression.

Discussion

In this work, we asked if Netrin family genes, and more specifically NTN1, are involved in pluripotency regulation in primates as demonstrated in mouse, and could represent a new lever for the capture of bona fide primates' naïve pluripotency. Our main conclusion is that NTN1 expression, unlike in macaques and mouse, is not associated to pluripotency in human but instead triggers differentiation to late mesodermal lineages. NETRIN-1 antagonist DRAXIN, on the contrary, is found enriched in the human naïve epiblast and is a potential new candidate for human pluripotency regulation, shielding PSCs against NTN1 differentiating effect in vitro.

A. Netrin signaling in non-human primates' pluripotency

A.1 The NTN1/UNC5B/NEO1 axis is a hallmark of naïve macaque cells

The first question we asked is how Netrin family genes are expressed in non-human primates' PSCs in vivo and in vitro. We found in analyzing publicly available single-cell RNAseq data that, as for the mouse, the cynomolgus macaque's pre-implantation epiblast is enriched for NTN1, UNC5B and NEO1, and that NTN1-positive cells are associated with higher levels of pluripotency genes OCT4, SOX2 and NANOG. NTN1 antagonist DRAXIN, however, despite being expressed, is not associated with any difference in such markers' expression.

Through bulk-RNA seq and qPCR analysis, we revealed that upon reprogramming to naïvelike pluripotency, primed rhesus macaque PSCs show an increase of NTN1 and a converse decrease of DRAXIN while maintaining UNC5B and NEO1 expression. This is consistent with our findings and previous reports that, while primed EpiSCs express no Ntn1, bona fide mouse naïve cells express the Ntn1-Unc5b-Neo1 axis. By qPCR, we intriguingly observed in rhesus reprogrammed cell lines, along with NTN1, a co-upregulation of NTN3. Since NTN3 expression was not detected in both rhesus PSCs bulk RNA-seq and embryos single-cell RNA-seq, and that NTN1 and -3 genes share the strongest sequences homology of the family [START_REF] Van Raay | The NTN2L gene encoding a novel human netrin maps to the autosomal dominant polycystic kidney disease region on chromosome 16p13.3[END_REF], we hypothesize that this increase is due to a lack of specificity of the NTN3 primers used for our qPCR experiment rather than an actual upregulation of the gene.

A.2 NTN1 overexpression leads to pluripotency reinforcement of rhesus PSCs

Using a dox-inducible system, we showed that NTN1 overexpression in rhesus macaque primed PSCs leads to discrete morphological changes, with a flattening and thinning of colonies not associated with visible increases in cell death or proliferation rate. Along with this transformation, we observed by qPCR analysis an increase of several pluripotencyassociated genes in comparison to non-induced cells. [START_REF] Hackett | Regulatory principles of pluripotency: From the ground state up[END_REF][START_REF] Weinberger | Dynamic stem cell states: Naive to primed pluripotency in rodents and humans[END_REF].

A.3 NTN4 expression is associated to primed pluripotency and gastrulation in macaque and mouse.

In the cynomolgus macaque embryo, we noticed at the peri-gastrulation stages a switch of expression from NTN1 to another secreted netrin, NTN4, and an upregulation, in addition to UNC5B and NEO1, of another UNC5-family receptor, UNC5D. We also noticed that DRAXIN, despite being already expressed at the pre-implantation stages, is gradually increased upon implantation. Interestingly, we made consistent observations in rhesus PSCs. While NTN4 and UNC5D are expressed at similar levels in primed cells, they are both downregulated upon reprogramming (Figure 41). DRAXIN, strongly expressed in the primed state, also shows a sharp downregulation depending on the reprogramming method. In mouse, although we did not observe Unc5d expression in the EpiSCs dataset at our disposal, we found Ntn4 to be expressed, in contrast to the naïve 2i/LIF or epiblast cells. We thus speculate that, in the mouse and macaque, NTN1 and -4 represent antagonizing signals, the former regulating naïve, and the latter primed pluripotency or gastrulation. In the macaque, while NTN1 is likely to act through UNC5B and NEO1, NTN4 could act through UNC5D, co-expressed in the same context and embryonic stage. DRAXIN, strongly upregulated in primed cells in vivo and in vitro,

B.1 NTN1 expression is not a hallmark of human naïve pluripotency

By analyzing publicly available single-cell RNA-seq data, we investigated netrin family genes expression in the human pre-implantation embryo, and found that the naïve epiblast is enriched for DRAXIN, NEO1 and UNC5B, but devoid of NTN1 expression. NTN1-positive cells, contrary to the macaque, are not enriched but impoverished for pluripotency-associated genes.

Interestingly, we showed that DRAXIN-positive cells on the other hand, show an upregulation of OCT4 and NANOG. By exploring microarray data from the 5i/L/A and t2iLGöY protocols' original publications, we found that human PSCs reprogrammed with both methods display contrasting patterns of netrin family genes expression. We conclude that, contrary to the macaque or mouse, NTN1 upregulation is not a hallmark of human naïve pluripotency. On the contrary, DRAXIN enrichment in the naïve epiblast and its correlation with high OSN expression, along with the complete absence of NTN1 transcript in the same tissue, strongly suggest that NTN1 action is actively silenced in the human pre-implantation embryo. To verify this hypothesis, in situ observations of NTN1 and DRAXIN transcripts and protein allocation will be instrumental.

B.2 NTN1 overexpression leads to pluripotency exit of primed and naïve-like human PSCs.

Using the same tools as for rhesus macaque cells, we generated a human cell line, hSTAN1, expressing NTN1 in response to doxycycline. In primed hSTAN1 PSCs, we demonstrated that NTN1 triggers an unambiguous pluripotency collapse. At the transcript level, we observed downregulation of numerous pluripotency-associated genes, including OCT4, NANOG, and the primed surface marker CD24, which we confirmed at the protein level through immunostaining. Interestingly, we observed that OCT4, NANOG and CD24 decrease does not occur in a salt-and-pepper fashion, or by a homogenous reduction of signal in all cells, but is rather zone-specific, with clumps retaining expression of pluripotency genes in the central area of colonies. We noticed that this pattern anti-correlates with that of the NTN1-HA protein, aggregated in a net-like structure at the periphery of the colonies and impoverished in the middle. After reprogramming to the naïve-like state, we found that TL2i-hSTAN1 cells undergo similar changes, indicating that NTN1 affects self-renewal in the pluripotent state, whether primed or naïve. These observations were confirmed by our single-cell RNA-seq analysis of TL2i-hSTAN1 cells, through which we showed widespread downregulation of numerous pluripotency markers upon dox induction, and a clear-cut anti-correlation of NTN1 and NANOG expression. We have moreover noticed that uninduced TL2i cells express primed and formative pluripotency markers, and exhibit heterogeneous expression of naïve markers such as KLF4. Bona fide naïve cells, to the opposite of this pattern, are expected to express none of the former, and the latter in a homogeneous fashion. In preceding reports, it was already shown that human PSCs reprogrammed with different protocols exhibit transcriptional heterogeneity and expression of early lineage/ primed markers [START_REF] Han | Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing[END_REF][START_REF] Mccracken | Transcriptional dynamics of pluripotent stemcellderived endothelial cell differentiation revealed by single-cell RNA sequencing[END_REF][START_REF] Nguyen | Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations[END_REF]. This is consistent with the current knowledge that naïve-like human PSCs exhibit incomplete naïve characteristics [START_REF] Aksoy | Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells[END_REF], however, our findings reveal that the statement might be true to a greater extent than previously thought.

Incidentally, a surprisingly low number of single-cell RNA-sequencing studies have been published on naïve-like human or non-human primates' cells to further confirm or infirm these observations, account being taken of the amount of efforts deployed by several groups to publish numerous reprogramming methods in the recent years. Despite our finding that both primed hSTAN1 and naïve-like TL2i-hSTAN1 cells undergo pluripotency exit upon NTN1 induction, we could not demonstrate any commitment to early lineages through qPCR analyses. This is a compelling observation, given that pluripotency and differentiation are two sides of a same coin, exit from the former triggering engagement in the latter. Surprisingly, we found that TL2i-hSTAN1 expressing autocrine NTN1, instead of committing to early lineages, express genes enriched for striated muscle cell differentiation, a late mesodermal lineage, and striated muscle structures such as myofibrils, sarcomeres and muscle filament. Muscle systems development, as any type of adult tissue differentiation, is a stepwise process during which cells of restricted potential gradually advance toward complete specification. In the case of striated muscle, three types of cells, myogenic progenitors (Expressing the PAX3 and PAX7 factors), myoblasts (Expressing MYF5 and MYOD1) and myocytes (Expressing MYOG1) precede the formation of more complex structures, the myotubes, myofibrils and myofibers, expressing the Major Histocompatibility Complex I (MHC-I) [START_REF] Jiwlawat | Current progress and challenges for skeletal muscle differentiation from human pluripotent stem cells using transgene-free approaches[END_REF] (Figure 42A). When myocytes differentiation is directed from human iPSCs, the process takes up to 6 weeks in transgene-free conditions, reclaiming use of several pharmacological inhibitors and growth factors, and reclaims up to the double in duration to obtain myofibers [START_REF] Jiwlawat | Differentiation and sarcomere formation in skeletal myocytes directly prepared from human induced pluripotent stem cells using a sphere-based culture[END_REF][START_REF] Shelton | Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells[END_REF]). In our system, dox induction is applied for 72h, a strikingly shorter period of time, with a single protein being overexpressed. Furthermore, cells of origin are in a naïve-like state, i.e., further away from differentiation in the development timeline than the primed human iPSCs used for said studies. When exploring expression of striated muscle precursors markers in TL2i-hSTAN1 cells, we found ~25% of the population to express the myogenic progenitor PAX3 upon induction, versus ~90% expressing strong levels of B2M, coding for the Beta-2-Microglobulin protein, a member of the MHC-I complex (Figure 42B). We also noted that the 'net-like' aggregates observed in both primed and naïve-like hSTAN1 cells expressing NTN1 share structural similarities with myotubes formed by myocytes differentiated from human iPSCs (Figure 42C).

In mouse, it was demonstrated that Wnt activation (or GS3β inhibition) is necessary but not sufficient for myogenic differentiation [START_REF] Suzuki | WNT/β-catenin signaling regulates multiple steps of myogenesis by regulating step-specific targets[END_REF]. FGF2 (MEK/ERK activation), Hedgehog (Hh) activation and TGFβ inhibition are thus additionally used in numerous protocols to trigger efficient commitment [START_REF] Brennan | Transforming growth factor β represses the actions of myogenin through a mechanism independent of DNA binding[END_REF][START_REF] Scata | FGF receptor availability regulates skeletal myogenesis[END_REF].

Interestingly, TL2i-hSTAN1 cells are cultured in both GSK3β and MEK/ERK inhibition conditions, and we showed that upon induction, NTN1-negative cells, contrary to their NTN1positive counterparts, are enriched for TGF-β/NODAL signaling genes. This suggests that NTN1 overexpression allows to override MEK/ERK blockade and triggers TGF-β signaling inhibition, allowing progression through striated muscle commitment. These findings need to be further demonstrated at the protein and transcript level, but offer promising perspectives for muscle cells differentiation from human PSCs. An interesting approach in future studies will be to use recombinant NTN1 in a transgene-free PSCs culture system.

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B.3.2. Paracrine NTN1 induces ossification and osteoblast differentiation

By comparing NTN1-zero TL2i-hSTAN1 in induced conditions to non-induced cells, we found a downregulation of pluripotency genes and upregulation of genes enriched for processes similar to NTN1-exo cells (i.e., muscle filament and actinmyosin filament sliding), however, we also found the "ossification" and "osteoblast differentiation" terms to be enriched. We confirmed this finding by inspecting the expression of RUNX2 and SOX9, both markers of osteoblast commitment, in TL2i-hSTAN1 [START_REF] Rutkovskiy | Osteoblast Differentiation at a Glance[END_REF]. We found the former to be strongly upregulated in a dox dose-dependent manner upon induction, and the latter to be upregulated, with a peak at 10ng/mL (Figure 42D). Collectively, these findings show that paracrine NTN1 has an effect only partially overlapping with autocrine NTN1 and triggers a distinct phenotype. Two hypotheses could explain this observation: 1) Paracrine NTN1 reaches to neighboring cells in residual quantities compared to autocrine NTN1, resulting in a low-dose effect triggering a different commitment. Incidentally, such an effect could not be tested with our inducible system, because of its surprisingly greater than expected efficiency, triggering strong NTN1 overexpression with the low dox dose of 10 ng/mL. 2) NTN1 signal acts differently when captured from neighboring cells versus when secreted. Whether one, the other, or both of the hypotheses are true, autocrine and paracrine signals do not trigger opposed phenotypes, but differentiation into lineages belonging to the late mesodermal lineage. It is also worth noting that osteoblast, as striated muscle differentiation, reclaims Wnt activation or GSRβ inhibition; a major difference however, is the necessity of TGF-β signaling activation instead of inhibition. This suggests that, in our system, only autocrine NTN1 is able to trigger this pathway's inhibition, explaining at least partially the different outcomes observed in lineage engagement.

B.3.3. NTN1 signal triggers morphological changes in hSTAN1 cells.

In addition to differentiation, we found that hSTAN1 exposed to NTN1, whether primed or naïve, undergo morphological transformations. We noticed at the highest dox doses a thinning of colonies accompanied by an apparent fusion of cells, making colonies appear as syncytia. During skeletal muscle differentiation, cell fusion is a mandatory step leading to the formation of myotubes, our finding is thus consistent in that context. We moreover found enrichment in NTN1-exo cells for terms confirming a morphological transformation, such as regulation of actin cytoskeleton, cellular component assembly involved in morphogenesis, and regulation of cell-substrate adhesion. A compelling finding, however, is the observation of a similar morphological change, although to a lesser extent, in rhesus STAN1 cells for which we demonstrated a reinforcement of pluripotency rather than differentiation. A possible explanation is that NTN1 itself, being an extracellular matrix and laminin-related protein, aggregates in macrostructures when overexpressed and induces mechanical pressure on the surrounding cells. In both NTN1-exo and NTN1-zero cells from the induced conditions (undergoing paracrine NTN1), we found enrichment for terms associated for cilium assembly and organization. Primary cilia are microtubule-based structures functioning as sensory organelles in most adult cell types of vertebrates [START_REF] Alaiwi | Primary cilia: Highly sophisticated biological sensors[END_REF]. In addition to the detection of mechanical stresses, they have been shown to be paramount to the capture and processing of molecular signals including Hedgehog, Wnt and TGF-β [START_REF] Bodle | Concise Review : Primary Cilia : Control Centers for Stem Cell Lineage Specification and Potential Targets for Cell-Based Therapies[END_REF] So is done by the gathering along the cilium of receptors and downstream effectors of a given pathway [START_REF] Clement | TGF-β Signaling Is Associated with Endocytosis at the Pocket Region of the Primary Cilium[END_REF]. Disruption of ciliogenesis has been shown to severely impair numerous processes, including proliferation and differentiation of adult stem cells and progenitors [START_REF] Yuan | Ciliary IFT80 balances canonical versus non-canonical hedgehog signalling for osteoblast differentiation[END_REF].

Interestingly, ciliogenesis has also been linked to the cell cycle, anti-correlating with mitosis [START_REF] Plotnikova | Primary cilia and the cell cycle[END_REF]. It was indeed shown that cilia are assembled during the G1 (or G0) phase, last through the S phase, and are disassembled during G2 before entering the M phase. In our system, ciliogenesis in NTN1-positive cells could thus be explained by several scenarios: 1) NTN1 itself triggers cilia assembly, allowing enhanced capture of TGF-β ligands and growth factors, 2) NTN1 triggers differentiation toward lineages reclaiming pathways controlled by cilia, resulting in a cilium assembling as a feedback loop, or 3) Ciliogenesis is the reflect of NTN1-positive cells being differentiated, thus going through a longer G1 phase than the rapidly proliferating naïve-like NTN1-negative population. Regardless of the correct hypothesis, ciliogenesis and primary cilia in stem cell research have been so far overlooked considering the pathways to which they are tightly linked. Further investigations in this area will be instrumental in better understanding self-renewal and differentiation processes. We further demonstrated that DRAXIN-positive cells in +dox conditions do not only retain pluripotency, but exhibit the highest expression levels of pluripotency and NODAL-signaling markers among the NTN1-negative fraction, thus qualifying as the most pluripotent compartment of the whole population. Most interestingly, this is also true for control TL2i-hSTAN1, where DRAXIN-positive cells exhibit the highest levels of NANOG, OCT4, SOX2, DPPA4 and SALL4, though to a lesser extent than in the induced conditions. The antagonizing capacity of Draxin toward Ntn1 has been clearly demonstrated in the mouse, zebrafish and more recently human, and remains to this day this only characterized function of the molecule [START_REF] Gao | A floor-plate extracellular protein-protein interaction screen identifies Draxin as a secreted Netrin-1 antagonist[END_REF][START_REF] Islam | Draxin, a repulsive guidance protein for spinal cord and forebrain commissures[END_REF]. Although correlation between DRAXIN expression and naïve pluripotency maintenance in TL2i-hSTAN1 cells does not represent a causal link per se, we therefore conclude that DRAXIN expression here actively protects against NTN1 mesodermal differentiation effects (Figure 43).

B.4.2 DRAXIN is associated with naïve pluripotency in the human preimplantation embryo

As mentioned in part B.1, DRAXIN, but not NTN1, is enriched in the human naïve-epiblast. We also demonstrated that, contrary to the macaque, DRAXIN-positive cells display higher expression levels of OCT4, NANOG, and the naïve pluripotency markers SUSD2 and DPPA4. We also found NODAL and the GDF3, LEFTY1 and -2 related genes to be enriched. KEGG pathway and GO analyses further demonstrated enrichment for signaling pathways regulating pluripotency along with oxidative phosphorylation, the preferential metabolic mode of naïve pluripotent stem cells. We moreover showed that DRAXIN is specifically enriched in the epiblast but absent from all other tissues, to the exception of a discrete expression in the primitive endoderm.

We found, however, that the PE marker GATA6 is the most downregulated gene of the DRAXIN population, indicating that DRAXIN is indeed a naïve-epiblast marker in human. DRAXIN enrichment in the human epiblast has no relevance in the current state of knowledge, where its only function is to antagonize NTN1 signal. If this is the case, the only explanation for this finding would be that NTN1 is exclusively devoted to differentiation in human, and is thus actively (and disproportionately) countered in vivo to avoid potential deleterious effects of a stochastic expression of NTN1 by a few cells. A second hypothesis, however, is that DRAXIN has a more potent role than previously characterized, and is either a driver of naïve pluripotency, maybe acting itself as a ligand, or prevents the action of one or several unreported molecules. One observation supporting this scenario is the fact that uninduced TL2i-hSTAN1 cells widely and strongly express DRAXIN, despite 8% of the population expressing, at low levels, its NTN1 interactant.

B.5 NTN1 partially acts through NEO1, but not UNC5B to trigger pluripotency exit

In TL2i-hSTAN1 cells, we demonstrated upon induction a switch from a majority UNC5B to NEO1 netrin receptors expression. More specifically, we found the NEO1+UNC5B-configuration to be predominant at the highest dox doses. By isolating the NEO1-positive population in all induced conditions, we showed that these cells are downregulated for pluripotency and NODAL-signaling and undergo ciliogenesis, thus exhibiting an overlapping phenotype with the NTN1-exo cells. In analyzing UNC5Bpositive cells in a similar way, we instead found enrichment for some, but not all pluripotency and NODAL-associated markers (e.g., OCT4, ESRG, TDGF1 NODAL and LEFTY2; but not NANOG, SOX2 and GDF3). We further found enrichment for signaling pathways regulating pluripotency and ribosome biogenesis, a term reported to be consensual in naïve-like human cells. This suggests that, in our system, NEO1 and UNC5B-positive cells are respectively associated to differentiation and naïvepluripotency maintenance. NTN1 might thus be a bipotent signal inducing two distinct outcomes depending the receptor bound. This hypothesis was suggested in the mouse by Huyghe and colleagues, in a mirror scenario where a majority activation of NEO1 promotes self-renewal versus a MAPK activation/ differentiation by UNC5B [START_REF] Huyghe | Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling[END_REF]. Another hypothesis is that human NTN1 has a stronger affinity for NEO1 than for UNC5B, thus triggering differentiation in cells expressing the former, but barely affecting the background pluripotent phenotype in those expressing the latter. We attempted to answer this question by assessing NEO1 and UNC5B activation effect in our induced populations. Surprisingly, we found that NTN1 autocrine signal alone is responsible for significantly greater transcriptional changes than the simultaneous presence of NTN1/NEO1 or NTN1/UNC5B. In what we termed NEO1-activated cells, however, we found a downregulation of OCT4, and an upregulation of H19, COL1A1 and MALAT1, NTN1-exo 2 nd , 8 th and 38 th top markers, respectively. This represents a 6% of genes upregulated with an average Log2Fc above 0,25 in cells expressing NTN1 alone. Conversely, UNC5B activation leads to no upregulation of NTN1-exo markers, but does lead to the upregulation of the NODAL receptor coding gene TDGF1. We conclude that NTN1 overexpression effect in TL2i-hSTAN1 cells is in a minor way due to NEO1 binding, leading to OCT4 downregulation and to H19, COL1A1 and MALAT1 upregulation (Figure 44A), but remains largely unexplained. The most likely hypothesis is that NTN1 is able to bind in our system one or several receptors different from the UNC, DCC/NEO1 and DSCAM families, and does so with greater affinity. Consistently with our findings in TL2i-hSTAN1 cells, we found in the human preimplantation embryo that NEO1-positive cells, despite being ubiquitously expressed at all stages and in all tissues including the naïve epiblast, are impoverished for pluripotency and NODAL-signaling markers. Interestingly, we also found in this population upregulation of genes enriched for ciliogenesis. In the mouse, primary cilia first appear at E6 in epiblast cells, and further propagate in early lineages, but are absent from extra-embryonic tissues [START_REF] Lyu | The Multifaceted Roles of Primary Cilia in the Regulation of Stem Cell Properties and Functions[END_REF]. Upon implantation, the overall number of ciliated cells increases, correlating with progression in embryogenesis and thus differentiation/specification. If this paradigm is similar in human, NEO1-positive cells are hence not trophoblastic, and likely epitomize a fraction of cells undergoing specification.

Contrary to NEO1, we found that UNC5B is strongly enriched in the early epiblast and PE, but absent at all other stages. Further analysis demonstrated that UNC5B-positive cells are, contrary to their NEO1-positive counterparts, enriched for signaling pathways regulating pluripotency and oxidative phosphorylation, and exhibit expression of naïve pluripotency and NODAL-signaling markers. Despite its presence in the PE, we found that UNC5B anti-correlates with GATA6 expression, demonstrating its human naïve epiblast marker status. We consider this finding of the utmost significance, because UNC5B enrichment cannot be explained by the presence of NTN1, which expression fades out at the 8-cell stage. Missing its putative ligand, we therefore hypothesize that UNC5B binds in the human embryo another "X" ligand (Figure 44B). This is supported by the fact that UNC5B, being a dependence receptor, should trigger apoptosis in absence of ligand binding, and thus epiblast collapse in the embryo in absence of ligand binding. A potential candidate for this unknown molecule is NTN4, expressed in the early ICM, EPI and PE, although no enrichment for pluripotency can be observed for NTN4-positive compared to NTN4-negative cells.

Another possibility is that UNC5B binds other molecules from families distinct of Netrins, and for which interaction has not been thus far reported. C. To sleep or not to sleep, discrepancies of Netrin-1 effect in the mouse, macaque and human.

In this work, we showed important discrepancies between Netrin family members involvement in pluripotency regulation of the mouse and primates. In the mouse, promotion of self-renewal and naïve pluripotency through the Ntn1-Unc5b-Neo1 axis has been clearly demonstrated in vitro, and all three genes are found concomitantly expressed in the naïve epiblast, suggesting a similar role. In the macaque, we showed a conservation of the NTN1-UNC5B-NEO1 axis in vivo, and a correlation between NTN1 expression and pluripotency.

Accordingly, we demonstrated that NTN1 overexpression in primed macaque PSCs leads to an increase of pluripotency genes expression. Interestingly, we made drastically different findings in humans. First, we showed a complete absence of NTN1 transcript after the 8-cell stage in vivo, and an impoverishment of pluripotency genes expression in NTN1-positive cells.

Second, we demonstrated that NTN1 overexpression rapidly triggers late mesodermal differentiation of naïve-like human PSCs. We thus showed that, while NTN1 promotes pluripotency in the mouse and macaque, it does precisely the opposite in human, where naïve pluripotency is quite contrarily associated with the NTN1 antagonist DRAXIN. Interestingly, DRAXIN expression in the early embryo is a major difference between the mouse and primates. Despite a strong homology between genes of the two groups and a similar putative function, Draxin is completely absent from the mouse early embryo, but is enriched in both the human and macaque's.

As mentioned in introduction of this work, one major difference between the mouse and primates that has been proposed to account for pluripotency regulation discrepancies is embryonic diapause. In eutherian mammals, this phenomenon, also known as discontinuous development, is the process by which a blastocyst enters a reversible dormant state upon unfavorable environmental conditions [START_REF] Lopes | Embryonic diapause and its regulation[END_REF]. In mouse, this mechanism has been linked to the LIF/gp130/STAT3 and mTOR pathways, c-Myc activity, and has been shown to be a generally facilitating factor to mESCs derivation from blastocysts [START_REF] Brook | The origin and efficient derivation of embryonic stem cells in the mouse[END_REF][START_REF] Bulut-Karslioglu | Inhibition of mTOR induces a paused pluripotent state[END_REF][START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF][START_REF] Scognamiglio | Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause[END_REF]. In primates, embryonic diapause has not yet been reported [START_REF] Ptak | Embryonic diapause is conserved across mammals[END_REF], however, we speculate that genes coding for an ancestral molecular tooling might remain in regions silenced during early embryogenesis. If so is the case for macaques, it is possible that NTN1 belongs to this vestigial apparatus and thus has a pro-pluripotency effect, although dampened compared to the mouse. This would imply that the human genome, contrary to its non-human primates' counterparts, has completely lost this heritage, only retaining the later functions of NTN1 in development, i.e., neurogenesis and late mesodermal lineages differentiation. It seems, however, that Netrin family genes in general are not entirely disconnected from pluripotency regulation, as both DRAXIN and UNC5B associate with it in human. It is possible that what is observed with the Ntn1-Unc5b-Neo1 axis in the mouse occurs in humans through a X-UNC5B-DRAXIN axis, X being an UNC5B ligand remaining to be identified. It is worth noting that, in attempts to identify potential candidates for X, we noticed several guiding molecules from families distinct of Netrins (Semaphorins, Slits and Repulsive Guidance Molecules) to be enriched in the naïve human epiblast or uninduced TL2i-hSTAN1 cells (Supplementary Figure S3). This strongly suggests that, as Netrins, these other guiding molecules have functions broader than their involvement and neurogenesis, and may constitute a new research avenue in early embryogenesis and pluripotency understanding.

D. Further studies D.1 NETRIN1 effect.

In order to further understand Netrins and receptors function in primates' pluripotency regulation, several experiments will be instrumental. It will be first necessary to confirm that NTN1 exerts the effects observed in this work in other human and macaque cell lines; notably, NTN1 overexpression effect in naïve-like rhesus PSCs has not been tested here. Assessing NTN1 effect through other means that the XLone system, i.e., using other inducible systems or a transgene-free system with recombinant NTN1, would be of great interest. Incidentally, Netrin family genes expression in primed human PSCs remains to be overviewed and will reveal if, as for the macaque, NTN4 and UNC5D are the primed components of the family.

D.2 DRAXIN

In humans, we showed the central position of DRAXIN in the naïve pluripotency context. To establish if DRAXIN expression has a causal link with pluripotency maintenance, a DRAXIN knockout and converse overexpression in human PSCs will be critical, and will allow to characterize potential underlying mechanisms. Another paramount information will be to establish if DRAXIN is expressed at the protein level in the human epiblast.

D.3 UNC5B and NEO1

In this work, we demonstrated a distinct effect of NEO1 and UNC5B expression in human both in vivo and in vitro, however, both receptors function remains elusive. Whether other ligands are involved, and if so, what is the nature of these has to be elucidated. This could be addressed using mutant forms of NTN1, able to bind one but not the other, or none of the receptors. Complete knockouts of NEO1 and UNC5B would also be instructive. We also suggested that, in vivo, UNC5B binds an unidentified ligand termed "X" therefore promoting naïve pluripotency. If this hypothesis is correct, the identification of X will be critical, and a complete KO of UNC5B in human PSCs should lead to pluripotency disruption.

Quantitative PCR

RNA was extracted using RNAeasy kits with on-column DNAse digestion and reverse transcription carried out with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's recommendations. qRT-PCR was performed using specific primers (listed in Supplemental Table S3) and Fast SYBR® Green Master Mix on the StepOnePlus™ system (Applied Biosystems).

Bioinformatics Single-cell RNA sequencing

Cell suspension (1200 cells per µl) was added to 10x Genomics Chromium Single Cell Controller (10x Genomics) to achieve 2500 encapsulated cells per condition (4 samples). The next steps for cDNA synthesis and library preparation were done following the manufacturer's instructions (chemistry V3). All Libraries have been sequenced simultaneously using the Novaseq 6000 platform (Illumina) in order to target 150k reads per cell. Cell Ranger version 5.0.1 was used to align reads on the mouse reference genome GRCm38 mm10 to exclude MEFs and the human reference genome GRCh38, and to produce the count matrix.

Analysis of single-cell RNA-sequencing data

Quality control and filtering. We filtered 2414, 2333, 2065 and 1835 cells for dox0, dox10, dox30 and dox1000, respectively, based on two-quality control criteria: the number of genes per cell and the fraction of counts from mitochondrial genes per cell. Cells with <4500 genes or >10000 and with >10% mitochondrial genes fraction were removed.

Clustering analysis. Filtering and Data analysis were performed using the R package Seurat (version 4). First, genes expressed in less than 3 cells were removed in each dataset. Gene expression was normalized using sctransform workflow in which the 3000 most variable genes were identified. Then, the 4 independent datasets were aggregated, scaled with cell cycle and mitochondria expression regression and used for PCA at 50 dimensions. We then performed clustering (30 PCs; resolution = 0.5) yielding 10 final clusters. The clusters were visualized in two dimensions using the RunUMAP() function (minimum distance = 0.3; n_neighbors = 30 L; umap.method = 'umap-learn'; metric = 'correlation'). We finally performed differential expression analysis using the Findmarkers() function of Seurat.

Volcano plots from differentially expressed genes were generated using the Enhanced Volcano package (v3.13) for R. Gene Ontology, KEGG pathway and Gene Set Enrichment analyses were carried out using the ClusterProfiler (v3.18.1) and DOSE (v3.16) packages.

Figures 1 . 2 .

 12 Figures and Tables Index ...................................................................... Introduction ............................................................................................ I/ The Pluripotency continuum -Lessons from the mouse model .......................................... 1. Pluripotency in vivo ............................................................................................................... a) Molecular regulation of the ICM onset ........................................................................ b) Emergence of the epiblast ............................................................................................ 2. Pluripotency in vitro -Pluripotent Stem Cells derivation .................................................. a) Conventional Embryonic Stem Cells (ESCs) ............................................................. b) 2i/LIF ESCs ..................................................................................................................... c) Epiblast-derived Stem Cells (EpiSCs)......................................................................... d) Induced Pluripotent Stem Cells (iPSCs) .....................................................................

3 . 2 .

 32 From Naïve to Primed: The Continuum of Pluripotency .................................................. a) Discrepancies of ESCs and EpiSCs ............................................................................ b) Epigenetic landscape of ESCs and EpiSCs ............................................................... c) Cell Cycle of ESCs and EpiSCs ................................................................................... d) The Metabolic Switch of PSCs ..................................................................................... e) The Naïve, the Formative and the Primed.................................................................. f) Refining Culture Conditions .......................................................................................... f.1. The Ground State, Holy Grail of Pluripotency ......................................................... f.2. WiEpiSCs ..................................................................................................................... g) The Pluripotency Gene Network .................................................................................. II/ Pluripotency in Primates........................................................................................................... 1. Human Embryonic Stem Cells (hESCs) ......................................................................... The naïve state of primates' pluripotency in vivo........................................................... a) Early embryonic development of the mouse and primates ...................................... b) The hallmarks of the naïve primates' epiblast ............................................................ b.1 ERK independence ..................................................................................................... b.2 Apolarity ........................................................................................................................ b.3 DNA Hypomethylation ................................................................................................ b.4 X Chromosome Activation.......................................................................................... b.5 Primate-specific naïve network ................................................................................. B.2 NTN1 overexpression leads to pluripotency exit of primed and naïve-like human PSCs. ........................................................................................................................................ B.3 NTN1 triggers commitment of naïve-like human PSCs to late mesodermal lineages. ................................................................................................................................................... B.3.1. Autocrine NTN1 induces striated muscle cell differentiation of TL2i-hSTAN1 cells........................................................................................................................................ B.3.2. Paracrine NTN1 induces ossification and osteoblast differentiation ................. B.3.3. NTN1 signal triggers morphological changes in hSTAN1 cells. ......................... B.3.4. Cells exposed to NTN1 form cilia. .......................................................................... B.4 DRAXIN is a marker of human naïve pluripotency....................................................... B.4.1 DRAXIN shields TL2i-hSTAN1 cells against NTN1 pro-differentiation effect and preserves naive pluripotency ............................................................................................. B.4.2 DRAXIN is associated with naïve pluripotency in the human pre-implantation embryo .................................................................................................................................. B.5 NTN1 partially acts through NEO1, but not UNC5B to trigger pluripotency exit ...... B.6 UNC5B and NEO1 are respectively associated to naive pluripotency and differentiation in the human pre-implantation embryo. ....................................................... D. Further studies ........................................................................................................................ D.1 NETRIN1 effect. ................................................................................................................ D.3 UNC5B and NEO1 ........................................................................................................... Cell culture .................................................................................................................................... Primed cells .............................................................................................................................. TL2i ............................................................................................................................................ hSTAN1 cell line creation ....................................................................................................... Western Blots ............................................................................................................................... Immunofluorescence ................................................................................................................... Quantitative PCR ......................................................................................................................... Bioinformatics ............................................................................................................................... Single-cell RNA sequencing ................................................................................................... Analysis of single-cell RNA-sequencing data ......................................................................

Introduction

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .ResultsFigure 14 .Figure 15 .Figure 16 .Figure 18 .Figure 22 .Figure 27 .Figure 28 .Figure 29 .Figure 36 .Figure 37 .Figure 38 .Figure 39 .Figure 40 .DiscussionFigure 41 .Figure 42 .Figure 44 .

 12345678914151618222728293637383940414244 Figure 1. Pre-and peri-implantation stages of mouse embryonic development……..……..p16 Figure 2. Molecular regulation of the mouse epiblast emergence……………………...…….p19 Figure 3. Extrinsic factors regulating mouse pluripotency……………………………………..p21Figure 4. Discrepancies of mouse ESCs and EpiSCs………………………………………….p24 Figure 5. The different types of mouse PSCs and their in vivo equivalent -The pluripotency continuum……………………………………………………………………………………..….…p30 Figure 6. The mouse pluripotency gene network……………………………………..………...p33 Figure 7. Early embryonic development of the mouse and human……………………..……p37 Figure 8. Hallmarks of naïve epiblast cells in mouse and primates……………………..……p42 Figure 9. Molecular targets of human naïve-reprogramming protocols………………….…..p50 Figure 10. Netrin-1 drives commissural axonogenesis during vertebrates' neural development………………………………………………………………………………..……….p57 Figure 11. The Netrin Protein Family…………………………………………………………….p59 Figure 12. The Netrin receptors DCC, Neo1 and UNC5A-D are Dependence Receptors…………………………………………………………………………………….……..p62 Figure 13. Netrin-1 positively regulates survival and self-renewal of mouse PSCs………...p64

  In a recent study, Lynch and colleagues targeted CDK8/19 in hESCs in the aim of promoting extended human PGN expression and reprogram hESCs to naïve pluripotency.Combination of CDK8/19i with 2i yielded cells with features of naïve pluripotency including morphology, naïve-ERVs and naïve-specific transcription factors expression[START_REF] Lynch | Global hyperactivation of enhancers stabilizes human and mouse naive pluripotency through inhibition of CDK8/19 Mediator kinases[END_REF]. Interestingly, factors of the MEK signaling pathway (RAF, SRC, PKC, p38, JNK, MEK, and ERK) targeted in different naïve-reprogramming protocols as detailed in part b.1 and b.2, have all been shown to regulate CDK8 activity[START_REF] Mcdermott | Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer[END_REF][START_REF] Staab | CDK8 as the STAT1 serine 727 kinase? JAK-STAT[END_REF]

  although this capacity has not been yet verified for cleaved and colleagues incorporated in their Reset-reprogramming protocol study a Principal Component Analysis of single-cell RNA sequencing (scRNA-seq)

  [START_REF]7 Hippo pathway inhibition/ YAP activation[END_REF] epiblast, accordingly with what was described in previous work. Upon implantation, however, Ntn1 switches from the EPI to visceral endoderm (VE), while Unc5b and Neo1 remain expressed in EPI, VE and ExE (Extraembryonic ectoderm) (Figure14B and C). In line with these observations, we found that mouse PSCs cultured in conditions supporting naïve pluripotency express the same three members of the netrin family as the EPI cells: Ntn1, Unc5b and Neo1 (Figure14D). Interestingly, ESCs cultured in 2i/LIF or alternative 2i/LIF (SRC inhibition instead of ERK inhibition), both epitomizing the ground state (homogenous naïve state) of pluripotency, display higher levels of Ntn1 and Neo1 compared to their serum/LIF

B. 3

 3 NTN1 triggers commitment of naïve-like human PSCs to late mesodermal lineages

B. 3 . 4 .

 34 Cells exposed to NTN1 form cilia.

B. 4

 4 DRAXIN is a marker of human naïve pluripotency B.4.1 DRAXIN shields TL2i-hSTAN1 cells against NTN1 pro-differentiation effect and preserves naive pluripotency Upon induction, we noticed that TL2i-hSTAN1 cells exhibit anti-correlative NTN1 and DRAXIN expression and a converse correlation between NANOG and DRAXIN.

B. 6

 6 UNC5B and NEO1 are respectively associated to naive pluripotency and differentiation in the human pre-implantation embryo.

e) The Naïve, the Formative and the Primed

  Esrrb[START_REF] Zhou | HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition[END_REF]. This suggest that the metabolic mode is not only a consequence to ESCs and EpiSCs respective states, but also an intrinsic driver of such states.

	inducible factor 1α (HIF1α) in conventional ESCs leads to their conversion in cells
	morphologically and metabolically similar to EpiSCs, with notably a drastic downregulation of
	ESCs marker ESCs and EpiSCs are equivalent in their differentiation potential (i.e. pluripotent) but, as
	developed above, they also exhibit a considerable number of differences. This suggests that
	they are not strictly in the same state, but rather in two distinct states of pluripotency. ESCs
	epitomize the pre-implantation epiblast in vivo, a tissue of unbiased potential, of uncondensed,
	opened to transcription chromatin. EpiSCs on the other hand, epitomize the peri-implantation
	epiblast, poised to gastrulate and, as a matter of fact, already expressing differentiation factors
	(I.g. Fgf5, Eomes, Brachyury, Gata6 and FoxA2). Consistently with their tissue of origin,
	EpiSCs are of reduced plasticity compared to ESCs, and biased in their differentiation
	potential. This led to name the pluripotent states associated to ESCs and EpiSCs respectively
	"Naïve" and "Primed" (Nichols and Smith, 2009).
	Embryonic development is progressive, and tissues in vivo gradually change identity. For
	this reason, it is unlikely that pluripotency behaves as a binary switch from naïve to primed.
	Naïve cells (ESCs) are derived from the mouse embryo between E3.75 and 4.75, while primed
	cells (EpiSCs) are obtained between E6.0-6.5 and further. In between, pluripotency is neither
	naïve nor primed as precedingly defined. During 24 to 30h post-implantation, epiblast cells
	expand and stop expressing naïve markers, however, global gene expression analysis shows
	that they diverge from both the naïve pre-implantation epiblast and the gastrulating post-
	implantation epiblast (Smith, 2017).
	Two Consistent with this observation, several pluripotency states have been captured in vitro
	modes of cellular energy production exist: glycolysis, yielding 2 molecules of ATP per molecule that can be qualified of intermediates between naïve and primed. 2i/LIF ESCs passed in
	of glucose, and oxidative phosphorylation (OXPHOS), yielding ~38 molecules of ATP per Activin/FGF with fibronectin and 1% of KnockOut Serum (KOSR) for instance, start to
	molecule of glucose (Zhang et al., 2012). In conventional conditions, ESCs are able to use downregulate naïve markers after 2 days, such as Stella, Klf4 and Rex1, but yet do not
	both OXPHOS and glycolysis metabolic pathways, though relying mostly on OXPHOS, more overexpress early lineage markers such as Brachyury, FoxA2 or Sox1. These cells were
	cost-effective, for faster proliferation. This ability is referred to as metabolic bivalency. As they named Epiblast-Like Cells (EpiLCs), and represent a pluripotent state just prior to gastrulation
	shift toward differentiation or are converted into EpiSCs though, ESCs gradually lose their in vivo, slightly ahead of the primed state. Interestingly, EpiLCs hold the capacity, when
	bivalency to rely exclusively on glycolysis. This phenomenon is termed metabolic switch, and exposed to BMP4, to differentiate into Primordial Germ Cells-Like Cells (PGCLCs)
	is consistent with the lengthening of G1 phase observed between ESCs and EpiSCs and the
	associated difference in proliferation speed. Interestingly, forcing expression of the hypoxia-

Figure 5). f) Refining Culture Conditions f.1. The Ground State, Holy Grail of Pluripotency

	the endogenous secretion of contradictory signals by conventional ESCs: LIF, that sustains
	self-renewal, and FGF, that promotes differentiation. In conventional conditions (i.e.
	Serum/LIF), the exogenous supplementation of LIF allows most cells to ultimately balance
	toward self-renewal, but does not prevent a part of the population to be on the edge of
	differentiation (Morgani et al., 2017). In the 2i/LIF condition, FGF pro-differentiating effect is
	countered by direct inhibition of the MAPK/MEK pathway through use of PD0032. This
	inhibition of differentiation is, in parallel, reinforced by GSK3 inhibition with CHIR99021 (Ying
	et al., 2008).
	2i/LIF ESCs, unlike their conventional counterparts, are widely homogenous in their
	transcriptomic signature and functional capacity of chimeric formation. For this reason, they
	intermediate state can be captured from the combination of both Activin and Fgf2 are considered as "true" naïve cells, representing more accurately pre-implantation epiblast
	to one of the 2i inhibitors of the 2i/LIF condition, CHIR. These intermediate Pluripotent Stem cells than conventional ESCs. This in vitro captured state was named "ground state" of
	Cells (INTPSCs) have, as their name judiciously suggests, mixed naïve and primed pluripotency, the pinnacle of the pluripotency spectrum. Rather than a different state per se,
	characteristics (Tsukiyama and Ohinata, 2014). INTPSCs conserve expression of some naïve the ground state is a homogenous naïve state, devoid of any intermediate subpopulations and
	markers (e.g. Rex1, Esrrb and Klf4), retain a naïve morphology of tight dome-shaped colonies, differentiation bias, corresponding to the tabula rasa of development in vivo.
	have two active X chromosomes in female lines and remain able to colonize a pre-implantation
	blastocyst. However, they also exhibit primed characteristics, though to a lesser extent than
	EpiSCs, such as increased expression of peri-implantation epiblast markers (e.g Otx2 or Fgf5)
	(Morgani et al., 2017). Compared to EpiLCs, INTPSCs do not epitomize the formative state as
	precedingly defined, but are rather slightly upstream, between naïve and formative.
	EpiLCs, INTPSCs and in vivo observations illustrate the gradually changing nature of
	development, and demonstrate that, accordingly, pluripotency is not a coin with two sides. The
	naïve and primed states are rather the endpoints of a continuum, starting with epiblast
	establishment and ending with gastrulation (In conventional culture conditions, ESCs exhibit important transcriptional
	heterogeneity, showing subpopulations with signature of pre-implantation epiblast, endoderm
	or peri-implantation epiblast. If such populations are isolated and cultured de novo in
	conventional Serum/LIF conditions, heterogeneity of the initial population is soon restored,
	demonstrating that these subpopulations are not representing stable fractions of cells, but
	rather a dynamic landscape of interconverting states. One explanation to this phenomenon is

  EpiSCs exclusively rely on glycolysis. Although this distinction is very clear in vitro, the in vivo, complex embryonic microenvironment is not yet fully recapitulated by any culture conditions, and oxygen consumption rate as well as metabolites accumulation measurements are challenging to perform in vivo. The metabolic bivalency observed for mouse in vitro has been described in hESCs reprogrammed to naïve-like pluripotency(Wu and Belmonte, 2015a)

	c) Questioning Hallmarks
	c.1 Metabolism
	As mentioned in part I, metabolism is a discriminating aspect of pluripotency in mouse.
	While mESCs are metabolically bivalent, i.e. able to switch from glycolysis to OXPHOS and
	vice-versa,

(see part II/3.b)), however, this is not observed in all naïve-like reprogrammed hESCs, and does not result from a in vivo observation, therefore remaining as a questionable hallmark of the naïve primates epiblast.

  These changes, despite being collectively in favor of pluripotency, are faint. Several factors could explain this observation: first, we used for these experiments the human NTN1 gene, exhibiting a 99.34% sequence homology with its macaque counterpart. Although NTN1 binding sites to its receptors are here preserved, this could lead, for unknown reasons, to a weakening of the phenotype compared to rhesus NTN1. Second, we found that NTN1, as in the mouse, is associated in the macaque with the naïve epiblast, and to naïve-like reprogrammed PSCs rather than primed ones. It is a possibility that NTN1, in a context different from naïve pluripotency, does not trigger a pro-selfrenewal effect or does so to a lesser extent. Such behavior would not be surprising, as several signals and pathways act in contrasting if not opposed fashion depending on the context;

FGF/MEK/ERK, for example, is a potent differentiation axis in a naïve context, but is critical to the maintenance of pluripotency and self-renewal in the primed state of both primates and mouse

Acknowledgments/ Remerciements

Acknowledgments/ Remerciements ...................................................... Abbreviations .........................................................................................

Abbreviations b.6 CDK8/19 inhibition

Mediator is a transcription complex located nearby potent enhancers called superenhancers (SEs), able to recruit RNA pol II in the vicinity of their associated genes. One specific module of Mediator is composed of the cyclin-dependent kinases CDK8 and its paralog CDK19. When active, CDK8/19 prevent RNA pol II recruitment by Mediator and thus, are transcription repressors [START_REF] Allen | The Mediator complex: A central integrator of transcription[END_REF]. Moreover, CDK8/19 are able to IV. Single-cell RNA sequencing of TL2i-hSTAN1 cells.

To further investigate NTN1 overexpression effect on human naïve-like cells, we decided to perform single-cell RNA sequencing on TL2i-hSTAN1 cells. We selected a clone (c15) that displayed the strongest morphological changes in response to induction, and treated it with, low (+10, +30ng/mL), high (+1000), or no dox for 72h. After trypsinization, cells were sorted out and collected by fluorescence-activated cell sorting (FACS), and a cDNA library preparation followed by sequencing were performed using the 10X genomics Chromium Single-cell 3' end protocol (Figure 26A). Because TL2i-hSTAN1 are cultured on mouse embryonic fibroblasts, we then mapped out and removed cells aligning with mouse genome (Figure 26B), and filtered out apoptotic debris and mitochondrial residues in the remaining population (Figure 26C). We thus obtained a final dataset comprised of an average 2100 cells per condition, with a mean read counts of 52000 (Figure 26D). DRAXIN+ cells retain pluripotency upon induction and are shielded from NTN1 prodifferentiation effects. might here solely act to extinguish NTN1 signal in favor of NTN4. An element supporting this hypothesis is the important structural differences between NTN4, belonging to βsubfamily of netrins, compared to the other secreted netrins (NTN1, -3 and -5) belonging to the γgroup [START_REF] Rajasekharan | Protein family review The netrin protein family[END_REF]. It is worth noting that, although interaction between the latter and receptors UNC5 and NEO1/DCC has been demonstrated, a map or hierarchy of affinity remains to be established. This knowledge will be critical in future characterization of netrins and receptors' role in early mouse and primates' embryogenesis.

B. Netrin signaling in human pluripotency

Methods

Cell culture

All macaque and human cells were cultured at 37°C in 5% CO2, 5% O2 on 0.1% gelatin-coated dishes with murine embryonic fibroblasts (MEFs) growth-inactivated with mytomycine (Sigma) for 2 to 3 hours. All media used for culture were refreshed daily, and ROCK inhibitor Y27632 (Calbiochem) was used at 10µM for 24h following passages.

Brightfield pictures were taken using Nikon's Eclipse Ti-S microscope.

Primed cells

Primed human (F-OS3-10) and macaque (LyonES) cells were cultured on 35mm dishes with 180 000 and 200 000 growth-inactivated MEFs per 35mm, respectively, using KnockOut Dulbecco's Modified Eagle's Medium (KO-DMEM) supplemented with 20% Knock Out Serum Replacement (KOSR, Gibco), 1% non-essential amino acids (NEAA, Gibco) 1mM glutamine (Gibco), 0.1mM β-Mercaptoethanol (Sigma), and 5ng/mL of FGF2 (Interchim) were used for routine culture, with daily medium refreshing. LyonES colonies were manually split and passaged every 4 to 5 days; F-OS3-10 every week.

TL2i

Primed to naïve conversion of F-OS3-10 cells was performed using the TL2i protocol from [START_REF] Chen | Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency[END_REF]. Basal culture medium for primed cells (described above) was supplemented with 10,000 U/mL hLIF, 250nM 4'-OHT, 3 mM CHIR99021 and 1 mM PD325901(Stemgent). TL2i medium was applied on primed cells with ROCKi during 24h; ROCKi was subsequently removed and cells were grown for a week, then routinely passaged by single-cell dissociation with trypsin-EDTA (Gibco) every 3 to 4 days. Stabilized TL2i-OS3 cells were cultured on 280 000 MEFs or 640 000 MEFS on 35 or 60mm dishes, respectively.

hSTAN1 cell line creation

F-OS3-10 cells were transfected with 2.5 µg for ~100 000 cells of the XLone-NTN1-HA transgene (obtained after modification of the XLone-GFP plasmid; Addgene #96930) using the Neon electroporation system (Invitrogen; 1050v, 20ms, 2 pulses). The hSTAN1 cells thus obtained were subsequently plated on fresh MEFs (similar seeding to TL2i-OS3) and grown 2 weeks in medium supplemented with 250ng/mL of neomycin (G418). After two weeks of selection, cells were routinely cultured as TL2i-OS3, with permanent addition of Neomycin.

Inductions were performed using doxycycline (Sigma) at 10, 30, 100, 300, 500, 1000 or 2000 ng/mL.

Western Blots

Cells used for western blots were pelleted through centrifugation and subsequently lysed in RIPA buffer, complemented with protease and phosphatase inhibitors. Lysates were then cleared by centrifugation (14 000 rpm for 30 min at 4°C) and protein concentration was assessed with the Bradford method. 15µg of proteins were used for subsequent steps. After SDS-PAGE and electroblotting on polyvinylidene difluoride, membranes were incubated with a HA specific primary antibody (Sigma), diluted at 1 :1 000. Blots were incubated with horseradish peroxidase-coupled anti-rabbit immunoglobulin G (Jackson ImmunoResearch, diluted at 1 : 10 000) and developed with Clarity Western ECL Substrate (BIO-RAD).

Immunofluorescence

Cells were fixed on coverslips with 4% Paraformaldehyde for 30min at room temperature (RT), and permeabilized with 0.4% Triton X-100 (Sigma) for 10min. Non-specific binding sites were blocked with PBS containing 5% of donkey serum (Biowest) and 0.1% Triton X-100 for 30min at RT. Cells were incubated overnight at 4°C with primary antibodies raised against HA (Sigma), NANOG (R&D Systems), OCT4 (CliniSciences), SOX2 (Bio-Techne), CD24 (Beckton & Dickintson Biosciences), diluted at a concentration of 1:200 in the blocking solution. After four rinses with PBS, cells were subsequently stained with secondary antibodies conjugated to Alexa , and diluted at 1/1000th in the blocking solution for 1 hour at RT in the dark. Nuclear staining was performed by applying DAPI (Invitrogen ; diluted at 1:8000 in the blocking solution) on the cells for 8min at RT. After 3 rinses in PBS, coverslips were mounted on microscope slides and analyzed using confocal laser scanning microscopy system (Leica Microsystems, SP5). All incubations were performed under continuous gentle agitation.

Supplementary Data