The conversion of primed pluripotent stem cells to a naive-like state has emerged as a critical strategy for enhancing developmental potential and broadening applications in regenerative medicine. Conditioned media (CM)-based approaches provide a supportive microenvironment enriched with secreted factors that may facilitate this state transition without extensive genetic or chemical manipulation. In this study, we investigated the potential of human Wharton’s Jelly-derived mesenchymal stem cell-conditioned media (hWJ-MSCs-CM) and mouse embryonic fibroblasts CM (MEFs-CM) to support the conversion of primed rhesus monkey embryonic stem cells (rhESCs) into a naive-like state. The rhESCs were cultured under feeder-free and feeder conditions using both hWJ-MSCs-CM and MEFs-CM, exhibiting distinct morphological changes during conversion. Immunofluorescence analysis demonstrated the expression of pluripotency and naive markers under both conditions. Gene expression analysis further confirmed the upregulation of naive-specific genes and downregulation of primed markers, with statistically significant differences between groups. Additionally, epigenetic reprogramming was assessed, revealing differential effects of the CM sources on the reversion to a naive state. These findings highlight the potential of hWJ-MSCs-CM as a supportive system for naive-like state induction in primate ESCs.

Unraveling how gene regulations are remodeled during evolution is central to understanding how biological processes evolve. We explored this question in the frame of X-chromosome inactivation, a process under strong selective constraint, governed by the XIST lncRNA and its cis-regulators. Using functional approaches on closely related primate species, we show that XIST regulation has uniquely diverged over a short evolutionary timescale. In human and marmoset embryonic stem cells (ESCs), the JPX lncRNA gene is a major regulator of XIST expression. In contrast, JPX has a minor effect on XIST in macaque ESCs, where it acts together with a macaque-specific enhancer. This occurs within a reshuffled 3D organization of the XIST neighborhood triggered by the insertion of a HERVK transposon in the macaque lineage. Retrospective sequence comparisons revealed that many XIST regulators are not evolutionarily constrained, supporting the hypothesis that neutrally evolving noncoding elements harbor adaptive potential. These results illuminate how evolutionary recent elements are integrated into preexisting regulatory landscapes.

This study aimed to examine the potential of human Wharton’s Jelly-derived mes-enchymal stem cells (hWJ-MSCs) in supporting the conversion of the primed state of rhesus monkey embryonic stem cells (rhESCs) to a naïve-like state. The rhESCs were cultured under feeder-free and feeder conditions using hWJ-MSC-conditioned media (hWJ-MSCs-CM) and mouse embryonic fibroblasts CM (MEFs-CM) exhibited distinct morphological changes during conversion. Immunofluorescence analysis demonstrated the expression of pluripotency and naïve markers under both condi-tions. Gene expression analysis further confirmed the upregulation of naïve-specific genes and downregulation of primed markers, with statistically significant differ-ences between groups. Additionally, epigenetic reprogramming was assessed, re-vealing differential effects of the CM sources on the reversion to a naïve state. These findings highlight the potential of hWJ-MSCs-CM as a supportive system for na-ïve-state induction in primate ESCs.

We evaluated the potential of Fourier-transform infrared (FTIR) microspectroscopy for non-invasive biochemical profiling of rhesus macaque embryonic stem cells (rhESCs) cultured in either conventional FGF2/KOSR medium or a novel formulation, ALGöX. Cells from both conditions were analyzed by immunocytochemistry, RNA sequencing, and high-resolution FTIR profiling. Molecular marker expression patterns and transcriptional profiles revealed that rhESCs maintained in FGF2/KOSR were in the primed pluripotent state, whereas those cultured in ALGöX adopted a naïve-like state. FTIR spectra showed consistent differences in protein, lipid, and nucleic acid signatures, with ALGöX-cultured cells displaying higher amide I/II and nucleic acid absorbance and FGF2/KOSR-cultured cells exhibiting stronger lipid-associated bands. Principal component analysis (PCA) separated the two groups along PC−1 (64% variance), and partial least squares discriminant analysis (PLS-DA) classified samples with 100% specificity and 100% sensitivity. These findings demonstrate that FTIR microspectroscopy can reliably discriminate pluripotent state–specific biochemical features in non-human primate PSCs, providing a rapid and label-free approach for monitoring stem cell identity and quality.

Transcriptional activation of the embryonic genome (EGA) is a major developmental landmark enabling the embryo to become independent from maternal control. The magnitude and control of transcriptional reprogramming during this event across mammals remains poorly understood. Here, we developed Smart-seq+50 for high sensitivity, full-length transcript coverage and simultaneous capture of 50 transcript information from single cells and single embryos. Using Smart-seq+50, we profiled 34 developmental stages in 5 mammalian species and provide an extensive characterization of the transcriptional repertoire of early development before, during, and after EGA. We demonstrate widespread transposable element (TE)-driven transcription across species, including, remarkably, of DNA transposons. We identify 19,657 TE-driven genic transcripts, suggesting extensive TE co-option in early development over evolutionary timescales. TEs display similar expression dynamics across species and species-specific patterns, suggesting shared and divergent regulation. Our work provides a powerful resource for understanding transcriptional regulation of mammalian development.

How extracellular cues are temporally integrated to regulate self-renewal and differentiation propensities across the cell cycle remains largely unresolved. We identify a ligand/receptor trafficking clock in rodent and human pluripotent stem cells (PSCs) in which the cyclic turnover of Netrin-1 and its receptors Neo1 and Unc5b (NNU) governs self-renewal capacity and abscission dynamics. In G1, NNU complexes undergo Clathrin-mediated internalization and lysosomal degradation, a process required for timely post-mitotic bridge abscission. At later stages of the cycle, NNU activate Src within early endosomes, inducing a genome-wide redistribution of the transcriptional co-activator Yap1. This reshapes gene regulatory networks by activating stemness- and ectoderm-associated transcriptional programs enriched for Sox2/Nanog binding and by repressing mesodermal- and cell cycle-related targets enriched for Sox2 and Tcf3. Functionally, recombinant Netrin-1 reduces functional heterogeneity and enhances clonogenicity in G1, uncovering a tractable strategy to canalize stem cell behavior. Collectively, our results reveal cell cycle-dependent ligand/receptor trafficking as a temporal clock that directly links membrane dynamics to epigenetic regulation and stem cell fate, opening new avenues for regenerative medicine. ### Competing Interest Statement The authors have declared no competing interest.

Leukemia inhibitory factor (LIF)/STAT3 signaling is central to maintaining naïve pluripotency in mouse embryonic stem cells (mESCs). We identify Asgard , a previously uncharacterized long non-coding RNA, as a direct STAT3 target required for efficient self-renewal. Asgard is rapidly induced by LIF, enriched in the epiblast, and its depletion reduces alkaline phosphatase–positive colony formation while enhancing differentiation. Mechanistically, Asgard fulfils a dual role: it acts as the primary transcript for the differentiation-promoting microRNA Odin , while also functioning as a sponge to sequester Odin and related miRNAs. This dual mechanism enables Asgard to both generate and buffer pro-differentiation signals, thereby stabilizing the pluripotent state while preserving responsiveness to lineage cues. Our work reveals a new paradigm in RNA-mediated control of stem cell identity, where a single STAT3-regulated lncRNA couples microRNA production with competitive inhibition to safeguard naïve pluripotency. ### Competing Interest Statement The authors have declared no competing interest. Fondation ARC pour la Recherche sur le Cancer, PJA-20151203436 Fondation pour la Recherche MM-CM-)dicale, EQU202303016295 LabEx REVIVE, ANR-10-LABX-73 LabEx DEVweCAN, ANR-10-LABX-0061 LabEx CORTEX, ANR-11-LABX-0042 Investissements dM-BM-^RAvenir: University of Lyon, ANR-11-IDEX-0007

The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.

Development of new 2D and 3D models of human development such as trophoblast stem cells, gastruloids or blastoids widened possibilities to study early timepoints of development and brightened up ever so slightly the black box of human development. While opening new horizons, the cell sources of those models need proper benchmarking to clarify which hallmark is associated with which lineage and developmental stage. Here, we propose a thorough characterization of pluripotent and trophoblastic stem cell models by transcriptomic, proteomic, epigenetic and metabolic approaches. Extended pluripotent stem cells are similar to primed pluripotent stem cells for most criteria, except metabolic activity, which might explain their ability to convert directly into trophoblast stem cells. We show that trophoblast stem cells are hypo-methylated and that they have a high metabolic activity. Our results clarify the fact that hallmarks of pluripotency are not predictive of each other and have to be used in combination. Multiplying hallmarks alleviate stage matching bias.

Unravelling how gene regulatory networks are remodelled during evolution is crucial to understand how species adapt to environmental changes. We addressed this question for X-chromosome inactivation, a process essential to female development that is governed, in eutherians, by the XIST lncRNA and its cis -regulators. To reach high resolution, we studied closely related primate species, spanning 55 million years of evolution. We show that the XIST regulatory circuitry has diversified extensively over such evolutionary timeframe. The insertion of a HERVK transposon has reshuffled XIST 3D interaction network in macaque embryonic stem cells (ESC) and XIST expression is maintained by the additive effects of the JPX lncRNA gene and a macaque specific enhancer. In contrast, JPX is the main contributor to XIST expression in human ESCs but is not significantly involved in XIST regulation in marmoset ESCs. None of these entities are however under purifying selection, which suggests that neutrally evolving non-coding elements harbour high adaptive potentials.### Competing Interest StatementThe authors have declared no competing interest.

For nearly three decades, more than 80 embryonic stem cell lines and more than 100 induced pluripotent stem cell lines have been derived from New World monkeys, Old World monkeys, and great apes. In this comprehensive review, we examine these cell lines originating from marmoset, cynomolgus macaque, rhesus macaque, pig-tailed macaque, Japanese macaque, African green monkey, baboon, chimpanzee, bonobo, gorilla, and orangutan. We outline the methodologies implemented for their establishment, the culture protocols for their long-term maintenance, and their basic molecular characterization. Further, we spotlight any cell lines that express fluorescent reporters. Additionally, we compare these cell lines with human pluripotent stem cell lines, and we discuss cell lines reprogrammed into a pluripotent naive state, detailing the processes used to attain this. Last, we present the findings from the application of these cell lines in two emerging fields: intra- and interspecies embryonic chimeras and blastoids.

In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.

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

Inter-species chimeras are both fantastic and monstrous creatures from Greek or Egyptian mythology, and a long-established research tool. Recent advances in the field of pluripotent stem cells have made it possible to extend the repertoire of inter-species chimeras to "systemic" chimeras, in which the mixing of cells from both species involves all organs including the germline. These chimeric embryos and fetuses open up new research avenues and potential medical applications. We will review the latest advances in the field. We will discuss the concepts of developmental complementation and developmental equivalence. We will discuss the methodological hurdles to be unlocked, as well as the biological and ethical limits of these new technologies.Les chimères « systémiques » homme/animal.Les chimères inter-espèces sont à la fois les créatures fantastiques et monstrueuses des mythologies grecque ou égyptienne, et un outil de recherche établi de longue date. Des avancées récentes dans le domaine des cellules souches pluripotentes ont permis d’élargir le répertoire des chimères inter-espèces aux chimères « systémiques » dans lesquelles le mélange des cellules des deux espèces concerne tous les organes, y compris la lignée germinale. Ces embryons et fœtus chimériques ouvrent de nouvelles voies de recherches et des applications médicales potentielles. Dans cette revue, nous ferons le point sur les dernières avancées dans ce domaine. Nous discuterons les concepts de complémentation et d’équivalence développementale. Nous évoquerons également les verrous méthodologiques à débloquer, ainsi que les limites biologiques et éthiques de ces nouvelles techniques.

Les chimères inter-espèces sont à la fois les créatures fantastiques et monstrueuses des mythologies grecque ou égyptienne, et un outil de recherche établi de longue date. Des avancées récentes dans le domaine des cellules souches pluripotentes ont permis d’élargir le répertoire des chimères inter-espèces aux chimères « systémiques » dans lesquelles le mélange des cellules des deux espèces concerne tous les organes, y compris la lignée germinale. Ces embryons et fœtus chimériques ouvrent de nouvelles voies de recherches et des applications médicales potentielles. Dans cette revue, nous ferons le point sur les dernières avancées dans ce domaine. Nous discuterons les concepts de complémentation et d’équivalence développementale. Nous évoquerons également les verrous méthodologiques à débloquer, ainsi que les limites biologiques et éthiques de ces nouvelles techniques.

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

Inter-species chimeras are both fantastic and monstrous creatures from Greek or Egyptian mythology, and a long-established research tool. Recent advances in the field of pluripotent stem cells have made it possible to extend the repertoire of inter-species chimeras to "systemic" chimeras, in which the mixing of cells from both species involves all organs including the germline. These chimeric embryos and fetuses open up new research avenues and potential medical applications. We will review the latest advances in the field. We will discuss the concepts of developmental complementation and developmental equivalence. We will discuss the methodological hurdles to be unlocked, as well as the biological and ethical limits of these new technologies.

Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF-MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF-MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity.