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

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

Global hyperactivation of enhancers stabilizes human and mouse naïve pluripotency through inhibition of CDK8/19 Mediator kinases

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

The FunGenES Database: A Genomics Resource for Mouse Embryonic Stem Cell Differentiation

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells

Identification et caractérisation fonctionnelle des gènes cibles de la voie LIF/STAT3 impliqués dans le contrôle de l autorenouvellement des cellules souches embryonnaires de souris

LIF activates the transcription factor STAT3, resulting in the maintenance of mouse pluripotent ES cells in the undifferentiated state by inhibiting mesodermal and endodermal differentiation. To unravel the mechanisms of LIF/STAT3-dependant pluripotency, we identified target genes of this pathway and characterize their role in the inhibition of differentiation. We identified 58 targets of STAT3. Functional analysis showed that 22 of them showed an increase of differentiated colonies in a self-renewal assay, and an elevation of early differentiation markers upon knockdown. Pim1 and Pim3 were shown to protect ES cells from differentiation induced by LIF starvation when overexpressed, demonstrating their role in the maintenance of pluripotency. The 22 STAT3 target genes analysed fell into two categories, one suppressing mesoderm and the other endoderm differentiation, as evidenced by the differential expression of mesoderm and endoderm markers after knockdown. Two genes, Klf4 and Klf5 were examined in more details. We demonstrated that knockdown of Klf4 induced ectopic expression of endodermal regulators and resulted in an increase of cells differentiating into extraembryonic and definitive endoderm. Whereas Klf5 knockdown induced expression of the panmesodermal regulator Brachyury and an increase of cells differentiating into mesoderm. Our work shows that the LIF/STAT3 pathway maintains the undifferentiated state of ES cells by activating the expression of target genes involved in the suppression of mesoderm or endoderm differentiation. And it highlights the central role played by Klf4 and Klf5 in self-renewal and commitment of mouse ES cells into mesoderm and endodermLYON1-BU.Sciences (692662101) / SudocSudocFranceF

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

Cinquante nuances de pluripotence

Depuis la dérivation des premières lignées de cellules souches embryonnaires pluripotentes chez la souris au début des années 1980, une pléthore de lignées a été obtenue chez diverses espèces de mammifères, dont les rongeurs, les lagomorphes

Single-cell qPCR analysis of pluripotency in rabbit embryos

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

Single-cell qPCR analysis of pluripotency in rabbit embryos

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Deciphering the pluripotency of rabbit pre-implantation embryos

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Deciphering the epigenetic context of pluripotency in rabbit pre-implantation embryos

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