Primed to naïve-like conversion of the common marmoset embryonic stem cells
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PROVIDER: PRJNA577867 | ENA |
REPOSITORIES: ENA
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Project description:Mammalian pluripotent stem cells are thought to exist in two states: naïve and primed states. Generally, unlike those in rodents, pluripotent stem cells in primates including humans are regarded as being in the primed pluripotent state. Recently, several groups reported the existence of naïve pluripotent stem cells in humans. In this study, we report the conversion of primed state embryonic stem cells from common marmoset, a New World monkey, to the naïve state by using transgenes. The cells showed typical naïve state features including dome-like colony morphology, growth factor requirement, gene expression profile, X chromosome activation state, and energy metabolic status. Moreover, interspecies chimeric embryo formation ability with mouse embryos was increased in the naïve state. This technique can be applied in basic medical research using non-human primates such as preclinical use of naïve pluripotent stem cells and generating genetically modified primates.
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Project description:We derived primed pluripotent stem cell cultures (PSCs) from marmoset blastocyst using conventional human PSC culture conditions (KSR/bFGF on feeders). Naïve marmoset PSCs were established by chemical resetting. In addition, we generated forebrain-derived cells from the frontal lobe of a marmoset neonate. Individual cells were manually isolated with a mouth pipette and subjected to single-cell full-lengths transcriptome profiling using a modified version of Smart-Seq2.
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Project description:Deciphering the regulatory network for human naïve and primed pluripotency is of fundamental theoretical and applicable significance. Here, by combining quantitative proteomics, phosphoproteomics and acetylproteomics analyses, we revealed RNA processing and translation as the most differentially-regulated processes between naïve and primed human embryonic stem cells (hESCs). While glycolytic primed hESCs rely predominantly on eIF4E-mediated cap-dependent pathway for protein translation, naïve hESCs with reduced mTORC1 activity are more tolerant to blockage of eIF4E-dependent translation, and their bivalent metabolism allows for translating selective mRNAs via both eIF4E-dependent and eIF4E-independent/eIF4A2-dependent pathways to form a more compact naïve proteome. Globally up-regulated proteostasis system and down-regulated post-translational modification system help to further refine and maintain the naïve proteome that is compatible with the more rapid cycling of naïve hESCs, where CDK1 plays an indispensable coordinative role.
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Project description:Translational control plays a central role in regulation of gene expression and can lead to significant divergence between mRNA- and protein-abundance. The translational landscape of early mammalian development and its impact on cellular proteome, however, remains largely un-explored. Here we used genome-wide approaches combined with time-course analysis to measure the mRNA-abundance, mRNA-translation rate and protein expression during the transition of naïve into primed embryonic stem cells (ESCs). We found that the ground state ESCs cultured with GSK3- and MEK-inhibitors and LIF (2iL) display higher ribosome density on a selective set of mRNAs. These mRNAs show reduced translation during the exit from ground state pluripotency and transition to serum/LIF (SL) culture or upon commitment to primed epiblast-like stem cells (EpiLSCs). Strikingly, integrative analysis with cellular proteome indicate a strong translational buffering of this set of mRNAs in 2iL-ESCs leading to stable protein expression levels. Our data reveal that the global alteration of cellular proteome is largely accompanied by transcriptional rewiring. Furthermore, we identified a set of genes (including UHRF1 and KRAS) that undergo selective post-translational regulation during the transition of naïve into primed pluripotency and linked the observed changes to upstream GSK- and MEK/MAPK-signaling pathways using single inhibitor treated ESCs. Thus, we provide a comprehensive and detailed overview of the global changes in gene expression during the transition of naïve to primed pluripotency and dissect the relative contributions of RNA-transcription, translation and regulation of protein stability in controlling protein abundance.
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Project description:The common marmoset (marmoset; Callithrix jacchus) has been attracting much attention especially in the research fields of biomedical science and neuroscience, because of its potential to recapitulate the complex and multidimensional phenotypes of human diseases. Taking these advantages, several transgenic models have been reported so far. However, there remain several issues such as (i) it takes years to generate the late-onset disease models, and (ii) the onset age and severity of phenotypes can vary among individual animals due to their differences in the genetic background. In the present study, we established an efficient and rapid direct neuronal induction method (induced Neuron; iN) from embryonic and adult marmoset fibroblasts. This iN method is a minimally invasive approach to investigate cellular-level phenotypes in the marmoset brain. We overexpressed microRNA-9/9*-124 and a transcription factor ASCL1 in fibroblasts with a small molecule cocktail that ameliorates neuronal induction. The resultant iN cells from both embryonic and adult marmoset fibroblasts showed standard neuronal characteristic within 2 weeks, including neuronal specific gene expression and neuronal membrane spontaneous firing activity. Thus, this novel neuronal induction method may offer a new tool for investigating neurological phenotypes in vitro without collecting living brain tissue in non-human primate neurological disease models.
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Project description:Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic techniques, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E8.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Thus, we established the efficacy of the method for the derivation of marmoset EPSCs
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