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Novel culture conditions facilitating the induction of naïve pluripotency in human embryonic stem cells

ABSTRACT: It is now well recognized that human embryonic stem cells (hESCs)1 closely resemble mouse epiblast stem cells (mEpiSCs)2-5 exhibiting primed pluripotency unlike mouse ESCs (mESCs) which acquire a naïve pluripotent state4-8. Efforts have been made to trigger naïve pluripotency in hESCs9-11 for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines12. We report here a novel culture medium facilitating rapid induction of naïve pluripotency in established hESCs. Our medium also allows derivation of naïve mESCs from blastocyst stage which has not been shown earlier. The established naïve hESCs could survive long-term single cell passaging, maintain a normal karyotype, exhibit upregulation of naïve pluripotency genes and were dependent on signaling pathways similar to naïve mESCs. Also, they undergo global DNA demethylation, cluster together with previously described naïve hESCs13 and show a distinctive long non-coding RNA profile. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs which is fast, reproducible, can be employed to derive naïve mESCs and can induce efficient differentiation. Three primed and matching naive human embryonic stem cell lines were profiled in duplo.

ORGANISM(S): Homo sapiens

SUBMITTER: Pieter mestdagh

PROVIDER: E-GEOD-57271 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Novel culture conditions facilitating the induction of naïve pluripotency in human embryonic stem cells

Project description:It is now well recognized that human embryonic stem cells (hESCs)1 closely resemble mouse epiblast stem cells (mEpiSCs)2-5 exhibiting primed pluripotency unlike mouse ESCs (mESCs) which acquire a naïve pluripotent state4-8. Efforts have been made to trigger naïve pluripotency in hESCs9-11 for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines12. We report here a novel culture medium facilitating rapid induction of naïve pluripotency in established hESCs. Our medium also allows derivation of naïve mESCs from blastocyst stage which has not been shown earlier. The established naïve hESCs could survive long-term single cell passaging, maintain a normal karyotype, exhibit upregulation of naïve pluripotency genes and were dependent on signaling pathways similar to naïve mESCs. Also, they undergo global DNA demethylation, cluster together with previously described naïve hESCs13 and show a distinctive long non-coding RNA profile. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs which is fast, reproducible, can be employed to derive naïve mESCs and can induce efficient differentiation.

2015-06-08 | GSE57271 | GEO

The functional role of OGDH for maintaining mitochondrial respiration and identity of primed human embryonic stem cells

Project description:Human embryonic stem cells (hESCs) can self-renew infinitely or differentiate into the 3 germ layer lineages in vitro with certain cues, holding great promise to model early human embryo development ex vivo. It is wildly accepted that hESCs take advantage of both glycolysis and mitochondrial respiration to favor the naïve pluripotency, while prefer glycolysis to support the primed pluripotency. Thus, the function of mitochondrial respiration in primed hESCs has been underestimated for a long time, and has not been fully understood yet. Herein, we report that the adenosine triphosphate (ATP) production rate is comparable between mitochondrial respiration and glycolysis, suggesting that mitoATP may serve as one of the major sources of the ATP pool in primed hESCs. To further reveal the function of mitochondrial respiration, we deployed inducible CRISPRi method to inhibit OGDH expression with high efficiency, resulting in the TCA cycle blockade as well as the diminishment of mitochondrial respiration activity and total ATP level. Of note, OGDH deficiency led to the exit of primed pluripotency accompanied by cell death. Furthermore, the treatment with small molecule inhibitors to block electron transport chain (ETC) can phenocopy the loss-of-function of OGDH in hESCs. Therefore, genetic and pharmacological perturbations on mitochondrial respiration can impair the stemness of primed hESCs. Collectively, the current study unveils that OGDH acts as a key regulator to fine-tune the abundance of TCA cycle metabolites to ensure the mitochondrial respiration activity in primed hESCs, and highlights the pivotal roles of mitochondrial respiration in terms of ATP production and the maintenance of primed pluripotency. Our findings may provide insights into the linkage between pluripotent states and energy metabolism in hESCs.

2022-03-03 | MTBLS4223 | MetaboLights

AMPK activation reverts mouse epiblast stem cells to naïve state

Project description:Reversion from primed to naïve pluripotent status has been achieved by various signaling manipulation, but it is still unclear what signaling is the actual driving force to get over the hurdle from primed to naïve pluripotency. We previously reported that activation of AMP kinase (AMPK) contributed to maintenance of naïve pluripotency. Here, we further show that AMPK activators, AICAR, A769662 or metformin, can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to naïve pluripotent state. Primed mEpiSCs in our naïve cell culture condition with leukemia inhibitory factor (LIF) and 2 kinase inhibitors (2i) (2iL) never gave rise to naïve state cells. Addition of AICAR alone even in the absence of 2iL or either of AMPK inhibitors with LIF induced appearance of naïve-like cells from primed mEpiSCs. Through maintenance and passages of these cells in 2iL condition, clear naïve-like morphology colonies were purely obtained. They showed core naïve protein expression, and global naïve gene expression profiles. These cells contributed to chimeric mice including germline transmission. Inhibition of p38 signaling abolished the AMPK-elicited reversion and forced activation of p38 in primed mEpiSCs partially reproduced the naïve cell induction, suggesting that p38 is one of the critical downstream in AMPK activation. AMPK pathway should be a novel critical driving force in reversion of primed to naïve pluripotency.

2021-07-21 | GSE157087 | GEO

Epigenomic comparison of distinct pluripotent stem cell states reveals a new class of enhancers with roles throughout mammalian development (RNA-seq)

Project description:Naïve mouse embryonic stem cells (mESCs) and primed epiblast stem cells (mEpiSCs) represent successive snapshots of pluripotency during embryogenesis. Using transcriptomic and epigenomic mapping, we show that a small fraction of transcripts are differentially expressed between mESCs and mEpiSCs and these genes show expected changes in chromatin at their promoters and enhancers. Unexpectedly, the cis-regulatory circuitry of genes that are expressed at identical levels between these cell states also differs dramatically. In mESCs, these genes are associated with dominant proximal enhancers and dormant distal enhancers, which we term seed enhancers. In mEpiSCs, the naïve-dominant enhancers are lost, and the seed enhancers take up primary transcriptional control. Seed enhancers have increased sequence conservation and show preferential usage in downstream somatic tissues, often expanding into super enhancers. We propose that seed enhancers ensure proper enhancer utilization and transcriptional fidelity as mammalian cells transition from naïve pluripotency to a somatic regulatory program.

2014-06-04 | GSE57403 | GEO

Epigenomic comparison of distinct pluripotent stem cell states reveals a new class of enhancers with roles throughout mammalian development (DNase-seq)

2014-06-04 | GSE57406 | GEO

Epigenomic comparison of distinct pluripotent stem cell states reveals a new class of enhancers with roles throughout mammalian development (ChIP-seq)

2014-06-04 | GSE57407 | GEO

Characterization of distinct states of mouse naïve and primed pluriopotency

Project description:Pluripotency of mammalian stem cells is stabilized at different status depending upon the extracellular stimuli of their environment. Now, there is a consensus that mouse embryonic stem cells (mESCs) and iPSCs (miPSCs) represent undifferentiated status very close to ground-state of mammalian development when cultured in optimal condition. Since they have not been activated to commit into any lineages, they are stated as “naïve-state stem cells”. In contrast, human ESCs (hESCs), hiPSCs, and mEpiSCs are considered to possess partially differentiated characteristics compared with mESCs and miPSCs. Thus, they are stated as “primed-state stem cells”. Investigation of the prerequisites for establishment of the naïve-state is significantly important to fully understand mammalian development and to extrapolate the technologies developed with mouse ES/iPS cells for human. We used expression data to understand the gene expression profile and identified distinct characteristics on pluripotent state.

2017-12-12 | GSE107889 | GEO

Characterization of distinct states of human naïve and primed pluriopotency

2017-12-13 | GSE107969 | GEO

AMPK activation reverts mouse epiblast stem cells to naïve state

Project description:Reversion from primed to naïve pluripotent status has been achieved by various signaling manipulation, but it is still unclear what signaling is the actual driving force to get over the hurdle from primed to naïve pluripotency. We previously reported that activation of AMP kinase (AMPK) contributed to maintenance of naïve pluripotency. Here, we further show that AMPK activators, AICAR, A769662 or metformin, can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to naïve pluripotent state. Primed mEpiSCs in our naïve cell culture condition with leukemia inhibitory factor (LIF) and 2 kinase inhibitors (2i) (2iL) never gave rise to naïve state cells. Addition of AICAR alone even in the absence of 2iL or either of AMPK inhibitors with LIF induced appearance of naïve-like cells from primed mEpiSCs. Through maintenance and passages of these cells in 2iL condition, clear naïve-like morphology colonies were purely obtained. They showed core naïve protein expression, and global naïve gene expression profiles. These cells contributed to chimeric mice including germline transmission. Inhibition of p38 signaling abolished the AMPK-elicited reversion and forced activation of p38 in primed mEpiSCs partially reproduced the naïve cell induction, suggesting that p38 is one of the critical downstream in AMPK activation. Single cell RNA-seq analysis under AICAR stimulation successfully demonstrated the reversion process with appearance of intermediate naïve-like population. AMPK pathway should be a novel critical driving force in reversion of primed to naïve pluripotency.

2021-07-21 | GSE175610 | GEO

Translational and Post-translational LC-MS of Naïve and Primed hESCs

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.

2021-12-10 | PXD030230 | Pride

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