Reduced MEK inhibition confers a growth advantage and reduces genomic instability in naïve human ES cells (RRBS-seq data set)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE111018 | GEO | 2018/07/10
REPOSITORIES: GEO
Similar Datasets
Project description:Human embryonic stem cells (hESCs) can be captured in either a primed state resembling the post-implantation epiblast or in a naïve state resembling the pre-implantation epiblast. Naïve culture conditions allow study of pre-implantation development ex vivo but reportedly lead to chromosomal abnormalities, compromising their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naïve state, here we show that reduced MEK inhibition facilitates the establishment and maintenance of naïve hESCs that retain naïve-specific transcriptional and DNA methylation patterns yet accrue fewer chromosomal abnormalities. We further show that hESCs cultured under these modified conditions are less prone to apoptosis and proliferate more rapidly. Our results point to the importance of residual MAPK signaling and provide a simple modification to current protocols to enable robust growth and genomic stability in naïve hESCs.
2018-07-10 | GSE110805 | GEO
Project description:Human embryonic stem cells (hESCs) typically exhibit "primed" pluripotency, analogous to stem cells derived from the mouse post-implantation epiblast. This has led to a search for growth conditions that support self-renewal of hESCs akin to hypomethylated naïve epiblast cells in human pre-implantation embryos. We have discovered that reverting primed hESCs to a hypomethylated naïve state or deriving a new hESC line under naïve conditions results in the establishment of Stage Specific Embryonic Antigen 4 (SSEA4) negative hESC lines with a transcriptional program resembling the human pre-implantation epiblast. In contrast, we discovered that the methylome of naïve hESCs in vitro is distinct from the human epiblast in vivo with loss of DNA methylation at primary imprints and a lost "memory" of the methylation state of the human oocyte. This failure to recover the naïve epiblast methylation landscape appears to be a consistent feature of self-renewing hypomethylated naïve hESCs in vitro.
2016-02-04 | GSE76970 | GEO
Project description:Background: Naïve human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional “primed” hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Methods:Here we performed high-throughput screening using a library of >3,000 well-annotated compounds to identify essential signaling requirements for naïve human pluripotency. Results:We report that MEK1/2 inhibitors can be replaced during maintenance of naïve human pluripotency by inhibitors targeting either upstream (FGFR, RAF1) or downstream (ERK1/2) kinases. Naïve hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naïve resetting in combination with PKC, ROCK, and TNKS inhibitors and Activin A. Conclusions: This work demonstrates that induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements.
2021-06-15 | GSE153213 | GEO
Project description:Background: Naïve human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional “primed” hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Methods:Here we performed high-throughput screening using a library of >3,000 well-annotated compounds to identify essential signaling requirements for naïve human pluripotency. Results:We report that MEK1/2 inhibitors can be replaced during maintenance of naïve human pluripotency by inhibitors targeting either upstream (FGFR, RAF1) or downstream (ERK1/2) kinases. Naïve hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naïve resetting in combination with PKC, ROCK, and TNKS inhibitors and Activin A. Conclusions: This work demonstrates that induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements.
2021-06-15 | GSE153212 | GEO
Project description:The transition of pluripotent stem cells (PSCs) from primed to naïve states constitutes a prototypical example of cellular plasticity. The naïve state can be stabilized by defined chemical cocktails that block extracellular signals, notably including the MEK pathway. However, little is known regarding the underlying transcriptional mechanisms. Here, we report that the transcriptional landscape of the naïve state can be mimicked in mouse and human PSCs by stimulating transcriptional enhancers. This is attained by inhibiting the CDK8 and CDK19 kinases, which are negative regulators of Mediator, a critical component of enhancer function. Mechanistically, CDK8/19i triggers a global increase in the recruitment of RNA Pol II at promoters and enhancers, hyperactivating enhancers and their target genes. Lastly, the emergence of naïve pluripotency in the pre-implantation epiblast coincides with a marked reduction in CDK8/19 activity, and CDK8/19i blocks its subsequent developmental progression. These findings suggest that naïve pluripotency during development includes hyperactivation of enhancers and can be captured in vitro, either by blunting extracellular signaling, or by stimulating enhancer-driven transcription. These principles may apply to other cellular transitions.
2020-09-29 | PXD009200 | Pride
Project description:The self-renewing pluripotent state was first captured in mouse embryonic stem cells (mESCs) over two decades ago. The standard condition requires the presence of serum and LIF, which provide growth promoting signals for cell expansion. However, there are pro-differentiation signals which destabilize the undifferentiated state of mESCs. The dual inhibition (2i) of the pro-differentiation Mek/Erk and Gsk3/Tcf3 pathways in mESCs is sufficient to establish an enhanced pluripotent “ground state” which bears features resembling the pre-implantation mouse epiblast. Gsk3 inhibition alleviates the repression of Esrrb, a transcription factor that can substitute for Nanog function in mESCs. The molecular mechanism that is mediated by Mek inhibition is however not clear. In this study, we investigate the pathway through which Mek inhibition operates to maintain ground state pluripotency. We have found that in mESCs, Kruppel-like factor 2 (Klf2) is a protein target of the Mek/Erk pathway; and that Klf2 protein is phosphorylated by Erk2 and subsequently degraded through the proteosome. It is therefore by Mek-inhibition through PD0325901 or 2i that enables the stabilization and accumulation of Klf2 to sustain ground state pluripotency. Importantly, we found that Klf2-null mESCs, while viable under LIF/Serum conditions, cannot be maintained and eventually gradually die within a few passages. Our result thus demonstrates that Klf2 is an essential factor of ground state pluripotency. Collectively, our study defines the Mek/Klf2 axis that cooperates with the Gsk3/Esrrb pathway in mediating ground state pluripotency.
2014-06-04 | E-MTAB-2365 | biostudies-arrayexpress
Project description:The self-renewing pluripotent state was first captured in mouse embryonic stem cells (mESCs) over two decades ago. The standard condition requires the presence of serum and LIF, which provide growth promoting signals for cell expansion. However, there are pro-differentiation signals which destabilize the undifferentiated state of mESCs. The dual inhibition (2i) of the pro-differentiation Mek/Erk and Gsk3/Tcf3 pathways in mESCs is sufficient to establish an enhanced pluripotent “ground state” which bears features resembling the pre-implantation mouse epiblast. Gsk3 inhibition alleviates the repression of Esrrb, a transcription factor that can substitute for Nanog function in mESCs. The molecular mechanism that is mediated by Mek inhibition is however not clear. In this study, we investigate the pathway through which Mek inhibition operates to maintain ground state pluripotency. We have found that in mESCs, Kruppel-like factor 2 (Klf2) is a protein target of the Mek/Erk pathway; and that Klf2 protein is phosphorylated by Erk2 and subsequently degraded through the proteosome. It is therefore by Mek-inhibition through PD0325901 or 2i that enables the stabilization and accumulation of Klf2 to sustain ground state pluripotency. Importantly, we found that Klf2-null mESCs, while viable under LIF/Serum conditions, cannot be maintained and eventually gradually die within a few passages. Our result thus demonstrates that Klf2 is an essential factor of ground state pluripotency. Collectively, our study defines the Mek/Klf2 axis that cooperates with the Gsk3/Esrrb pathway in mediating ground state pluripotency.
2014-06-04 | E-MTAB-2366 | biostudies-arrayexpress
Project description:<p>Naïve human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naïve hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901, a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy, DNA damage, and apoptosis. Remarkably, MitoQ, a mitochondria-targeted antioxidant, effectively restored the pluripotency and proliferation of PRODH-knockdown naïve hESCs, indicating that PRODH maintains naïve pluripotency by preventing excessive ROS production. Concomitantly, PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus, we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production, and thereby safeguarding naïve pluripotency of hESCs.</p><p><br></p><p><strong>Untargeted metabolomics</strong> - H9N PLKO.1 vs H9P PLKO.1 / H9P shPRODH vs H9P PLKO.1 / H9N shPRODH vs H9N PLKO.1 - is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS7840' rel='noopener noreferrer' target='_blank'><strong>MTBLS7840</strong></a>.</p><p><strong>Targeted metabolomics</strong> (amino acids and derivatives) - H9P (Primed) VS H9N (Naïve) - is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS7832' rel='noopener noreferrer' target='_blank'><strong>MTBLS7832</strong></a>.</p>
2024-02-12 | MTBLS7840 | MetaboLights
Project description:<p>Naïve human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation, but their mitochondrial regulators are poorly understood. Here we report that, proline dehydrogenase (PRODH), a mitochondria-localized proline metabolism enzyme, is dramatically upregulated in naïve hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901, a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy, DNA damage, and apoptosis. Remarkably, MitoQ, a mitochondria-targeted antioxidant, effectively restored the pluripotency and proliferation of PRODH-knockdown naïve hESCs, indicating that PRODH maintains naïve pluripotency by preventing excessive ROS production. Concomitantly, PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus, we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production, and thereby safeguarding naïve pluripotency of hESCs.</p><p><br></p><p><strong>Targeted metabolomics</strong> (amino acids and derivatives) - H9P (Primed) VS H9N (Naïve) - is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS7832' rel='noopener noreferrer' target='_blank'><strong>MTBLS7832</strong></a>.</p><p><strong>Untargeted metabolomics</strong> - H9N PLKO.1 vs H9P PLKO.1 / H9P shPRODH vs H9P PLKO.1 / H9N shPRODH vs H9N PLKO.1 - is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS7840' rel='noopener noreferrer' target='_blank'><strong>MTBLS7840</strong></a>.</p>
2024-02-12 | MTBLS7832 | MetaboLights
Project description:The epiblast is the first cell type that forms apical-basal polarity de novo as the mouse embryo implants into the maternal uterus, while the extraembryonic neighbours of the epiblast - trophectoderm and primitive endoderm - retain their pre-established polarity beyond implantation [1]; however, it is still unclear how the epiblast establishes apical-basal polarity de novo. Here, we focused on Rap1 signaling pathway, which is activated during the transition of the epiblast from the naïve to primed state of pluripotency during implantation [2]. Through the preestablished in vitro three-dimensional culture system [3], genetic knockouts and proximity-biotinylation analyses, we found that Rap1 integrates multiple signals that contribute to de novo formation of apical-basal polarity. Importantly, formation of apical-basal polarity in the epiblast is essential for its correct patterning and proper communication with the extraembryonic lineages. Altogether, these results not only dissect molecular details of de novo apical-basal polarity formation, but also have broader implications for epithelial polarity and development.
2022-06-14 | PXD028315 | Pride