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NaÃ¯ve pluripotency is associated with global DNA hypomethylation

ABSTRACT: Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Importantly, inhibition of Erk and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs. EGC lines were derived from E8.5 mouse embryos using three different protocols: FCS+LIF on MEFs (FCS, n = 4)), FCS+LIF on MEFs for 48 hours followed by 2i+LIF (2is, n = 4), and direct derivation into 2i+LIF (2i, n = 4). ESC lines were derived in either FCS+LIF on MEFs or in 2i+LIF conditions and once established the cell lines were also switched between the two culture environments (n = 5 for each culture condition). All cell lines were derived from genetically identical embryos.

ORGANISM(S): Mus musculus

SUBMITTER: Kirsten McEwen

PROVIDER: E-GEOD-43398 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Naïve pluripotency is associated with global DNA hypomethylation

Project description:Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Importantly, inhibition of Erk and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.

2013-02-17 | GSE43398 | GEO

Integrative analysis of the acquisition of pluripotency in PGCs

Project description:To elucidate the molecular mechanisms by which PGCs become pluripotent cells, we performed whole transcriptome analysis during the conversion of PGCs into EGCs. There are 3 groups of gene expression profiles, time course analysis of PGCs to EGCs, forced expression of germ cell genes in ESCs, and chemical treatment of PGCs at day2 of the culture

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Derivation of ground-state female ESCs maintaining gamete-derived DNA methylation [gene expression]

Project description:Preimplantation embryos undergo a transient wave of genome-wide demethylation with the exception of imprinted genes that are critical for fetal development. Here we show that the derivation of female mouse embryonic stem cells (ESCs) in the presence of inhibitors of MEK1/2 and Gsk3 (2i-ESCs), known as ‘2i’ or “ground-state” culture conditions, results in a widespread loss of DNA methylation including a massive erasure of genomic imprints. In this study, we analyzed global gene expression profile and global DNA methylation status in 2i-ESCs and 2i-ESCs derived differentiated cells. S-ESCs are ESCs established under serum-containing medium. 2i_S_ESCs are ESCs established in 2i-containing medium, followed by maintenance in serum-containing medium.

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Continuous suppression of Mek1/2 impairs the developmental potential of mouse embryonic stem cells

Project description:Co-suppression of Gsk3 and Mek1/2 signaling (“2i culture”) in mouse embryonic stem cells (ESCs) induces a naïve state that more closely resembles the inner cell mass (ICM) of the pre-implantation embryo, including global DNA hypomethylation. While the ICM exists only transiently in vivo, it remains unclear how continuous suppression of Mek1/2 and Gsk3 signaling and associated hypomethylation affect the stability and functionality of ESCs in vitro. Here we show that, compared to ESCs maintained in conventional serum/LIF media, culturing male ESCs in 2i/LIF for >6 passages results in the erosion of genomic imprints and a concomitant impairment in the ability to generate adult, entirely ESC- derived mice (all-ESC mice). We further demonstrate that female ESCs cultured in serum/LIF phenocopy male ESCs cultured in 2i/LIF, including global hypomethylation, imprint erosion and impaired developmental potential. Unexpectedly, culture of female ESCs in serum/LIF and male ESCs in 2i/LIF for >16 passages causes recurrent chromosomal aberration that have been associated with a selective growth advantage. We provide evidence that inhibition of Mek1/2, but not Gsk3, is responsible for hypomethylation, imprint loss and chromosomal instability in ESCs, and identify alternative conditions that preserve epigenetic integrity. Taken together, our data suggest that while short-term suppression of Mek1/2 in ESCs captures an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise developmental potential.

2017-07-27 | GSE97995 | GEO

Derivation of ground-state female ESCs maintaining gamete-derived DNA methylation

Project description:Inhibitors of MEK1/2 and Gsk3b, known as '2i' culture conditions, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency in rodents 1,2. Here we show that the derivation of female mouse ESCs in the presence of 2i (2i-ESCs) results in a widespread loss of DNA methylation including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, the early-passage 2i-ESCs efficiently differentiate into somatic cells and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in the 2i-ESC-derived differentiated cells. Consistently, 2i-ESCs exhibit impairment of autonomous embryonic and placental development by tetraploid embryo complementation and nuclear transplantation. We identified the derivation conditions of female ESCs that display 2i-ESC-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ESCs exhibit ICR demethylation, regardless of culture conditions. Our results provide insights into derivation of female ESCs reminiscent of ICM of preimplantation embryos. S-ESCs are ESCs established under serum-containing medium. 2i_S_ESCs are ESCs established in 2i-containing medium, followed by maintenance in serum-containing medium.

2017-07-27 | GSE84164 | GEO

Extended suppression of Mek1/2 impairs the developmental potential of mouse embryonic stem cells

Project description:Simultaneous inhibition of Gsk3α/β and Mek1/2 activity in the presence of LIF (2i/L) induces a naïve state in mouse embryonic stem cells (ESCs) that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how extended propagation of naïve ESCs in vitro affects their stability and functionality. Here we show that extended culture of male ESCs in 2i/L results in the progressive erosion of genomic imprints, loss of H2A.X binding, and accumulation of chromosomal aberrations. Consistent with these observations, we find that the developmental potential of ESCs cultured in 2i/L is impaired. Mechanistically, we demonstrate that Mek1/2 inhibition is predominantly responsible for these effects, in part through downregulation of DNA methyltransferases. Additionally, we demonstrate that female ESCs cultured in conventional serum/LIF media phenocopy male ESCs cultured in 2i/L, including the aforementioned epigenetic and developmental abnormalities. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as developmental potential of ESCs. Taken together, our data suggest that, while short-term suppression of Mek1/2 in ESCs maintains an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

2017-07-27 | GSE97954 | GEO

Continuous suppression of Mek1/2 impairs the developmental potential of mouse embryonic stem cells (H2A.X pattern)

Project description:Simultaneous inhibition of Gsk3α/β and Mek1/2 signaling in the presence of LIF (2i/L) induces a naïve state in mouse embryonic stem cells (ESCs) that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how continuous propagation of naïve ESCs in vitro affects their stability and functionality. Here, we show that extended culture of male ESCs in 2i/L results in the progressive erosion of genomic imprints, loss of H2A.X binding, and accumulation of chromosomal aberrations. Consistent with these observations, we find that ESCs propagated in 2i/L have an impaired developmental potential. Mechanistically, we demonstrate that Mek1/2 inhibition is predominantly responsible for these effects, in part through downregulation of DNA methyltransferases. We further provide evidence that female ESCs cultured in conventional serum/LIF media phenocopy male ESCs cultured in 2i/L, including the aforementioned epigenetic and developmental abnormalities. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as developmental potential of ESCs. Taken together, our data suggest that, while suppression of Mek1/2 in ESCs maintains an ICM-like epigenetic state, continuous suppression results in irreversible changes that compromise their developmental potential.

2017-07-27 | GSE98771 | GEO

Continuous suppression of Mek1/2 impairs the developmental potential of mouse embryonic stem cells

Project description:Simultaneous inhibition of Gsk3α/β and Mek1/2 activity in the presence of LIF (2i/L) induces a naïve state in mouse embryonic stem cells (ESCs) that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how continuous propagation of naïve ESCs in vitro affects their stability and functionality. Here we show that extended culture of male ESCs in 2i/L results in the progressive erosion of genomic imprints, loss of H2A.X binding, and accumulation of chromosomal aberrations. Consistent with these observations, we find that the developmental potential of ESCs cultured in 2i/L is impaired. Mechanistically, we demonstrate that Mek1/2 inhibition is predominantly responsible for these effects, in part through downregulation of DNA methyltransferases. Additionally, we demonstrate that female ESCs cultured in conventional serum/LIF media phenocopy male ESCs cultured in 2i/L, including the aforementioned epigenetic and developmental abnormalities. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as developmental potential of ESCs. Taken together, our data suggest that, while suppression of Mek1/2 in ESCs maintains an ICM-like epigenetic state, continuous suppression results in irreversible changes that compromise their developmental potential.

2017-07-27 | GSE93103 | GEO

Gene-expression profile of human embryonic germ cells

Project description:Gene-expression profile of human embryonic germ cells (EGCs), primordial germ cells (PGCs), embryonal carcinoma cells (ECCs), pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (IPSCs)

2018-06-28 | E-MTAB-6851 | biostudies-arrayexpress

PGC reversion to pluripotency involves erasure of DNA methylation from imprinting control centers followed by locus-specific re-methylation

Project description:Primordial germ cells (PGCs) are fate restricted to differentiate into gametes in vivo. However when removed from their embryonic niche PGCs undergo reversion to generate pluripotent embryonic germ cells (EGCs) in vitro. One of the major differences between EGCs and embryonic stem cells (ESCs) involves variable methylation at imprinting control centers (ICCs), a phenomenon that is poorly understood. In the current study we show that reverting PGCs to EGCs involves ICC methylation erasure, which remain stably hypomethylated at Snrpn, Igf2r and Kcnqot1. In contrast, the H19/Igf2 ICC undergoes almost complete de novo remethylation. Using the same approach for PGCs differentiated in vitro from ESCs we show that the Snrpn ICC is erased however the hypomethylated state is highly unstable. We also discovered that when the H19/Igf2 ICC is abnormally hypermethylated in ESCs, ICC methylation is not erased with differentiation into PGCs. This highlights the importance of not only launching germline differentiation with correctly methylated ESC lines but also the need to better stabilize the hypomethylated state in the in vitro derivatives following ICC erasure.

2015-06-01 | GSE69354 | GEO

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