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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE98771 | GEO | 2017/07/27

SECONDARY ACCESSION(S): PRJNA386173

REPOSITORIES: GEO

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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.

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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.

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

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NaÃ¯ve 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. 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.

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