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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE84164 | GEO | 2017/07/27

SECONDARY ACCESSION(S): PRJNA328171

REPOSITORIES: GEO

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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.

2017-07-27 | GSE82313 | GEO

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.

2013-02-17 | E-GEOD-43398 | biostudies-arrayexpress

Integrative proteomics reveals a protective role of PRC2 for the ground state pluripotent epigenome

Project description:The ground state of pluripotency is defined as a basal proliferative state free of epigenetic restriction, represented by mouse embryonic stem cells (ESCs) cultured with two kinase inhibitors (so-called “2i”). Through comparison with serum-grown ESCs, we identify epigenetic features characterizing 2i ESCs by proteome profiling of chromatin including post-translational histone modifications. The most prominent difference is H3K27me3 and its enzymatic writer complex PRC2 that are highly abundant on eu- and heterochromatin in 2i ESCs, with H3K27me3 redistributing outside canonical PRC2 targets in a CpG-dependent fashion. Using PRC2-deficient 2i ESCs, we identify epigenetic crosstalk with H3K27me3, including significant increases in H4 acetylation and DNA methylation. This suggests that the unique H3K27me3 configuration protects 2i ESCs from preparation to lineage priming. Interestingly, removal of DNA methylation in PRC2-deficient 2i ESCs lacking H3K27me3 using 5-azacytidine hardly affected ESC viability and transcriptome, showing that ESCs are independent of both major repressive epigenetic marks.

2018-11-21 | PXD007154 | Pride

Transcriptome profiles of mouse embryonic stem cells cultured in different conditions by RNA-seq analysis

Project description:Pluripotent mouse embryonic stem cells (ESCs) were originally derived and stably maintained on feeder cells such as inactivated mouse embryo fibroblasts, and can generate complete ESC-pups by tetraploid embryo complementation (TEC), the most stringent functional test of naive pluripotency. Remarkably, 2i (inhibitors of Mek and Gsk3β signaling) medium with LIF in the absence of serum and feeders was developed to achieve ground state of mouse ESCs, and also has been successfully used for derivation of germline competent ESCs in other species such as rat. Notably, 2i-culture gives rise to transcriptional profiles and epigenetic landscapes quite distinct from serum-based ESCs. To better understand transcriptional landscape and signal transductions, we performed RNA-seq analysis of ESCs cultured in four different conditions: serum without feeder, serum with feeder, serum with feeder and 2i, and N2B27+2i.

2018-01-20 | GSE109418 | GEO

Identify Tfcp2l1-regulated genes in mouse embryonic stem cells grown in 2i medium

Project description:Mouse embryonic stem cells (ESCs) grown in the 2i (an ERK inhibitor, PD0325901, and a GSK-3b inhibitor, CHIR99021) mimic pluripotent epiblasts in preimplantation blastocysts. ESCs grown in FBS containing medium are more heterogenous in morphology and expression of stem cell factors. Tfcp2l1 is a transcription factor highly expressed in ESCs grown in the 2i medium, suggesting that it plays in important role in ESC biology. To search for the putative role(s) of Tfcp2l1 in ESCs, we plan to use an integrated genomic approach--combing RNA-seq and ChIP-seq--to identify the direct targets of Tfcp2l1 in ESCs. RNA-seq is used to identify the transcripts regulated by Tfcp2l1 in ESCs grown in the 2i medium.

2021-03-01 | GSE159937 | GEO

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

Identify genomic binding sites of Tfcp2l1 in mouse embryonic stem cells grown in 2i medium

Project description:Mouse embryonic stem cells (ESCs) grown in the 2i (an ERK inhibitor, PD0325901, and a GSK-3b inhibitor, CHIR99021) mimic pluripotent epiblasts in preimplantation blastocysts. Tfcp2l1 is a transcription factor highly expressed in ESCs grown in the 2i medium, suggesting that it plays in important role in ESC biology. To search for the putative role(s) of Tfcp2l1 in ESCs, we plan to use an integrated genomic approach--combing RNA-seq and ChIP-seq--to identify the direct targets of Tfcp2l1 in ESCs. ChIP-seq is used to identify the genomic bindings sites of Tfcp2l1 in ESCs grown in the 2i medium.

2021-03-01 | GSE159877 | GEO

Activin A and BMP4 Signaling Expands Potency of Mouse Embryonic Stem Cells in Serum Free Media

Project description:Inhibitors of Mek1/2 and Gsk3b, known as 2i, and together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show prolonged Mek1/2 suppression impairs the developmental potential of ESCs, and is rescued by serum (S/L-ESCs). In spite of the unclear roles of growth factors in serum, the coculture system has remained the gold standard. Here we show that culturing ESCs in Activin A, BMP4 and in the absence of MEK1/2 inhibitor (ABC/L medium) establishes stable ESCs, named advanced pluripotent stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full term chimeras and generated esASCs-derived mice by tetraploid complementation. We show that in contrast to 2i/L-ESCs, these esASCs display distinct molecular signatures and stable hypermethylated epigenome which is reversible and similar to S/L-ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. These blASCs are persisting at an intermediate state between naïve and primed state of pluripotency and stable hypermethylated epigenome. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in a chemically defined medium.

2020-01-07 | GSE119985 | GEO

Naïve pluripotency is associated with global DNA hypomethylation

2013-02-17 | GSE43398 | GEO

Comparison of transcriptional profiles between naive, primed and ground state pluripotency in mouse embryonic stem cells of the same genetic background

Project description:Derivation of naive state of mouse embryonic stem cells (mESCs) in LIF+serum (LS) culture condition is strain dependent, whereas derivation of ground state mESCs is readily possible from all strains tested so far in “2i” culture condition. ESCs can be derived from the post-implantation stage mouse embryos (EpiSCs), showing primed characteristics. In the present study, we characterized and compared the transcriptional profile of naïve, primed and ground state mESCs. Considering the importance of genetic background of mouse model for ESCs derivation in conventional culture conditions, all ESCs lines used in the study were derived from the same strain of mice. We found distinct transcriptional profiles between naive, primed and ground state mESCs. Primed state mESCs exhibit lower expression of pluripotency markers along with higher expression of lineage specific markers compared to naive and ground state mESCs. We also demonstrate that the differentiation propensity of ESCs to specific germ layer varies depending on the pluripotency state of ESCs.

2018-04-12 | GSE78144 | GEO

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