A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity (BeadChip)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE58656 | GEO | 2016/03/31
SECONDARY ACCESSION(S): PRJNA253113
REPOSITORIES: GEO
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Project description:A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity
| PRJNA319265 | ENA
Project description:Analysis of genes involved in the mESC maintainance between different conditions of growth. The hypothesis tested in the present study was that Myc can sustain mESC self renewal and pluripotency. Results provide important information on the mechanism by which Myc supports mESC self renewal, in comparison with the routinely used LIF+serum culture condition, such as a Myc-dependent specific transcriptional program, which involves induction of an alternative Core Regulatory Network, modulation of signallign pathways (Wnt/bcat) and repression of developmental genes. Total RNA obtained from mESC growth 3 days in the indicated condition compared to the routinely used LIF+serum culture condition. Total RNA obtained from EpiSC growth 3 days in standard condition were also used as control for primed stem cell state.
2016-03-31 | E-GEOD-58656 | biostudies-arrayexpress
Project description:Epiblast stem cells (EpiSC) are pluripotent stem cells derived from mouse postimplantation embryos between E5.5 to E7.5, a time window in which gastrulation commences. Therefore, EpiSC represent a valuable tool for studying mammalian postimplantation development in vitro. Beyond their pluripotent features, EpiSC can also be reprogrammed into a mouse embryonic stem cell-like (mESC) state. Published reports showed that EpiSC reversion requires transcription factor overexpression, while others suggest that applying stringent mESC culture conditions alone was sufficient to revert EpiSC to mESC-like cells. In an effort to clarify these discrepancies, we systematically compared a panel of independent EpiSC lines. Indeed, the different lines shared a number of defining EpiSC characteristics such as teratoma formation ability, regardless of the day of isolation from the embryo. However, despite being grown in identical, self-renewal-promoting conditions, some lines displayed strong expression of genes associated with mesendodermal differentiation. Strikingly, these same lines were not revertable to a mESC-like state by applying stringent mESC culture conditions. Moreover, expression of mesendodermal marker genes correlated in a negative manner with the ability to efficiently undergo neural induction. Overall, our data suggest that different EpiSC lines may self-renew in distinct developmental ground states, which has important consequences for functional readouts such as revert ability to a mESC-like state and directed differentiation. For EpiSC lines derivation E5.5 embryos were plated onto irradiated mouse embryonic feeder cells (MEF cells) seeded at low density. Outgrowths were picked and expanded manually. Basal medium consisted of Knock-Out-DMEM, 20% Knock-Out Serum Replacement, Non Essential Amino Acids, L-Glutamine and beta-Mercaptoethanol (all Gibco). For cultivation on feeders 5ng/ml bFGF (Peprotech) was added to the medium to yield EpiSC medium. For preparation of mouse embryonic fibroblast-conditioned medium, EpiSC medium incubated on inactivated MEFs, followed by addition of another 5ng/ml bFGF (CM+). For feeder free culture, EpiSCs were seeded onto FCS-coated dishes. EpiSC were routinely passaged using 1mg/ml Collagenase IV (Invitrogen). Mouse ESC were cultured in standard conditions - Knock-Out-DMEM, 15% Knock-Out Serum Replacement (both Gibco), 5% fetal calf serum (Biowest), Non Essential Amino Acids, L-Glutamine and beta-Mercaptoethanol (all Gibco), supplemented with either homemade or commercially available LIF (ESGRO) at 1000U/ml. Some experiments were carried out in chemically defined N2B27 medium 11 in the presence of 10ng/ml bFGF. For reversion experiments, EpiSC were harvested using Accutase (Invitrogen) and seeded as single cells at approximately 16.000 cells / well of a 6-well plate (Sarstedt) in CM+. After 24h medium was changed to PCL medium (basal medium, LIF (1000U/ml), PD0325901 (Axon Medchem 1µM), and CHIR99021 (Axon Medchem 3µM). After 48h treatment, reversion medium was further supplemented with 10µM SB431542 (Sigma) to eliminate remaining EpiSC.
2012-08-10 | GSE28270 | GEO
Project description:Embryonic stem cells (ESCs), which are derived from the primitive ectoderm of pre-implantation blastocysts, are pluripotent cells and can thus contribute to the formation of all somatic cell lineages in chimeric animals. Similarly, epiblast stem cells (EpiSCs), which are derived from epiblast tissue of post-implantation embryos, are also pluripotent and can give rise to derivatives of all three germ layers in teratoma assays. Introduction of the four transcription factors Oct4/Sox2/Klf4/c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs). iPSCs exhibit ESC-like properties and are virtually identical to ESCs with respect to a number of charactertistics. However, generation of EpiSC-like cells by direct reprogramming of somatic cells using these transcription factors has not been shown to date. Here we show that Yamanaka’s four transcription factors can be used to directly generate EpiSC-like iPS cells (ePSCs) under EpiSC culture conditions. These ePSCs are identical to EpiSCs with respect to morphology, gene expression pattern, epigenetic status, and chimera formation. This is the first study to demonstrate that the culture environment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We could therefore envision that we eventually are able to shape the identity of a directly reprogrammed cell at will simply by modulating the culture conditions.
2010-12-08 | GSE21516 | GEO
Project description:Embryonic stem cells (ESCs), which are derived from the primitive ectoderm of pre-implantation blastocysts, are pluripotent cells and can thus contribute to the formation of all somatic cell lineages in chimeric animals. Similarly, epiblast stem cells (EpiSCs), which are derived from epiblast tissue of post-implantation embryos, are also pluripotent and can give rise to derivatives of all three germ layers in teratoma assays. Introduction of the four transcription factors Oct4/Sox2/Klf4/c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs). iPSCs exhibit ESC-like properties and are virtually identical to ESCs with respect to a number of charactertistics. However, generation of EpiSC-like cells by direct reprogramming of somatic cells using these transcription factors has not been shown to date. Here we show that Yamanaka’s four transcription factors can be used to directly generate EpiSC-like iPS cells (ePSCs) under EpiSC culture conditions. These ePSCs are identical to EpiSCs with respect to morphology, gene expression pattern, epigenetic status, and chimera formation. This is the first study to demonstrate that the culture environment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We could therefore envision that we eventually are able to shape the identity of a directly reprogrammed cell at will simply by modulating the culture conditions. RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates. 24 samples were analyzed: MEF: Female mouse embryonic fibroblast, 2 biological rep ESC OG2: OG2 female embryonic stem cell, 2 biological rep EpiSC OG2: OG2 female EpiSC grown as single cells with conditioned medium, 2 biological rep EpiSC GOF18: GOF18 EpiSC grown as single cells with conditioned medium, 2 biological rep EpiSC T9b: T9 EpiSC on MEFs in activin-containing CDM, harvested w/o feeders, subconfluent, 2 biological rep ePSC L4: EpiSC-like iPS Cells L4, MEF reprogrammed to EpiSCs with 4F, line L4, grown with LIF and activin, 2 biological rep ePSC L7: EpiSC-like iPS cells L7, MEF reprogrammed to EpiSCs with 4F, line L7, grown with LIF and activin, 2 biological rep ePSC C1: EpiSC-like iPS Cells C1, MEF reprogrammed to EpiSCs with 4F, line C1, grown with LIF and activin, 2 biological rep ePSC C3: EpiSC-like iPS Cells C3, MEF reprogrammed to EpiSCs with 4F, line C3, grown with LIF and activin, 2 biological rep ePSC-R: EpiSC-like iPS Cells reverted, L4 ePSC cells reverted to an ESC-like state using Klf4 virus, 2 biological rep ePSC-RC1: EpiSC-like iPS Cells reverted, L4 ePSR Cre1, Cre-treated L4 ePSR cells, line 1, 2 biological rep ePSC-RC2: EpiSC-like iPS Cells reverted, L4 ePSR Cre2, Cre-treated L4 ePSR cells, line 2, 2 biological rep
2010-12-08 | E-GEOD-21516 | biostudies-arrayexpress
Project description:We profiled gene expression and splicing changes in MCF-10A human mammary epithelial cells expressing MYC fused to a portion of estrogen receptor (MYC-ER) (Eilers et al., 1989; Littlewood et al., 1995). We performed RNA-seq, in triplicate, on 3D-grown MCF-10A MYC-ER cells at 0, 8, and 24 hours (h) after 4-OHT-induced MYC activation. As a control for 4-OHT-induced effects, 3D-grown parental MCF-10A cells lacking the MYC-ER fusion protein were treated with 4-OHT at the same timepoints.
2022-07-11 | GSE181968 | GEO
Project description:Myc is a master transcription factor that has been demonstrated to be required for embryonic stem cell (ESC) pluripotency, self-renewal, and inhibition of differentiation. Although recent works identified several Myc-targets in ESC the list of Myc binding sites is largely incomplete due to the low sensitivity and specificity of the antibodies available so far. To systematically identify Myc binding sites in mouse ESCs here we used a stringent streptavidin based genome-wide chromatin immunoprecipitation (ChIP-Seq) of a biotin-tagged Myc (Bio-Myc) as well as a ChIP-Seq of the Myc partner Max. This analysis identified 4273 Myc binding sites of which more than 85% co-occupied by Max, overlap with H3K4me3 positive promoters and active enhancers of transcriptional regulators, chromatin modifiers, and genes involved in stem cell self-renewing. The new sites identified were validated experimentally. This study provides a new Myc and Max binding reference in mouse ESCs. ChIP-seq of bio-Myc and Max in E14 and respective controls; Bio-Myc ChIP-seq was performed in a stable clone of E14 mouse embryonic stem cell expressing Biotin-tagged Myc; Mock, Max and IgG were performed in parental wt E14 mESC.
2014-03-05 | E-GEOD-48175 | biostudies-arrayexpress
Project description:To characterize molecular features of new EpiSC lines established by the Wnt inhibition method, global gene expression profiles of the EpiSC lines were determined by microarray, and compared to those of EpiSC lines established by other group using the conventional method. Epiblasts, the source of the EpiSCs, and mESCs were also analyzed. EpiSCs and mESC were maintained as undifferentiated state on feeder layers. The stem cells were then separated from feeders, and RNAs were extracted from the stem cell lines. Embryonic tissues were manually dissected out from mouse embryos of E5.5, E6.5 or E7.5, from which RNAs were extracted.
2014-08-01 | E-GEOD-58735 | biostudies-arrayexpress
Project description:The comparison of mouse ESCs and ESD-EpiSCs was done using three biological replicates for each cell type. ESC samples (ESR1 p10, p20, ESR1/E p15) and ESD-EpiSC samples (EpiSC-7 p20, EpiSC-5 p22, and EpiSC-7 p25) were used for microarray analysis. Total RNA was extracted using Trizol and labeled and hybridized to Agilent Whole Mouse Genome Oligo 4X44K Microarrays (one-color platform) according to the manufacturers protocols.
2010-05-12 | E-MEXP-2691 | biostudies-arrayexpress
Project description:Macrophages change their phenotype in response to complex (reinforcing or opposing) environmental cues. The molecular components providing the epigenomic basis of progressive or reinforcing polarization are largely unknown. Here we show that the normally ligand-activated nuclear receptor, Peroxisome Proliferator-Activated Receptor gamma (PPARγ), is a predominantly ligand-insensitive, epigenomic driver of a robust phenotypic change in macrophages uniquely upon repeated/reinforcing IL-4 stimulation. Ligand-insensitive PPARγ recruits P300, RAD21 and establishes a permissive chromatin environment, conferring transcriptional memory by facilitating the binding of STAT6 and RNAPII leading to more robust eRNA production upon IL-4 restimulation. PPARγ-mediated transcriptional memory allows the progressive, functional transition of macrophages upon repeated cytokine exposure by controlling the expression of an extracellular matrix remodeling related gene network, also expressed in macrophages from a mouse model of muscle regeneration and show progressive upregulation during the course of regeneration after muscle injury, coinciding with the appearance of IL-4 and PPARγ. Collectively, in contrast to the current prevailing view, alternative polarization gives rise to a predominantly ligand-insensitive PPARγ:RXR cistrome, regulating progressive/reinforcing macrophage polarization.
2018-06-20 | GSE115502 | GEO