Transcriptome of ESCs, EpiSCs and reverted ES by MM401 (Mll1 inhibitor) #2
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE73993 | GEO | 2016/05/21
SECONDARY ACCESSION(S): PRJNA298683
REPOSITORIES: GEO
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Project description:This study describes the transcriptome profiling of: 1) mouse ES cells and EpiSCs in LIF/serum-free (KSR) medium; 2) E14Tg2a (E14) ES cells in LIF/Serum with or without MM401 treatment; 3) rES reverted form EpiSC by MM401/LIF KSR treatment at P6, P30. RNA-Seq profiling on mouse pluripotent cells. Biological duplicates of each sample are labled as rep1/2.
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2014-12-24 | E-GEOD-62205 | 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.
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Project description:Epiblast stem cells (EpiSCs) were derived from the epiblast or the ectoderm (epi/ect) of pre-gastrula stage to late-bud stage mouse embryos. To identify if the EpiSCs retain any original stage specific characteristics or which developmental stage of epi/ect they most closely related to, we performed microarray analysis to compare the gene expression profile of multiple EpiSC lines with that of epi/ect of 7 different stages.
2013-09-15 | GSE46227 | GEO
Project description:This study describes the transcriptome profiling of: 1) Mll1fl/fl mESCs in LIF/Serum medium as control; 2) Mll1fl/fl mESCs with 4-OHT (Mll1-/-), MM-401 (Mll1i) in LIF/serum medium ; 2) Mll1fl/fl mESCs with 2i in LIF/serum-free (KSR) medium.
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Project description:We compared the transcriptomes of EpiSCs maintained in the presence or absence of Wnt pathway inhibitor IWP2. We screened also our gene expression data for potential markers for genuine EpiSCs, maintained in the presence of Wnt inhibition and compared with ESC expression data. We compared the transcriptomes of EpiSCs maintained in the presence or absence of IWP2. The high level of Wnt-induced differentiation occurring in conventional EpiSC cultures may have interfered with the analysis of their characteristics. By applying Wnt inhibitors we are now able to establish the properties of genuine EpiSCs.
2014-12-24 | GSE62155 | 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
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