Global gene expression profiling reveals similarities and differences among mouse pluripotent stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE5914 | GEO | 2007/05/11
SECONDARY ACCESSION(S): PRJNA97355
REPOSITORIES: GEO
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Project description:We present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6). Multiple ESC and EGC cell lines were cultured without feeders on gelatin coated, 6-well plates, 100,000 cells/well (10,000 cells/cm2) in 3 conditions: 1) in complete ES medium: DMEM, 15% FBS; LIF (ESGRO) 1000 u/ml; 1mM sodium pyruvate; 0.1 mM NEAA, 2 mM glutamate, 0.1 mM beta-mercapto ethanol, and penicillin/streptomycin (50U/50ug per ml); 2) in ES medium without LIF; 3) in complete ES medium plus 1 uM RA. Cells were incubated at 37 0C; 5% CO2. Medium was changed daily. On the day 3 Trizol was added, RNA extracted using Phase lock gelTM (Eppendorf) columns according to manufacturer protocol. RNA was precipitated with isopropanol, washed with 70% ethanol and dissolved in DEPC dH2O.
2010-06-11 | E-GEOD-5914 | biostudies-arrayexpress
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
Project description:To examine the mechanisms by which EGCs affect the immune CRC compartment with particular regard to the TAMs, we first investigated their transcriptional adaptations upon CRC onset. To this end, we established an in vitro setup to study EGC-CRC interactions able to mimic the response of EGCs to the factors secreted by the TME. These EGCs, from now onwards defined as TME-conditioned medium-treated EGCs (TME-CM EGCs), were generated by stimulating primary EGCs with the CM of digested murine MC38 orthotopic tumor tissues (Figure 2A). Bulk RNA sequencing (RNA-seq) was performed to determine transcriptional differences in TME-CM EGCs, compared to unstimulated and healthy conditioned medium-treated (H-CM) primary EGCs, at different time points.
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Project description:Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs. Cell fusion experiments further reveal that the ratio of X-chromosomes to autosomes dictates methylation levels, with female hybrids being hypomethylated and male hybrids being hypermethylated. We show that the X-linked MAPK phosphatase DUSP9 is upregulated in female compared to male ESCs, and its heterozygous loss in female ESCs leads to male-like methylation levels. Notably, male and female blastocysts are similarly hypomethylated, indicating that sex-specific methylation differences arise in culture. Collectively, our data demonstrate the epigenetic similarity of sex-matched ESCs and EGCs and identify DUSP9 as a regulator of female-specific hypomethylation.
2017-05-18 | GSE94481 | GEO
Project description:Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs. Cell fusion experiments further reveal that the ratio of X chromosomes to autosomes dictates methylation levels, with female hybrids being hypomethylated and male hybrids being hypermethylated. We show that the X-linked MAPK phosphatase DUSP9 is upregulated in female compared to male ESCs, and its heterozygous loss in female ESCs leads to male-like methylation levels. However, male and female blastocysts are similarly hypomethylated, indicating that sex-specific methylation differences arise in culture. Collectively, our data demonstrate the epigenetic similarity of sex-matched ESCs and EGCs and identify DUSP9 as a regulator of female-specific hypomethylation.
2017-04-12 | GSE68733 | GEO
Project description:Porcine embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibilities to establish porcine EGC lines in the domestic breed pig more efficiently and from earlier embryonic stages than reported to date. In vitro culture of PGC from both pooled and individual embryos at days 17-24 of gestation resulted in the successful derivation of putative EGC lines from days 20-24 with high efficiency, while no lines could be established from days 17-18. The EGC-like colonies had characteristic morphology and electron microscopy revealed tight junctions and presence of primary cilia on the cell surfaces. The cells formed simple embryoid bodies in suspension culture and further differentiated into epithelial-like, mesenchymal-like, and neuronal-like cells. Our results show that putative porcine EGC can be derived from migrating PGC with high efficiency using individual embryos from different genetic backgrounds.
2011-03-24 | GSE24220 | GEO
Project description:Porcine embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibilities to establish porcine EGC lines in the domestic breed pig more efficiently and from earlier embryonic stages than reported to date. In vitro culture of PGC from both pooled and individual embryos at days 17-24 of gestation resulted in the successful derivation of putative EGC lines from days 20-24 with high efficiency, while no lines could be established from days 17-18. The EGC-like colonies had characteristic morphology and electron microscopy revealed tight junctions and presence of primary cilia on the cell surfaces. The cells formed simple embryoid bodies in suspension culture and further differentiated into epithelial-like, mesenchymal-like, and neuronal-like cells. Our results show that putative porcine EGC can be derived from migrating PGC with high efficiency using individual embryos from different genetic backgrounds. RNA-Seq profiling of 2 different in vitro cultures of pig embryonic cells. The cells, both the pig Embryonic Germ cells (pEGCs) and the pig Fetal Fibroblasts (pFF) show properties of pluripotency and self renewal.
2011-03-24 | E-GEOD-24220 | biostudies-arrayexpress
Project description:To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). We found 11 genes ≥1.5-fold overexpressed in EGCs. None of the genes identified were Yamanaka's genes, but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGL), which differentiate into cells of the three germ layers in vitro, and contribute to the ICM in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1? stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signalling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. The data suggests that the properties of stemness, pluripotency and selfrenewal, are differentially regulated in primordial germ cell reprogramming induced by hypoxia. 8.5 dpc Oct4-GFP PGCs cultured in hypoxia conditions for 6 days were isolated by FACS. To eliminate the possibility of differences due solely to anaerobic respiration, TGC-10 EGC line was exposed to the hypoxia-mimetic agent Cl2Co at 150 microM for four hours prior to RNA isolation. RNA samples to be analyzed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Tetu-Bio), which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer's instructions. After washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of RBioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clustering of genes and samples was performed with one minus correlation metric and the unweighted average distance (UPGMA) (also known as group average) linkage method. 14 samples were analyzed EG: pluripotent Embryonic Germ Cells (EGCs), 2 replicates hiEGL1: hypoxia-induced EGC-like cells (hiEGL), 2 replicates hiEGL2: hypoxia-induced EGC-like cells (hiEGL), 2 replicates ESCm: Embryonic Stem Cells (ESCs) male, 2 replicates ESCf: Embryonic Stem Cells (ESCs) female, 2 replicates EpiT9: Epiblast Stem Cells T9, 1 replicate EpiE3: Epiblast Stem Cells E3, 1 replicate PGC11.5: unipotent Primordial Germ Cells (PGCs) from dpc 11.5 , 2 replicates
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Project description:The species Staphylococcus (S.) aureus harbors 19 superantigen gene loci, six of which are located in the enterotoxin gene cluster (egc). While these egc superantigens are far more prevalent in clinical S. aureus isolates than non-egc superantigens, they are not a prominent cause of toxic shock. Moreover, neutralizing antibodies against egc superantigens are very rare, even among carriers of egc-positive S. aureus strains. In search of an explanation we have tested two non-exclusive hypotheses: 1) egc and non-egc superantigens have unique intrinsic properties and drive the immune system into different directions; 2) egc and non-egc-superantigens are released by S. aureus under different conditions, which shape the immune response. A comparison of three egc (SEI, SElM and SElO) and three non-egc superantigens (SEB, SElQ, TSST-1) revealed that both induced proliferation of human PBMC with comparable potency and elicited similar Th1/Th2-cytokine signatures. This was supported by gene expression analysis of PBMC stimulated with one representative superantigen from each group (SEI and SEB). They induced very similar transcriptional changes, especially of inflammation-associated gene networks, corresponding to a very strong Th1- and Th17-dominated immune response. In contrast, the regulation of superantigen release differed markedly between both superantigen groups. Egc-encoded proteins were secreted by S. aureus during exponential growth, while non-egc superantigens were released in the stationary phase. We conclude that the distinct biological behavior of egc and non-egc superantigens is not due to their intrinsic properties, which are very similar, but caused by their differential release by S. aureus.
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