Serum-based culture conditions provoke gene expression variability in mouse embryonic stem cells as revealed by single cell analysis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE75804 | GEO | 2015/12/09
SECONDARY ACCESSION(S): PRJNA305407
REPOSITORIES: GEO
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Project description:Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. Using novel computational approaches we define highly variable gene clusters; and reveal their distinct epigenetic characteristics. We show that bivalent genes are prone to expression variation. At the same time, we identify an ESC priming pathway that initiates the exit from the naïve ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum free culture reduces cellular heterogeneity and transcriptome variation in ESCs. Single cell mRNA-seq analysis of ES cells cultured with serum
2015-12-09 | E-GEOD-75804 | biostudies-arrayexpress
Project description:Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra- and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking for GBM and the majority of patients survive less than one year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models to use in target identification and drug screening. The current state of the art method to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 230 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The establishment of maintainable SF cultures (SFCs) was most frequent, but for a subgroup of GBM specimens a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of GSCs, i.e. extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that had evaded modeling under SF conditions provide unique cell models of GBM intertumor heterogeneity.
2019-12-20 | GSE116488 | GEO
Project description:Naïve mouse ESCs exhibit full term developmental competence thus hold great potential in regenerative medicine. Maintaining genome stability is essential for potential application of ESCs in stem cell therapy. While ESC potency fluctuate with retrovirus activity, it is unclear whether long-term cultures affect retrotransposition and genomic stability. We compared retrotransposons in naïve ESCs using various approaches. We show that feeder-serum based culture conditions and small molecule LCDM maintain appropriate expression of retrovirus activity following long-term cultures, whereas cultures under 2iLif and a2i result in aberrant upregulation of retrotransposons. This leads to high frequent retrotransposition. Moreover, naïve ESCs on feeder-serum based culture conditions and small molecule LCDM still generate complete ESC pups by TEC assay, functional test of pluripotency, following long-term cultures, despite that all culture conditions maintain high expression levels of pluripotent genes such as Oct4, Nanog, Klf4 and Sox2. Meanwhile, long telomeres are maintained in ESCs under cultures in Feeder-serum and LCDM conditions but telomeres shortened in other conditions with increasing passages. These data provide insights into retrotransposition, genomic stability and pluripotency of naïve ESCs.
2023-09-12 | GSE157875 | GEO
Project description:Naïve mouse ESCs exhibit full term developmental competence thus hold great potential in regenerative medicine. Maintaining genome stability is essential for potential application of ESCs in stem cell therapy. While ESC potency fluctuate with retrovirus activity, it is unclear whether long-term cultures affect retrotransposition and genomic stability. We compared retrotransposons in naïve ESCs using various approaches. We show that feeder-serum based culture conditions and small molecule LCDM maintain appropriate expression of retrovirus activity following long-term cultures, whereas cultures under 2iLif and a2i result in aberrant upregulation of retrotransposons. This leads to high frequent retrotransposition. Moreover, naïve ESCs on feeder-serum based culture conditions and small molecule LCDM still generate complete ESC pups by TEC assay, functional test of pluripotency, following long-term cultures, despite that all culture conditions maintain high expression levels of pluripotent genes such as Oct4, Nanog, Klf4 and Sox2. Meanwhile, long telomeres are maintained in ESCs under cultures in Feeder-serum and LCDM conditions but telomeres shortened in other conditions with increasing passages. These data provide insights into retrotransposition, genomic stability and pluripotency of naïve ESCs.
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Project description:Naïve mouse ESCs exhibit full term developmental competence thus hold great potential in regenerative medicine. Maintaining genome stability is essential for potential application of ESCs in stem cell therapy. While ESC potency fluctuate with retrovirus activity, it is unclear whether long-term cultures affect retrotransposition and genomic stability. We compared retrotransposons in naïve ESCs using various approaches. We show that feeder-serum based culture conditions and small molecule LCDM maintain appropriate expression of retrovirus activity following long-term cultures, whereas cultures under 2iLif and a2i result in aberrant upregulation of retrotransposons. This leads to high frequent retrotransposition. Moreover, naïve ESCs on feeder-serum based culture conditions and small molecule LCDM still generate complete ESC pups by TEC assay, functional test of pluripotency, following long-term cultures, despite that all culture conditions maintain high expression levels of pluripotent genes such as Oct4, Nanog, Klf4 and Sox2. Meanwhile, long telomeres are maintained in ESCs under cultures in Feeder-serum and LCDM conditions but telomeres shortened in other conditions with increasing passages. These data provide insights into retrotransposition, genomic stability and pluripotency of naïve ESCs.
2023-09-12 | GSE157873 | GEO
Project description: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.
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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
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
Project description:H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in ESCs. However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of SET1/MLL complexes, biochemically modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4me3 throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and H3K4 methylation at key developmental loci. Our results provide strong experimental evidence for the hypothesis that H3K4 methylation is an essential functional component of the bivalent mark during activation of developmental genes in ESCs. Total RNAs from control or knockdown cells before and after RA-mediated differentiation were subjected to Illumina microarray analyses. ChIP-enriched DNA from mouse ES cells was analyzed by Solexa sequencing.
2011-02-01 | E-GEOD-26136 | biostudies-arrayexpress
Project description:H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in ESCs. However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of SET1/MLL complexes, biochemically modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4me3 throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and H3K4 methylation at key developmental loci. Our results provide strong experimental evidence for the hypothesis that H3K4 methylation is an essential functional component of the bivalent mark during activation of developmental genes in ESCs.
2011-02-01 | GSE26136 | GEO