Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Serum-Based Culture Conditions Provoke Gene Expression Variability in Mouse Embryonic Stem Cells as Revealed by Single-Cell Analysis.

ABSTRACT: 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. We define highly variable gene clusters with distinct chromatin states and 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 naive 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.

SUBMITTER: Guo G

PROVIDER: S-EPMC4740311 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Serum-Based Culture Conditions Provoke Gene Expression Variability in Mouse Embryonic Stem Cells as Revealed by Single-Cell Analysis.

Guo Guoji G Pinello Luca L Han Xiaoping X Lai Shujing S Shen Li L Lin Ta-Wei TW Zou Keyong K Yuan Guo-Cheng GC Orkin Stuart H SH

Cell reports 20160121 4

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. We define highly variable gene clusters with distinct chromatin states and show that bivalent genes are prone to expression variation. At the same time, we identify an ESC-primin ...[more]

PMID: 26804902

Similar Datasets

Serum-based culture conditions provoke gene expression variability in mouse embryonic stem cells as revealed by single cell analysis

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 naÃ¯ve 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

Serum-based culture conditions provoke gene expression variability in mouse embryonic stem cells as revealed by single cell analysis

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.

2015-12-09 | GSE75804 | GEO

Totipotent embryonic stem cells arise in ground-state culture conditions.

Project description:Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.

| S-EPMC3701323 | biostudies-literature

Direct effect of chenodeoxycholic acid on differentiation of mouse embryonic stem cells cultured under feeder-free culture conditions.

Project description:Chenodeoxycholic acid (CDCA), a farnesoid X receptor (FXR) ligand, is a member of the nuclear receptor family and is probably involved in regulating the cellular activities of embryonic stem (ES) cells. Recently, although it was reported that the FXR ligand can mediate differentiation, apoptosis, and/or growth arrest in several cell types, it is still not well known how CDCA mediates effects in ES cells. Therefore, we investigated the direct effect of CDCA on mES cells. Feeder-free mES cells were treated in a dose-dependent manner with CDCA (50, 100, and 200? ?M) for 72?h, and then a 100? ?M CDCA treatment was performed for an additional 72?h. We analyzed the morphology, cell growth, cell characteristics, immunocytochemistry, and RT-PCR. In CDCA-treated cells, we observed the disappearance of pluripotent stem cell markers including alkaline phosphatase, Oct4, and Nanog and a time- and dose-dependent increase in expression of nestin, PAX6, and ?-smooth muscle actin, but not ?-fetoprotein. The 100? ?M CDCA-treated cells in their second passage continued this differentiation pattern similar to those in the controls. In conclusion, these results suggest that CDCA can guide mES cells by an FXR-independent pathway to differentiate into ectoderm and/or mesoderm, but not endoderm.

| S-EPMC3591134 | biostudies-literature

Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition.

Project description:The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products.

| S-EPMC2559871 | biostudies-literature

3D mouse embryonic stem cell culture for generating inner ear organoids.

Project description:This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16-20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.

| S-EPMC6453571 | biostudies-literature

Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells.

Project description:Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.

| S-EPMC8155104 | biostudies-literature

Short-term serum-free culture reveals that inhibition of Gsk3β induces the tumor-like growth of mouse embryonic stem cells.

Project description:Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3β. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3β, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3β in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3β governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.

| S-EPMC3121758 | biostudies-literature

Defined conditions for propagation and manipulation of mouse embryonic stem cells.

Project description:The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.

| S-EPMC6451320 | biostudies-literature

Stochastic promoter activation affects Nanog expression variability in mouse embryonic stem cells.

Project description:Mouse embryonic stem cells (mESCs) are self-renewing and capable of differentiating into any of the three germ layers. An interesting feature of mESCs is the presence of cell-to-cell heterogeneity in gene expression that may be responsible for cell fate decisions. Nanog, a key transcription factor for pluripotency, displays heterogeneous expression in mESCs, via mechanisms that are not fully understood. To understand this variability, we quantitatively analyzed Nanog transcription and found that Nanog was both infrequently transcribed, and transcribed in a pulsatile and stochastic manner. It is possible that such stochastic transcriptional activation could contribute to the heterogeneity observed in Nanog expression as "intrinsic noise." To discriminate the effects of both intrinsic noise from other (extrinsic) noise on the expression variability of Nanog mRNA, we performed allele-specific single-molecule RNA fluorescent in situ hybridization in a reporter cell line and found that intrinsic noise contributed to approximately 45% of the total variability in Nanog expression. Furthermore, we found that Nanog mRNA and protein levels were well correlated in individual cells. These results suggest that stochastic promoter activation significantly affects the Nanog expression variability in mESCs.

| S-EPMC4238020 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data