Guided differentiation of mouse embryonic stem cell
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE3528 | GEO | 2005/11/01
SECONDARY ACCESSION(S): PRJNA104825
REPOSITORIES: GEO
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Project description:During in vitro differentiation, pluripotent stem cells undergo extensive remodeling of their gene expression profiles. While studied extensively at the transcriptome level, much less is known about protein dynamics, which might differ significantly from their mRNA counterparts. Here, we present deep proteome-wide measurements of protein levels during the differentiation of embryonic stem cells.
2017-01-10 | MSV000080461 | MassIVE
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Project description:Mechanistic Model-Guided Study of Embryonic Morphogenesis
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Project description:During mammalian pre-implantation development, the cells of the blastocyst’s inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived extra-embryonic endoderm (XEN) cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and extra-embryonic endoderm differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the re-organization of membrane trafficking machinery and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.
2015-06-13 | PXD001901 | Pride
Project description:The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used <sup>1</sup>H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that <sup>1</sup>H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.
2023-05-24 | MTBLS4854 | MetaboLights
Project description:During embryogenesis, many key transcription factors are used repeatedly, achieving different outcomes depending on cell type and developmental stage. The epigenetic modification of the genome functions as a memory of a cell’s developmental history, and it has been proposed that such modification shapes the cellular response to transcription factors. To investigate the role of DNA methylation in the response to transcription factor Gata4, we examined expression profiles of Dnmt3a-/-Dnmt3b-/- ES cell-derived mesoderm cells cultured for 4 days with or without Gata4 activation, as well as the wild-type counterparts, using Affymetrix microarrays. Wild-type and Dnmt3a-/-Dnmt3b-/- (DKO) ES cell clones stably expressing dexamethasone (Dex)-inducible Gata4 were generated by introducing an expression plasmid for Gata4 fused with the ligand-binding domain of the human glucocorticoid receptor (Gata4GR) driven by the CAG promoter, by electroporation. For mesoderm differentiation, 1 x 10^5 ES cells were plated on a type IV collagen-coated 10-cm dish and cultured for 4 days. Cultured cells were harvested and collected as single-cell suspensions using 0.25% trypsin-EDTA, followed by incubation in differentiation medium at 37˚C to allow cell-surface markers to recover. Flk1(+)/PDGFRalpha(+) double-positive (DP) cells, which are considered equivalent to the lateral plate mesoderm, were sorted by FACS-Aria (BD Biosciences). Sorted DP cells were further cultured on type IV collagen-coated culture dishes in the absence or presence of 100 nM Dex for 4 days. For reference data, primitive endoderm (PE) cells were directly differentiated from undifferentiated ES cells by addition 100 nM Dex in ES cell-maintenance medium.
2013-06-24 | E-GEOD-36814 | biostudies-arrayexpress
Project description:Nuclear pore complexes (NPCs) are established players in cell division and differentiation. However studies on the contribution of individual NPC subunits to these processes are still scarce. Here we have used mouse embryonic stem cells (mESCs) to characterize the role of structural components of the NPCs, focusing on the short arm of the Y-complex that comprises Nup85, Seh1 and Nup43. We show that Seh1 and Nup43, although dispensable at the pluripotent stage, are required for normal cell growth rates at that stage and for mESC viability upon differentiation. Lack of Seh1 or Nup43 in mESCs is associated with a mild reduction of NPC density that is also observed when Seh1 interaction with Nup85 is impaired. Nevertheless, mESC proliferation and differentiation are not altered in these ∆E2-GFP-Nup85 mutants, indicating that it is the integrity of the Y-complex, rather than the number of NPCs, that is critical to ensure these processes.
2021-05-20 | PXD022190 | Pride
Project description:Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short long, and paired-end sequencing In order to examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of human embryonic stem cells (hESCs) into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation, N1 (early initiation), N2 (neural progenitor), and N3 (early glial-like) were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call “isoform specialization.” During neural differentiation, we observed differential expression of many types of genes including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, NPC identity maintenance and the transition from a predominantly neuronal state into one with increased gliogenic potential
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Project description:Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of non-coding ORFs during differentiation, we mapped new functions to well annotated long non-coding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.
2019-07-23 | PXD004652 | Pride
Project description:Investigate the genome-wide DNA methylation and gene expression changes during human embryonic stem cell differentiation. hESCs (H1 and H13) DNA samples for 3 different differentian stage (D0, undifferentiated;D21, 21 days after undirected differentiation; D90, 90 days after undirected differentiation); two-color experiment, paired comparison (differentiated vs undifferentiated; A & B: two biological replicates). Differentian-stage variation in methylation profiling of human DNA samples were detected by MSAM. 2µg of DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification. Analysis of differentian-stage variation in gene expression of human mRNA samples were implementd following standard Agilent protocol.
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