RNAseq and ChIPseq on Mouse Embryonic Stem Cells and Sorted Mesendoderm Progenitor Cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE103263 | GEO | 2017/10/10
SECONDARY ACCESSION(S): PRJNA400639
REPOSITORIES: GEO
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Project description:We mapped the enhancer and long non-coding transcriptional landscape during mesendoderm specification. Mesendodermal progenitors were sorted from differentiating ESCs according to Eomes expression. Enhancer usage was coordinated with mesendoderm-specific expression of key lineage-determining transcription factors. We demonstrated that many of these enhancers are associated with the expression of lncRNAs.
2017-10-10 | GSE103262 | GEO
Project description:We mapped the enhancer and long non-coding transcriptional landscape during mesendoderm specification. Mesendodermal progenitors were sorted from differentiating ESCs according to Eomes expression. Enhancer usage was coordinated with mesendoderm-specific expression of key lineage-determining transcription factors. We demonstrated that many of these enhancers are associated with the expression of lncRNAs.
2017-10-10 | GSE103261 | GEO
Project description:RNAseq and ChIPseq on Mouse Embryonic Stem Cells and Sorted Mesendoderm Progenitor Cells
| PRJNA400639 | ENA
Project description:Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3' untranslated region (3' UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.
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Project description:We developed an allele-specific assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to genotype and profile active regulatory DNA across the genome. Using a mouse hybrid F1 system, we found that monoallelic DNA accessibility across autosomes was pervasive, developmentally programmed and composed of several patterns. Genetically determined accessibility was enriched at distal enhancers, but random monoallelically accessible (RAMA) elements were enriched at promoters and may act as gatekeepers of monoallelic mRNA expression. Allelic choice at RAMA elements was stable across cell generations and bookmarked through mitosis. RAMA elements in neural progenitor cells were biallelically accessible in embryonic stem cells but premarked with bivalent histone modifications; one allele was silenced during differentiation. Quantitative analysis indicated that allelic choice at the majority of RAMA elements is consistent with a stochastic process; however, up to 30% of RAMA elements may deviate from the expected pattern, suggesting a regulated or counting mechanism.
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Project description:SummaryShort reads sequencing technology has been used for more than a decade now. However, the analysis of RNAseq and ChIPseq data is still computational demanding and the simple access to raw data does not guarantee results reproducibility between laboratories. To address these two aspects, we developed SeqBox, a cheap, efficient and reproducible RNAseq/ChIPseq hardware/software solution based on NUC6I7KYK mini-PC (an Intel consumer game computer with a fast processor and a high performance SSD disk), and Docker container platform. In SeqBox the analysis of RNAseq and ChIPseq data is supported by a friendly GUI. This allows access to fast and reproducible analysis also to scientists with/without scripting experience.Availability and implementationDocker container images, docker4seq package and the GUI are available at http://www.bioinformatica.unito.it/reproducibile.bioinformatics.html.Contactbeccuti@di.unito.it.Supplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Neural crest is a source of diverse cell types, including the peripheral nervous system. The transcription factor Sox10 is expressed throughout early neural crest. We exploited Sox10 reporter and selection markers created by homologous recombination to investigate the generation, maintenance and expansion of neural crest progenitors. Sox10-GFP-positive cells are produced transiently from mouse embryonic stem (ES) cells by treatment with retinoic acid in combination with Fgf8b and the cytokine leukaemia inhibitory factor (Lif). We found that expression of Sox10 can be maintained using noggin, Wnt3a, Lif and endothelin (NWLE). ES cell-derived Sox10-GFP-positive cells cultured in NWLE exhibit molecular markers of neural crest progenitors. They differentiate into peripheral neurons in vitro and are able to colonise the enteric network in organotypic gut cultures. Neural crest cells purified from embryos using the Sox10 reporter also survive in NWLE, but progressively succumb to differentiation. We therefore applied selection to eliminate differentiating cells. Sox10-selected cells could be clonally expanded, cryopreserved, and multiplied for over 50 days in adherent culture. They remained neurogenic in vitro and in foetal gut grafts. Generation of neural crest from mouse ES cells opens a new route to the identification and validation of determination factors. Furthermore, the ability to propagate undifferentiated progenitors creates an opportunity for experimental dissection of the stimuli and molecular circu that govern neural crest lineage progression. Finally, the demonstration of robust enteric neurogenesis provides a system for investigating and modelling cell therapeutic approaches to neurocristopathies such as Hirschsprung's disease.
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Project description:TGF-β family proteins including Nodal are known as central regulators of early development in metazoans, yet our understanding of the scope of Nodal signaling's downstream targets and associated physiological mechanisms in specifying developmentally appropriate cell fates is far from complete. Here, we identified a highly conserved, transmembrane micropeptide-NEMEP-as a direct target of Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells (mESCs), and this micropeptide is essential for mesendoderm differentiation. We showed that NEMEP interacts with the glucose transporters GLUT1/GLUT3 and promotes glucose uptake likely through these interactions. Thus, beyond expanding the scope of known Nodal signaling targets in early development and showing that this target micropeptide augments the glucose uptake during mesendoderm differentiation, our study provides a clear example for the direct functional impact of altered glucose metabolism on cell fate determination.
| S-EPMC9271079 | biostudies-literature
Project description:BACKGROUND:Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. METHODS:We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. RESULTS:Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors. CONCLUSIONS:The methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.
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Project description:MicroRNAs (miRNAs) belonging to the evolutionary conserved miR-302 family play important functions in Embryonic Stem Cells (ESCs). The expression of some members, such as the human miR-302 and mouse miR-290 clusters, is regulated by ESC core transcription factors. However, whether miRNAs act downstream of signaling pathways involved in human ESC pluripotency remains unknown. The maintenance of pluripotency in hESCs is under the control of the TGF? pathway. Here, we show that inhibition of the Activin/Nodal branch of this pathway affects the expression of a subset of miRNAs in hESCs. Among them, we found miR-373, a member of the miR-302 family. Proper levels of miR-373 are crucial for the maintenance of hESC pluripotency, since its overexpression leads to differentiation towards the mesendodermal lineage. Among miR-373 predicted targets, involved in TGF? signaling, we validated the Nodal inhibitor Lefty. Our work suggests a crucial role for the interplay between miRNAs and signaling pathways in ESCs.
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