Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse embryonic stem cells in vitro - FunGenES: IMM-1: Neural Differentiation of Embryonic Stem Cells in vitro: a Road Map to Neurogenesis in the Embryo

ABSTRACT: This experiment is part of the FunGenES project (FunGenES - "Functional Genomics in Embryonic Stem Cells" partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org). The experiment was conducted at the "Instituto de Medicina Molecular,Faculdade de Medicina de Lisboa", Portugal. Goal of the experiment is the in vitro generation and characterisation of neurons from embryonic stem (ES) cells. This is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages try to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors (NPs) to their terminal differentiation into neurons and glial cells, is required. In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer system [1]. We established improved conditions to routinely produce highly homogeneous cultures of NPs, which display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. We show that the transition to neuronal production is linked to the organization of NPs into neural tube-like rosettes, where these cells divide and give rise to neurons. In order to characterize the global gene activity correlated with each particular stage of neural differentiation, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five synexpression groups that correlate with successive stages in the path from ES cells to neurons. This resulted in the identification of unique gene signatures for each cell stage, predicting in addition some of the molecular pathways associated with the transitions between these stages. Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.

ORGANISM(S): Mus musculus

SUBMITTER: Oliver Hummel

PROVIDER: E-TABM-717 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo.

Abranches Elsa E Silva Margarida M Pradier Laurent L Schulz Herbert H Hummel Oliver O Henrique Domingos D Bekman Evguenia E

PloS one 20090721 7

<h4>Background</h4>The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and m ...[more]

PMID: 19621087

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Project description:DNA hydroxymethylation (5hmC) represents a new layer of epigenetic regulation in addition to DNA methylation (5mC). The genome-wide patterns of 5hmC distribution in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, the DNA hydroxymethylome data generated by the conventional hMeDIP-seq method can not be used for direct quantitative comparison across different samples. In this study, we report a new quantitative hMeDIP-seq method, which uses barcode technology to label different DNA samples and performs hMeDIP-seq for multiple samples in one reaction system. Using this new method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESCs-derived neural progenitor cells (NPCs). 5hmC levels around TSS in either ESCs or NPCs had negative correlation with gene expression levels，while 5hmC levels at gene body regions had different correlation with gene expression, depending on cell types. We identified differential hydroxymethylated regions (DHMRs) by comparing the 5hmC density of all 5hmC peaks between ESCs and NPCs. Many selected DHMRs (e.g., Ankrd23, Hist1h2aa, Fbxw7, and Epha2) were validated by hMeDIP-qPCR. Furthermore, we analyzed the relationship between the alteration of DNA hydroxymethylation and the gene expression change during neural differentiation, and our data suggest that both up- and down-regulated genes exhibited dramatic decrease in 5hmC during neural differentiation while the alteration of 5hmC had weak positive correlation with the changes in gene expression. Taken together, our data demonstrate that quantitative hMeDIP-seq is a powerful approach for genome-wide comparison of DNA hydroxymethylation across multiple samples. Importantly, the DHMRs between ESCs and NPCs uncovered in this study may provide clues for further investigation of the function of 5hmC in gene regulation and neural differentiation. Comparision of the genome-wide distribution of 5hmC in mouse embryonic stem cells and mouse ES-derived neural progenitor cells.

Project description:It remains controversial whether the routes from differentiated cells to iPSCs are related to the reverse order of normal developmental processes or independent of them. Here, we generated iPSCs from mouse astrocytes by three (Oct3/4, Klf4 and Sox2 (OKS)), two (OK), or four (OKS plus c-Myc) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently upregulated during reprogramming and Sox1-positive cells become iPSCs. The upregulation of Sox1 is essential for OK-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during MEF reprogramming, neither Sox1 upregulation nor an increase in neurogenic potential occurs. Thus, astrocytes are reprogrammed through an NSC-like state, suggesting that reprogramming partially follows the retrograde pathway of normal developmental processes. To investigate the gene expression profile of intermediate-state cells during astrocyte reprogramming, we performed genome-wide gene expression analysis in five samples; starting astrocytes, intermediate-state cells expressing Sox1-GFP, NSCs, iPSCs established from astrocytes, and iPSCs established from MEFs (iPS-MEF-Ng-20D-17) that had previously been reported (Okita, K. et al. Nature 448: 313-317 (2007)). Two (NSCs, iPSCs from astrocytes and MEFs) or three (astrocytes, intermediate-state cells) biological replicates were prepared for microarray samples. Total RNA was extracted with an RNeasy kit (Qiagen). cDNA synthesis and transcriptional amplification were performed using 50-100 ng of total RNA with the GeneChip WT PLUS Reagent Kit (Affymetrix). Fragmented and biotin-labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) according to the manufacturerâ??s protocol. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.

Dataset Information

Transcription profiling of mouse embryonic stem cells in vitro - FunGenES: IMM-1: Neural Differentiation of Embryonic Stem Cells in vitro: a Road Map to Neurogenesis in the Embryo

Publications

Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo.

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