Dataset Information

Transcriptomic characterization of C57BL/6 mouse embryonic stem cell differentiation and its modulation by developmental toxicants.

ABSTRACT: The Tox21 program calls for transforming toxicology testing from traditional in vivo tests to less expensive and higher throughput in vitro methods. In developmental toxicology, a spectrum of alternative methods including cell line based tests has been developed. In particular, embryonic stem cells (ESCs) have received widespread attention as a promising alternative model for developmental toxicity assessment. Here, we characterized gene expression changes during mouse ESC differentiation and their modulation by developmental toxicants. C57BL/6 ESCs were allowed to differentiate spontaneously and RNA of vehicle controls was collected at 0, 24, 48, 72, 96, 120 and 168 h after embryoid body (EB) formation; RNA of compound-exposed EBs were collected at 24 h. Samples were hybridized to Affymetrix Mouse Gene 2.0 ST Array; using stringent cut-off criteria of Bonferroni-adjusted p<0.05 and fold change >2.0, a total of 1996 genes were found differentially expressed among the vehicle controls at different time points. Gene ontology (GO) analysis showed these regulated genes were mostly involved in differentiation-related processes such as development, morphogenesis, metabolism, cell differentiation, cell organization and biogenesis, embryonic development, and reproduction. Biomarkers of all three germ layers or of their derivative early cell types were identified in the gene list. Principal component analysis (PCA) based on these genes showed that the unexposed vehicle controls appeared in chronological order in the PCA plot, and formed a differentiation track when connected. Cultures exposed to thalidomide, monobutyl phthalate, or valproic acid deviated significantly from the differentiation track, manifesting the capacity of the differentiation track to identify the modulating effects of diverse developmental toxicants. The differentiation track defined in this study may be further exploited as a baseline for developmental toxicity testing, with compounds causing significant deviation from the differentiation track being predicted as potential developmental toxicants.

SUBMITTER: Gao X

PROVIDER: S-EPMC4172731 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Transcriptomic characterization of C57BL/6 mouse embryonic stem cell differentiation and its modulation by developmental toxicants.

Gao Xiugong X Yourick Jeffrey J JJ Sprando Robert L RL

PloS one 20140923 9

The Tox21 program calls for transforming toxicology testing from traditional in vivo tests to less expensive and higher throughput in vitro methods. In developmental toxicology, a spectrum of alternative methods including cell line based tests has been developed. In particular, embryonic stem cells (ESCs) have received widespread attention as a promising alternative model for developmental toxicity assessment. Here, we characterized gene expression changes during mouse ESC differentiation and th ...[more]

PMID: 25247782

Similar Datasets

Project description:BackgroundEmbryonic Sertoli cells (eSCs) play an important role in sex determination and in male gonad development which makes them a very useful cell type for therapeutic applications. However, the deriving mechanism of Sertoli cells has been unclear and challenging to create a large number of quality eSCs. Therefore, this study aimed to create the eSCs induced from mouse embryonic stem (mES) cells by regulating defined factors and to explore the relevant regulatory mechanism.MethodsSix inducing factors, Sry, Sox9, SF1, WT1, GATA4, and Dmrt1, were respectively transduced into mES cells by lentiviral infection according to the experimental design. The test groups were identified by development stage-specific markers, AMH, Emx2, SF1, and FasL, using flow cytometry. Induced eSCs were determined by FasL and AMH biomarkers under immunofluorescence, immunocytochemistry, and flow cytometry. Moreover, the pluripotency markers, gonad development-related markers, epithelial markers and mesenchymal markers in test groups were transcriptionally determined by qPCR.ResultsIn this study, the co-overexpression of all the six factors effectively produced a large population of eSCs from mES cells in 35 days of culturing. These eSCs were capable of forming tubular-like and ring-like structures with functional performance. The results of flow cytometry indicated that the upregulation of GATA4 and WT1 contributed to the growth of somatic cells in the coelomic epithelium regarded as the main progenitor cells of eSCs. Whereas, SF1 facilitated the development of eSC precursor cells, and Sry and Sox9 promoted the determination of male development. Moreover, the overexpression of Dmrt1 was essential for the maintenance of eSCs and some of their specific surface biomarkers such as FasL. The cellular morphology, biomarker identification, and transcriptomic analysis aided in exploring the regulatory mechanism of deriving eSCs from mES cells.ConclusionConclusively, we have elucidated a differentiation roadmap of eSCs derived from mES cells with a relevant regulatory mechanism. Through co-overexpression of all these six factors, a large population of eSCs was successfully induced occupying 24% of the whole cell population (1 × 105 cells/cm2). By adopting this approach, a mass of embryonic Sertoli cells can be generated for the purpose of co-culture technique, organ transplantation, gonadal developmental and sex determination researches.

Project description:BackgroundLittle is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC) differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5) undergoing undirected differentiation into embryoid bodies (EBs) over a period of two weeks.ResultsWe identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1), our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2) that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set.ConclusionOur analysis profiles for the first time gene expression at a very early stage of mESC differentiation, and identifies a functional and phylogenetic signature for the genes involved. The data generated constitute a valuable resource for further studies. All DNA microarray data used in this study are available in the StemBase database of stem cell gene expression data 1 and in the NCBI's GEO database.

Dataset Information

Transcriptomic characterization of C57BL/6 mouse embryonic stem cell differentiation and its modulation by developmental toxicants.

Publications

Transcriptomic characterization of C57BL/6 mouse embryonic stem cell differentiation and its modulation by developmental toxicants.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets