Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Development of gene expression signatures for practical radiation biodosimetry

ABSTRACT: Genome-wide analysis of miRNA expression was performed in Activin A and Wnt3a-treated mouse ESCs during the different stages of DE differentiation to identify candidate miRNAs likely to be involved in Wnt3a and Activin A induced DE formation. Our analysis exhibited a distinct miRNA expression finger print. Furthermore, we found that forced expression of a subset of synergistically regulated miRNAs could partially mimic the roles of Wnt3a and Activin A. Pathway analyses also revealed the involvement of histone acetylation in Activin A/Wnt3a-driven DE differentiation, which is further confirmed by treating the cells with small molecular weight HDAC inhibitors as well as ChIP experiments. Our study established a regulatory cascade from extracellular cytokine treatment to miRNA expression to histone modification in cell nucleus during DE differentiation. ESCs were treated with 100ng/ml Activin A, or 50ng/ml Wnt3a, or 100ng/ml Activin A plus 50ng/ml Wnt3a, and the samples were collected at 1 day, 3 days, and 5 days after treatment, respectively. Cells treated with the same medium without growth factors were used as the negative controls for each time point.

ORGANISM(S): Mus musculus

SUBMITTER: Fu Shijun

PROVIDER: E-GEOD-29093 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Involvement of histone acetylation of Sox17 and Foxa2 promoters during mouse definitive endoderm differentiation revealed by microRNA profiling.

Fu Shijun S Fei Qi Q Jiang Hua H Chuai Shannon S Shi Song S Xiong Wen W Jiang Lei L Lu Chris C Atadja Peter P Li En E Shou Jianyong J

PloS one 20111123 11

Generation of hepatocyte from embryonic stem cells (ESCs) holds great promise for hepatocyte replacement therapy to treat liver diseases. Achieving high efficiency of directed differentiation of ESCs to hepatocyte is of critical importance. Previously, Wnt3a has been reported to promote Activin A-induced human definitive endoderm (DE) differentiation, the early stage of hepatocyte differentiation. However, the underlying molecular mechanisms are not clear. Growing evidence demonstrated that micr ...[more]

PMID: 22132182

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Project description:The aim of the study was to define the specific gene expression signature of pancreatic endoderm cells (PECs), by designing a strategy for generating putative PECs (PDX1+/NKX6-1+, and posterior foregut endoderm (PFG) cells (PDX1+/NKX6-1-). Cell line: PDX1-eGFP reporter cell line (PDXeG clone 170-3) generated using the HUES-4 embryonic stem cell line. Human embryonic stem cells (hESCs) were maintained on irradiated mouse embryonic fibroblasts (MEFs). Generation of a hESC-derived PDX1-eGFP reporter cell line: A PDX1-targeting vector was constructed by inserting an eGFP-pSV40-NeoR reporter cassette into the 5â² untranslated region of the PDX1 gene upstream of the PDX1 start codon (ATG), resulting in an GFP-tagged PDX1 allele. The GFP cassette was flanked by a 12.5kb homologous arm on the 5â direction and by a 3,5kb homologous arm on the 3 direction. The final targeting construct was verified by PCR, restriction analysis and sequencing. After approximately 2 weeks emerging clones were picked, expanded and analyzed by PCR. The Neomycin cassette was deleted by using a CRE expression vector (NLS-CRE-IRES-Puro) that was transiently co-transfected with a DsRED or GFP plasmid into selected targeted cloned. 24 hours post electroporation, GFP/DsRED positive single cells were FACS sorted and plated into 96-well plates. Clones were expanded and characterized for Neo excision by PCR. Differentiation protocol: hESCs cultured on MEFs were grown until 90% confluency and differentiated into definitive endoderm (DE) by using a modified version of the DâAmour protocol (D'Amour et al., 2005). The first day of differentiation, hESCs were cultured in the presence of 100ng/ml human Activin A (Peprotech) and 25ng/ml Wnt3a (R&D systems) in RPMI medium (Life Technologies). The four following days 100ng/ml human Activin A was added together with 1x B27-insulin in RPMI medium (Life Technologies). To generate posterior foregut cells, DE cells were treated with 64 ng/ml bFGF for additional 12 days. To generate pancreatic endoderm cells, DE cells were first treated with 2uM retinoic acid (RA, Sigma Aldrich) in DMEM/F12 medium containing 1x B27 +insulin (Life Technologies) for three days and then finally treated with 64 ng/ml FGF2 in combination with 100 ng/ml Noggin for the remaining 9 days.