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RNA-sequencing analysis of NGN3-GFP positive and negative cells purified from differentiating human embryonic stem cells

ABSTRACT: NGN3-eGFP could be used to mark endocrine progenitor cells and their progeny in differentiating hESCs, suggesting that profiling analysis of NGN3-eGFP+ cells could lead to the discovery of novel gene participation. Six batches of NGN3-eGFP+ and NGN3-eGFP -cells purified by FACS from a reporter hES cell line cell cultures at stage 4 were pooled together, respectively. RNA-seq-based analysis of the two cell fractions identified a total of 3976 differentially expressed genes (P-value<0.05; Fold >2), of which 72% are enriched in NGN3-GFP+ cells. As expected, all the genes expressed in pancreatic progenitor cells are enriched in NGN3-eGFP-cells; while all endocrine-associated genes are enriched in NGN3-eGFP+ cells. Among the 3976 genes, 195 have potential transcriptional factor activity and 453 are potential non-coding RNA genes, most of which have not been well investigated in pancreatic endocrine cells. The gene ontology term ‘integral to membrane’ GO: 0016021 identified 1003 differentially expressed genes that encoded ion channels, adhesion molecules and transporters.

ORGANISM(S): Homo sapiens

PROVIDER: GSE54879 | GEO | 2014/09/12

SECONDARY ACCESSION(S): PRJNA237955

REPOSITORIES: GEO

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RNA-sequencing analysis of NGN3-GFP positive and negative cells purified from differentiating human embryonic stem cells

Project description:NGN3-eGFP could be used to mark endocrine progenitor cells and their progeny in differentiating hESCs, suggesting that profiling analysis of NGN3-eGFP+ cells could lead to the discovery of novel gene participation. Six batches of NGN3-eGFP+ and NGN3-eGFP -cells purified by FACS from a reporter hES cell line cell cultures at stage 4 were pooled together, respectively. RNA-seq-based analysis of the two cell fractions identified a total of 3976 differentially expressed genes (P-value<0.05; Fold >2), of which 72% are enriched in NGN3-GFP+ cells. As expected, all the genes expressed in pancreatic progenitor cells are enriched in NGN3-eGFP-cells; while all endocrine-associated genes are enriched in NGN3-eGFP+ cells. Among the 3976 genes, 195 have potential transcriptional factor activity and 453 are potential non-coding RNA genes, most of which have not been well investigated in pancreatic endocrine cells. The gene ontology term M-bM-^@M-^Xintegral to membraneM-bM-^@M-^Y GO: 0016021 identified 1003 differentially expressed genes that encoded ion channels, adhesion molecules and transporters. NGN3-eGFP Postive and negtive cells were purified at stage 4 and subjected to RNA-sequncing

2014-09-12 | E-GEOD-54879 | biostudies-arrayexpress

Transcription profiling of mouse Ngn3-expressing pancreatic cells in a time series through embryonic development and adult islet cells

Project description:Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor which is expressed in scattered cells in the embryonic pancreas. Mice deficient for Ngn3 fail to produce any pancreatic endocrine cells and die shortly after birth. Expression of transcription factors critical to pancreatic development (Isl1, NeuroD, Pax4, and Pax6) is missing and endocrine precursors are absent in mutant pancreatic epithelium. This study utilizes mice which were engineered to contain EGFP (Enhanced Green Fluorescent Protein) under the control of the Ngn3 promoter. In this way, cells which express Ngn3 can be FACS sorted and studied throughout embryonic development (E13.5, E14.5, E15.5, E16.5, and E17.5). EGFP positive cells from the embryonic time-points in addition to adult islets (no cells expressing Ngn3) were hybridized to the BCBC PancChip 6.0 expression microarray, thus generating a profile of gene expression during this critical stage of development of the endocrine pancreas.

2007-12-15 | E-CBIL-40 | biostudies-arrayexpress

Expression data of Sox9+ and Ngn3+ mouse pancreas cells at different stages of development

Project description:Genes specific to Sox9+ pancreatic progenitors were identified by comparing the gene expression in embryonic and adult Sox9+ cells. We used microarray analysis to detail the global changes in gene expression as Sox9 positive embryonic pancreatic progenitors differentiatiate into adult ductal cells or the endocrine lineage. GFP positive cells from Sox9-EGFP mouse pancreas were isolated by FACS at different stages of development (e10.5, e15.5, and p23) for RNA extraction and hybridization to Affymetrix microarrays. To obtain populations highly enriched in Sox9 expression, we collected only GFP Hi populations for analysis. To identify gene expression changes specific to the differentiation of progenitors to ductal cells or endocrine cells, we also isolated and analyzed the gene expression profile of GFP negative cells isolated at p23, as well as GFP positive cells isolated from Ngn3-EGFP mouse pancreas at e15.5. These two populations allow the identification of genes whose expression is associated with the newly differentiated endocrine progeny in the embryo (Ngn3-GFP positive) and adult acinar and endocrine cells at p23.

2012-08-01 | E-GEOD-34060 | biostudies-arrayexpress

Identifying new interactors of NGN3 through IP-MS

Project description:NGN3 is a transcription factor whose transient expression during pancreatic development is vital for the generation of endocrine pancreatic cells, including beta cells. NGN3 stabilisation has been shown to induce exocrine-to-endocrine cell plasticity in the murine pancreas, making it a viable target for therapies aiming to replenish beta cells after immune-mediated destruction in type 1 diabetes patients. Here, we set out to identify new interactors of NGN3 that could play a role in its post-translational regulation. We transfected HEK293A cells with HA-tagged NGN3 and carried out immunoprecipitation of the HA-tag, followed by analysis of co-immunoprecipitated interactors via LC-MS/MS.

2023-03-27 | PXD033691 | Pride

Induced overexpression of Pdx1 and Ngn3 in a mouse ES cell-derived endoderm population induces pancreatic differentiation

Project description:Induced overexpression of Pdx1 in activin-induced endoderm population resulted in the upregulation of pancreas-related genes such as insulin 1 and 2 at day 20. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we next expressed neurogenin3 (Ngn3) together with Pdx-1. Induced overexpression of Pdx1 together with Ngn3 dramatically increased Insulin 1 mRNA by day 9 differentiation. The levels of insulin 1 mRNA present in the induced EBs represented approximately 100 % of that found in insulinoma cell line, betaTC6. We also confirmed insulin and C-peptide staining by immunohistochemistry. These cells process and secrete insulin and respond to various insulin secretagogues. These inductive effects were restricted to c-kit+ endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions at the level of pancreatic specification of endoderm in this model. Microarray analysis showed that Pdx1/Ngn3 regulated the expression of a broad spectrum of pancreatic endocrine cell-related genes.

2010-08-24 | GSE23752 | GEO

Transcription profiling of mouse Ngn3 expressing cells and endocrine pancreatic cells from wild type and Ngn3 mutant animals at day 15 of embryonic development

Project description:The basic helix-loop-helix transcription factor Neurogenin3 (Ngn3/Neurog3) is expressed in endocrine progenitor cells in the embryonic mouse pancreas. Ngn3 controls endocrine cell fate decisions. Ngn3 deficient mice do not develop any pancreatic endocrine cells, including insulin producing beta cells, and die postnatally from diabetes. Therefore, the characterization of gene expression in Ngn3-expressing cells and their progeny is of particular interest for the development of novel strategies for cell replacement therapies in type-1 diabetes. Here we describe two studies. In the first study (8 assays) we used mice where the EYFP (Enhanced Yellow fluorescent Protein) is expressed under the control of Ngn3 regulatory elements (knock add on strategy). EYFP-positive, Ngn3-expressing cells, were FACS sorted from embryonic pancreas at day 15.5 (E15.5), as well as EYFP-negative cells. In the second study (6 assays) we compared wild-type and Ngn3 mutant pancreas at E15.5. All samples were hybridized to Affymetrix GeneChip Mouse Genome 430.2.0 array.

2009-12-03 | E-CBIL-48 | biostudies-arrayexpress

Expression data from Xenopus endoderm at stage 15 following four hours of Ngn3-GR overexpression

2012-04-01 | GSE29017 | GEO

Transcriptional profiling of Ngn3-dependent pancreatic endocrine differentiation at E15.5

Project description:This experiment used RNA-Seq technology to explore gene expression in mouse Ngn3^GFP/+ [het] FACS sorted pancreatic cells at E15.5 (commited endocrine progenitor cells) and in Ngn3^GFP/GFP [null] at E15.5 (defective endocrine progenitor cells). This experiment is designed to understand the gene expression alteration in the endocrine lineage at different embryonic days. The aim is to understand both Ngn3 dependent and independent gene expression profiles so as to reveal the instructive signals that specfy the collective endocrine islet cell fate or specific islet cell type.

2015-10-01 | E-MTAB-3352 | biostudies-arrayexpress

Induced overexpression of Pdx1 and Ngn3 in a mouse ES cell-derived endoderm population induces pancreatic differentiation

2010-08-24 | E-GEOD-23752 | biostudies-arrayexpress

Expression data from Xenopus endoderm at stage 15 following four hours of Ngn3-GR overexpression

Project description:Ngn3 is a master regulator of pancreatic endocrine development. It is necessary for the creation of all endocrine cells in mice. Little is known about the genes that act downstream of the transcription factor Ngn3 in pancreas endocrine development to specify each of the endocrine lineages. As a consequence, little is known about the genes involved in early development and the specification of the beta cell. We used microarrays to identify Ngn3 downstream genes that are involved in early and ectopic beta cell development in Xenopus laevis. We overexpressed Ngn3 in the Xenopus early endoderm and analyzed the genes that are upregulated four hours after. Xenopus laevis embryos were injected with Ngn3-GR mRNA at the eight-cell stage in the two dorsal vegetal blastomeres. Embryos at stage 12 were either treated with dexamethasone (+DEX) for four hours or no DEX (-DEX). The endoderm was dissected at stage 15. We looked for genes upregulated in the +DEX samples compared to the -DEX samples.

2012-03-31 | E-GEOD-29017 | biostudies-arrayexpress

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