Inducible Ngn3 Embryonic Stem Cells - Study GBCO2800
Genomics Study Specifications
|Study Name||Inducible Ngn3 Embryonic Stem Cells|
|Contact Name||Nathan R. Treff (RMA of NJ)|
|My Strategies||Return to My Strategies page|
|Classification||Targets and roles of transcriptional regulators; Cell differentiation; Differentiation of insulin-producing cells|
|BCBC Release Date||November 28, 2006|
|Public Release Date||November 28, 2006|
|Citation||Treff NR, Vincent RK, Budde ML, Browning VL, Magliocca JF, Kapur V, Odorico JS. Differentiation of embryonic stem cells conditionally expressing neurogenin 3. Stem Cells. 2006. 24:2529-37|
16 embryonic stem cell (ESC) samples (8 groups in duplicate) were processed for microarray analysis using the Affymetrix Mouse Genome 430 2.0 GeneChip. The samples included the parental Ainv15 ESC line (1) undifferentiated (Ainv15 ESC), (2) differentiated for 3 days as embryoid bodies (Ainv15 EB3) or (3) for 10 days as embryoid bodies (Ainv15 EB10). The remaining samples included tetracycline inducible Ngn3 ESC line derived from the parental Ainv15 ESC line (4) after differentiation and addition of doxycycline for 3 days without embryoid body formation (Ngn3 ES3 ON), (5 and 6) after differentiation as embryoid bodies for 3 days with (Ngn3 EB3 ON) or without (Ngn3 EB3 OFF) doxycycline, and (7 and 8) after differentiation as embryoid bodies for 10 days with (Ngn3 EB10 ON) or without (Ngn3 EB10 OFF) doxycycline. This study corresponds to GEO accession GSE3653.
To better characterize the molecular events regulated by Ngn3 during development, expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3 were determined.
An mESC line was created in order to induce Ngn3 by adding doxycycline (DOX) to the culture medium. Microarray analysis was performed on Affymetrix GeneChip Mouse Genome 430 2.0 arrays to identify genes regulated by Ngn3 in a variety of contexts, including undifferentiated ESCs, ES cells treated with DOX for 3 days (ES3), and differentiating embryoid bodies treated with DOX for 3 (EB3) or 10 days (EB10). Subsets of genes identified as significantly regulated by Ngn3 induction and relevant to pancreatic islet cell development were validated using QPCR. Venn diagram analysis was performed on probe sets representing genes with significant differential expression relative to undifferentiated parental ESCs from each of three Ngn3 induction contexts (ES3, EB3, and EB10). The intersection contained 757 probe sets which were evaluated for significant enrichment of signaling and metabolic pathways as well as network processes using MetaCore (a gene ontology tool).
Microarray analysis revealed Notch signaling as the most significantly regulated signaling pathway (p = .009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and, conversely, more sensitive to Notch signaling inhibition. Sim1, known to play an important role in the differentiation of neuroendocrine cells of the hypothalamic-pituitary axis was up-regulated in EB10 cells and is likely to be a direct target based on finding Ngn3 binding sites in the Sim1 promoter. EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor (a Notch signaling inhibitor) when compared with uninduced EBs. Ngn3 induction in differentiating ESCs was also found to result in significant increases in insulin, glucagon, and somatostatin expression.
Simultaneous Notch signaling inhibition and Ngn3 activation resulted in significantly increased pancreatic hormone transcripts, including insulin and somatostatin, indicating that the Ngn3 ESC line may recapitulate some important molecular mechanisms that occur during the development of islet hormone-producing cells.
|Platform types||Expression, Expression microarray|
Show platform Affymetrix Mouse430_2
|Study Design Type||
|Study Factors||Show study factors|
|Study Assays||Show study assays|
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Last modified on Jan 17, 2012
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