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)

Publication

http://www.ncbi.nlm.nih.gov/pubmed/16809427

My Strategies

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Classification

Targets and roles of transcriptional regulators; Cell differentiation; Differentiation of insulin-producing cells

Links

Biomaterials Graph

ArrayExpress

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

Synopsis

Study Description
Goals
Approaches
Results
Conclusions
Related Studies

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.

ES cell differentiation: 2800, 3365, 3425, 3668, 4216, 4314, 4574,
Ngn3: 1110, 1470, 2520, 2800, 3021, 3100, 3425, 3733, 3795, 4195, 4374,

Platform types

Expression, Expression microarray

Platforms

Show platform Affymetrix Mouse430_2

Study Design Type

compound_treatment_design
genetic_modification_design
growth_condition_design

Study Factors

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Study Assays

Show study assays

ID	Assay name	Study factor name	Study factor value
9705	Ainv15 EB10 replicate 1	Genetic Modification	Wild Type
9705	Ainv15 EB10 replicate 1	Embryoid Body Status	Embryoid Body
9705	Ainv15 EB10 replicate 1	Number of Days Cells were Grown	10
9705	Ainv15 EB10 replicate 1	Doxycycline treatment duration	0
9706	Ainv15 EB10 replicate 2	Genetic Modification	Wild Type
9706	Ainv15 EB10 replicate 2	Embryoid Body Status	Embryoid Body
9706	Ainv15 EB10 replicate 2	Number of Days Cells were Grown	10
9706	Ainv15 EB10 replicate 2	Doxycycline treatment duration	0
9707	Ainv15 EB3 replicate 1	Genetic Modification	Wild Type
9707	Ainv15 EB3 replicate 1	Embryoid Body Status	Embryoid Body
9707	Ainv15 EB3 replicate 1	Number of Days Cells were Grown	3
9707	Ainv15 EB3 replicate 1	Doxycycline treatment duration	0
9708	Ainv15 EB3 replicate 2	Genetic Modification	Wild Type
9708	Ainv15 EB3 replicate 2	Embryoid Body Status	Embryoid Body
9708	Ainv15 EB3 replicate 2	Number of Days Cells were Grown	3
9708	Ainv15 EB3 replicate 2	Doxycycline treatment duration	0
9709	Ainv15 ES0 replicate 1	Genetic Modification	Wild Type
9709	Ainv15 ES0 replicate 1	Embryoid Body Status	Not Applicable
9709	Ainv15 ES0 replicate 1	Number of Days Cells were Grown	0
9709	Ainv15 ES0 replicate 1	Doxycycline treatment duration	0
9710	Ainv15 ES0 replicate 2	Genetic Modification	Wild Type
9710	Ainv15 ES0 replicate 2	Embryoid Body Status	Not Applicable
9710	Ainv15 ES0 replicate 2	Number of Days Cells were Grown	0
9710	Ainv15 ES0 replicate 2	Doxycycline treatment duration	0
9711	Ngn3 EB10 OFF replicate 1	Genetic Modification	Ngn3-D6
9711	Ngn3 EB10 OFF replicate 1	Embryoid Body Status	Embryoid Body
9711	Ngn3 EB10 OFF replicate 1	Number of Days Cells were Grown	10
9711	Ngn3 EB10 OFF replicate 1	Doxycycline treatment duration	0
9712	Ngn3 EB10 OFF replicate 2	Genetic Modification	Ngn3-D6
9712	Ngn3 EB10 OFF replicate 2	Embryoid Body Status	Embryoid Body
9712	Ngn3 EB10 OFF replicate 2	Number of Days Cells were Grown	10
9712	Ngn3 EB10 OFF replicate 2	Doxycycline treatment duration	0
9713	Ngn3 EB10 ON replicate 1	Genetic Modification	Ngn3-D6
9713	Ngn3 EB10 ON replicate 1	Embryoid Body Status	Embryoid Body
9713	Ngn3 EB10 ON replicate 1	Number of Days Cells were Grown	10
9713	Ngn3 EB10 ON replicate 1	Doxycycline treatment duration	3
9714	Ngn3 EB10 ON replicate 2	Genetic Modification	Ngn3-D6
9714	Ngn3 EB10 ON replicate 2	Embryoid Body Status	Embryoid Body
9714	Ngn3 EB10 ON replicate 2	Number of Days Cells were Grown	10
9714	Ngn3 EB10 ON replicate 2	Doxycycline treatment duration	3
9715	Ngn3 EB3 OFF replicate 1	Genetic Modification	Ngn3-D6
9715	Ngn3 EB3 OFF replicate 1	Embryoid Body Status	Embryoid Body
9715	Ngn3 EB3 OFF replicate 1	Number of Days Cells were Grown	3
9715	Ngn3 EB3 OFF replicate 1	Doxycycline treatment duration	0
9716	Ngn3 EB3 OFF replicate 2	Genetic Modification	Ngn3-D6
9716	Ngn3 EB3 OFF replicate 2	Embryoid Body Status	Embryoid Body
9716	Ngn3 EB3 OFF replicate 2	Number of Days Cells were Grown	3
9716	Ngn3 EB3 OFF replicate 2	Doxycycline treatment duration	0
9717	Ngn3 EB3 ON replicate 1	Genetic Modification	Ngn3-D6
9717	Ngn3 EB3 ON replicate 1	Embryoid Body Status	Embryoid Body
9717	Ngn3 EB3 ON replicate 1	Number of Days Cells were Grown	3
9717	Ngn3 EB3 ON replicate 1	Doxycycline treatment duration	3
9718	Ngn3 EB3 ON replicate 2	Genetic Modification	Ngn3-D6
9718	Ngn3 EB3 ON replicate 2	Embryoid Body Status	Embryoid Body
9718	Ngn3 EB3 ON replicate 2	Number of Days Cells were Grown	3
9718	Ngn3 EB3 ON replicate 2	Doxycycline treatment duration	3
9719	Ngn3 ES3 ON replicate 1	Genetic Modification	Ngn3-D6
9719	Ngn3 ES3 ON replicate 1	Embryoid Body Status	Not Applicable
9719	Ngn3 ES3 ON replicate 1	Number of Days Cells were Grown	3
9719	Ngn3 ES3 ON replicate 1	Doxycycline treatment duration	3
9720	Ngn3 ES3 ON replicate 2	Genetic Modification	Ngn3-D6
9720	Ngn3 ES3 ON replicate 2	Embryoid Body Status	Not Applicable
9720	Ngn3 ES3 ON replicate 2	Number of Days Cells were Grown	3
9720	Ngn3 ES3 ON replicate 2	Doxycycline treatment duration	3