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| Status |
Public on Aug 24, 2010 |
| Title |
Induced overexpression of Pdx1 and Ngn3 in a mouse ES cell-derived endoderm population induces pancreatic differentiation |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
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.
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| Overall design |
On day 4 of a multiday differentiation protocol, EBs were dissociated and cultured in serum-free growth medium supplemented with differentiation facots, with or without Dox (1 ug/ml). On day 6, EBs were replated on gelatin in 12-well low-cluster dishes (Nunc) to obtain non-adherent floating EBs with or without Dox (1 ug/ml) and cultured out to day 13, at which time RNA was harvested for microarray analysis.
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| Contributor(s) |
Kubo A, Stull R, Takeuchi M, Bonham K, Gouon-Evans V, Iwano M, Sho M, Saito Y, Keller G, Snodgrass R |
| Citation(s) |
21931641 |
| Submission date |
Aug 23, 2010 |
| Last update date |
Mar 25, 2013 |
| Contact name |
Robert A Stull |
| E-mail(s) |
bobstullca@gmail.com
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| Phone |
(650)244-9990
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| Organization name |
VistaGen Therapeutics
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| Department |
Genomics
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| Street address |
384 Oyster Point Blvd, Suite #8
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platforms (1) |
| GPL2897 |
GE Healthcare/Amersham Biosciences CodeLink™ Mouse Whole Genome Bioarray |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA130741 |
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