Induced overexpression of Pdx1 and Ngn3 in a mouse ES cell-derived endoderm population induces pancreatic differentiation
Ontology highlight
ABSTRACT: 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. 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.
ORGANISM(S): Mus musculus
SUBMITTER: Robert Stull
PROVIDER: E-GEOD-23752 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
ACCESS DATA