The overall goal of this project is to determine culture conditions that promote differentiation of embryonic stem (ES) cells to pancreatic beta-cells. Various protocols reported to date are largely inefficient and not reproducible. Recent progress in establishing culture conditions to direct differentiation of ES cells to definitive endoderm as a first critical step has advanced the field. In this application, we propose to conduct stepwise optimization of culture conditions to direct differentiation of endoderm to pancreatic beta-cells to mimic the pancreatic development in vivo. To this end, we will utilize a unique mouse ES cell line in which pancreatic beta-cells are endogenously marked with green fluorescent protein (GFP) under the control of mouse insulin I promoter (MIP), in combination with sequential expression of stage-specific reporter genes. This novel system will enable us to study the process of beta-cell formation in real-time thereby allowing us to readily quantify beta-cells at each stage without destroying the culture, which will accelerate the identification of culture conditions that promote beta-cell differentiation. Furthermore, as our in vivo assessment of differentiation of MIP-GFP ES cells by renal transplantation in mice demonstrates, endocrine cell differentiation occurs forming the epithelial tubular structure that recapitulates fetal endocrine cell neogenesis, suggesting that the differentiation takes place in three-dimensions and is controlled by the environmental cues. Here we propose to define the environmental cues and signaling pathway components during fetal pancreatic development in Specific Aim 1. In parallel in Specific Aim 2, we will test the hypothesis that ES cell differentiation to pancreatic beta-cells mimics the fetal beta-cell differentiation. Followed by Specific Aim 3, we will aim at establishing the minimal artificial in vitro environment to direct differentiation of ES cells/definitive endoderm to beta-cells. This application has three specific aims.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
High Priority, Short Term Project Award (R56)
Project #
5R56DK081527-02
Application #
7650211
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Sato, Sheryl M
Project Start
2008-07-01
Project End
2011-06-30
Budget Start
2009-07-01
Budget End
2011-06-30
Support Year
2
Fiscal Year
2009
Total Cost
$231,000
Indirect Cost
Name
University of Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Wang, Xiaojun; Zielinski, Mark C; Misawa, Ryosuke et al. (2013) Quantitative analysis of pancreatic polypeptide cell distribution in the human pancreas. PLoS One 8:e55501
Kilimnik, German; Jo, Junghyo; Periwal, Vipul et al. (2012) Quantification of islet size and architecture. Islets 4:167-72
Jo, Junghyo; Kilimnik, German; Kim, Abraham et al. (2011) Formation of pancreatic islets involves coordinated expansion of small islets and fission of large interconnected islet-like structures. Biophys J 101:565-74
Kilimnik, German; Zhao, Billy; Jo, Junghyo et al. (2011) Altered islet composition and disproportionate loss of large islets in patients with type 2 diabetes. PLoS One 6:e27445
Kim, Abraham; Kilimnik, German; Guo, Charles et al. (2011) Computer-assisted large-scale visualization and quantification of pancreatic islet mass, size distribution and architecture. J Vis Exp :
Steiner, Donald J; Kim, Abraham; Miller, Kevin et al. (2010) Pancreatic islet plasticity: interspecies comparison of islet architecture and composition. Islets 2:135-45
Kim, Abraham; Kilimnik, German; Hara, Manami (2010) In situ quantification of pancreatic beta-cell mass in mice. J Vis Exp :
Kilimnik, German; Kim, Abraham; Steiner, Donald F et al. (2010) Intraislet production of GLP-1 by activation of prohormone convertase 1/3 in pancreatic ?-cells in mouse models of ß-cell regeneration. Islets 2:149-55
Kilimnik, German; Kim, Abraham; Jo, Junghyo et al. (2009) Quantification of pancreatic islet distribution in situ in mice. Am J Physiol Endocrinol Metab 297:E1331-8
Uchizono, Yuji; Baldwin, Aaron C; Sakuma, Hiroya et al. (2009) Role of HNF-1alpha in regulating the expression of genes involved in cellular growth and proliferation in pancreatic beta-cells. Diabetes Res Clin Pract 84:19-26

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