Previously, we have demonstrated the ability to generate insulin-producing cells following in vitro manipulation of adult stem cells from rat liver. While these cells clearly produce insulin, many of their cellular properties have yet to be established. These properties include the optimal in vitro condition to generate insulin-producing cells, the kinetics of insulin production in response to glucose stimulation, and the capacity for these cells to reverse overt diabetes in vivo. In this project, we will determine the optimal culture conditions to generate insulin producing cells, to test the hypothesis that oval stem cell-derived insulin producing cells represent functional precursors that require an in vivo environment, potentially one that is hyperglycemic, in order for them to become fully functional (i.e., insulin secreting), hormone-specific beta cells. To test this hypothesis I propose three specific aims.
Aim 1 : To optimize the in vitro differentiation of hepatic oval stem cells into pancreatic islet-like cells and determine their biological characteristics.
Aim 2 : Functional characterization of in-vitro-generated islet-like cells using newly developed immunoassay and electrochemical probes. We will evaluate the secretory and metabolic function of hepatic oval cell-derived insulin-producing cells and compare them with normal rat islets by simultaneously monitoring intracellular Ca2+ and insulin secretion from a single islet-like cluster or monolayer cells with high temporal resolution.
Aim 3 : To determine the ability of oval cell-derived islet-like insulin producing precursor cells to reverse hyperglycemia in a diabetic animal model and define the in vivo glycemic state necessary to support cellular maturation into fully functional, insulin secreting cells. Characterization of the functionality of islet like cells generated from hepatic oval cells represents an important step towards the use of non-pancreatic sources of adult autologous stem cells for cell therapies aimed at curing type 1 diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08DK064054-04
Application #
7079294
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Hyde, James F
Project Start
2003-07-01
Project End
2008-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
4
Fiscal Year
2006
Total Cost
$123,366
Indirect Cost
Name
University of Florida
Department
Pathology
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Tang, Dong-Qi; Wang, Qiwei; Burkhardt, Brant R et al. (2012) In vitro generation of functional insulin-producing cells from human bone marrow-derived stem cells, but long-term culture running risk of malignant transformation. Am J Stem Cells 1:114-127
Donelan, William; Koya, Vijay; Li, Shi-Wu et al. (2010) Distinct regulation of hepatic nuclear factor 1alpha by NKX6.1 in pancreatic beta cells. J Biol Chem 285:12181-9
Li, Shi-Wu; Koya, Vijay; Li, Yi et al. (2010) Pancreatic duodenal homeobox 1 protein is a novel beta-cell-specific autoantigen for type I diabetes. Lab Invest 90:31-9
Koya, Vijay; Lu, Shun; Sun, Yu-Ping et al. (2008) Reversal of streptozotocin-induced diabetes in mice by cellular transduction with recombinant pancreatic transcription factor pancreatic duodenal homeobox-1: a novel protein transduction domain-based therapy. Diabetes 57:757-69
Yang, Li-Jun (2006) Liver stem cell-derived beta-cell surrogates for treatment of type 1 diabetes. Autoimmun Rev 5:409-13
Tang, Dong-Qi; Cao, Li-Zhen; Chou, Wayne et al. (2006) Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation. Lab Invest 86:829-41
Tang, Dong-Qi; Lu, Shun; Sun, Yu-Ping et al. (2006) Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors. Lab Invest 86:83-93
Tang, Dong-Qi; Cao, Li-Zhen; Burkhardt, Brant R et al. (2004) In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow. Diabetes 53:1721-32
Cao, Li-Zhen; Tang, Dong-Qi; Horb, Marko E et al. (2004) High glucose is necessary for complete maturation of Pdx1-VP16-expressing hepatic cells into functional insulin-producing cells. Diabetes 53:3168-78