Hepatocyte transdifferentiation of into functional insulin-producing cells (IPCs) by transcription factor-(TF-) mediated genetic reprogramming is a promising option for cell replacement and gene therapy, one that exploits the common embryonal origin of liver and pancreas cells.
In Specific Aim -1, we propose to investigate the molecular mechanisms of the liver-to-endocrine-pancreas transdifferentiation using gene knock-in or knockout strategies, and we will determine the most effective combination of a-cell TFs with external factors in controlling the process of the transdifferentiation using stably transfected hepatic cell lines. Hypotheses to be tested: (1) Pdx1 expression alone is insufficient for selective transdifferentiation of hepatic cells into endocrine pancreatic IPCs; and (2) Additional factors including other TFs downstream of Pdx1 and external factors are needed to reprogram hepatocytes into the pathway of pancreatic IPCs.
In Specific Aim -2, we propose to determine factors critical for an in vivo selective transdifferentiation of liver cells into IPCs in mice using a hydrodynamic-based tail vein delivery system, portal vein infusion, or direct intrahepatic injection. Hypotheses to be tested: (1) The in vivo diabetic microenvironment plays a permissive role in facilitating Pdx1 protein in the hepatocytes to activate insulin gene expression to produce insulin, and in turn, reducing blood glucose level; (2) Pdx1 expression alone (even its modified form Pdxl-VP16) in the hepatocytes, may not be sufficient to specify the pancreatic endocrine differentiation due to the lack of appropriate protein partners such as other TFs related to pancreatic endocrine development; and (3)The short lifespan of the insulin-producing cells in the liver may result from the transfection of terminally differentiated hepatocytes by Pdx1gene injection, while transfection/transduction of hepatic stem cells with target genes may produce long lasting effects of reversing hyperglycemia in diabetic mice.
In Specific Aim -3, we propose to transdiferentiate primary hepatocytes into IPCs to learn whether the liver-derived IPCs can escape the autoimmune attack in nonobese diabetic (NOD) type 1 diabetes (T1D) mouse model. Hypothesis to be tested: Primary hepatocytes and hepatic stem oval cells can be converted into IPCs by genetic modifications, and these liver-derived-genetically modified-IPCs may reduce or prevent the autoimmune destruction and reverse T1D in NOD animal models.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK071831-01
Application #
6957730
Study Section
Clinical and Integrative Gastrointestinal Pathobiology Study Section (CIGP)
Program Officer
Sato, Sheryl M
Project Start
2005-09-15
Project End
2010-08-31
Budget Start
2005-09-15
Budget End
2006-08-31
Support Year
1
Fiscal Year
2005
Total Cost
$259,074
Indirect Cost
Name
University of Florida
Department
Pathology
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Donelan, William; Wang, Hai; Li, Shi-Wu et al. (2013) Novel detection of pancreatic and duodenal homeobox 1 autoantibodies (PAA) in human sera using luciferase immunoprecipitation systems (LIPS) assay. Int J Clin Exp Pathol 6:1202-10
Wang, Qiwei; Wang, Hai; Sun, Yu et al. (2013) The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation. J Cell Sci 126:3638-48
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; Sun, Yuping; Donelan, William et al. (2010) Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris. Protein Expr Purif 72:157-61
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
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