1. Liver to pancreas transdifferentiation by pancreatic transcription factors Shiraz Gefen-Halevi Ph.D Proposal Dr. Sarah Ferber Prof. Jacob Shoham

2. The Pancreas Diabetes Type I Autoimmune distruction of  cells No Insulin production Hyperglicemia related complications

3.  Pancreas/islets cells transplantation Achieves good glycemic control Necessitates life long immunosupression Shortage of organs Todays Clinical therapies and complications  Insuin administration Hazards of hypoglycemia Diabetic complications

4. Number of adult islet allografts done in the last 4 yeats Cell replacement therapy for diabetic patients will become widely available only when new sources of islets or  cells are found.

5. Liver & Pancreas Originate from the primitive foregut endoderm. Originate from the primitive foregut endoderm. Share common characteristics, such as glucose sensing ability, due to GLUT-2 & GK expression. Share common characteristics, such as glucose sensing ability, due to GLUT-2 & GK expression. Share the expression of several unique transcription factors expression. Share the expression of several unique transcription factors expression. PDX-1+

6. Ad-CMV-PDX-1 PDX-1 induces transdifferentiation of liver to pancreas both in vivo and in vitro Pancreatic hormone gene expression Ameliorates hyperglycemia in diabetic mice Pancreatic Phenotype Insulin production and accumulation in liver In-vitro In-vivo Gene expressionProtein productionFunction * Ferber et al Nature Medicine 2000 & Ber et al J. Biol. Chem 2003

7. Pancreatic precursor Endocrine precursor  cell specific Pancreatic transcription factors cascase in pancreas development cascade

8. Aims To use liver tissue as a source of ectopic pancreas To re-evaluate the functional hierarchy of transcription factors by gain of function study. To examine whether other pancreatic transcription factors (NGN3 and Nkx6.1) besides PDX-1 can induce liver to pancreas transdifferentiation To examine whether NGN3 and/or Nkx6.1 augments PDX-1 induced liver to pancreas transdifferentiation. To examine the sequential effects of the transcription factor ectopic expression

9. Ectopic expression of pancreatic transcription factors in embryonic hepatocytes Results GF+Pdx - 1+Ngn3 beta - TC Control GF GF+Pdx - 1 GF+Nkx6.1 GF+Ngn3 GF+Pdx - 1+Nkx6.1 GF+Ngn3+Nkx6.1 GF+Pdx - 1+Ngn3+Nkx6.1 Ins RT - Ectopic PDX-1 Ectopic NKX6.1 Endogenic β-ACTIN Ectopic NGN3 GF= EGF + Nicotinamide

10. Quantitative Insulin gene expression Somatostatin GF+Pdx - 1+Ngn3+Nkx6.1 β-actin Glucagon Control GF GF+Pdx - 1 GF+Ngn3 GF+Nkx6.1 GF+Pdx - 1+Ngn3 GF+Pdx - 1+Nkx6.1 GF+Ngn3+Nk x6.1 Ins Insulin

11. Quantitative Glucagon and Somatostatin gene expression Somatostatin GF+Pdx - 1+Ngn3+Nkx6.1 β-actin Glucagon Control GF GF+Pdx - 1 GF+Ngn3 GF+Nkx6.1 GF+Pdx - 1+Ngn3 GF+Pdx - 1+Nkx6.1 GF+Ngn3+ Nk6.1 Ins Insulin

12. Insulin secretion Only PDX-1 alone can induce Insulin production and secretion Combinations with other transcription factors didn’t increase Insulin secretion

13. Somatostatin GF+Pdx - 1+Ngn3+Nkx6.1 β-actin Glucagon Control GF GF+Pdx - 1 GF+Ngn3 GF+Nkx6.1 GF+Pdx - 1+Ngn3 GF+Pdx - 1+Nkx6.1 GF+Ngn3+ Nk6.1 Ins Insulin Elastas e Quantitative exocrine gene expression

14. Summary: 1. PDX-1 induces a pancreatic phenotype in human embryonic liver cells 2. Other pancreatic transcription factors (NGN3 and Nkx6.1) can not induce a pancreatic phenotype in liver cells as does PDX-1 3. Nkx6.1 supports PDX-1 in the transdifferentiation process by increasing Insulin, Glucagon and Somatostatin gene expression 4. NGN3 subtracts from PDX-1 induced liver to pancreas transdifferentiation by decreasing Insulin, Glucagon and Somatostatin gene expression 5. Co expression of PDX-1 and Nkx6.1 seems to increase Elastase gene expression

15. Quantitative gene expression of pancreatic transcription factors  NKX2.2 is not expresseed by embryonic liver cells  PDX-1 increases NKX2.2 expression  NGN3 and NKX6.1 do not increase NKX2.2 expression Isl1

16. Quantitative gene expression of pancreatic transcription factors Isl1  ISL1 is expressed by embryonic liver cells  PDX-1 increases ISL1 gene expression by 2 folds  NGN3 and NKX6.1 hardly increase ISL1 expression  NKX6.1 + PDX-1 increases ISL1 expression by 3 folds

17. Quantitative gene expression of pancreatic transcription factors  Neuro D in not expressed by embryonic liver cells  NGN3 is the main inducer of Neuro D gene expression Neuro D

18. Quantitative gene expression of pancreatic transcription factors  NKx6.1 is not expressed by embryonic liver cells  Pdx-1 increases Nkx6.1 gene expression  Ectopic Nkx6.1 abolishes its own endogenic expression

19. Summary: 1. Nkx2.2, Isl1 and Nkx6.1 endogenic expression seem to be involved in the pancreatic phenotype induced in the liver 2. Neuro D seems not to be involved in the pancreatic phenotype induced in the liver 3. NGN3 induces Neuro D gene expression 4. PDX-1 induces Nkx2.2, Isl1 and Nkx6.1 gene expression 5. Nkx6.1 seems to increase Isl1 gene expression induced by PDX-1 and to decrease NKX2.2 and endogenic Nkx6.1 gene expression induced by PDX-1 ?

20. pPAC-CMVIPF-IRES6.1 12059 bp SV40pA Ad 5 Amp Ad 5 NotI 11344 NcoI 11881 EcoRI 1 KpnI 10 BamHI 1580 SalI 3310 HindIII 3320 NotI 3750 NcoI 4086 XhoI 6000 IRES-nkx6.1 IPF-PA CMV Construction of an Ad-CMV-IPF-1-IRES-NKX6.1

21. pCMV-IPF-1-IRES-NKX6.1 expression in cells m  -ACTIN haNKX6.1 PDX-1 293 Transfected 293 β-TC RNA Mouse pancrease aML+ Ad-PDX1 293 Transfected 293 pAC-PDX1 Transfected 293 pAC-PDX1- NKX6.1 PDX-1 Nkx6.1 293 Transfected 293 pAC-PDX1- NKX6.1  ML aML+ Ad-NKX6.1 Human Hepatocytes Human Hepatocytes + Ad-NGN3 + Ad- NKX6.1 Mouse pancrease Protein

22. The best pancreatic phenotype will be determined following these questions: 1. Is there an endocrine pancreatic phenotype? And to what extent? RIP Promoter activation (Ad Rip-GFP) Hormone gene expression (quantitative RT-PCR) Hormone secretion (RIA) Hormone content (RIA) 2. Is there an exocrine pancreatic phenotype? And to what extent? Exocrine enzyme gene expression (quantitative RT-PCR) Exocrine enzyme protein (western/Immunofluorescense) 3. Are the cells functional (in vitro and in vivo)? Glucose regulated insulin secretion Cell transplantation into SCID-NOD mice What to do next…..?

23. 4. What is the distribution of the hormones in the cells? Hormone cell distribution (Immunohistochemistry) 5. Can the bi-gene adenovirus cure diabetic mice better than PDX-1 or Nkx6.1 alone? Injection of the virus to diabetic mice 6. What are the downstream pancreatic transcription factors activated or delayed by the different treatments? Pancreatic transcriptin factors gene expression (quantitative RT-PCR) DNA microarray

24. Thank you for your attention…

25. Taqman Quantative RT-PCR

26. Glucose regulated Insulin secretion

27. Schematic representation

31. Taqman Real Time PCR

32. Pancreatic Transcription factors cascade in pancreas development Nkx6.1 Nkx2.2

33. Diabetes mellitus Diabetes is a disease in which the body does not produce or properly use insulin The cause of diabetes continues to be a mystery, although both genetics and environmental factors appear to play roles Type 1 Diabetes (5-10%) Is usually diagnosed in children and young adults The body does not produce insulin The insulin-producing cells in the pancreas have been destroyed Type 2 Diabetes (90-95%) The most common form of diabetes Either the pancreas does not produce enough insulin or the cells fail to properly use insulin (insulin resistance) 'maturity onset' diabetes because it usually appears in middle-aged or elderly people