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Liver to pancreas transdifferentiation by pancreatic transcription factors

Liver to pancreas transdifferentiation by pancreatic transcription factors. Shiraz Gefen-Halevi Ph.D Proposal. Dr. Sarah Ferber Prof. Jacob Shoham. The Pancreas. Diabetes Type I. Autoimmune distruction of b cells No Insulin production

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Liver to pancreas transdifferentiation by pancreatic transcription factors

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  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 b cells No Insulin production Hyperglicemia related complications

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

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

  6. Ad-CMV-PDX-1 PDX-1 induces transdifferentiation of liver to pancreas both in vivo and in vitro In-vivo In-vitro Pancreatic Phenotype Gene expression Protein production Function Pancreatic hormone gene expression Insulin production and accumulation in liver Ameliorates hyperglycemia in diabetic mice * Ferber et al Nature Medicine 2000 & Ber et al J. Biol. Chem2003

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

  8. Aims • To use liver tissue as a source of ectopic pancreas • 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 • To re-evaluate the functional hierarchy of transcription factors by gain of function study.

  9. Results GF= EGF + Nicotinamide 1+Ngn3+Nkx6.1 1+Nkx6.1 1+Ngn3 GF+Ngn3+Nkx6.1 1 - - - - TC GF+Nkx6.1 GF+Ngn3 GF+Pdx GF+Pdx GF+Pdx GF+Pdx - Control - beta RT GF Ins Ectopic PDX - 1 Ectopic NGN 3 Ectopic NKX6.1 Endogenic β - ACTIN Ectopic expression of pancreatic transcription factors in embryonic hepatocytes

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

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

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

  13. Elastase 1+Ngn3+Nkx6.1 1+Nkx6.1 1+Ngn3 GF+Ngn3+Nk6.1 1 - GF+Nkx6.1 - - - GF+Ngn3 GF+Pdx GF+Pdx GF+Pdx GF+Pdx Control GF Ins Insulin Glucagon Somatostatin β - actin 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. Isl1 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

  16. Isl1 Quantitative gene expression of pancreatic transcription factors • 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. Neuro D 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

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

  21. RNA Transfected 293 Transfected 293 Mouse aML+ pAC - PDX1 - NKX6.1 293 Ad - PDX1 pAC - PDX1 pancrease Human PDX - 1 Hepatocytes Transfected 293 Mou se Human + Ad - NGN3 aML+ pAC - PDX1 - NKX6.1 293 a pancrease Hepatocytes ML Ad - NKX6 .1 + Ad - NKX6.1 Nkx6.1 Transfected Protein 293 β - TC 293 PDX-1 .1 haN KX6 b m - ACTIN pCMV-IPF-1-IRES-NKX6.1 expression in cells

  22. What to do next…..? 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

  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

  28. Taqman Real Time PCR

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

  30. Type 2 Diabetes (90-95%) Type 1 Diabetes (5-10%) 'maturity onset' diabetes because it usually appears in middle-aged or elderly people Is usually diagnosed in children and young adults The body does not produce insulin The most common form of diabetes The insulin-producing cells in the pancreas have been destroyed Either the pancreas does not produce enough insulin or the cells fail to properly use insulin (insulin resistance) 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

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