1 / 34

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

yale
Download Presentation

Liver to pancreas transdifferentiation by pancreatic transcription factors

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related