1. Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

2. DIFFERENTIATION AND REGENERATION IN THE PANCREAS Dr. Péter Balogh and Dr. Péter Engelmann Transdifferentiation and regenerative medicine – Lecture 9 Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

3. TÁMOP-4.1.2-08/1/A-2009-0011 I Structure and function of pancreas I Pancreas is an exocrine and endocrine gland of the digestive system. The exocrine part represents 95-99% of the total pancreatic mass. It consists of serous acini of cells producing digestive enzymes (lipase, amylase,, phospholipase) as well as pro-enzymes (pepsinogen, elastase, procarboxypeptidase, trypsinogen, deoxyribonuclease, ribonuclease), which are stored in zymogen granules.

4. TÁMOP-4.1.2-08/1/A-2009-0011 II Structure and function of pancreas II The endocrine pancreas is composed of Langerhans islets representing 1-5% of the pancreas. production of specific hormones: Adult islets are composed of different cell types characterized by the production of specific hormones: Glucagon insulin somatostatinpancreatic polypeptide ghrelin Glucagon by  -cells, insulin by  -cells, somatostatin by  -cells and pancreatic polypeptide by PP-cells. A rare fifth endocrine cell type, the  -cell, secreting ghrelin, represents about 1% of the embryonic endocrine pancreas, but disappears after birth. Insulin and glucagon control blood glucose levels, whereas PP and ghrelin are appetite stimulant (orexigenic) hormones and somatostatin regulates the secretion of insulin, glucagon and PP.

5. TÁMOP-4.1.2-08/1/A-2009-0011 Pancreas phylogeny First, apperance of pancreas happened in agnathan fishes (lamprey) representing a collection of  -cells around the bile duct in connection to the duodenum. This endocrine organ is composed of 99%  -cells and 1 % somatostatin producing  -cells. Later, in the ancient cartilagous fishes (skates) we can found  -cells are joined by exocrine tissue and  -cells. From sharks, pancreas has also the islet PP- cell compartments.

6. TÁMOP-4.1.2-08/1/A-2009-0011 I Specification of the pancreas I liver formationThe heart promotes and notochord inhibits liver formation pancreas formationThe notochord promotes, and the heart inhibits pancreas formation ??? Pdx1Pdx1 (pancreatic and duodenal homeobox 1) expression provides the digestive tube with the ability to form liver or pancreas

7. TÁMOP-4.1.2-08/1/A-2009-0011 II Specification of the pancreas II Notochord activates pancreas development by repressing Shh expression in the endoderm –Shh is expressed throughout the endoderm but repressed where pancreas will develop FGF2 and activinFGF2 and activin are secreted in this region by the notochord which are able to down regulate expression of Shh Pdx1After establishing the Shh pattern of expression, Pdx1 becomes expressed in the pancreatic epithelium.

8. TÁMOP-4.1.2-08/1/A-2009-0011Human Mouse Embryonic pancreas development e4.5e5.5e6.5e7.5e8.5e9.5e10.5e11.5e12.5e13.5e14.5 1WD2WD3WD4WD5WD6WD Oct4 Sox2 Nanog Brachyury T Gsc Gata5 Sox17 Pdx1 Foxa2 Hnf4a Hhex Mnx1 Ngn3 Nkx6.1 Nkx2.2 Pax6 Neurod1 Pax4 Insm1 MafAPtf1aExocrine Sox9Duct Hnf1bDuct Onescut1Duct

9. TÁMOP-4.1.2-08/1/A-2009-0011 I Pancreas development I Once pancreatic rudiments are initiated, they begin to form both Exocrine tissueExocrine tissue –Produces amylase and  -fetoprotein Endocrine tissueEndocrine tissue –Produces insulin, glucagon and somatostatin Follistatin The ratio of exocrine and endocrine cells is regulated by Follistatin – protein secreted by pancreatic mesenchyme (which inhibits BMP4 and activin) promotes the development of exocrine cells and represses the formation of endocrine cells.

10. TÁMOP-4.1.2-08/1/A-2009-0011 II Pancreas development II Pax6 is associated with Pdx1. Mice without Pax6 are deficient of pancreatic hormone production and have malformed islets. Pax6Pax4  cells insulinCells with Pax6 and Pax4 become  cells of the islets of Langerhans, and they produce insulin Pax6  -cells glucagonThose islet cells that down-regulate Pax4 and synthesize only Pax6 become the  -cells that secrete glucagon

11. TÁMOP-4.1.2-08/1/A-2009-0011 Maintenance of β cell identity TGF-  signalling MafA BETA2/NeuroD Pdx1 Hedgehog signalling

12. TÁMOP-4.1.2-08/1/A-2009-0011 Maintenance of α cell identity Brn4 Pax6 Isl1

13. TÁMOP-4.1.2-08/1/A-2009-0011 Maintenance of exocrine identity Pdx1 Ptf1a Mist1 Wnt/  -catenin signaling Notch signaling TGF-  signaling

14. TÁMOP-4.1.2-08/1/A-2009-0011 Diabetes epidemiology Diabetes mellitus is affecting approx. 200 million people worldwide. There are more than 37 million diabetic children and adults in North America. In Europe more than 55 million people suffers in diabetes.

15. TÁMOP-4.1.2-08/1/A-2009-0011 Main types of diabetes Type 1 Diabetes Type 2 Diabetes LADA (latent autoimmune diabetes of adulthood)

16. TÁMOP-4.1.2-08/1/A-2009-0011 Pathogenesis of type 1 diabetes and β cells Insulin dependent diabetes mellitus (IDDM)Insulin dependent diabetes mellitus (IDDM) It can affect children or adults, but most frequently children, that’s why earlier terminology referred it as juvenile diabetes. Loss of insulin producing beta cells by immune mechanisms. Hyperglycemia, ketosis Autoimmune process mediated by the cellular components of immune system. Autoantibodies (GAD65, IA2, Insulin, etc) T-cell mediated, Th1/Th2 balance affected, Th1, Tc, macrophage

17. TÁMOP-4.1.2-08/1/A-2009-0011 β cell and autoimmune processes of diabetes Viruses, endogenous ligands? Cytokines INF-α and INF- β Apoptotic β cell  cell MHC class I T-cell + + + + --T-cellTNF IL-1 β INF-  INF-  and INF- β Macrophage Dendritic cell Chemokines Cytokines Chemokines Cytokines TLR3/4, RIG-I, MDA5, other receptors Cytokine receptor signalling  STAT-1,  NF  B,  IRF3, others (?) ↑JunB  Presentation of modified antigens  Cell death  MHC class I ER stress  Apoptotic signalling  Apoptotic signalling  Chemokines  Cytokines  Chemokines  Cytokines

18. TÁMOP-4.1.2-08/1/A-2009-0011 Process of type I diabetes Genetic background Immunological malfunctions T1DM Metabolic malfunction s Trigerring mechanism Autoantibodie s, insulitis Normal insulin secretion Decreased insulin secretion Normal blood sugar level Insulin C- peptide present s C-peptide - C-peptide - Age β cell mass (%) 100 HLA-DR3/4

19. TÁMOP-4.1.2-08/1/A-2009-0011 Type 2 diabetes Non-insulin dependent diabetes mellitus or adult onset diabetes.Non-insulin dependent diabetes mellitus or adult onset diabetes. Factors parctipate in the disease is life style and genetic background. Insulin resistance Renal failure, coronary artery disease, retinal damage

20. TÁMOP-4.1.2-08/1/A-2009-0011 LADA (latent autoimmune diabetes) 20% of patients diagnosed with type 2 diabetes actually has LADA. Low, although sometimes moderate, levels of C-peptide Autoantibody testing is essential.

21. TÁMOP-4.1.2-08/1/A-2009-0011 Regenerative capacity of pancreas and β cells Islet transplantation: Through 1 year many patients are insulin independent, however after 5 years of transplantation only <10% of the recipients remain insulin independent. β-cell proliferation in adult humans is extremely low, and greatly enlarged islets are rarely found. Stem cells (embryonic and iPS) could be forced to generate functional β- cells.

22. TÁMOP-4.1.2-08/1/A-2009-0011 Differentiation of insulin producing β cells from ES cellsHuman ES cell Oct4 Nanog Sox2 E-cadMesendoderm Bra Fgf4 Wnt3 N-cadDefinitiveendoderm Sox17 Cer FoxA2 Cxcr4Primitive gut tube Hnf1b Hnf4aPosteriorforegut Hnf6 Pdx1 Hlxb9Endocrineprogenitor Ngn3 Nkx2.2 Pax4 Nkx6.1 Activin A WntFgf10Cyclopamine Fgf11Cyclopamine Retinoid acid DAPTExendin-4 Exendin-4IGF-1HGF Immatureendocrine Ins Glu Ghr Som PP Human ES cell Oct4 Nanog Sox2 E-cadMesendoderm Bra Fgf4 Wnt3 N-cadDefinitiveendoderm Sox17 Cer FoxA2 Cxcr4Primitive gut tube Hnf1b Hnf4aPosteriorforegut Hnf6 Pdx1 Prox1 Sox9Pancreaticendoderm/Endocrineprecursors Nkx6.1 Ptf1a Nkx2.2 Ngn3 Activin A Wnt Keratinocyte growth Factor NogginCyclopamine Retinoid acid In vivo milieu Endocrine MafA Ins Glu Ghr Som PP

23. TÁMOP-4.1.2-08/1/A-2009-0011 Possible sources of β-cells for cell replacement therapy β-cells might be generated from existing β-cells through purification and in vitro expansion. β-cells might be generated via a pancreatic stem cell that could be purified, expanded and differentiated in vitro to generate β-cells. β-cells might be differentiated in vitro from embryonic stem cells. β-cells might be directly reprogrammed from patient somatic cells using expression of pancreatic β-cell transcription factors.

24. TÁMOP-4.1.2-08/1/A-2009-0011 β-cells generated from existing β-cells through purification and in vitro expansion Adult  -cell mass is not static, but fluctuates in response to changing physiological conditions, such as pregnancy and insulin resistance. Following partial pancreatectomy, or during pregnancy, neonatal growth, insulin resistance, new  -cells arise from pre-existing  -cells. It is possible to force beta cell to proliferate in vitro. Several other studies suggested alternative origins for  -cells during pancreas regeneration

25. TÁMOP-4.1.2-08/1/A-2009-0011 β-cells generated via a pancreatic stem cell that is purified, expanded and differentiated in vitro to generate β-cells The ductal compartment seemingly represents the site where stem/progenitor cells at least transiently reside. The progeny of pancreatic duct cells following birth showed that carbonyc anhydrase II (CAII) expressing cells can give rise to both endocrine and exocrine cells. Besides the ductal lining, intra-islet precursor cells as well as acinar cells were suggested to contribute to beta-cell neogenesis.

26. TÁMOP-4.1.2-08/1/A-2009-0011 β-cells differentiated in vitro from embryonic stem cells First attempts were rather unsuccessfull claiming ES cells were differentiated into insulin secreting beta cells, because those cells were insulin immune-reactive, but no insulin mRNA or C-peptide was detected. It is likely, that ES cells consumed insulin from the culture media causing this discrepancy. Recently independent research groups were able to differentiate endocrine cells (including insulin production) from human ES cells copying the embryonic development. In these studies human ES cells can serve as a source of functional insulin-producing cells capable of maintaining glucose stably at normal levels in mice lacking their own beta-cells.

27. TÁMOP-4.1.2-08/1/A-2009-0011 β-cells reprogrammed from somatic cells by expression of pancreatic β-cell transcription factors Acinar cell culture with the cytokines like epidermal growth factor (EGF) and leukemia inhibitory factor (LIF) along with expression of Pdx1, Ngn3, MafA to generate functional  -cells. It is possible to induce the conversion of liver cells (hepatocytes, intra-/extrahepatic biliary epithelial cells, and gall- bladder epithelium) to pancreatic lineages. A sub-population of intrahepatic biliary epithelial cells (IHBECs) can be induced to a  -like phenotype.

28. TÁMOP-4.1.2-08/1/A-2009-0011 Summary Pancreas is a complex endodermal organ participating in exocrine and endocrine metabolic response. Great number of human population is suffering in diabetes and have a high risk for developing one of the form of the disease. In addition to pancreas/islet transplantation other  -cell replacement therapies are considered in clinical research. One of the promising applications for diabetic patients would be the use of hES or iPS cells to generate functional insulin secreting  - cells.