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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 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 Dr. Péter Balogh and Dr. Péter Engelmann Transdifferentiation and regenerative medicine – Lecture 9 DIFFERENTIATION AND REGENERATION IN THE PANCREAS TÁMOP-4.1.2-08/1/A-2009-0011 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. TÁMOP-4.1.2-08/1/A-2009-0011 Structure and function of pancreas II • The endocrine pancreas is composed of Langerhans islets representing 1-5% of the pancreas. • Adult islets are composed of different cell types characterized by the production of specific hormones: Glucagon by a-cells, insulin by b-cells, somatostatin by d-cells and pancreatic polypeptide by PP-cells. A rare fifth endocrine cell type, the e-cell, secreting ghrelin, represents about 1% of the embryonic endocrine pancreas, but disappears after birth. TÁMOP-4.1.2-08/1/A-2009-0011 Pancreas phylogeny • First, apperance of pancreas happened in agnathan fishes (lamprey) representing a collection of b-cells around the bile duct in connection to the duodenum. This endocrine organ is composed of 99% b-cells and 1 % somatostatin producing d-cells. • Later, in the ancient cartilagous fishes (skates) we can found b-cells are joined by exocrine tissue and a-cells. • From sharks, pancreas has also the islet PPcell compartments. Specification of the pancreas I TÁMOP-4.1.2-08/1/A-2009-0011 • The heart promotes and notochord inhibits liver formation • The notochord promotes, and the heart inhibits pancreas formation ??? • Pdx1 (pancreatic and duodenal homeobox 1) expression provides the digestive tube with the ability to form liver or pancreas Specification of the pancreas II TÁMOP-4.1.2-08/1/A-2009-0011 • 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 activin are secreted in this region by the notochord which are able to down regulate expression of Shh • After establishing the Shh pattern of expression, Pdx1 becomes expressed in the pancreatic epithelium. Embryonic pancreas development e4.5 e5.5 1WD e6.5 e7.5 2WD Oct4 Sox2 Nanog Brachyury T Gsc Gata5 Sox17 Pdx1 Foxa2 Hnf4a TÁMOP-4.1.2-08/1/A-2009-0011 e8.5 e9.5 e10.5 e11.5 e12.5 e13.5 e14.5 3WD 4WD 5WD 6WD Hhex Mnx1 Ptf1a Sox9 Hnf1b Onescut1 Ngn3 Nkx6.1 Nkx2.2 Pax6 Neurod1 Pax4 Insm1 Mouse Human Exocrine Duct Duct Duct MafA TÁMOP-4.1.2-08/1/A-2009-0011 Pancreas development I Once pancreatic rudiments are initiated, they begin to form both • Exocrine tissue – Produces amylase and a-fetoprotein • Endocrine tissue – Produces insulin, glucagon and somatostatin 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 TÁMOP-4.1.2-08/1/A-2009-0011 Pancreas development II • Pax6 is associated with Pdx1. • Mice without Pax6 are deficient of pancreatic hormone production and have malformed islets. • Cells with Pax6 and Pax4 become b cells of the islets of Langerhans, and they produce insulin • Those islet cells that down-regulate Pax4 and synthesize only Pax6 become the a-cells that secrete glucagon Maintenance of β cell identity • • • • • TGF-b signalling MafA BETA2/NeuroD Pdx1 Hedgehog signalling TÁMOP-4.1.2-08/1/A-2009-0011 Maintenance of α cell identity • Brn4 • Pax6 • Isl1 TÁMOP-4.1.2-08/1/A-2009-0011 TÁMOP-4.1.2-08/1/A-2009-0011 Maintenance of exocrine identity • • • • • • Pdx1 Ptf1a Mist1 Wnt/b-catenin signaling Notch signaling TGF-b signaling 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. 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) TÁMOP-4.1.2-08/1/A-2009-0011 Pathogenesis of type 1 diabetes and β cells • 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 β cell and autoimmune processes of diabetes TÁMOP-4.1.2-08/1/A-2009-0011 iruses, endogenous ligands? Cytokines TNF IL-1β INF- Macrophage INF-a and INF-β b cell TLR3/4, RIG-I, MDA5, other receptors Cytokine receptor signalling T-cell STAT-1, NFB, IRF3, others (?)↑JunB MHC class I ER stress INF-α and INF-β + Apoptotic signalling Chemokines Cytokines Chemokines Cytokines + + + Presentation of modified antigens Dendritic ce Cell death MHC class I T-cell Apoptotic β ce Process of type I diabetes Trigerring mechanism 100 β cell mass (%) Normal Insulin blood sugar CAutoantibodie level peptide s, insulitis present s Immunological malfunctions Age Metabolic malfunction s - Genetic background Decreased insulin secretion C-peptide HLA-DR3/4 Normal insulin secretion TÁMOP-4.1.2-08/1/A-2009-0011 T1DM TÁMOP-4.1.2-08/1/A-2009-0011 Type 2 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 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. 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 βcells. Differentiation of insulin producing β cells from ES cells TÁMOP-4.1.2-08/1/A-2009-0011 Activin A Human ES cell Oct4 Nanog Sox2 E-cad Mesendoderm Definitive Bra endoderm Fgf4 Sox17 Wnt3 Cer N-cad FoxA2 Cxcr4 Activin A Wnt Human ES cell Oct4 Nanog Sox2 E-cad Activin A Wnt Fgf11 Cyclopamine Fgf10 DAPT CyclopamineRetinoid acid Exendin-4 Primitive gut tube Hnf1b Hnf4a Posterior foregut Hnf6 Pdx1 Hlxb9 Noggin Keratinocyte Cyclopamine Retinoid acid Activin A growth Factor Mesendoderm Definitive Bra endoderm Fgf4 Sox17 Wnt3 Cer N-cad FoxA2 Cxcr4 Primitive gut tube Hnf1b Hnf4a Posterior foregut Hnf6 Pdx1 Prox1 Sox9 Exendin-4 IGF-1 HGF Endocrine progenitor Ngn3 Nkx2.2 Pax4 Nkx6.1 Immature endocrine Ins Glu Ghr Som PP In vivo milieu Pancreatic endoderm/ Endocrine precursors Nkx6.1 Ptf1a Nkx2.2 Ngn3 Endocrine MafA Ins Glu Ghr Som PP Possible sources of β-cells for cell replacement therapy TÁMOP-4.1.2-08/1/A-2009-0011 • 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 β-cells generated from existing β-cells through purification and in vitro expansion TÁMOP-4.1.2-08/1/A-2009-0011 • Adult b-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 b-cells arise from pre-existing b-cells. • It is possible to force beta cell to proliferate in vitro. • Several other studies suggested β-cells generated via a pancreatic stem cell that is purified, expanded and differentiated in vitro to generate β-cells TÁMOP-4.1.2-08/1/A-2009-0011 • 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 β-cells differentiated in vitro from embryonic stem cells TÁMOP-4.1.2-08/1/A-2009-0011 • 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 β-cells reprogrammed from somatic cells by expression of pancreatic β-cell transcription factors TÁMOP-4.1.2-08/1/A-2009-0011 • 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 b-cells. • It is possible to induce the conversion of liver cells (hepatocytes, intra-/extrahepatic biliary epithelial cells, and gallbladder epithelium) to pancreatic lineages. • A sub-population of intrahepatic 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 b-cell replacement therapies are considered in clinical research. • One of the promising applications for diabetic patients would be the use of hES or