Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

DIFFERENTIATION AND REGENERATION IN THE PANCREAS

Dr. Péter Balogh and Dr. Péter EngelmannTransdifferentiation and regenerative medicine – Lecture 9

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

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.

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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.

• 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.

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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 PP-cell compartments.

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Specification of the pancreas I• 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

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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 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.

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HumanMouse

Embryonic pancreas development

e4.5 e5.5 e6.5 e7.5 e8.5 e9.5 e10.5 e11.5 e12.5 e13.5 e14.51WD 2WD 3WD 4WD 5WD 6WD

Oct4Sox2Nanog

Brachyury TGscGata5Sox17Pdx1Foxa2Hnf4a

HhexMnx1

Ngn3Nkx6.1Nkx2.2Pax6Neurod1

Pax4Insm1

MafA

Ptf1a ExocrineSox9 DuctHnf1b DuctOnescut1 Duct

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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 endocrine cells.

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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

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Maintenance of β cell identity• TGF-b signalling• MafA• BETA2/NeuroD• Pdx1• Hedgehog signalling

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Maintenance of α cell identity• Brn4• Pax6• Isl1

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Maintenance of exocrine identity• Pdx1• Ptf1a• Mist1• Wnt/b-catenin signaling• Notch signaling• TGF-b signaling

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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.

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Main types of diabetes

• Type 1 Diabetes• Type 2 Diabetes• LADA (latent autoimmune diabetes of

adulthood)

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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

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β cell and autoimmune processes of diabetes

Viruses, endogenous ligands? Cytokines

INF-α andINF-β

Apoptotic β cell

b cell

MHC class I T-cell

+

+

+

+

- -

T-cell

TNFIL-1β INF-

INF-a andINF-β

Macrophage

Dendritic cell

ChemokinesCytokines

TLR3/4, RIG-I, MDA5, other receptors Cytokine receptor signalling

STAT-1, NFB, IRF3, others (?) ↑JunB

Presentation of modified antigens

Cell death

MHC class I ER stress Apoptoticsignalling

ChemokinesCytokines

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Process of type I diabetes

Genetic background Immunological malfunctions

T1DM

Metabolic malfunctions

Trigerring mechanism

Autoantibodies, insulitis

Normal insulin

secretion

Decreased insulin

secretionNormal blood sugar level

InsulinC-

peptide presents

C-peptide -

Age

β ce

ll m

ass (

%)

100

HLA-DR3/4

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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

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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.

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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.

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Differentiation of insulin producing β cells from ES cells

HumanES cellOct4

NanogSox2E-cad

MesendodermBraFgf4Wnt3N-cad

Definitiveendoderm

Sox17Cer

FoxA2Cxcr4

Primitivegut tubeHnf1bHnf4a

Posteriorforegut

Hnf6Pdx1Hlxb9

Endocrineprogenitor

Ngn3Nkx2.2Pax4

Nkx6.1

Activin AActivin A

WntFgf10

Cyclopamine

Fgf11CyclopamineRetinoid acid

DAPTExendin-4

Exendin-4IGF-1HGF

Immatureendocrine

InsGluGhrSomPP

Human ES cellOct4

NanogSox2E-cad

MesendodermBraFgf4Wnt3N-cad

Definitiveendoderm

Sox17Cer

FoxA2Cxcr4

Primitivegut tubeHnf1bHnf4a

Posteriorforegut

Hnf6Pdx1Prox1Sox9

Pancreaticendoderm/Endocrineprecursors

Nkx6.1Ptf1a

Nkx2.2Ngn3

Activin AWnt Activin A

Keratinocytegrowth Factor

NogginCyclopamineRetinoid acid In vivo milieu

EndocrineMafAInsGluGhrSomPP

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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.

TÁMOP-4.1.2-08/1/A-2009-0011β-cells generated from existing β-cells through purification and in vitro expansion • 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 alternative origins for b-cells during pancreas regeneration

TÁMOP-4.1.2-08/1/A-2009-0011β-cells generated via a pancreaticstem 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.

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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.

TÁMOP-4.1.2-08/1/A-2009-0011β-cells reprogrammed from somaticcells 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 b-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 b-like phenotype.

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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 iPS cells to generate functional insulin secreting b- cells.