at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
DIFFERENTIATION AND REGENERATION IN THE PANCREAS
Dr. Péter Balogh and Dr. Péter Engelmann
Transdifferentiation and regenerative medicine – Lecture 9
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
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.
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.
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.
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
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.
Human Mouse
Embryonic pancreas development
e4.5 e5.5 e6.5 e7.5 e8.5 e9.5 e10.5 e11.5 e12.5 e13.5 e14.5
1WD 2WD 3WD 4WD 5WD 6WD
Oct4 Sox2 Nanog
Brachyury T Gsc Gata5 Sox17 Pdx1 Foxa2 Hnf4a
Hhex Mnx1
Ngn3
Nkx6.1 Nkx2.2 Pax6
Neurod1 Pax4
Insm1
MafA
Ptf1a Exocrine
Sox9 Duct
Hnf1b Duct
Onescut1 Duct
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.
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
Maintenance of α cell identity
• Brn4
• Pax6
• Isl1
Maintenance of exocrine identity
• Pdx1
• Ptf1a
• Mist1
• Wnt/b-catenin signaling
• Notch signaling
• TGF-b signaling
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.
Main types of diabetes
• Type 1 Diabetes
• Type 2 Diabetes
• LADA (latent autoimmune diabetes of adulthood)
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
diabetes
Viruses, endogenous ligands? Cytokines
INF-α and INF-β
Apoptotic β cell
b cell
MHC class I
T-cell
+
+
+
+
- -
T-cell TNF
IL-1β INF-
INF-a and INF-β Macrophage
Dendritic cell
Chemokines Cytokines 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 Apoptotic signalling
Chemokines
Cytokines
Process of type I diabetes
Genetic background Immunological malfunctions
T1DM Metabolic
malfunctions Trigerring
mechanism
Autoantibodies, insulitis Normal insulin
secretion
Decreased insulin secretion
Normal blood sugar level
Insulin C-peptide
presents C-peptide -
Age
β cell mass (%)
100
HLA-DR3/4
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
diabetes)
• 20% of patients diagnosed with type 2 diabetes actually has LADA.
• Low, although sometimes moderate, levels of C- peptide
• Autoantibody testing is essential.
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.
cells from ES cells
Human ES cell
Oct4 Nanog
Sox2 E-cad
Mesendoderm Bra
Fgf4 Wnt3 N-cad
Definitive endoderm
Sox17 Cer FoxA2
Cxcr4
Primitive gut tube Hnf1b Hnf4a
Posterior foregut
Hnf6 Pdx1 Hlxb9
Endocrine progenitor
Ngn3 Nkx2.2
Pax4 Nkx6.1 Activin A
Activin A Wnt
Fgf10 Cyclopamine
Fgf11 Cyclopamine Retinoid acid
DAPT Exendin-4
Exendin-4 IGF-1
HGF
Immature endocrine
Ins Glu Ghr Som
PP
Human ES cell
Oct4 Nanog
Sox2 E-cad
Mesendoderm Bra
Fgf4 Wnt3 N-cad
Definitive endoderm
Sox17 Cer FoxA2
Cxcr4
Primitive gut tube Hnf1b Hnf4a
Posterior foregut
Hnf6 Pdx1 Prox1
Sox9
Pancreatic endoderm/
Endocrine precursors
Nkx6.1 Ptf1a Nkx2.2
Ngn3 Activin A
Wnt Activin A
Keratinocyte growth Factor
Noggin Cyclopamine
Retinoid acid In vivo milieu
Endocrine MafA
Ins Glu Ghr Som
PP
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.
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
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.
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.
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 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.
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.
