Transcript pregnancy in vitro

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
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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.
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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 PPcell compartments.
Specification of the
pancreas I
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• 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
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• 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
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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
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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
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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
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
β cell and autoimmune
processes of diabetes
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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, NFB, 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
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T1DM
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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 βcells.
Differentiation of insulin
producing β cells from ES
cells
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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
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• 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
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• 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
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• 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
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• 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
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• 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
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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