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

LIVER REGENERATION FROM STEM CELLS

Dr. Péter Balogh and Dr. Péter EngelmannTransdifferentiation and regenerative medicine – Lecture 8

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

Glutamine synthetase +(1-3 cells)

Centrilobular(8-10 cells)Limiting plate

Periportal(6-8 cells)

Structure of the hepatic lobe

Portal tracts(triads)

Central vein

Bile duct

Bile canaliculi

Sinusoids

Branch portalvein

Branch hepaticartery

Central vein

Portal tract

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Clinical necessity of liver regeneration• Shortage of livers for orthotopic liver

transplantation• Liver cell transplantation – limited amount• Choice of stem cell candidates – variable

success in experimental conditions

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Stem cells(c-kit, c-met, CXCR4)

Main phases of liver regeneration

3 ClearanceGadolinium chloride/monocrotaline

Immunosuppression

EncapsulationCo-transplantation

Effector cells

Central veinKupffer cells(phagocytosis)

Dead cell

Central veinVasodilatatorsAlteration of blood flow

VEGFHGFTGFFGF

MMP-9MMP-2

MT1-MMP

Cell loss of 70-80%

2 Integration

Gap junctionsVariable in vivo cell phenotype

Organ damageSinus endothelpermeability

Central veinMMP-9SDF-1HGF(SCF)

Organ damage

Recruitment

1 Migration

Monocrotaline

DoxorubicinHepatic injuryVEGF

Physical/chemical/genetical stimulus

TÁMOP-4.1.2-08/1/A-2009-0011Developmental relationship between hepatic-pancreatic differentiation

Oval cell progenitor

Hepatic oval cell

Bile duct Hepatocyte

Pancreatic oval cell

Endocrine cell Acinar cellPancreatic duct

Pancreatic progenitor(s)?

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Transcriptional control of hepatoblast development

Hepatocyte maturation cords

HepatocyteCore transcription

factor network:

HNF-1

LRH-1HNF-6Foxa2

HNF-4

HNF-1

Jagged

Cholangiocyte

Parenchyma PeriportalHNF-1

Sox9

HNF-6/OC-2 TGF

Hex C/EBP

Hepatoblast

HNF-6

HNF-1

Notch2HNF-4

C/EBP

Tbx3

Albumin

HGF

Cholangiocyte maturation ducts

?

WntBMP+FGFFoxM1B

ECM

ECM

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Oval cells – adult liver stem/progenitor cells• Origin: debated (their precursors are

associated with the biliary tree)• Bipotential differentiation: hepatocyte and

cholangiocyte• Phenotype: shared markers with adult

hepatocytes (albumin, cytokeratins 8 and 18), bile duct cells (cytokeratins 7 and 19, OV-6, A6), fetal hepatoblasts (AFP), and haematopoietic stem cells (Thy -1, Sca-1, c-kit).

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Cellular targets for hepatic regeneration• Hepatocytes: metabolic activity of the liver• Cholangiocytes: formation of bile ducts• Both derive from embryonic endodermal

epithelium.

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Stages and forms of liver regeneration• Surgical partial hepatectomy – from hepatocytes

(often polyploid cells)• Possible sources: hepatocytes, oval cells and

extrahepatic stem cells (HSC?)• Assessment of lineage commitment: albumin,

glucose-6-phosphatase, transferrin and transthyretin (hepatic).

• Fibrotic regeneration: transformation of fibrocytes into myofibroblasts

• Parenchymal regeneration: regeneration of hepatocytes

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Sequence of parenchymal regeneration of the liver• Stem cell migration into the liver parenchyma is

directed by chemoattractive agents (as SDF-1, HGF and SCF) secreted by damaged liver cells

• Increased MMP-9 expression by host hepatocytes after injury, leading to ECM remodeling and increased vascular permeability

• Transformation of local microenvironment for the integration and proliferation of the transplanted cells, including local secretion of cytokines/growth factors (HGF, FGF, TGF). Dead cells will be phagocyted by Kupffer cells.

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Oval cell activation and expansion• Liver injury activates oval cells (their precursors in

the biliary tree?) AND other support cells (stellate cells, macrophages/Kupffer’s cells, NK cells, endothelium, etc)

• Homing/intrahepatic migration to the site of injury• Proliferation and bidirectional differentiation

(hepatocyte/cholangiocyte)

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Non-hepatic cells for liver regenerationAutologous: Bone marrow-derived/mesenchymal stem cells – fibroblastic regenerationAllogenic: Fetal-derived hepatocytes or embryonic stem cell-derived liver cells

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Differentiation of iPS cells into hepatocytes• Induction of iPS cells: transfection with TFs• Formation of embryoid bodies• Induction of endodermal commitment: treatment

with Activin A and bFGF• Differentiation into hepatocytes: treatment with

hepatocyte growth factor (HGF)• Assessment: gene expression, albumin secretion,

glycogen storage, urea production, and inducible cytochrome activity

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Summary

• Depending on the origin/type of liver damage, different regeneration processes operate, thus (a) in loss of liver mass, the regeneration is initiated from hepatocytes, whereas (b) in toxicity from hepato-cholangiocyte progenitors.

• Oval cells as adult-type hepatocyte/cholangiocyte progenitors are most likely to be facultative stem cells, although cells with stem cell activity from extrahepatic sources may also operate in liver regeneration.