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
LIVER REGENERATION FROM STEM CELLS
Dr. Péter Balogh and Dr. Péter Engelmann
Transdifferentiation and regenerative medicine – Lecture 8
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
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 portal vein
Branch hepatic artery
Central vein
Portal tract
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
Stem cells (c-kit, c-met, CXCR4)
Main phases of liver regeneration
3 Clearance
Gadolinium chloride/
monocrotaline
Immunosuppression Encapsulation Co-transplantation Effector cells
Central vein Kupffer cells
(phagocytosis)
Dead cell
Central vein
Vasodilatators Alteration of blood flow
VEGF HGF TGF
FGF MMP-9 MMP-2 MT1-MMP
Cell loss of 70-80%
2 Integration
Gap junctions
Variable in vivo cell phenotype Organ damage
Sinus endothel permeability
Central vein MMP-9
SDF-1 HGF (SCF)
Organ damage
Recruitment
1 Migration
Monocrotaline Doxorubicin Hepatic injury
VEGF Physical/chemical/genetical stimulus
Developmental relationship between hepatic-pancreatic differentiation
Oval cell progenitor
Hepatic oval cell
Bile duct Hepatocyte
Pancreatic oval cell
Endocrine cell Pancreatic duct Acinar cell Pancreatic progenitor(s)
?
Transcriptional control of hepatoblast development
Hepatocyte maturation cords
Hepatocyte
Core transcription factor network:
HNF-1
LRH-1
HNF-6 Foxa2 HNF-4
HNF-1
Jagged
Cholangiocyte
Parenchyma Periportal
HNF-1
Sox9
HNF-6/OC-2 TGF
Hex C/EBP
Hepatoblast
HNF-6 HNF-1
Notch2 HNF-4
C/EBP
Tbx3
Albumin HGF
Cholangiocyte maturation ducts
?
Wnt BMP+FGF
FoxM1B ECM
ECM
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).
Cellular targets for hepatic regeneration
• Hepatocytes: metabolic activity of the liver
• Cholangiocytes: formation of bile ducts
• Both derive from embryonic endodermal epithelium.
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
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.
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)
Non-hepatic cells for liver regeneration
Autologous: Bone marrow-derived/mesenchymal stem cells – fibroblastic regeneration
Allogenic: Fetal-derived hepatocytes or embryonic
stem cell-derived liver cells
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
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.
