1. 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

2. LIVER REGENERATION FROM STEM CELLS Dr. Péter Balogh and Dr. Péter Engelmann Transdifferentiation and regenerative medicine – Lecture 8 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

3. 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

4. TÁMOP-4.1.2-08/1/A-2009-0011 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

5. TÁMOP-4.1.2-08/1/A-2009-0011 Stem cells (c-kit, c-met, CXCR4) Main phases of liver regeneration 3 Clearance Gadolinium chloride/ monocrotaline Gadolinium chloride/ monocrotaline Immunosuppressi on Encapsulation Co- transplantation Immunosuppressi on 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 Monocrotal ine Doxorubici n Hepatic injury VEGF Monocrotal ine Doxorubici n Hepatic injury VEGF Physical/chemical/genetical stimulus

6. TÁMOP-4.1.2-08/1/A-2009-0011 Developmental relationship between hepatic-pancreatic differentiation Oval cell progenitor Hepatic oval cell Bile ductHepatocyte Pancreatic oval cell Endocrine cellAcinar cellPancreatic duct Pancreatic progenitor(s) ?

7. TÁMOP-4.1.2-08/1/A-2009-0011 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 ParenchymaPeriportal 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

8. TÁMOP-4.1.2-08/1/A-2009-0011 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).

9. TÁMOP-4.1.2-08/1/A-2009-0011 Cellular targets for hepatic regeneration Hepatocytes: metabolic activity of the liver Cholangiocytes: formation of bile ducts Both derive from embryonic endodermal epithelium.

10. TÁMOP-4.1.2-08/1/A-2009-0011 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

11. TÁMOP-4.1.2-08/1/A-2009-0011 Sequence of parenchymal regeneration of the liver Stem cell migrationStem 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 expressionIncreased MMP-9 expression by host hepatocytes after injury, leading to ECM remodeling and increased vascular permeability Transformation of local microenvironmentTransformation 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.

12. TÁMOP-4.1.2-08/1/A-2009-0011 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)

13. TÁMOP-4.1.2-08/1/A-2009-0011 Non-hepatic cells for liver regeneration Autologous: Autologous: Bone marrow- derived/mesenchymal stem cells – fibroblastic regeneration Allogenic: Allogenic: Fetal-derived hepatocytes or embryonic stem cell-derived liver cells

14. TÁMOP-4.1.2-08/1/A-2009-0011 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

15. TÁMOP-4.1.2-08/1/A-2009-0011 Summary (a) (b)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.