Falk Research Workshop

Liver Regeneration, Stem Cells and Transdifferentiation January 19–20, 2006 Gewandhaus zu Leipzig Germany

Abstracts Poster Abstracts

Abstracts of Invited Lectures Poster Abstracts

Falk Research Workshop

LIVER REGENERATION, STEM CELLS AND TRANSDIFFERENTIATION

Leipzig (Germany) January 19 - 20, 2006

Scientific Organization: B. Christ, Halle (Germany) W.E. Fleig, Halle (Germany) R. Gebhardt, Leipzig (Germany)

CONTENTS

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Stem cells and liver Chair: W.E. Fleig, Halle D. Tosh, Bath Embryonic stem cell differentiation into hepatic phenotypes A.M. Wobus, Gatersleben

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Concepts of genomic plasticity and of the hepatic stem cell niche N.D. Theise, New York

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Bipotential mouse embryonic liver cell lines are a tool for liver stem cell biology H. Strick-Marchand, Paris

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Hepatocytes from stem cells B. Christ, Halle

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Liver repopulation Chair: N.D. Theise, New York C. Trautwein, Hannover Transcriptional networks in progenitor (oval) cell-mediated liver regeneration H.C. Bisgaard, Copenhagen Liver repopulation after hepatocellular transplantation S. König, Göttingen Fetal liver cells for cell therapy S. Sumitran-Holgersson, Stockholm

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18 - 19

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Recruitment of extrahepatic stem cells for liver tissue repair M. Ott, Hannover

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Transdifferentiation of cells from liver and pancreas Chair: V. Keim, Leipzig A.M. Wobus, Gatersleben Conversion of pancreatic cells to hepatocytes D. Tosh, Bath

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The mechanism of liver to pancreas transdifferentiation S. Ferber, Tel Hashomer

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Generation of hepatocyte-like cells from human extrahepatic stem and precursor cells J.G. Hengstler, Leipzig

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Selected short poster presentations Transdifferentiation of hepatocytes to metastatic myofibroblasts during late stage liver tumorigenesis M. Mikula, Vienna

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Identification of new surface markers for fetal liver stem cells D. Nierhoff, New York

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Expression of hepatocytic markers after differentiation of mouse embryonic stem cells into the pancreatic lineage A. Rolletschek, Gatersleben

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The contribution of bone marrow cells to liver progenitor cells in CDE diet and murine hepatitis virus induced injury J.N. Tonkin, Perth

List of Speakers, Moderators and Scientific Organizers

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33 - 34

Poster Abstracts 1.

Transdifferentiation of pancreatic AR42J-B13 cells to hepatocyte and ductal phenotypes A. Al-Adsani, K.L. Ralphs, D. Tosh (Bath, GB)

2.

Induction of periportal- and prevenous-like hepatocytes from pancreatic cells Z.D. Burke (Bath, GB)

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In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells T. Cantz, M. Bleidißel, H.R. Schöler (Münster, D)

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Homeobox transcription factor Prox1 in rat models of liver damage and regeneration J. Dudas, T. Mansuroglu, A. Elmaouhoub, D. Batusic, K. Tron, B. Saile, J. Wilting, G. Ramadori (Göttingen, D)

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Common morphology, metabolism and signal transduction in primary hepatocytes and blood monocyte derived neo-hepatocytes from patients with alcoholic liver disease S. Ehnert, U. Böcker, K. Breitkopf, M.V. Singer, A. Nüssler, S. Dooley (Mannheim, Bad Homburg, D)

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Ultrastructural hepatic changes after bone marrow transplantation in murine schistosomiasis N. Elkhafif, H. Yehia, O. Hammam, S. Mahmoud, H. Helmy, S. Boehm, B. Voss, S. Mansy, M. Akl, G. El Fandy (Giza, ET; Bochum, D)

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Induction of maturation in human fetal hepatoblasts under different culture conditions E.C.S. Ellis, K. Bodin, H. Cai, S.C. Strom (Pittsburgh, USA; Stockholm, S)

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Characterization of embryonic hepatic cells during rat liver development A. Elmaouhoub, J. Dudas, G. Ramadori (Göttingen, D)

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Erythropoietin stimulates liver regeneration E. Hogrebe, J.G. Hengstler, A. Tannapfel, A. Bauer, W. Jelkmann, J.P. Hauss, M.R. Schön (Leipzig, Bochum, Lübeck, D)

10. An acellularized vascularized matrix: Scaffold for the development of a liver cell modul and for stem cell differentiation studies K. Linke, J. Schanz, H. Mertsching (Stuttgart, D) 11. Expression of the homeobox transcription factor Prox1 in human chronic liver disease and in hepatocellular/cholangiocellular carcinoma T. Mansuroglu, J. Dudas, B. Saile, T. Armbrust, F. Moriconi, J. Wilting, T. Pieler, L. Füzesi, G. Ramadori (Göttingen, D)

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12. Ultrastructural effects of taurine treatment in experimental liver fibrosis M.R. Mas, I. Tasci, A.T. Isik (Etlik, TR) 13. Stem cell characteristics of amniotic epithelial cells T. Miki, T. Lehmann, H. Cai, D.B. Stolz, S.C. Strom (Pittsburgh, USA) 14. Transdifferentiation of hepatocytes to metastatic myofibroblasts during late stage liver tumorigenesis M. Mikula, E. Fuchs, W. Mikulits (Vienna, A) 15. Remodeling of hepatocyte calcium signaling during liver regeneration in the rat is linked to cell cycle progression A. Nicou, V. Serrière, S. Prigent, M. Hilly, L. Combettes, G. Guillon, T. Tordjmann (Orsay, Montpellier, F) 16. Identification of new surface markers for fetal liver stem cells D. Nierhoff, L. LeVoci, R. Norel, L.E. Rogler, D.A. Shafritz (Bronx, USA) 17. Cell-cell competition: A new way to generate liver tissue from fetal liver stem/progenitor cells M. Oertel, A. Menthena, M.D. Dabeva, D.A. Shafritz (Bronx, USA) 18. Liver regeneration in a retrorsine/CCI4-induced acute liver failure model: Do bone marrow-derived cells contribute? F.C. Popp, P. Slowik, P. Piso, H.J. Schlitt, M.H. Dahlke (Regensburg, D) 19. Transdifferentiated hepatocytes: An in vitro model for liver function K.L. Ralphs, Z.D. Burke, D. Tosh (Bath, GB) 20. TIMP-1 antagonism reduces toxic-induced hepatic fibrosis but not biliary hepatic fibrosis in mouse models M. Roderfeld, R. Weiskirchen, S. Hillebrandt, F. Lammert, A.M. Gressner, M.-L. Berres, S. Matern, E. Roeb (Gießen, Aachen, Bonn, D) 21. Expression of hepatocytic markers after differentiation of mouse embryonic stem cells into the pancreatic lineage A. Rolletschek, I.S. Schröder, A. Daniel-Wojcik, A.M. Wobus (Gatersleben, D) 22. Influence of demethylating agents on in vitro differentiation of adult liver stem cells into the hepatocytic lineage I.S. Schröder, V.M. Factor, J.-S. Lee, S.S. Thorgeirsson (Gatersleben, D; Bethesda, USA) 23. Immune-mediated hepatitis drives low-level fusion between hepatocytes and adult bone marrow cells P. Slowik, F.C. Popp, P. Piso, H.J. Schlitt, M.H. Dahlke (Regensburg, D) 24. High expression of matrilin-2 by stem cells in liver regeneration E. Szabo, C. Lódi, E. Korpos, I. Kiss, F. Deak, S. Paku, P. Nagy, Z. Schaff, A. Kiss (Budapest, Szeged, H)

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25. Two-stage hepatectomy in huge right lobe liver tumors A. Szijártó, B. Hargitai, P. Kupcsulik (Budapest, H) 26. Liver fibrosis regresses better with ursodeoxycholic acid treatment than spontaneous recovery I. Tasci, M.R. Mas, N. Mas (Etlik, TR) 27. Relationships between haematopoietic and epithelial lineages in human fetal liver J.D. Terrace, I.S. Currie, N.M. Masson, R.W. Parks, J.A. Ross (Edinburgh, D) 28. Transdifferentiation of hepatocytes into pancreatic β-cells S. Thowfeequ, W.-C. Li, M. Hornsey, D. Eberhard, J.M.W. Slack, D. Tosh (Bath, GB) 29. Immortalised bipotential cell lines as a tool for oval cell biology J.E.E. Tirnitz-Parker, J. Olynyk, G.C.T. Yeoh (Crawley, Perth, AUS) 30. The contribution of bone marrow cells to liver progenitor cells in CDE diet and murine hepatitis virus induced injury J.N. Tonkin, D. Curtis, G.C.T. Yeoh (Perth, Melbourne, AUS) 31. Oval cells compensate for premature senescence mediated inhibition of proliferation of hepatocytes in liver-specific, tetracycline regulated p16INK4a transgenic mice E. Ueberham, R. Bittner, R. Gebhardt, U. Ueberham (Leipzig, D) 32. Enhanced expression of hepatotrophic mitogen ALR (Augmenter of Liver Regeneration) in hepatocellular carcinoma and cirrhosis H. Wagner, W.E. Thasler, F. Bataille, A. Hartmann, K.-W. Jauch, H.-J. Schlitt, T.S. Weiss (Regensburg, Munich, D) 33. Partial hepatectomy induces regeneration with high levels of telomerase activity in pig livers H. Wege, A. Nunez, L. Müller, A. Müller, C. Hillert (Hamburg, D) 34. C/EBPα and C/EBPβa expression in the developing mouse embryo A. Westmacott, Z.D. Burke, J.M.W. Slack, D. Tosh (Bath, GB) 35. Expression and localization of hepatocyte growth factor (HGF), its receptor cMet and HGF activator (HGFA) in human liver cell cocultures maintained in 3D multicompartment bioreactors K. Zeilinger, K. Mutig, P. Schrade, P. Neuhaus, S. Bachmann, J.C. Gerlach (Berlin, D)

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Stem cells and liver

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Embryonic stem cell differentiation into hepatic phenotypes Anna M. Wobus Leibniz Institute of Plant Genetics (IPK), D-06466 Gatersleben, Germany, E-mail: [email protected] Due to the high incidence of liver-related diseases and the lack of suitable donor cells and tissues for transplantation, alternative sources for generating transplantable hepatic cells are needed. We established novel protocols for the generation of functional pancreatic or hepatic cells from embryonic stem (ES) cells via multilineage precursor and committed lineage-specific (pancreatic or hepatic) progenitor cells. Here, we describe the ES cell differentiation into the hepatic lineage. ESderived committed hepatic progenitor and differentiated hepatic phenotypes were characterized by RT-PCR, immunohistochemistry and enzyme assays. During hepatic differentiation, ES-derived cells transiently co-expressed nestin and albumin (ALB) as well as nestin and α-fetoprotein (AFP), respectively, but not cytokeratin 19 (CK 19) suggesting that ES cell differentiation in vitro involves different intermediate filament proteins compared to hepatic progenitors in vivo. These data allowed us to postulate a progenitor cell population that develops during hepatic differentiation of ES cells (Nestin+/ALB+/AFP+/CK19-). Following terminal differentiation, hepatic cells in vitro expressed liver-specific genes and proteins and acquired a functional hepatic phenotype as demonstrated by glycogen synthesis and albumin secretion. Experiments are in progress to increase the level of endodermal precursor and hepatic progenitor cells by extra-cellular factors and epigenetic modification.

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Concepts of genomic plasticity and of the hepatic stem cell niche N.D. Theise Beth Israel Medical Center, Division of Digestive Diseases, New York, NY 10003, USA In recent years, the existence of hepatic stem and progenitor cells has not only become generally accepted, but both intra- and extrahepatic pools of such cells are now recognized. Extrahepatic progenitors may contribute by several possible, posited routes: direct differentiation as hepatocytes, direct differentiation as hepatobiliary cells of ductular reactions (“oval cells” in rodent models), cell-cell fusion with pre-existing hepatocytes, with or without nuclear fusion, and possibly followed by reductive cell division. Intrahepatic progenitor cells are posited to exist in four possible locations: periportal hepatocytes (the “streaming liver” model), canals of Hering, cholangiocytes within bile ducts, and periductal cells which are cytokeratin negative and often lack any other stem/progenitor cell specific markers (“null cells”). In vivo assays for infrequently cycling progenitor cells, identified as “label retaining cells”, can be used to verify the tissue location of stem cells. Approaches to applying such assays to the liver and preliminary results of such experiments will be presented. Identifying the intrahepatic stem cell niche in this fashion also allows for definition of cell-cell and cell-matrix interactions which are likely to influence activation of stem cells in response to different types of injury and to direct differentiation down hepatocyte or cholangiocyte lineages in response to physiologic requirements.

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Bipotential mouse embryonic liver cell lines are a tool for liver stem cell biology Helene Strick-Marchand, Serban Morosan*, Graham Hayhurst, Catherine Deschatrette, Tereza Imaizumi-Scherrer, Daniela Faust, Pierre Charneau, Dina Kremsdorf*, Mary C. Weiss Institut Pasteur, 25, rue du Dr. Roux, F-75724 Paris France *Hopital Necker U370 Inserm, 156, rue de Vaugirard, F-75015 Paris, France In the embryo, hepatoblasts respond to growth and differentiation signals which lead to the development of the two major specialized cell types of the liver: hepatocytes and bile duct cells. The growth factors and cell-cell interactions involved in these processes are not fully understood. We have established a simple and reproducible method to isolate bipotential cell lines from mouse embryonic livers. In culture these BMEL (bipotential mouse embryonic liver) cell lines express liver-enriched transcription factors (HNF1, HNF3, HNF4) without expressing hepatocyte or bile duct cell functions. BMEL cell lines are composed of diploid and tetraploid cells that can be subcloned and are not transformed. The expression of hepatocyte functions (production of serum proteins, metabolic enzymes, detoxifying enzymes) can be induced by changing the culture conditions. Likewise, BMEL cells cultured on Matrigel differentiate into bile duct cells. Stem cells are able to differentiate into more than one cell type in vitro and in vivo, where they are required to respond to signals of growth and differentiation to participate in organ repair when needed. In the Alb-uPA/Scid mouse model of liver regeneration, the Alb-uPA deleterious transgene induces cell death specifically in the liver of newborn mice. Rare hepatocytes which excise the transgene progressively restore the liver's architecture and mass by proliferating and differentiating within a few weeks. To determine whether BMEL cells are stem cells which participate in liver regeneration, they were injected intrasplenically into Alb-uPA/ Scid mice. Analysis of the transplanted livers showed that BMEL cells had indeed differentiated in vivo as hepatocytes or as bile duct cells. Cell-type specific genes, such as cytokeratins (CK7, CK19), liver-enriched transcription factors (HNF4), and metabolic enzymes (glutamine synthetase, CPSI), were found to be correctly expressed by BMEL cells in vivo according to the zonal distribution of the liver. Thus BMEL cells respond appropriately to growth and differentiation signals in the regenerating liver. One of the characteristics of hematopoietic stem cells and of freshly isolated adult hepatocytes is their capacity to repopulate a damaged tissue after serial transplantations. BMEL cells retrieved from a repopulated Alb-uPA/ Scid mouse were also able to participate in the regeneration of a secondary recipient. BMEL cell lines isolated from embryos of knock-out mice, as well as cell lines isolated from adult mice, have been tested for their ability to proliferate and differentiate in the Alb-uPA/ Scid mouse model. Taken together, the results obtained with the different cell lines allow us to have a clearer understanding of which cells are able to proliferate and differentiate in vivo in the context of liver regeneration.

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Hepatocytes from stem cells Bruno Christ Universitätsklinik und Poliklinik für Innere Medizin I, Bereich Molekulare Hepatologie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany The limited number and quality of marginal human donor organs for the isolation of human hepatocytes restrict the outcome of the clinical application of hepatocyte transplantation as an alternative to orthotopic liver transplantation. Therefore, alternative cell resources have to be explored in order to generate hepatocytes of adequate quality for clinical use. Different kinds of stem cells from various sources have been investigated for their potential to differentiate into hepatocyte-like cells in vitro and in vivo featuring selected markers and functions of differentiated adult hepatocytes. Based on their multiple differentiation potential and their proliferative capacity, mesenchymal stem cells (MSC) may serve as a well suitable source to generate hepatocytes for the use in humans. Here, MSCs were isolated from human bone marrow and, in response to specified growth factors gained hepatocyte-specific morphology and functions in vitro. These comprised the expression of hepatocyte epithelial cell markers, plasma proteins, metabolic enzymes, and proteins specific for hepatocyte cell-cell communication. Differentiated MSC stored glycogen, synthesised urea and featured the active hepatocyte-specific gene promotor of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into immunodeficient mice, cells integrated into the host parenchyma in periportal regions of the recipient liver and retained prominent qualities of hepatocytes after their regional integration. Thus, human bone marrow-derived MSC may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.

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Liver repopulation

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Transcriptional networks in progenitor (oval) cell-mediated liver regeneration Hanne Cathrine Bisgaard Department of Medical Biochemistry and Genetics, The Panum Institute, Bldg. 6.5.40, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark, E-mail: [email protected] Generation of new cells in the injured adult liver depends primarily on a two-tier cell system comprised of mature, pre-existing liver cells and endogenous liver progenitor cells. What is unique to the liver is that pre-existing mature cells constitute the primary option of response to injury, while progenitor cells function as a reserve compartment that is activated when the regenerative capacity of mature cells is compromised. The cellular and molecular responses orchestrating regeneration from mature or progenitor cells appear to depend on the hepatic injury inflicted and therefore on the molecular microenvironment created in the liver by a certain insult. In recent years, much has been learned about the molecular responses governing regeneration from mature cells. However, the molecules and pathways regulating the activity of hepatic progenitor cells are still incompletely understood. Elucidating the molecular responses and subsequently the mechanism(s) that control the activation and expansion of progenitor cells in the hepatic niche is of fundamental importance because it is a prerequisite for understanding the progenitor cell-mediated liver regeneration and the potential development of novel pharmacological and/or transplantation based approaches to treatment of liver diseases. We have exploited a number of functional genomics and computational pathway analysis tools to examine gene expression changes that occur in progenitor cell-mediated regeneration using the rat as our experimental model system with the aim of understanding the biological processes and specific regulatory networks involved. This has allowed us to identify large networks of genes that are either up-regulated or down-regulated at the mRNA level in vivo when progenitor cells accumulate to aid liver regeneration. The two largest gene networks identified comprise genes whose main function is in the immune system (Network 1: these genes are mainly up-regulated) and liver metabolism (Network 2: these genes are mainly down-regulated). The importance of these networks for progenitor cell-mediated regeneration will be discussed.

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Liver repopulation after hepatocellular transplantation Sarah König Dept. of General Surgery, Georg-August University of Göttingen, Germany At present, whole organ transplantation is the only procedure available for the advanced stage of many serious liver diseases, and the need for suitable donor livers is continuously rising (1). However, there is only limited availability of such organs. The search for an alternative to correct acute or chronic liver failure and genetically related defects is therefore of central importance (2, 3). A promising option is the functional repopulation of the diseased organ through the transplantation of isolated cells (4, 5). Experimental transplantation of hepatic cells aiming to repopulate the host liver has two fundamental requirements: Firstly, donor cells need to be identified within the recipient tissue. Secondly, selective proliferation stimulus is necessary for the transplanted cells to proliferate in preference to host cells. The Dipeptidylpeptidase IV (DPPIV)-mutant/Fischer 344 rat liver cell transplantation model implementing retrorsine and partial hepatectomy as selective stimulus, which has recently been investigated by the workgroup (6), fulfils both conditions: Transplanted hepatocytes integrate rapidly into the accustomed liver tissue and proliferate extensively, finally repopulating the host liver. The reconstitution of organ mass in this transplantation model is remarkably fast, homogeneous and reproducible. Within 3 to 5 days, transplanted cells are fully integrated and start to proliferate in the host parenchyma, where the release of growth hormones and cytokines ensuing from the partial hepatectomy guarantees ideal conditions. Hybrid membrane domains (connexin 32) form between host and donor cells, as well as polarised membrane domains (DPPIV) on transplanted cells and descendents, providing strong evidence for tissue integrity and intercellular communication. During the process of repopulation, the host architecture of the liver is completely respected by the expanding donor hepatocytes. This rat liver cell transplantation model is exceptionally reliable when tracking the fate of engrafted cells. However, the retrorsine model is not really suited to human application, as the reagent is an alkaloid with systemic toxic and carcinogenic side effects. Therefore, less harmful stimuli as an alternative to prime the host liver have to be studied. It was shown recently that liver irradiation may provide a promising alternative to retrorsine pre-treatment (7, 8). The capacity of adult hepatocytes to proliferate und repopulate the liver is outstanding. Even so, the availability of mature hepatocytes is as much restricted as are whole donor organs. The focus is therefore shifting towards progenitor and stem cells as the regenerative resource for cell therapy and organ replacement in many prospective studies. Nevertheless, we do not know yet if liver precursor cells repopulate the recipient liver more rapidly and effectively than adult hepatocytes. The basic key for any successful cell transplantation would certainly be repopulation without any selective stimulus. The question remains as to whether this can be achieved by precursor cells.

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References: 1. Thalheimer U.; Capra F. Liver transplantation: making the best out of what we have. Dig Dis Sci. 2002; 47: 945-953 2. Markus P.M.; Krause P.; Becker H.; Röher H.D. Hepatocyte transplantation - an alternative to orthotopic liver transplantation? Z Tx Med. 1994; 6: 138-140 3. Sterling R.K.; Fisher R.A. Liver transplantation. Living donor, hepatocyte, and xenotransplantation. Clin Liver Dis. 2001; 5: 431-460 4. Allen, K.J.; Soriano HE. Liver cell transplantation: the road to clinical application. J Lab Clin Med. 2001; 138: 298-312 5. Gupta S. Hepatocyte transplantation. J Gastroenterol Hepatol. 2002; 17: S287S293 6. Koenig S, Stoesser C, Krause P et al. Liver repopulation after hepatocellular transplantation: integration and interaction of transplanted hepatocytes in the host. Cell Transplant. 2005; 14: 31-40 7. Guha C, Sharma A, Gupta S et al. Amelioration of radiation-induced liver damage in partially hepatectomized rats by hepatocyte transplantation. Cancer Res. 1999; 59: 5871-5874 8. Guha C, Parashar B, Deb NJ et al. Liver irradiation: a potential preparative regimen for hepatocyte transplantation. Int J Radiat Oncol Biol Phys. 2001; 49: 451-457

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Fetal liver cells for cell therapy Suchitra Holgersson Karolinska Institute, Stockholm, Sweden Liver disease is an exceptionally common cause of morbidity and mortality worldwide. Despite the high incidence of diseases that result in liver dysfunction and failure, major advances in medical therapies are currently limited to the prevention and treatment of certain forms of viral hepatitis. Acute and chronic liver diseases are still treated with supportive rather than curative approaches. Orthotopic liver transplantation and hepatocyte cell transplantation have so far been the only available therapy for patients with end-stage liver failure. Unfortunately, the availability of donor organs is limited and many patients die each year waiting for liver transplants. Cellular therapy with stem cells and their progeny is a promising new approach to this largely unmet medical need. The achievement of positive outcomes in many clinical protocols involving liver progenitor cells has been handicapped by the limited numbers of liver repopulating cells available to actually bring about therapy. The mammalian fetal liver contains a compartment of colony-forming cells with highproliferative potential. The fetal liver is therefore an attractive source for isolation of hepatic progenitors for transplantation. We have meticulously phenotyped and characterized the various cell types present in developing human livers. For this purpose, we analysed 94 human fetal livers in gestation weeks between 5 and 18. We demonstrated that the liver is essentially a “hematopoietic organ” in the first trimester followed by a shift to a “hepatopoietic organ” during the second trimester. We also demonstrated that human fetal cells expressing CD117/CD34 can in vitro differentiate to hepatic cells expressing albumin and CK19. In a subsequent study, we identified a defined population of progenitor cells from human fetal livers expressing the markers CD117+/CD34+/Lin- that could be successfully expanded ex vivo by more than 4,000-fold over the input numbers (GUT 2005, In press). These cells could be maintained with stable morphology and phenotype for several passages. When cells in various passages were transplanted into animals with acute liver injury they exhibited functional differentiation into hepatocytes, cholangiocytes and sinusoidal cells. Furthermore, transplanted cells repopulated the hepatic compartments of acutely damaged livers. Thus, successful in vitro expansion of human liver progenitor cells (HLPC) provides a basis for developing cell therapy strategies, metabolic and toxicity testing systems, and may serve as a vehicle for gene therapy.

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Recruitment of extrahepatic stem cells for liver tissue repair Michael Ott, MD, Amar Deep Sharma, MD/PHD & Michael P. Manns, MD Hannover Medical School, Department of Gastroenterology, Hepatology and Endocrinology, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany The liver has a large capacity to regenerate after tissue loss or injury. Most of the regenerative capacity is provided by division the adult hepatocytes. When the regeneration of the liver tissue by hepatocytes is exhausted or experimentally inhibited, a facultative stem cell pool can be activated. Small oval shaped cells (termed “oval cells”) expand from ductular structures and differentiate into cholangiocytes and hepatocytes. Oval stem cells of the liver have been extensively studied in mice and rats and shown to express hepatocytic, cholangiocytic and hematopoietic markers. Recent experimental observations have suggested that not only hepatocytes and “oval cells”, but also extrahepatic stem cells participate in liver tissue regeneration. Petersen et al., Alison et al. and Theise et al. were among the first to show in rats, mice as well as in humans that hepatocytes and cholangiocytes could be generated from bone marrow. With Y-chromosome staining and liver specific markers they detected bone marrow derived hepatocytes in the liver of irradiated mice and humans after gender mismatched bone marrow transplantation indicating participation of extrahepatic stem cells in liver regeneration. These observations led to the conclusion, that the oval cell compartment of the liver might originate from extrahepatic (hematopoetic) stem cells. Understanding the mechanisms of transdifferentiation/plasticity of extrahepatic stem cells would provide important clues for the use of stem cells in organ repopulation and regeneration. Whether the phenomenon of stem cell plasticity exists at all in adult somatic stem cells, has recently become a controversially debated issue. The reports, which followed the initial experiments by Theise, Alison and Petersen, have either favoured or opposed the concept of differentiation of bone marrow cells into many different types of tissue cells. Lagasse et al. have shown that highly purified stem cells isolated from the bone marrow of adult mice rescued the liver defect in the fumaryl acetoacetate hydrolase FAH(-/-) mouse, an animal model of tyrosinemia type I, by restoring the biochemical function of its liver. The transplanted bone marrow cells were able to protect the mice from lethal irradiation and to generate functional hepatocytes in the liver. The generation of hepatocytes, however, was not the result of direct differentiation, but occurred by fusion of hematopoietic cells with recipient hepatocytes under the high selection pressure in this model. Krause et al. and Harris et al. injected highly purified bone marrow cells into irradiated mice and obtained engraftment in several organs, including skin, lung, and liver without apparent signs of cell fusion. In contrast to these experiments, other groups including ours failed to show a significant contribution of bone marrow derived cells in liver regeneration of mice. After reconstitution with either EGFP- or β-galactosidasetransgenic hematopoietic stem cells only a very few marker gene positive nonhematopoietic cells were detected in the recipient livers.

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Umbilical cord blood contains (hematopoietic) stem cells, which differ in some aspects from bone marrow hematopoietic stem cells and may have a higher plasticity than the adult somatic (hematopoietic) stem cells. It has been reported that intravenous administration of human UCBs in the mouse model of amyotrophic lateral sclerosis replaced damaged neurones and also produced primitive neuropoietic progenitors. Transplantation of hUCB into fetal sheep resulted in human hepatocyte formation in a non-injury animal model without signs of fusion. One study has shown expression of a variant AFP transcript in human umbilical cord blood cells that might suggest the presence of some non-hematopoietic primitive progenitors, which may have the potential to differentiate into cells of hepatic as well as hematopoietic phenotype. Human umbilical cord blood is highly enriched for hematopoietic stem cells and can partially repopulate the bone marrow of NOD-SCID mice. Few recent articles have highlighted the transdifferentiation potential and plasticity of human cord blood cells and the generation of human hepatocytes from transplanted cord blood cells in NODSCID mice. We were able to show that human cord blood cells give rise to hepatocyte like cells after transplantation into NOD-SCID mice in response to CCl4 induced liver injury, whereas mouse bone marrow cells rarely formed hepatocyte-like cells in similar experimental conditions. To the best of our knowledge we show for the first time that human cord blood cells derived hepatocyte-like cells expressing human albumin and human hepatocyte specific antigen Hep Par 1 do not express human cytokeratins 8 and 18, but express their murine counterpart as demonstrated for cytokeratin 18.

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Transdifferentiation of cells from liver and pancreas

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Conversion of pancreatic cells to hepatocytes David Tosh Centre for Regenerative Medicine, Department of Biology & Biochemsitry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom Transdifferentiation means the conversion of one cell type to another and demonstrates that embryological commitments can be reversed under pathological and experimental conditions. Although a number of examples of transdifferentiation exist in the literature, little is known of the cellular and molecular mechanisms underlying the switch in phenotype. We have developed two models for the transdifferentiation of pancreatic cells to hepatocytes. Both models are based on culture with the synthetic glucocorticoid dexamethasone. The first model is the rat pancreatic cell line AR42J and the second is mouse embryonic pancreas. Using the AR42J system we have shown that the hepatocytes arise directly from pancreatic exocrine cells and that the transcription factor C/EBPβ is required for transdifferentiation. Following addition of dexamethasone to cultured AR42J, a number of liver cell types are induced. For example, cells with a biliary phenotype (cytokeratin 7 and 19 positive) are induced as well as sub-populations of hepatocytes (periportal and perivenous hepatocytes) based on the reciprocal expression of carbamoylphosphate synthetase and glutamine synthetase. Lastly, we have examined the properties of the transdifferentiated hepatocytes and found that they exhibit features typical of bona fide hepatocytes. For example, following addition of glucocorticoid the cells are responsive to hormones and xenobiotics and can replicate hepatitis B virus. These results suggest that transdifferentiated liver cells are a good model for studying liver function.

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The mechanism of liver to pancreas transdifferentiation Sarah Ferber, PhD Chaim Sheba Medical Ctr. and Tel-Aviv University, Tel Hashomer, Israel It has long been thought that mammalian cells once committed to a specific lineage, generally, can no longer change their fate and thus become "terminally differentiated". Recent studies performed in numerous labs, suggest the capacity to endow adult liver cells with pancreatic characteristics and function using ectopic expression of pancreatic transcription factors. Sapir et al. (PNAS 2005) demonstrated that about 25% of adult human liver cells, treated by PDX-1 and soluble factors, start to produce insulin, process it and secrete it in a glucose regulated manner. Moreover these cells ameliorated hyperglycemia upon implantation in diabetic SCID-NOD mice. Here we disclose the mechanism of liver to pancreas transdifferentiation induced by ectopic expression of PDX-1. Using DNA microarray analysis we demonstrate that ectopic expression of PDX-1 in adult human liver cells induces wide changes in the gene expression profile. In addition to the induction of the pancreatic repertoire, PDX1 turns off the mature hepatic repertoire that includes the expression of Albumin, ADH1b and α-anti-trypsin inhibitor genes. Importantly, hepatic dedifferentiation was not associated with accelerated cell proliferation, in-vitro, and was unique to Pdx-1 but not to other pancreatic transcription factors expression in liver. The identification of key events in the transdifferentiation process will allow us to increase the therapeutic merit of using liver as a source of pancreatic. Using adult liver as pancreatic progenitor tissue, may allow the diabetic patient of being also the donor of his own therapeutic tissue. Therefore, this approach overcomes both the shortage in tissue availability for allo-transplantation, and the need for life-long, immune suppression.

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Generation of hepatocyte-like cells from human extrahepatic stem and precursor cells Jan G. Hengstler1, Maren Ruhnke2, Hendrik Ungefroren2, Fred Fändrich2, Marc Brulport1, Matthias Hermes1, Wiebke Schormann1, Alexander Bauer1 and Andreas Nüssler3 1 Center for Toxicology, Institute of Legal Medicine and Rudolf-Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Germany; 2Department of General and Thoracic Surgery, University Hospital Schleswig-Holstein, Campus Kiel, Germany; 3Fresenius Biotech, Bad-Homburg, Germany Primary human hepatocytes are well established in drug metabolism research. In addition transplantation of isolated hepatocytes represents a promising strategy for treatment of acute and chronic liver disease. The major limitation working with human hepatocytes is availability. Although fresh and cryopreserved human hepatocytes are meanwhile commercially available, the extremely high costs limit their application in research laboratories. Unfortunately, an efficient proliferation of human hepatocytes in vitro has not yet been achieved. Recently, several articles reported about generation of “hepatocyte-like cells” from different types of extrahepatic stem or precursor cells. At a first glance this seems to open exciting new possibilities for Pharmacology and Toxicology, since some types of stem cells proliferate efficiently in vitro and therefore might help to generate an unlimited supply of human hepatocytes. Without doubt the possibility of unlimited human hepatocyte generation would be an important breakthrough for drug metabolism research and would also open new perspectives for the treatment of liver disease. In the present study we examined three different types of extrahepatic human stem and precursor cells, namely (i) nestin positive hepatopancreatic precursor cells, (ii) dedifferentiated human blood monocytes and (iii) adherently proliferating cells isolated from human cord blood. We studied the fate of these cells after transplantation into the livers of SCID/NOD mice. Three weeks after transplantation we observed hepatocyte-like cells expressing human albumin in the mouse livers. This encouraged us to establish in vitro protocols for differentiation of these extrahepatic cell types to hepatocyte-like cells. We achieved expression of several hepatocellular functions with similar activities compared to primary human hepatocytes, such as albumin and urea secretion as well as several phase I and II metabolizing enzymes. However, not all activities were expressed in a hepatocytelike manner. For instance basal as well as rifampicin induced activities of CYP3A4 were much lower in the stem cell derived cells compared to cultured primary human hepatocytes. Therefore, the studied cell types showed a partial but not a complete hepatocellular differentiation. Presently, it is not clear whether these limitations are due to a limited differentiation capacity of our stem cells or to a suboptimal in vitro microenvironment. To come closer to an answer we established a triple transgenic conditional mouse model that allows a doxycycline controlled EGFP marking of haematopoietic stem cells in vivo during precisely defined time periods. After repeated induction of severe liver damage by 2-acetylaminofluorene and CCl4 in mice with EGFP-tagged haematopoietic cells EGFP-marked hepatocytes were observed only extremely rarely.

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In conclusion, we have shown that expression of several hepatocellular markers and functions can be induced in extrahepatic stem cells, although a complete and homogeneous transdifferentiation of extrahepatic cell populations to genuine hepatocytes has not yet been achieved.

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Selected short poster presentations Transdifferentiation of hepatocytes to metastatic myofibroblasts during late stage liver tumorigenesis Mario Mikula, Eva Fuchs and Wolfgang Mikulits Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria Changes in epithelial cell plasticity associated with a dramatic increase in malignancy are frequently observed in various types of carcinomas. Here we present for the first time evidence that transforming growth factor (TGF)-beta1 induces transdifferentiation of malignat hepatocytes to myofibroblastoid cells in vivo. We established a murine tumor model based on cooperation of Ras with TGF-beta1, the latter representing a hallmark in hepatocellular carcinoma (HCC). Orthotopic transplantation of immortalized and genetically modified hepatocytes allows us to faithfully reflect the progression of HCC, and to monitor signaling pathways responsible for tumor invasion and metastasis. Upon crosstalk between TGF-beta and Ras, expression profiling revealed upregulation of proteins associated with fibrosis and metastasis. Experimentally generated tumors strongly increase in size which was accompanied by reduced survival of mice. This increase in malignancy could be correlated with downregulation of E-cadherin and cytokeratin as well as with nuclear accumulation of PCNA, Smad-2 and beta-catenin. Most importantly, exogenous hepatocytes exhibit an intra and peritumoral localization of alpha-SMA and showed fibrotic marker expression such as fibulin 2, GFAP and desmin. This transdifferentiation could be completely blocked by overexpression of Smad-7 which abrogates TGF-beta signaling. This indicates that a subpopulation of hepatocytes transdifferentiates into myofibroblastoid derivatives during the acquisition of invasive and metastatic properties in a TGF-beta dependent manner. Taken together these data provide evidence for a direct link between fibrosis and liver tumor progression which might be taken into consideration for the pathological assessment of and therapeutic intervention in liver carcinoma progression.

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Identification of new surface markers for fetal liver stem cells Nierhoff D., LeVoci L., Norel R., Rogler L.E. and Shafritz D.A. Albert Einstein College of Medicine, Marion Bessin Liver Research Center, Bronx, NY, USA Disclosures: The following authors have indicated they have no relationships to disclose: Dirk Nierhoff, Lauretta LeVoci, Raquel Norel, Leslie Rogler, David Shafritz. We have previously shown that murine fetal liver epithelial cells (FLEC) at embryonic day (ED) 12.5 are a homogeneous population of Alb+/Afp+/Pancytokeratin+/Sca1+ hepatoblasts that can be purified using the specific surface markers E-cadherin, Liv2 or Dlk and show high repopulation capacity in vivo (Nierhoff et al. 2005). To identify additional surface markers expressed by murine hepatoblasts, we performed a cDNA microarray analysis using the AECOM 27K mouse cDNA array, comparing purified hepatoblasts isolated from ED13.5 mouse fetal liver with adult liver. Hepatoblasts were purified based on their expression of E-cadherin, using MACS bead technology, and purity was estimated to be 90% by subsequent FACS analysis and staining with an epithelial-specific pancytokeratin marker on cytospins. 404 genes were significantly upregulated in purified hepatoblasts versus adult liver, while 788 genes were upregulated in the adult liver versus purified hepatoblasts. Further analysis concentrated on extracellular membrane proteins within the group of genes upregulated in purified hepatoblasts, such as CD24a, Nope, Mfge8, Cntnap2, Gpc3, Pcsk9, Emp1, Efnb3 and Igf2r. Among these genes, the expression of the signal transducer CD24a had been shown in the ventral endoderm at ED 9.5 and may have a pivotal role in cell differentiation. Double-labeling experiments and FACS analysis confirmed the expression of CD24a in E-cadherin positive ED13.5 hepatoblasts. Further studies will focus on the use of CD24 and other identified surface markers for isolation of stem cells from the fetal or adult liver.

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Expression of hepatocytic markers after differentiation of mouse embryonic stem cells into the pancreatic lineage Alexandra Rolletschek, Insa Schröder, Ania Daniel-Wojcik, and Anna M. Wobus In Vitro Differentiation Group, IPK, Corrensstr. 3, D-06466 Gatersleben, Germany The organogenesis of pancreas and liver are highly correlated with the pancreas emerging from the dorsal and ventral regions of the foregut, while the liver develops solely from the ventral endoderm. However, little is known about the signaling mechanisms that control the specification and early differentiation of these endoderm-derived organs. Therefore we developed in vitro protocols to analyze lineage commitment and differentiation of mouse ES cells into either pancreatic or liver cells. ES cells were cultured as embryoid bodies (EBs) and after plating allowed to differentiate spontaneously into a progenitor population representing cells of all three primary germ layers. Cells were then subjected to hepatocyte culture medium (HGM) to induce hepatic differentiation or differentiation medium containing nicotinamide and laminin (N2) to promote pancreatic differentiation. Hepatic differentiation of the ES cells led to the generation of glycogen- and albuminproducing hepatocyte-like cells with induced TTR and TAT expression. Pancreatic differentiation gave rise to insulin releasing islet-like clusters with increased expression of Pdx-1, Pax4, insulin and IAPP. Microarray analysis using Affimetrix chips revealed that during pancreatic differentiation hepatocytic markers like Foxa1 and TTR were upregulated. Further analysis including real time PCR as well as immunocytochemistry are currently performed to elucidate potential functional implications.

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The contribution of bone marrow cells to liver progenitor cells in CDE diet and murine hepatitis virus induced injury Tonkin J.N.1,2, Curtis D.3, and Yeoh G.C.1,2 1 Western Australia Institute for Medical Research, Perth, WA. 2School of Biomedical Biomolecular and Chemical Sciences, University of Western Australia, Perth, WA. 3 Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Victoria, Australia Liver progenitor oval cells (OC) have great potential to treat patients with liver disease but their use is limited by the number of donor livers from which the cells can be retrieved. Bone marrow may provide a readily available source of these cells. To investigate the contribution of bone marrow cells to the OC pool, we tracked the movement of bone marrow cells containing a lac Z transgene over a six-week timecourse of chronic liver injury induced by a choline-deficient, ethionine supplemented (CDE) diet and murine hepatitis virus (MHV) infection. Bone marrow derived OC were identified by X-gal staining combined with immunohistochemistry for two markers, A6 and CK19. Both viral hepatitis and the CDE diet induced an OC response. OC originating from bone marrow were present in both injury models but represented a minor percentage of the OC compartment (0-2.3%). Similar levels of bone marrow derived hepatocytes and cholangiocytes, the differentiated products of OC, were observed and increased over time. The combination of MHV infection with the CDE diet produced more severe liver damage with large numbers of proliferating OC. This did not increase the proportion of OC that were generated from bone marrow (0-3%). In all models only rare, individual bone marrow OC were observed. We hypothesised that the bone marrow cells may be a source for replenishing OC that are expended over long periods of injury and regeneration, however experiments with recurrent liver injury failed to induce proliferation of the bone marrow OC. Studies are underway to transdifferentiate bone marrow cells to OC in vitro.

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List of Speakers, Moderators and Scientific Organizers

Prof. Dr. H.C. Bisgaard University of Copenhagen Panum Institute Blegdamsvej 3 DK-2200 Copenhagen Denmark [email protected]

Prof. Dr. J.G. Hengstler Universität Leipzig Institut für Pharmakologie Härtelstr. 16/18 D-04107 Leipzig Germany [email protected]

Prof. Dr. B. Christ Molekulare Hepatologie Martin-Luther-Universität Halle Heinrich-Damenow-Str. 1 D-06097 Halle Germany [email protected]

Prof. Dr. V. Keim Innere Medizin II Universitätsklinikum Leipzig Philipp-Rosenthal-Str. 27 D-04103 Leipzig Germany [email protected]

Dr. S. Ferber Chaim Sheba Medical Center The Endocrine Institute 2 Sheba Road IL-52 621 Tel Hashomer Israel [email protected]

Dr. S. König Allgemein-Chirurgie Universitätskliniken Göttingen Robert-Koch-Str. 40 D-37075 Göttingen Germany [email protected]

Prof. Dr. W.E. Fleig Innere Medizin I Martin-Luther-Universität Halle-Wittenberg Ernst-Grube-Str. 40 D-06120 Halle Germany [email protected]

Dr. M. Mikula Universität Wien Institut für Tumorbiologie und Krebsforschung Borschkegasse 8A A-1090 Vienna Austria [email protected]

Prof. Dr. R. Gebhardt Institut für Biochemie Medizinische Fakultät Universität Leipzig Liebigstraße 16 D-04103 Leipzig Germany [email protected]

Dr. D. Nierhoff Albert Einstein College of Medicine Liver Research Center Laboratory DA Shafritz 1300 Morris Park Ave. Bronx, NY 10461 USA [email protected]

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Prof. Dr. M. Ott Gastroenterologie/Hepatologie Medizinische Hochschule Hannover Carl-Neuberg-Str. 1 D-30625 Hannover Germany [email protected] Frau A. Rolletschek IPK Gatersleben Pflanzengenetik und Kulturpflanzenforschung Corrensstr. 3 D-06466 Gatersleben Germany [email protected] Dr. H. Strick-Marchand Institut Pasteur 25, rue du Dr. Roux F-75724 Paris France [email protected] Dr. S. Sumitran-Holgersson Karolinska Institutet S-141 86 Stockholm Sweden [email protected] N.D. Theise, M.D. Beth Israel Medical Center Division of Digestive Diseases First Avenue at 16th Street New York, NY 10003 USA [email protected] Mrs. J.N. Tonkin Western Australia Institute for Medical Research Lab. for Cancer Med./Prof. Yeoh Level 5 & 6, MRF Building Rear 50 Murray Street Perth, WA 6000 Australia [email protected]

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Dr. D. Tosh University of Bath Claverton Down Bath, BA2 7AY UK [email protected] Prof. Dr. C. Trautwein Gastroenterologie/Hepatologie Medizinische Hochschule Hannover Carl-Neuberg-Str. 1 D-30625 Hannover Germany [email protected] Prof. Dr. A.M. Wobus Leibniz Institut Corrensstr. 3 D-06466 Gatersleben Germany [email protected]

POSTER ABSTRACTS Poster Numbers 1 - 35

1 Transdifferentiation of pancreatic hepatocyte and ductal phenotypes

AR42J-B13

cells

to

Amani Al-Adsani, Kate L. Ralphs and David Tosh Bath, United Kingdom Transdifferentiation is the loss of one phenotype and the acquisition of a new one. AR42J-B13 cells (B13) are derived from a rat pancreatic tumour and express both exocrine and neuroendocrine properties. It has been shown that B13 cells can transdifferentiate to hepatocytes when cultured with the synthetic dexamethasone (Dex). The aim of the present study was to determine whether during transdifferentiation, cells with the ductal phenotype were formed as well as hepatocytes. B13 cells were treated with Dex for up to 14 days. Cells were fixed at different time points (days 1, 3, 5, 7, 9 and 14) and immunostained for pancreatic, ductal and hepatocyte markers. We examined the expression of amylase and transferrin as markers of pancreatic and hepatocyte phenotypes. In the presence of Dex, B13 lost amylase and gained transferrin. In addition, we observed induction of the expression of ductal cell markers c-kit, CK7 and CK19 after Dex treatment. These results indicate that ductal cells are also induced during transdifferentiation of pancreatic B13 cells to hepatocytes.

2 Induction of periportal- and pervenous-like hepatocytes from pancreatic cells Z.D. Burke Bath, United Kingdom Transdifferentiation is the term used to describe the conversion of one differentiated cell type into another, completely different phenotype. We have developed a model for the transdifferentiation of pancreatic AR42J-B13 cells to hepatocytes based on culture with dexamethasone (Dex). Cells cultured with Dex lost the pancreatic exocrine marker amylase and gained an hepatocyte phenotype. Transdifferentiated hepatocytes express several markers that are representative of a range of hepatic functions including albumin, transferrin, Glucose 6 phosphatase, Apolipoprotein B, Cytochrome p450 2E1 and UDP-glucoronosyl transferase. In vivo, hepatocytes exhibit functional heterogeneity along the axis of the periportalperivenous tract. Perhaps the most marked zonation is in ammonia metabolism. For example the enzymes of the urea cycle (e.g. carbamoylphosphate synthase (CPS)) and glutamine synthetase (GS) are restricted to periportal and perivenous hepatocytes respectively in a complementary fashion. We determined the expression of CPS and GS following treatment with Dex. TD hepatocytes exhibit mutually exclusive expression of CPS and GS Here we present our observations on the effects of the GS inhibitor methionine sulphoximine on TD hepatocytes with respect to changes in the expression of enzymes involved in nitrogen metabolism and the transcription factors that may be involved in determining hepatic functional heterogeneity.

3 In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells Tobias Cantz, Martina Bleidißel, Hans R. Schöler Max-Planck-Institute for molecular Biomedicine, Münster, Germany Background: Recently, a new approach to reprogram somatic cells to pluripotent stem cells was shown by fusion of somatic cells with embryonic stem cells (ESC), avoiding generation of totipotent embryos. These fusion hybrids morphologically resemble normal ESC but have a tetraploid karyotype. Little is known about their differentiation potential using in vitro differentiation protocols and it has to be revealed whether tetraploid or segregated diploid cells (or both of them) has the capability to differentiate into hepatic cells. Methods: Mononuclear bone marrow cells from C3H and Rosa26 mice were fused with HM-1 (hypoxanthine-phosphoribosyltransferase-deficient) or OG2 (Oct4-GFP transgenic) ESC, respectively. Unfused ESC were eliminated by selection with hypoxanthine, aminopterine, thymidine (HAT) for C3H/HM-1 hybrids or G418 for Rosa26/OG2 hybrids and fusion-derived colonies could be subcloned. After cultivation in hanging drops for 5 days cells were plated on gelatin-coated dishes in IMDM, supplemented with alpha-thioglycerole, to facilitate endodermal differentiation. Results: Gene expression analyses by RT-PCR shows the presence of hepatic marker genes as albumin, alpha-fetoprotein, cytokeratin 18 and the conjugate export pump MRP2 (ABC C2), which are absent in undifferentiated ESC or unfused bone marrow cells. Preliminary transplantation studies of hepatic precursor cells, derived from reprogrammed somatic Rosa26 cells showed engraftment in immunosuppressed toxic milk mice, an established model for Wilson’s disease, without any sign of tumor development after 7 weeks. But more careful analyses need to be done to proof a therapeutical benefit and to exclude the risk of teratoma formation.

4 Homeobox transcription factor Prox1 in rat models of liver damage and regeneration Jozsef Dudas, Tümen Mansuroglu, Abderrahim Elmaouhoub, Danko Batusic, Kyrylo Tron, Bernhard Saile, Joerg Wilting1 and Giuliano Ramadori Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany, and 1Children’s Hospital, Pediatrics I, Georg-August-University Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany Background: Prox1, a homeobox transcription factor, is essential for embryonic liver development, and expressed in hepatoblasts and adult hepatocytes. Changes of Prox1 gene expression in liver damage and regeneration may be of interest to understand hepatocyte origin. Methods: Prox1 mRNA expression and immunohistochemical protein localisation was examined in normal rat liver and various models of rat liver damage: 1. Acute and chronic liver damage induced by application of carbon-tetra-chloride (CCl4), 2. Conventional 2/3 partial hepatectomy (PH) 3. PH in combination with 2-acetylaminofluorene (AAF) treatment (AAF/PH). Muscular turpentine oil injection and Sham operation was used as acute phase controls. Hepatoblasts were isolated from rat embryos at embryonic days (ED) 12-14. Results: In normal rat liver Prox1 remained a stable marker of hepatocytes, and was absent from bile ducts. In models of liver damage and regeneration: Prox1 was detectable in hepatocytes and in some scattered cells, which were also positive for OV-6 and cytokeratin 7 (CK-7). Additionally, cells, which were isolated by laser capture from AAF/PH livers expressed alpha-fetoprotein, whereas Prox1 mRNA was not detectable. In models of liver damage and regeneration Prox1 mRNA expression shows a transitional decrease and a restoration period. Isolated rat hepatoblasts (1214 ED) showed Prox1 and OV-6 immunoreactions. Conclusions: Prox1 is a stable marker of embryonic hepatoblasts and adult hepatocytes, but could be detected in some adult liver progenitor cells.

5 Common morphology, metabolism and signal transduction in primary hepatocytes and blood monocyte derived neohepatocytes from patients with alcoholic liver disease S. Ehnert,† U. Böcker,† K. Breitkopf,† M.V. Singer,† A. Nüssler,* †† and S. Dooley* † *contributed equally † Dept. of Medicine II, University Hospital Mannheim, Campus Mannheim, Mannheim, Germany; ††Dept. of Celltherapy, Fresenius Biotech GmbH, Bad Homburg, Germany End stage liver disease induced by chronic viral infection or compound intoxication, e.g., alcohol and aflatoxin, represented as cirrhosis and hepatocellular carcinoma is a leading cause of death, especially in Asia and Africa. Alcohol dependent liver damage is continuously increasing in developed countries, e.g., in Germany. Liver transplantation currently is the only definitive treatment for severe liver failure and the shortage of donor organs raises a growing interest in new therapeutic options. Adult stem cells provide a promising alternative, thus hepatocyte-like (NeoHep) cells derived by trans-differentiation of peripheral blood monocytes have been established recently intended to bridge liver function. In the present study, NeoHep cells were generated from monocytes of alcoholic patients as well as healthy controls and compared to primary human and mouse hepatocytes in terms of their biochemistry and metabolic behaviour. The isolated NeoHep cells resemble primary human hepatocytes with respect to morphology and many secretory and metabolic functions, e.g., formation of urea and glucose, and synthesis of the enzymes lactate-dehydrogenase, alanineaminotranferase and aspartate-transaminase. Furthermore, similarities in drug detoxification activities, e.g., metabolization of hydroxycoumarine, hydroxyresorufine, and 4-methyl-umbelliferone are present. Hepatocytes are strongly responsive to transforming growth factor (TGF)-β, a major cytokine in liver fibrogenesis. NeoHep cells stimulated with TGF-β display expression and activation of smads1, 2, 3, 4, 5, and 7 analogous to primary hepatocytes. Furthermore, we were able to establish protocols for NeoHep cell transfection and infection with GFP constructs. Our data show that NeoHep cells display both phenotypic features and metabolic functions that are comparable to human hepatocytes. It is planned to transplant NeoHep cells from patients with chronic liver damage in an animal fibrogenesis model and test their potential to improve liver function.

6 Ultrastructural hepatic changes after transplantation in murine schistosomiasis

bone

marrow

Nagwa Elkhafif1, Hoda Yehia1, Olfat Hammam1, Soheir Mahmoud1, Hazem Helmy1, Sabine Boehm2, Bruno Voss2, Soheir Mansy1, Maha Akl1, Gihan El Fandy1 1 Theodor Bilharz Research Institute, Giza, Egypt, 2BGFA, Ruhr-University Bochum, Germany The limited availability of tissues and organs for liver transplantation, the high risks and costs of this major surgical interference are main problems in treating hepatic failure. Recently, cell transplantation has been introduced as adjuvant therapy for overcoming these problems. Bone marrow transplantation studies have shown the potential of adult bone marrow stem cells to differentiate into various cell types in vitro, including hepatocytes. This study focuses on the ultrastructural hepatic changes in murine schistosomiasis after bone marrow transplantation. Unfractionated male bone marrow cells were transplanted into female mice chronically infected with schistosoma mansoni by intrahepatic injection. Two weeks after transplantation mice were sacrificed on a weekly basis for a period of 8 weeks. Histopathological examination showed an increase in newly formed bile ducts and the appearance of small sized hepatocytes. Electron microscopy showed a relative increase in hepatic regenerative changes in comparison to the control groups. Small cells with large nuclei were detected in between the hepatocytes facing the sinusoids, in the periportal areas and also in between the inflammatory cells in the perigranuloma areas. Accordingly, we suggest that the transplanted bone marrow cells may participate in the regeneration of the liver in schistosomiasis. Further extensive studies are needed to reveal the potential of the bone marrow stem cells in this regenerative process.

7 Induction of maturation in human fetal hepatoblasts under different culture conditions Ewa C.S. Ellis1, Karl Bodin2, Hongbo Cai1 and Stephen C. Strom1 1 Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, USA and 2Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden Many stem cell derived hepatocytes only show markers of early liver differentiation. Methods to induce full maturation of stem cells are not known. Bile acid synthesis and secretion are unambiguous markers of mature liver. Bile acid metabolism is a highly specialized function of well differentiated, polarized hepatocytes which is difficult to achieve in vitro. We examined the expression and function of CYP7A1, bile salt export pump (BSEP), sodium tautocholate cotransport polypeptide (NTCP), genes critical to the production uptake and excretion of bile acids as a measure of maturation. Bile acid transport was measured using 3H-taurocholate and visualized using cholylglycylamido-fluorescien. Hepatoblasts (18-22 weeks) were cultured in DMEM or William’s Medium E (WE) with, or without, matrigel overlay. As compared to DMEM media, WE and matrigel overlay greatly increase CYP7A1, NTCP and BSEP mRNA levels and bile acid transport. Uptake of taurocholate increased 5-fold, BSEP and NTCP expression increased 2-fold and 13-fold, respectively, in cells cultured in WE. Canalicular spaces filled with bile acid and BSEP activity could only be detected in cells cultured in WE. Expression of CYP7A1 mRNA, the rate limiting enzyme in bile acid synthesis, was increased 400% in cells cultured in WE and analysis of the cell culture media revealed secretion of both glycine- and taurineconjugated di- and tri- hydroxylated bile acids. These results indicate that under appropriate conditions, hepatoblasts mature into well differentiated hepatocytes that synthesize, conjugate and secrete bile acids. These culture conditions may be useful for differentiation and maturation of hepatic stem/progenitor cells.

8 Characterization of embryonic hepatic cells during rat liver development Elmaouhoub A., Dudas J., Ramadori G. Göttingen, Germany Background: The main molecular evidence for liver anlage is the expression of albumin and alpha-fetoprotein (AFP). The earliest time point of albumin/AFP gene expression at RNA and protein levels during embryonic development in the rat has not yet been established. Methods: Rat embryos from E10 and E12 were dissected, the ventral foregut and liver were removed respectively, then a single cell suspension was performed. Albumin/AFP-expression was studied in whole embryo and cultured cells. We used whole mount- and cryosection- in situ hybridization and real time PCR. Synthesis and secretion of albumin/AFP were measured by radioactive biosynthetic labeling followed by immunoprecipitation and SDS-PAGE. HNF4alpha, Foxa1, Foxa2, Thy1 and beta-catenin were analysed by immunostaining and real time PCR. Result: Cells isolated from ventral foregut were shown to synthesise and secrete albumin/AFP. E12 isolated hepatoblasts were shown to synthesise abundant amount of albumin/AFP and fibrinogen. HNF4alpha, Foxa1, Foxa2, Thy1 and beta-catenin were expressed in cultured cells. Ventral foregut in E10 embryos indicate AFPexpression. In situ section of E12 livers shown a strong positivity for albumin/AFP genes in hepatoblasts. Conclusion: In this study we demonstrate that isolated cells, which develop into liver synthesise and secrete albumin/AFP and fibrinogen. A positivity for Foxa1, Foxa2, HNF4alpha, Thy1 and beta-catenin, which play a crucial role in liver development confirm the identity of the hepatoblasts. The data establish E10 as the earliest time point for appearance of hepatoblasts during liver development in the rat.

9 Erythropoietin stimulates liver regeneration Esther Hogrebe1, Jan G. Hengstler2, Andrea Tannapfel3, Alexander Bauer2, Wolfgang Jelkmann4, Johann P. Hauss1, Michael R. Schön1 1 Klinik für Viszeral-, Transplantations-, Thorax- und Gefäßchirurgie, Universität Leipzig, Germany 2 Zentrum für Toxikologie, Institut für Medizin und Rudolf-Böhme-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Germany 3 Institut für Pathologie, Universität Leipzig, Germany 4 Instiut für Physiologie, Universität Lübeck, Germany Introduction: EPO plays an important role in myocardial and cerebral regeneration, the role of EPO in liver regeneration is unknown. We studied the influence of EPO on cultured hepatocytes and on liver regeneration using a laparoscopic porcine liver resection model. Material and Methods: Isolation and culture of mouse hepatocytes was performed according to standards. Immunoblot for phosphorylated and total Akt and ERK 1/2 was analysed. 18 German Landrace pigs underwent laparoscopic left hemihepatectomy and were observed postoperativley for two weeks. Group 1 served as a control group. In group 2 a fibrin-glue-EPO- mixture was sprayed topically on to the resection surface during surgery and in group 3 EPO was administered topically and systemically every two days following surgery. Results: EPO induces phosphorylation of Akt kinase (Akt/PKB). Phosphorylated Akt kinase (p-Akt) increased by a factor of 2.39, whereas only a slight increase in total Akt kinase was observed. Only a very small increase in phosphorylated MAP kinase (ERK1 and ERK2) was seen. β-actin staining demonstrates similar loading of the lanes. In group 3 liver volume 14 days after partial left sided hepatectomy was increased by 24.2 % compared to the control group (p = 0.043). EPO led to a significant increase in hepatocytes with Ki-67 positive nuclei 14 days after liver resection compared to group1 (p = 0.0176, p = 0.0173). Conclusions: 1. We identified a regenerative effect of EPO on cultured hepatocytes and after liver resection in a porcine model. 2. EPO has a direct effect on hepatocytes in addition to a previously described effect on angiogenic mechanisms. 3. We suggest that EPO is a first line candidate for clinical studies on liver regeneration.

10 An acellularized vascularized matrix: Scaffold for the development of a liver cell modul and for stem cell differentiation studies Kirstin Linke, Johanna Schanz, Heike Mertsching Fraunhofer Institute for Interfacial Engineering and Biotechnolgy (IGB), Nobelstr. 12, D-70569 Stuttgart, Germany Background: Essential for hepatocyte vitality and function in longterm culture in vivo are extracellular matrix (ECM) components and the co-culture with endothelial cells (EC). Therefore sandwich culture techniques were established. They don`t solve metabolic evacuation. We developed a vascularized matrix which admits ECM contact and provides a bloodvessel network for EC co-culture and the transport of nutrients, metabolites and gases by physiological perfusion. We hypothese that the in vivo liver regeneration potential will be achieved ex vivo with this system. Methods: EC and Endothelial precursor cells were seeded on the vascular structures of a decellularized porcine scaffold. During the cultivation the matrix was perfused with medium over the artery in a bioreactor. In a second step hepatocytes will be seeded on the matrix and co-cultured with EC. Results: The culture of hepatocytes on the matrix shows good results for cell growth and conservation of liver specific functions. EC and precursor cells were seeded successfully via the arterial inflow in the vascular bed of the matrix. The cells shows vitality and the expression of EC specific markers within 2 weeks. Perspective: Our aim is the development of a vascularized liver modul for different applications. For the first time a testing system should enable the arterial application of substances and the identification of metabolites in a venous system. In addition this system could be used for differentiation studies of liver stem cells and the analysis of liver regeneration.

11 Expression of the homeobox transcription factor Prox1 in human chronic liver disease and in hepatocellular/cholangiocellular carcinoma Tümen Mansuroglu, Jozsef Dudas, Bernhard Saile, Thomas Armbrust, Federico Moriconi, Joerg Wilting1, Thomas Pieler2, Laszlo Füzesi3 and Giuliano Ramadori Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany; 1Children’s Hospital, Pediatrics I, Georg-August-University Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany; 2Department of Developmental Biochemistry, Institute of Biochemistry and Molecular Cell Biology, Georg-AugustUniversity of Göttingen, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany; 3 Department of Gastroenteropathology, Georg-August University Göttingen, RobertKoch-Str. 40, D-37075 Göttingen, Germany Background: Prox1, a homoebox transcription factor, is expressed in hepatoblasts and adult hepatocytes, but not in biliary cells, which are supposed to derive from hepatoblasts. The aim of the study was to investigate the gene expression and immunohistological localisation of Prox1 in normal, cirrhotic and peritumoral liver samples as well as in hepatocellular and cholangiocellular carcinomas. Methods: Human liver samples were subjected to Prox1 messenger RNA quantification and to immunofluorescent double stainings with hepatocytes (HepPar1), biliary epithelial (CK-7; cytokeratin 7) and hepatic progenitor cell markers (OV-6). Results: Prox1 mRNA expression was detected in all liver samples, and increased gene expression compared to normal was found in peritumoral liver, in hepatocellular and cholangiocellular carcinoma. At protein level the hepatocytes were Prox1-positive in normal and diseased liver, whereas the bile duct epithelial cells were negative. Surprisingly, cells, which were positive for bile duct epithelial cell markers in cholangiocellular carcinomas: were Prox1 positive. Conclusions: The results might suggest a common precursor for both hepatocellular and cholangiocellular carcinomas.

12 Ultrastructural effects of taurine treatment in experimental liver fibrosis Mehmet Refik Mas, Ilker Tasci, A.Turan Isik Etlik, Turkey Liver fibrosis is characterized with increased production, accumulation and decreased degradation of collagen in liver. The main event initiating fibrosis process is activation and myofibroblast like transformation of hepatic stellate cells. Taurine is a semi essential sulphur amino acid which is found in almost all mammalian tissues. It is a potent antioxidant with hepatoprotective effects. We investigated the changes in hepatocyte ultrastructure with Taurine treatment in experimental rat liver fibrosis. Twelve male Sprague Dawley rats weighing 250-400 g. were divided into two groups. Group I (n = 6) was injected with CCl4 (s.c.) plus Taurine (i.p.), and Group II with CCl4 plus saline for 12 weeks. All the rats were killed at 12th week and tissue specimens were collected. Transmission electron microscopy and histological examinations were performed to determine ultrastructural and histological changes. \par Necrosis, fibrosis, fat accumulation and inflammation were all significantly lower in Grup I than Group II. Nucleus, mitochondria, rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) were examined according to a injury scoring system. Injury scores of nucleus, mitochondria and SER but not of RER were significantly lower in Group I than group III (p < 0.003, p < 0.001 and p < 0.001, respectively). In conclusion, Taurine significantly decreased severity of CCl4 induced liver fibrosis on organelle basis. With the other reported beneficial effects, these findings suggest that Taurine can be involved in antifibrotic strategies.

13 Stem cell characteristics of amniotic epithelial cells Toshio Miki1, Thomas Lehmann1, Hongbo Cai1, Donna B. Stolz2,3, and Stephen C. Strom1,3 Department of Pathology1, Cell Biology & Physiology2 and McGowan Institute for Regenerative Medicine3, University of Pittsburgh, Pittsburgh, PA 15261, USA Amniotic epithelial cells develop from the epiblast by 8 days after fertilization and prior to gastrulation opening the possibility that they might maintain the plasticity of pre-gastrulation embryo cells. We report that amniotic epithelial (AE) cells isolated from human term placenta express surface makers normally present on embryonic stem and germ cells including stage specific embryonic antigens (SSEA) 3 and 4 and Tumor rejection antigens (TRA) 1-60, 1-81 in addition to c-kit and Thy-1. AE cells express markers of pluripotency including the expression of octamer-binding protein 4 (Oct-4), and nanog. Based on immunohistochemical and genetic analysis, amniotic epithelial cells have the potential to differentiate to all three germ layers - endoderm (liver, pancreas), mesoderm (cardiomyocyte), and ectoderm (neural cells) in vitro. Under certain culture conditions, amniotic epithelial cells form spheroid structures which retained stem cell characteristics. Amniotic epithelial cells do not require other cell derived feeder layers to maintain Oct-4 expression, do not express telomerase and are non-tumorigenic upon transplantation. Amnion derived from term placenta following live birth may be a useful and non-controversial source of stem cells for cell transplantation and regenerative medicine.

14 Transdifferentiation of hepatocytes to metastatic myofibroblasts during late stage liver tumorigenesis Mario Mikula, Eva Fuchs and Wolfgang Mikulits Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria Changes in epithelial cell plasticity associated with a dramatic increase in malignancy are frequently observed in various types of carcinomas. Here we present for the first time evidence that transforming growth factor (TGF)-beta1 induces transdifferentiation of malignat hepatocytes to myofibroblastoid cells in vivo. We established a murine tumor model based on cooperation of Ras with TGF-beta1, the latter representing a hallmark in hepatocellular carcinoma (HCC). Orthotopic transplantation of immortalized and genetically modified hepatocytes allows us to faithfully reflect the progression of HCC, and to monitor signaling pathways responsible for tumor invasion and metastasis. Upon crosstalk between TGF-beta and Ras, expression profiling revealed upregulation of proteins associated with fibrosis and metastasis. Experimentally generated tumors strongly increase in size which was accompanied by reduced survival of mice. This increase in malignancy could be correlated with downregulation of E-cadherin and cytokeratin as well as with nuclear accumulation of PCNA, Smad-2 and beta-catenin. Most importantly, exogenous hepatocytes exhibit an intra and peritumoral localization of alpha-SMA and showed fibrotic marker expression such as fibulin 2, GFAP and desmin. This transdifferentiation could be completely blocked by overexpression of Smad-7 which abrogates TGF-beta signaling. This indicates that a subpopulation of hepatocytes transdifferentiates into myofibroblastoid derivatives during the acquisition of invasive and metastatic properties in a TGF-beta dependent manner. Taken together these data provide evidence for a direct link between fibrosis and liver tumor progression which might be taken into consideration for the pathological assessment of and therapeutic intervention in liver carcinoma progression.

15 Remodeling of hepatocyte calcium signaling during liver regeneration in the rat is linked to cell cycle progression Alexandra Nicou1, Valérie Serrière1, Sylvie Prigent1, Mauricette Hilly1, Laurent Combettes, Gilles Guillon2 and Thierry Tordjmann1 1 Unité de Recherche U442, Institut National de la Santé et de la Recherche Médicale, Université Paris Sud, bât. 443, 91405 Orsay, France, 2Unité de Recherche U, Institut National de la Santé et de la Recherche Médicale, F-34094 Montpellier, France Agonists that trigger Ca2+ signals in the liver contribute to a regulatory network of factors interacting with hepatocytes after partial tissue loss, although little is known about hepatocyte calcium signalling during liver regeneration. We observed two phases of deep reduction in hepatocyte responsiveness to Ca2+mobilizing agonists, peaking at 24 hours and at the fifth day after partial hepatectomy in the rat. Both waves of hepatocyte desensitization involved V1a vasopressin receptor and type 2 InsP3 receptor down-regulation (western blot). Although a 50-60% loss of type 2 InsP3 receptor was observed, InsP3-mediated Ca2+ mobilization studied in suspensions of permeabilized hepatocytes was not altered at 24 h nor at the fifth day after hepatectomy. However, single cell studies with flash photolysis of encaged InsP3 revealed that InsP3-mediated Ca2+ release was slowed-down in 24 hours and five days regenerating hepatocytes. Also, the temporal pattern of vasopressin-elicited intracelluar Ca2+ oscillations studied on fura2-loaded regenerating cells was altered, the duration of each Ca2+ peak being longer, as compared with control cells. Type 2 InsP3 receptor immunohistochemistry showed that its peri-canalicular localization was conserved after hepatectomy, and that the loss in receptor staining was predominantly observed in periportal areas. Finally, experiments performed on S phase-synchronized HepG2 and CHO cell lines provided evidence linking hepatocyte desensitization to progression through the cell cycle towards S phase. Taken together, our study supports the view that hepatocyte Ca2+ signalling is remodelled during liver regeneration, and that this remodelling is at least in part linked with cell cycle progression.

16 Identification of new surface markers for fetal liver stem cells Nierhoff D., LeVoci L., Norel R., Rogler L.E. and Shafritz D.A. Albert Einstein College of Medicine, Marion Bessin Liver Research Center, Bronx, NY, USA Disclosures: The following authors have indicated they have no relationships to disclose: Dirk Nierhoff, Lauretta LeVoci, Raquel Norel, Leslie Rogler, David Shafritz. We have previously shown that murine fetal liver epithelial cells (FLEC) at embryonic day (ED) 12.5 are a homogeneous population of Alb+/Afp+/Pancytokeratin+/Sca1+ hepatoblasts that can be purified using the specific surface markers E-cadherin, Liv2 or Dlk and show high repopulation capacity in vivo (Nierhoff et al. 2005). To identify additional surface markers expressed by murine hepatoblasts, we performed a cDNA microarray analysis using the AECOM 27K mouse cDNA array, comparing purified hepatoblasts isolated from ED13.5 mouse fetal liver with adult liver. Hepatoblasts were purified based on their expression of E-cadherin, using MACS bead technology, and purity was estimated to be 90% by subsequent FACS analysis and staining with an epithelial-specific pancytokeratin marker on cytospins. 404 genes were significantly upregulated in purified hepatoblasts versus adult liver, while 788 genes were upregulated in the adult liver versus purified hepatoblasts. Further analysis concentrated on extracellular membrane proteins within the group of genes upregulated in purified hepatoblasts, such as CD24a, Nope, Mfge8, Cntnap2, Gpc3, Pcsk9, Emp1, Efnb3 and Igf2r. Among these genes, the expression of the signal transducer CD24a had been shown in the ventral endoderm at ED 9.5 and may have a pivotal role in cell differentiation. Double-labeling experiments and FACS analysis confirmed the expression of CD24a in E-cadherin positive ED13.5 hepatoblasts. Further studies will focus on the use of CD24 and other identified surface markers for isolation of stem cells from the fetal or adult liver.

17 Cell-cell competition: A new way to generate liver tissue from fetal liver stem/progenitor cells Michael Oertel, Anuradha Menthena, Mariana D. Dabeva, & David A. Shafritz Marion Bessin Liver Research Center, Albert Einstein College of Medicine of Yeshiva University, New York, USA A critical property of stem cells is their ability to repopulate an organ or tissue under non-selective conditions. We obtained 23.5% replacement of total normal liver mass at 6 months by transplanting unfractionated DPPIV+ ED14 fetal liver cells (containing ~1x106 AFP+/CK-19+ stem/progenitor cells) into DPPIV- mutant F344 rats. The progeny of transplanted cells are morphologically and functionally indistinguishable from neighboring host hepatocytes, expressing many liver-specific genes, such as albumin, glucose-6-phosphatase, the asialoglycoprotein receptor, and UDPglucuronosyl transferase. Following two-thirds PH, the liver/body weight ratio returned to normal within 2-4 weeks, however, number and size of transplanted cell clusters continued to increase for up to one year. To investigate the mechanism of hepatic replacement by transplanted cells, we measured the proliferative activity and apoptotic rate of cells in DPPIV+ clusters vs. surrounding DPPIV- parenchyma. At both 2 and 6 months after cell transplantation, the proliferative index was 6-7 fold higher in transplanted cells vs. host hepatocytes, and at the same time, apoptosis was markedly reduced in transplanted cells (4-5 times lower than in host parenchyma). In contrast, apoptosis was increased in host hepatocytes at the junctional margin with transplanted cells. Thus, fetal liver stem/progenitor cells with a higher proliferative capacity repopulate the liver by killing more slowly proliferating, neighboring host hepatocytes, while maintaining total liver mass at a fixed size, a process referred to as cell-cell competition. Use of this developmental paradigm as a strategy to reconstitute functional organ tissue in the normal adult liver under non-selective conditions has broad therapeutic implications.

18 Liver regeneration in a retrorsine/CCl4–induced acute liver failure model: Do bone marrow-derived cells contribute? Felix C. Popp, Przemyslaw Slowik, Pompiliu Piso, Hans J.Schlitt and Marc H. Dahlke Regensburg, Germany It was recently reported that adult bone marrow contains progenitors capable of (trans-)differentiation into mature hepatocytes. In the present study a new liver failure model is introduced combining retrorsine pretreatment and exposure to carbon tetrachloride to evaluate whether bone marrow-derived progeny can contribute to liver regeneration after acute hepatotoxic liver failure. Intraperitoneal retrorsine injection was used to inhibit endogenous hepatocyte proliferation, before inducing acute liver failure by injection of carbon tetrachloride. Mixed bone marrow chimeras were generated before inducing liver failure to trace bone marrow-derived cells. Therefore, CD45 (RT7) and MHC class I (RT1) dimorphic rat models were applied. Early after acute liver failure a multilineage inflammatory infiltrate could be observed, mainly consisting of granulocytes. In long-term experiments small numbers of CD90+/CD45- cells of donor origin occurred in clusters associated with portal triads. Bone marrow cell infusion at the time of hepatic failure was not able to enhance liver regeneration. Cellular hypertrophy was the predominant way of liver mass regeneration in models applying retrorsine. In the present model of acute liver failure, retrorsine pretreatment did not affect sensitivity for carbon tetrachloride. A multilineage inflammatory infiltrate was observed in rats whether pretreated with retrorsine or not. Small numbers of donor cells co-expressing CD90 (THY 1) were present in recipient livers, which may resemble donor-derived hematopoietic progeny or oval cells. No other donor cells within liver parenchyma were detected. This is in contrast to other cell therapy models of acute cell death recently reported.

19 Transdifferentiated hepatocytes: An in vitro model for liver function Kate L. Ralphs, Zoë D. Burke and David Tosh Bath, United Kingdom Transdifferentiation is the conversion of one differentiated cell type to another with or without intervening cell division. It has previously been demonstrated by our lab that treatment of the pancreatic cell line, AR42J-B13 (B13) with the glucocorticoid Dexamethosone (Dex) induces transdifferentiation to hepatocytes with no apparent intervening cell division. In addition, we have previously demonstrated that a range of pancreatic markers are down regulated and liver markers are induced during transdifferentiation. The functional nature of the transdifferentiated hepatocytes has been analysed. The normal liver plays a major role in metabolic homeostasis, synthesis of plasma proteins, enzymes and cofactors and is crucial for detoxification. We determined the ability of the TD hepatocytes to synthesise urea, glycogen and metabolise low density lipoprotein. The results suggest transdifferentiated hepatocytes induced from pancreatic cells are a good model for studying liver function.

20 TIMP-1 antagonism reduces toxic-induced hepatic fibrosis but not biliary hepatic fibrosis in mouse models Martin Roderfeld (1), Ralf Weiskirchen (2), Sonja Hillebrandt (3), Frank Lammert (3), Axel M. Gressner (2), Marie-Luise Berres (4), Siegfried Matern (4), Elke Roeb (1) (1) Department of Internal Medicine II, Justus-Liebig University, D-35385 Giessen, Germany, (2) Institute of Clinical Chemistry and Pathobiochemistry, University Hospital RWTH Aachen, D-52074 Aachen, Germany, (3) Department of Internal Medicine I, University Hospital Bonn, D-53105 Bonn, Germany and (4) Department of Internal Medicine III, University Hospital RWTH Aachen, D-52074 Aachen, Germany Background: The tissue inhibitor of metalloproteinases-1 (TIMP-1), a molecule that inhibits the activity of matrix-degrading proteases, is effective in preventing apoptosis in activated hepatic stellate cells (HSC). To induce apoptosis in this liver cell subpopulation and to reduce fibrogenesis, we antagonized TIMP-1 by proteolytically inactive MMP-9 in toxic and cholestatic mouse models of hepatic fibrosis. Material and Methods: Toxic liver fibrosis was induced in BALB/c mice by administration of CCl4 while BALB/c mdr2 (Abcb4)-/- mice spontaneously developed hepatic lesions resembling primary sclerosing cholangitis. Recombinant adenoviruses coding for TIMP-1 antagonists were administered via tail vein. The effect on fibrogenesis was analysed by quantification of collagen deposition and αsmooth muscle actin (α-SMA) expression. Results: Treatment of toxically injured mice with adenoviruses coding for TIMP-1 antagonists reduced the stage of hepatic fibrosis and decreased expression of hepatic α-SMA. Interestingly, during fibrogenesis, α-SMA expression was not increased significantly in liver of untreated mdr2 knock out mice. Furthermore, the development of hepatic fibrosis in mdr2 knock out mice was not influenced by TIMP1 antagonism. Discussion: It could be demonstrated that proteolytically inactive MMP-9 mutants, acting as TIMP-1 antagonists, reduced the stage of hepatic fibrosis in toxicallyinduced hepatic fibrosis but not in BALB/c mdr2 knock out mice. The unchanged αSMA expression and the uneffectiveness of TIMP-1 antagonism suggests that hepatic fibrosis in BALB/c mdr2 knock out mice is not promoted by activated HSC or at least not by the mechanisms leading to hepatic fibrosis in CCl4-treated mice. Keywords: liver, hepatic fibrosis, stellate cells

21 Expression of hepatocytic markers after differentiation of mouse embryonic stem cells into the pancreatic lineage Alexandra Rolletschek, Insa Schröder, Ania Daniel-Wojcik, and Anna M. Wobus In Vitro Differentiation Group, IPK, Corrensstr. 3, D-06466 Gatersleben, Germany The organogenesis of pancreas and liver are highly correlated with the pancreas emerging from the dorsal and ventral regions of the foregut, while the liver develops solely from the ventral endoderm. However, little is known about the signaling mechanisms that control the specification and early differentiation of these endoderm-derived organs. Therefore we developed in vitro protocols to analyze lineage commitment and differentiation of mouse ES cells into either pancreatic or liver cells. ES cells were cultured as embryoid bodies (EBs) and after plating allowed to differentiate spontaneously into a progenitor population representing cells of all three primary germ layers. Cells were then subjected to hepatocyte culture medium (HGM) to induce hepatic differentiation or differentiation medium containing nicotinamide and laminin (N2) to promote pancreatic differentiation. Hepatic differentiation of the ES cells led to the generation of glycogen- and albuminproducing hepatocyte-like cells with induced TTR and TAT expression. Pancreatic differentiation gave rise to insulin releasing islet-like clusters with increased expression of Pdx-1, Pax4, insulin and IAPP. Microarray analysis using Affimetrix chips revealed that during pancreatic differentiation hepatocytic markers like Foxa1 and TTR were upregulated. Further analysis including real time PCR as well as immunocytochemistry are currently performed to elucidate potential functional implications.

22 Influence of demethylating agents on in vitro differentiation of adult liver stem cells into the hepatocytic lineage Insa S. Schröder1/2, Valentina M. Factor1, Ju-Seog Lee1, and Snorri S. Thorgeirsson1 1 Laboratory of Experimental Carcinogenesis, NCI, NIH, Bethesda, MD, USA 2 In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany Previously rat liver epithelial cells (RLE) were isolated from normal adult rat livers and established as a cell line (RLE-13). RLE cells resemble adult liver stem cells. They were used to address a) cellular and molecular mechanisms controlling lineage commitment of hepatic stem cells, and b) the influence demethylating agents like 5azadeoxycytidine (5ADC) and zebularine (ZEB) have in this process. To induce hepatic differentiation, protocols were developed consisting of 5ADC or ZEB in combination with FGF 1 and 2, oncostatin M, dexamethasone, and HGF. These treatments resulted in cell enlargement and increased organelle complexity. RT and real time PCR analysis revealed induction of early transcription factors like GATA4, HNF3α/3β and Hex, transcription factors associated with hepatic specification such as HNF1α/1β, HNF4 and HNF6, and mature hepatocyte markers like TAT, TTR and CX32. The PCR results were supported by cDNA microarray analysis. Demethylation was crucial for the successful differentiation of the RLE. ZEB had to be used in higher concentrations (400 µM) than 5ADC (1 µM), but led to higher induction of albumin, TAT, and G6P. Therefore the less cytotoxic and more stable ZEB offers a suitable alternative to 5ADC for the demethylation and hepatic differentiation of cell lines in vitro. We conclude that primitive rat epithelial cells can be successfully differentiated along the hepatocytic lineage using demethylating agents in combination with growth factors important in liver development. Further experiments will show the effectiveness of the differentiated RLE as a tool in regenerative medicine.

23 Immune-mediated hepatitis drives low-level fusion between hepatocytes and adult bone marrow cells Przemyslaw Slowik, Felix C. Popp, Pompiliu Piso, Hans J.Schlitt and Marc H. Dahlke Regensburg, Germany The role of adult bone marrow-derived cells (BMC) in hepatic regeneration is controversial. Both trans-differentiation of BMC as well as fusion with hepatocytes have been suggested in toxin-based and genetic selection models. We have developed a transgenic mouse model of immune-mediated hepatitis to clarify the role of BMC in liver regeneration following injury mediated by T cells. Repeated adoptive transfer of transgenic T cells into bone marrow chimeras resulted in multiple waves of hepatitis. Hepatocytes derived from donor bone marrow were identified using a self-protein that does not interfere with hepatocyte function and proliferation in recipient animals Some cells contained one recipient nucleus and another independent donor bone marrow-derived nucleus, suggesting that cellular fusion plays some role in liver repair after immune hepatitis. However despite pronounced infiltration by myeloid cells, the frequency of fusion was extremely low. This study provides a unique, clinically relevant model in which fusion hepatocytes can be purified and characterized by the expression of donor MHC antigen. It demonstrates that although fusion between BMC and hepatocytes occurs under conditions of inflammation that correspond to human disease, its frequency needs to be increased to be of any therapeutic value.

24 High expression of matrilin-2 by stem cells in liver regeneration Szabo E1, Lódi Cs1, Korpos E3, Kiss I3, Deak F3, Paku S2, Nagy P2, Schaff Zs1, Kiss A1. 1 II. Dept. of Pathology, Semmelweis University, Budapest, Hungary, 2I. Dept. of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary, 3Institue of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary Introduction: The matrilins are a recently discovered family of the extracellular matrix (EM) proteins, but their biologic role is still largely unknown. Matrilin-2 displays a broad tissue distribution in organs where the regeneration and developing processes are intensive, however, the type of cells producing it is not exactly clear. The aim of our study was to analyse the expression of matrilin-2 and to identify the cells producing matrilin-2 during liver regeneration. Methods: Liver regeneration was induced in rats by high dose 2-acetylaminofluorene (AAF) (5 mg/kg) followed by partial hepatectomy (PH). Formalin fixed, paraffin embedded tissue sections were used for immunohistochemistry applying rabbit polyclonal matrilin-2 antibody. The mRNA expression was studied by in situ hybridisation. For study the colocalization of matrilin-2 and laminin double fluorescence immunostaining was performed and analysed by confocal laserscanning microscopy. Results: Around the central viens, portal viens and bile ducts was detected a sligthly week positivity in the normal rat liver. Oval/stem cells located in the periportal areas of the AAF/PH treated rats, however, expressed high positivity for matrilin-2, which colocalized with laminin accompanying the primitive ductular structures. By in situ hybridisation, matrilin-2 mRNA was detected in oval/stem cells, but not in mature hepatocytes. Discussion/Conclusion: Our data show the first time that hepatic stem cells produce matrilin-2, an important EM protein, which probably participates in the reorganization of the trabecular architecture of the liver parenchyma during regeneration. The high expression of matrilin-2 in oval cells suggests that matrilin-2 participates in early phase of EM production. The project was supported by grants: Biol 14/2001, NKFP-1/0023/2002, ETT077/2003, OTKA-T037838

25 Two-stage hepatectomy in huge right lobe liver tumors Attila Szijártó, Bálint Hargitai, Péter Kupcsulik 1st Department of Surgery, Semmelweis University, Budapest, Hungary Background: Complete resection of hepatic tumors remains the first choice for curative treatment of primary and secondary liver malignancies. The reason for unresectability is that, often, the remnant liver is of insufficient volume to support postoperative liver function. The malfunction is still the principal cause of postoperative death after a major hepatectomy. Various procedures have been developed to induce liver regeneration because liver failure is related to the amount of remaining functional liver volume. Patients and methods: Data were collected during 3 years from 14 patients with primary (n = 3) or secondary (n = 11) liver tumors who underwent ligation of the right portal branch (first stage) before major liver resection. The median age of the patients was 54 years (range 25 to 72 years); 8 men and 6 women. ICGdensitometry was used to measure the hepatic function. In the pre-, and postoperative periods, US and CT-guided volumetry was performed to estimate the liver’s regeneration. Results: Liver resections were performed as a second stage in 8 cases (trisegmentectomy n = 4; right hepatectomy n = 3, non-anatomical resection n = 1). The rest of the patients were unsuitable for liver resection because of postoperative complications. Mortality was nil after the major resections. The ICG-densitometry is able to predict the postoperative liver failure. Conclusions: These results suggest that two-stage hepatectomy combined with portal ligation can be safely applied to selected patients, who initially are considered as unresectable cases. The postoperative liver failure and malfunction could be predicted by the preoperative ICG-densitometry and the 3D CT-volumetry procedures.

26 Liver fibrosis regresses better with ursodeoxycholic acid treatment than spontaneous recovery Ilker Tasci, Mehmet Refik Mas, Nuket Mas Etlik, Ankara, Turkey Fibrosis and cirrhosis are common complications of chronic liver diseases. We investigated the possible antifibrotic efficacy of ursodeoxycholic acid (UDCA). Liver fibrosis was established on 30 male Sprague-Dawley rats with CCl in 12 weeks. After cessation of CCL Group I (n = 15) was left for spontaneous recovery. Group II (n = 15) was treated with UDCA 25 mg/kg/day. All rats were killed at week 16. Histological fibrosis scores, tissue hydroxyproline, TIMP-1 and MMP-13 and hepatocellular apoptosis were determined. Fibrosis scores was lower in Group II than Group I (p < 0.05). Tissue hydroxyproline levels were significantly d ecreased in Group II when compared to Group I( p = 0.001). Lower liver TIMP-1 and higher MMP-13 levels were measured in Group III than Group I (p = 0.05, for TIMP, and p = 0.02 for MMP). Apoptosis was significantly increased in Group II when compared to Group I (p < 0.05). In conclusion, treatment with PEG-IFN and UDCA improves CCL induced rat liver fibrosis. Significantly higher effects can be obtained using these agents in combination.

27 Relationships between haematopoietic and epithelial lineages in human fetal liver John D. Terrace, Ian S Currie, Neil M. Masson, Rowan W. Parks, James A. Ross Tissue Injury and Repair Group, Clinical and Surgical Sciences (Surgery), Chancellor's Building, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom, Tel.: ++44 (0)131 242 6520, Fax: ++44 (0)131 242 6520, E-mail: [email protected] Introduction: In developing human liver, hepatoblasts generate hepatocytes and cholangiocytes. Haematopoietic progenitors generate lymphoid and myeloid precursors. Recent studies indicate that phenotypic haematopoietic stem cells may give rise to liver epithelial cells. This observation questions the lineage relationship between haematopoietic progenitors and hepatoblasts in developing liver. To examine this relationship, haematopoietic, epithelial and mesenchymal lineages were immunostained in human fetal liver. Methods: Monoclonal antibodies were applied to first trimester human fetal liver sections (week 8). Thy-1 and CD34 indicated putative haematopoietic stem cells and CD45 and glycophorin-A were used as blood lineage markers. Epithelial (albumin, cytokeratins 18 and 19) and mesenchymal (vimentin) lineages were also labelled. Results: Thy-1 labelled portal vein endothelium and portal tract mesenchyme. CD34 was expressed in portal and hepatic venous and sinusoidal endothelium. Intensely positive CD34-labelled cells were also scattered throughout the liver. Cytokeratin 18 labelled the epithelial compartment, whereas cytokeratin 19 and albumin staining was pronounced around portal structures. CD45 and glycophorin-A expression was seen within the presumptive sinusoidal compartment. Vimentin expression was pronounced on all endothelium and sinusoids. Conclusions: Phenotypic haematopoietic stem cells, which give rise to liver epithelium, may be derived from portal vein endothelium. Conversely, isolated CD34+ cells in the developing liver are probably haematopoietic stem cells. Common markers could reflect a common derivation of both cell types from embryonic haemangioblasts. This study has revealed a novel and unexpected portal endothelial compartment bearing haematopoietic stem cell markers in human fetal liver. Previous work suggests this compartment provides liver epithelial cells in vitro.

28 Transdifferentiation of hepatocytes into pancreatic β-cells Shifaan Thowfeequ, Wan-Chun Li, Mark Hornsey, Daniel Eberhard, Jonathan M. W. Slack & David Tosh Bath, United Kingdom Transplantation of islets of Langerhans has been used as a potential cure for patients with insulin-dependant diabetes who lack insulin secreting cells due to autoimmune attack. However, such approaches are hindered by the lack of donors and viable tissue. An alternative is to produce insulin-containing β-cells in vitro from other closely related tissue by inducing transdifferentiation. During development, hepatocytes and pancreatic cells share a common progenitor and differentiation into a hepatic or pancreatic fate is under the control of the master switch gene Pdx1. Now it is possible to isolate and maintain long term in vitro cultures of rat hepatocytes. In these cells multiple infections with adenoviruses containing Pdx1 and other genes such as Pax4, Pax6, Ngn3, Nkx2.2 and Nkx2.6 involved in β-cell lineage determination can result in the conversion of cultured hepatocytes into more mature β-cells. In addition to insulin, these β-cells express β-cell specific genes such as Kir6.2, IAAP, GLP-1, SUR1 and Chromogranin A. We have also investigated if multiple infections mimicking the temporal gene expression profile of the genes involved in in vivo pancreatic development would result in more morphologically and functionally mature β-cells.

29 Immortalised bipotential cell lines as a tool for oval cell biology Janina E.E. Tirnitz-Parkera,b, John Olynykb,c, George C.T. Yeoha,b a School of Biomedical, Biomolecular & Chemical Sciences, University of Western Australia, Crawley WA, Australia, bLaboratory for Cancer Medicine,Western Australian Institute for Medical Research, Perth, Australia, cSchool of Medicine and Pharmacology, University of Western Australia, Crawley WA, Australia Replacement of lost liver tissue following mechanical, chemical or immune related injury is usually accomplished by proliferation of remaining, healthy hepatocytes. When this process is impaired, the liver progenitor (oval) cell or adult liver stem cell compartment is induced to proliferate. In several rodent models, oval cells were not only shown to differentiate into hepatocytes or cholangiocytes, but also along intestinal and pancreatic lineages. They are linked to hepatocellular carcinoma and under some pathogenic circumstances might even be a cellular precursor. In the present study, we describe the isolation and primary culture of oval cells from mice fed a choline deficient, ethionine supplemented diet. Primary cultures were up to 80% pure at day 7 based on either A6 or CK19 staining. Cell lines were clonally derived from these cultures by spontaneous immortalisation. They were shown to express markers of both the hepatocytic and the biliary lineage and were therefore termed “bipotential murine oval liver” (BMOL) cells. Four lines were established from a mouse bearing a TAT GRE lac Z transgene. These BMOL-TAT cells facilitate assessment of their differentiation into the hepatocytic lineage as they stain positively with X-gal when TAT is expressed. BMOL or BMOL-TAT cells all undergo morphogenesis in Matrigel to form spheroids and bile duct structures. Under proliferating cell culture conditions, they expressed immature hepatic markers, whereas late markers were upregulated when the medium was changed to induce differentiation. All cell lines did not grow in semi-solid agar which suggests they are non-tumorigenic. These murine oval cell lines will be of great value in future studies. In particular the BMOL-TAT cells can be traced in transplantation experiments and provide a useful tool for investigating the contribution of oval cells to liver regeneration. They will be employed in future investigations to better understand their biology and to confirm their ability to repopulate injured mouse liver.

30 The contribution of bone marrow cells to liver progenitor cells in CDE diet and murine hepatitis virus induced injury Tonkin J.N.1, 2, Curtis D.3, and Yeoh G.C.T.1, 2 1 Western Australia Institute for Medical Research, Perth, WA. 2School of Biomedical Biomolecular and Chemical Sciences, University of Western Australia, Perth, WA. 3 Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Victoria, Australia Liver progenitor oval cells (OC) have great potential to treat patients with liver disease but their use is limited by the number of donor livers from which the cells can be retrieved. Bone marrow may provide a readily available source of these cells. To investigate the contribution of bone marrow cells to the OC pool, we tracked the movement of bone marrow cells containing a lac Z transgene over a six-week timecourse of chronic liver injury induced by a choline-deficient, ethionine supplemented (CDE) diet and murine hepatitis virus (MHV) infection. Bone marrow derived OC were identified by X-gal staining combined with immunohistochemistry for two markers, A6 and CK19. Both viral hepatitis and the CDE diet induced an OC response. OC originating from bone marrow were present in both injury models but represented a minor percentage of the OC compartment (0-2.3%). Similar levels of bone marrow derived hepatocytes and cholangiocytes, the differentiated products of OC, were observed and increased over time. The combination of MHV infection with the CDE diet produced more severe liver damage with large numbers of proliferating OC. This did not increase the proportion of OC that were generated from bone marrow (0-3%). In all models only rare, individual bone marrow OC were observed. We hypothesised that the bone marrow cells may be a source for replenishing OC that are expended over long periods of injury and regeneration, however experiments with recurrent liver injury failed to induce proliferation of the bone marrow OC. Studies are underway to transdifferentiate bone marrow cells to OC in vitro.

31 Oval cells compensate for premature senescence mediated inhibition of proliferation of hepatocytes in liver-specific, tetracycline regulated p16INK4a transgenic mice Elke Ueberham1, Renate Bittner1, Rolf Gebhardt1 and Uwe Ueberham2 1 Institut of Biochemie und 2Paul-Flechsig Institut for Brain Research, Medical Faculty, University of Leipzig, Leipzig, Germany Shortage of donors for orthotopic liver transplantation powers the search for alternative possibilties to transplant hepatocytes and/or hepatic stem cells. A number of cells comprising hepatocytes themselves, facultative liver stem cells (oval cells), and hematopoetic stem cells are considered as hepatic stem cells. So far, usage of oval cells suffers from insufficient numbers of progenitors in normal livers as well as from the lack of appropriate animal models enabling transfer of knowledge to the human system, because oval cell activation was mainly restricted to early stages of liver carcinogenesis induced in rodent models by different chemicals. Recently the application of this somatic stem cell in transplantation is rediscussed considering transgenic models without chemical induction of oval cell proliferation. Premature senescence circumventing hepatocyte proliferation is mediated by cell cycle inhibitors among them p16INK4a. Based on the tetracycline-regulated gene expression system a mouse model for hepatocyte-specific p16INK4a expression was established. Induction of transgenic p16INK4a does impair neither embryonic, perinatal nor adult liver development or function. However, provocation with various proliferative stimuli leads to activation of oval cells exclusively in p16INK4a expressing livers. These cells were identified by the expression of the A6-antigen and isolated by two-step collagenase perfusion/percoll density gradient. Oval cells were propagated over several passages, expanded and characterized regarding their expression of hepatocyte/stem cell markers in RT-PCR experiments.

32 Enhanced expression of hepatotrophic mitogen ALR (Augmenter of Liver Regeneration) in hepatocellular carcinoma and cirrhosis Hannah Wagner1,4, Wolfgang E. Thasler2, Frauke Bataille3, Arndt Hartmann3, KarlWalter Jauch2, Hans-Jürgen Schlitt1, Thomas S. Weiss1,4 1 Department of Surgery, University of Regensburg Hospital, Regensburg, Germany 2 Department of Surgery, Ludwig Maximillians University of Munich Hospital Großhadern, Munich, Germany 3 Department of Pathology, University of Regensburg Hospital, Germany 4 Center for Liver Cell Research, University of Regensburg Hospital, Germany ALR (Augmenter of Liver Regeneration) a member of the hepatotrophic growth factors was found to have specific liver regeneration enhancing effects in vitro and in vivo. Further it was proposed that ALR might act through auto- and paracrine mechanism during liver cell proliferation, but little is known about its expression in normal and cirrhotic or cancerous human liver tissue. Expression analysis of ALR was performed by immunohistochemistry on paraffin sections of human normal liver tissue (n = 18), cirrhotic livers (n = 66) and hepatocellular carcinomas (HCC) (n = 137) with various underlying diseases. Significantly enhanced ALR expression levels were observed in HCC and cirrhotic tissue compared to normal liver. Correlation study of ALR expression with grading or TNM classification revealed no statistically significant difference. Most interestingly, within the group of HCC tissue, samples from patients with an underlying hepatitis C virus infection had significantly elevated ALR levels compared to HCC samples of alcohol and hepatitis B virus aetiology. Furthermore ALR immunosignals were detected in the cytosol and in the perinuclear regions of hepatocytes and cholangiocytes. In non-parenchymal liver-cells no immunostaining were observed. These results were confirmed by analyzing mRNA levels of isolated liver cells (hepatocytes, bile duct-, Kupffer-, Ito-, endothelial cells) by quantitative RT-PCR. The expression pattern of increased hepatic ALR in cirrhotic livers and hepatocellular carcinomas indicates an important role of ALR in mitogenic altered cell proliferation. Further studies to elucidate a potential use of ALR for clinical diagnosis of liver cirrhosis and cancer, particularly of hepatitis aetiology, are currently under investigation.

33 Partial hepatectomy induces regeneration with high levels of telomerase activity in pig livers H. Wege1, A. Nunez1, L. Müller2, A. Müller2, C. Hillert2 1 First Department of Medicine and 2Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital Hamburg-Eppendorf, Germany Telomeres, specialized nucleoprotein structures at the ends of eukaryotic chromosomes, have to be maintained during extensive cellular proliferation to avoid chromosomal instability and early cell cycle arrest. Therefore, stem cells and expanding lymphocytes activate telomerase, a ribonucleoprotein that has the capacity to counterbalance replication-associated telomere erosion. Recently, mouse studies revealed high levels of telomerase activity in liver tissue following partial hepatectomy. However, man and mouse differ markedly in telomerase regulation, chromosome structure, and stringency of checkpoint arrest mechanisms. Thus, we evaluated telomerase levels in pig liver regeneration models. Samples from 22 pigs were analyzed (n = 5 control, n = 5 laparotomy, n = 6 partial hepatectomy, n = 6 partial hepatectomy with TIPS). Three days after surgery, liver samples were collected for microscopic confirmation of regeneration. RNA integrity was assessed by gel analysis. Finally, telomer! ase activity was measured using a commercially available telomeric repeat amplification protocol to determine the total product generated (TPG ± Stdev). In addition, we utilized a SYBR Green-based real-time assay with 293T cells as standards to calculate the relative telomerase activity per ?g of protein (RTA ± Stdev). Both assays showed a significant increase in telomerase activity comparing control animals to animals with partial hepatectomy (TPG: 2.7 ± 3.0 vs. 18.9 ± 15.3, p = 0.004; RTA 51.8 ± 23.2 vs. 229.4 ± 160.3, p = 0.002). Moreover, telomerase activity levels correlated significantly with the number of mitotic hepatocytes (R2 = 0.659 resp. R2 = 0.559). A difference in the degree of inflammation was not observed. In summary, the results indicate that telomerase activation might be an adaptive response to protect against excessive telomere loss associated with liver regeneration.

34 C/EBPα and C/EBPβ expression in the developing mouse embryo Adam Westmacott, Zoë D. Burke, Jonathan M.W. Slack, David Tosh Centre for Regenerative Medicine, Dept. Biology and Biochemistry, University of Bath, United Kingdom The molecular mechanisms that regulate liver differentiation are better understood compared to the molecular events involved in early liver specification. During embryogenesis, specification of the liver is dependent on the presence of various signalling molecules reaching the ventral foregut endoderm. In the absence of these signals, the developing endodermal tissue assumes a pancreatic fate. These observations suggest that neighbouring tissues may differ by the expression of only a few transcription factors known as master switch genes. CCAAT/enhancer binding proteins (C/EBPs) are known to regulate several important liver genes and may be important in distinguishing between the liver and pancreatic programmes of development. C/EBPs are basic-leucine zipper transcription factors that can influence expression via interaction with CCAAT motifs present in various gene promoters, for example, Hepatocyte Nuclear Factor 4 (HNF4). We studied the expression of two C/EBP family members, C/EBPα and β, as well as the regulatory transcription factor, HNF4. By in situ hybridisation we have determined the expression of C/EBPα, β, and HNF4 in both whole mount and embryonic sections. C/EBPα and β in the developing mouse embryo appeared liver-specific, and were identical to the expression patterns of HNF4. These results highlight the potential association of C/EBPs with a previously unidentified role in regulating liver development.

35 Expression and localization of hepatocyte growth factor (HGF), its receptor c-Met and HGF activator (HGFA) in human liver cell cocultures maintained in 3D multicompartment bioreactors Zeilinger K.1, Mutig K.2, Schrade P.2, Neuhaus P.1, Bachmann S.2, Gerlach J.C.1 Division of Experimental Surgery, Surgical Department1, Department of Anatomy2, Charité Campus Virchow-Klinikum, Universitätsmedizin Berlin, Germany Introduction: Within the scope of establishing a clinical extracorporeal liver support system, a 3D multicompartment bioreactor was developed that allows the coculture of primary human liver cells in a physiological-like environment, providing decentralized medium exchange and direct membrane oxygenation. Previous studies have provided evidence of liver cell reorganisation and cell proliferation in bioreactors. In this study we investigated the expression and localization of auto- and paracrine mediators known to enhance cell regeneration processes in vitro. Methods: Primary hepatocytes and nonparenchymal cells were isolated from human donor organs excluded from transplantation and cocultivated over 7 to 28 days in bioreactors. The 3D-arrangement of the cells was investigated using serial sections and specific cell markers for hepatocytes (CK 18, albumin), cholangiocytes (CK 19), endothelial cells (CD31) and progenitor cells (c-kit, AFP). In addition, the production and localization of hepatocyte growth factor (HGF), its receptor c-Met and HGFactivator (HGFA) were studied immunohistochemically. Results: Serial sections showed multiple ramified channel structures, outgrowing from biliary cells and/or progenitor cells. Positive signals for HGFA were regularly observed in these structures, near and within the luminal cell membrane, and also in hepatocytes. HGF-positive cells were often adjacent to vascular structures. C-Met showed positive staining of different intensity within the basolateral membrane region of parenchymal cells. Conclusion: The results indicate that the factors investigated in this study, HGF and HGFA may play an important role in the stimulation of cell proliferation in vitro, emphasizing the importance of auto- and paracrine regulation in cell regeneration processes.

Author Index to Poster Abstracts (Name - Poster Number) Akl, M. Al-Adsani, A. Armbrust, T.

6 1 11

Bachmann, S. Bataille, F. Batusic, D. Bauer, A. Berres, M.-L. Bittner, R. Bleidißel, M. Böcker, U. Bodin, K. Boehm, S. Breitkopf, K. Burke, Z.D.

35 32 4 9 20 31 3 5 7 6 5 2, 19, 34

Cai, H. Cantz, T. Combettes, L. Currie, I.S. Curtis, D.

7, 13 3 15 27 30

Dabeva, M.D. Dahlke, M.H. Daniel-Wojcik, A. Deak, F. Dooley, S. Dudas, J. Eberhard, D. Ehnert, S. El Fandy, G. Elkhafif, N. Ellis, E.C.S. Elmaouhoub, A.

17 18, 23 21 24 5 4, 8, 11 28 5 6 6 7 4, 8

Factor, V.M. Fuchs, E. Füzesi, L.

22 14 11

Gebhardt, R. Gerlach, J. Gressner, A.M. Guillon, G.

31 35 20 15

Hammam, O. Hargitai, B. Hartmann, A. Hauss, J.P. Helmy, H. Hengstler, J.G. Hillebrandt, S. Hillert, C. Hilly, M. Hogrebe, E. Hornsey, M.

6 25 32 9 6 9 20 33 15 9 28

Isik, A.T.

12

Jauch, K.-W. Jelkmann, W.

32 9

Kiss, A. Kiss, I. Korpos, E. Kupcsulik, P.

24 24 24 25

Lammert, F. LeVoci, L. Lee, J.-S. Lehmann, T. Li, W.-C. Linke, K. Lódi, C.

20 16 22 13 28 10 24

Mahmoud, S. Mansuroglu, T. Mansy, S. Mas, M.R. Mas, N. Masson, N.M. Matern, S. Menthena, A. Mertsching, H. Miki, T. Mikula, M. Mikulits, W. Moriconi, F. Müller, A. Müller, L. Mutig, K.

6 4, 11 6 12, 26 26 27 20 17 10 13 14 14 11 33 33 35

Nagy, P. Neuhaus, P. Nicou, A. Nierhoff, D. Norel, R. Nunez, A. Nüssler, A.

24 35 15 16 16 33 5

Tonkin, J.N. Tordjmann, T. Tosh, D. Tron, K.

30 15 1, 19, 28, 34 4

Ueberham, E. Ueberham, U.

31 31

Oertel, M. Olynyk, J.

17 29

Voss, B.

Paku, S. Parks, R. Pieler, T. Piso, P. Popp, F.C. Prigent, S. Ralphs, K.L. Ramadori, G. Roderfeld, M. Roeb, E. Rogler, L.E. Rolletschek, A. Ross Saile, B. Schaff, Z. Schanz, J. Schlitt, H.J. Schöler, H.R. Schön, M.R. Schrade, P. Schröder, I.S. Serrière, V. Shafritz, D.A. Singer, M.V. Slack, J.M.W. Slowik, P. Stolz, D.B. Strom, S.C. Szabo, E. Szijarto, A. Tannapfel, A. Tasci, I. Terrace, J.D. Thasler, W. Thorgeirsson, S.S. Thowfeequ, S. Tirnitz-Parker, J.E.E.

24 27 11 18, 23 18, 23 15 1, 19 4, 8, 11 20 20 16 21 27 4, 11 24 10 18, 23, 32 3 9 35 21, 22 15 16, 17 5 28, 34 18, 23 13 7, 13 24 25 9 12, 26 27 32 22 28 29

Wagner, H. Wege, H. Weiskirchen, R. Weiss, T.S. Westmacott, A. Wilting, J. Wobus, A.M. Yehia, H. Yeoh, G.C.T. Zeilinger, K.

6 32 33 20 32 34 4, 11 21 6 29, 30 35