- Email: [email protected]
298 ISOLATION AND CULTIVATION OF PURE HUMAN ADULT LIVER STEM CELL POPULATIONS FROM PATIENT LIVER RESECTATES
POSTERS 296 ISOLATION, CHARACTERIZATION AND HEPATOCYTE DIFFERENTIATION OF HUMAN ADULT PROGENITOR CELLS FROM LIVER AND PANCREAS C. Duret1 , I. Iankova1 , J. Ramos2 , S. Gerbal-Chaloin1 , J.-M. Fabre3 , A. Wojtusciszyn4 , P. Maurel1 , M. Daujat-Chavanieu1 . 1 IRB, INSERM U1040, 2 Service d’Anatomie Pathologique, CHU Gui de Chauliac, 3 Chirurgie Digestive, CHU St Eloi, 4 D´epartement d’Endocrinologie, Diab`ete et Nutrition, CHU Lapeyronie, Montpellier, France E-mail: [email protected] Introduction: During embryogenesis, liver and pancreas are generated from a common endodermic precursor. Furthermore, in vivo in humans and in rodents, and in pathological environment, adult hepatocyte clusters have been observed in pancreatic tumors. These hepatic cells could come from mature pancreatic cell transdifferentiation or from pancreatic progenitor cell differentiation towards the hepatocyte lineage. These observations raised the hypothesis that progenitors from endoderm developmental stage could persist during adult life (unidentified in humans). In this study we established a similar strategy for isolation, proliferation and differentiation of human progenitor cells from adult liver and pancreas. Results: Organ fragments were dissociated with collagenase and extracted cells were seeded in human adult hepatic progenitor cell proliferation medium. After 4–7 days, various cellular populations appeared. The proliferation medium was replaced at confluence by the hepatocyte differentiation medium. We observed an increased expression of the hepatocyte genes albumin and CYP3A4, and no detection of the pancreatic genes insulin and glucagon. At day 21, clusters of positive cells for albumin and cytokeratins 7 and 8/18 were observed. In a second set of experiments, we isolated a population negative for CD105 and CD90 antigens after 7–14 days of amplification. The populations from both liver and pancreas had an identical morphology. In the hepatocyte differentiation medium, the expression of albumin and CYP3A4 mRNAs increased whereas insulin and glucagon gene expression was not detected. At day 21 positive clusters for albumin and cytokeratins 7 and 8/18 were observed. Conclusion: In conclusion, we showed the feasibility to isolate an epithelial population containing progenitor cells with hepatocyte differentiation capacity from human adult liver and pancreas. These in vitro results strongly suggest the presence of cells with hepatocyte differentiation capacity in the pancreas, as observed in vivo under particular pathological conditions. In the future, these progenitor populations could be a model for basic research and a cell source for liver and/or pancreas biotherapy. 297 THE HEPATIC PROGENITOR B-13 CELL LINE ENGRAFTS INTO THE MOUSE LIVER AND TRANSDIFFERENTIATES IN VIVO E.A. Fairhall1 , C. Schwab2 , C. Harrison2 , K.A. Charlton1 , K. Wallace1 , M.C. Wright1 . 1 Institute or Cellular Medicine, 2 Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK E-mail: [email protected] Background and Aims: The AR42J-B-13 (B-13) cell line is a rat pancreatic acinar cell line capable of transdifferentiating into hepatocyte-like cells (B-13/H) in response to glucocorticoids. The transdifferentiation mechanisms have been shown to be dependent on a transient repression of WNT signalling upstream of an induction of liver enriched transcription factor C/EBPb [1,2]. B-13s thus have great potential in vitro, as an unlimited supply of functional hepatocytes, for a wide range of uses such as toxicological screening. Our aims were to investigate whether
the progenitor cells can engraft to the liver of severe-combined immuno-deficient (SCID) mice after paracetamol damage. Methods and Results: Cytogenetic analysis confirmed that B-13s carry the Y chromosome thus female SCID mice were dosed with paracetamol to induce liver damage prior to intra-venous injections of B-13 progenitor cells. Immuno-histochemistry, for the pancreatic marker amylase was performed in all tissues to determine if any B-13 cells were present. Clusters of amylase positive cells were found within the portal tracts of the liver but not in other tissues and showed hepatocyte morphology. To confirm the origin of amylase positive cells, fluorescent insitu hybridisation (FISH) was performed to probe for the rat Y chromosome and confirmed the presence of the B-13 cells within the mouse livers. Protein expression for the pancreatic marker confirmed the presence of amylase within the livers positive by immuno-histochemistry. More interestingly RT-PCR for rat specific amylase and CYP2E1 showed that B-13 cells only engrafted to the liver and pancreas and transdifferentiation was only observed within the liver suggesting that within the liver environment their hepatic-phenotype is promoted. Conclusions: These data show that the B-13 cell line is capable of engrafting to the damaged liver and differentiates into a hepatocyte-like morphology. This ability to transdifferentiate in vivo as well as in vitro brings new insights into the cell line and the isolation of a human equivalent would have great clinical potential for the treatment of liver disease. Acknowledgements: Supported by an MRC ITTP Studentship. Reference(s) [1] Wallace K et al, J Cell Sci. 2010 Jun 15; 123(pt12): 2103–10. [2] Wallace K et al, J Cell Sci. 2011 Feb 1; 124(pt3): 405–13.
298 ISOLATION AND CULTIVATION OF PURE HUMAN ADULT LIVER STEM CELL POPULATIONS FROM PATIENT LIVER RESECTATES 1 N. Fekete-Drimusz1 , I. Meder2 , U. Rudrich ¨ , K. Preis1 , R. Gutierrez1 , 1 2 1 1 M.P. Manns , F. Vondran , M. Bock . Department of Gastroenterology, Hepatology and Endocrinology, 2 Clinic for General, Abdominal and Transplant Surgery, Medizinische Hochschule Hannover, Hannover, Germany E-mail: [email protected] Background and Aims: Currently, freshly isolated or thawed human hepatocytes cannot be kept in vitro for reproducible experimentation or therapeutical use: They dedifferentiate rapidly in the absence of significant cell proliferation. Such a rapid loss of function makes it difficult to apply them for cell based therapies in vivo, or pharmacological and toxicological studies in vitro. This explains the need for the isolation and cultivation of liver stem cells from patient samples that can proliferate without signs of senescence and that could be differentiated to a desired state depending on their future application. Methods: Suspensions of liver cells are received from our clinical partners at MHH following a two-step collagenase perfusion treatment of liver biopsies obtained during liver surgery. After having tested a variety of different coating techniques and culture conditions we are now able to successfully enrich and purify populations of adult liver stem cells from patient specific liver cell suspensions using a highly modified ‘plate-and-wait’ method. The cells can be kept continuously in culture or be stored safely frozen in Nitrogen. Results: The populations obtained can be passaged at low ratios (1:3 every 7–10 days) and (so far) show no signs of senescence. They exhibit stunning morphologic plasticity and sensitivity to the slightest changes of culture conditions, strongly indicating an undifferentiated stem cell phenotype. When comparing gene expression levels of our stem cell populations to primary hepatocytes, the most striking difference
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POSTERS arises in the strong expression of EpCAM, which is upregulated 50fold in the stem cell populations. Expression levels of Albumin and KRT 8/18/7/19 can be rated as intermediate; AFP is barely detectable and basal levels of so-called liver enriched transcription factors (eg HNF1a, 1b, 4a, 6 and more) are largely similar to primary hepatocyte isolates. Conclusion: We have developed a robust method that allows for the enrichment and maintenance culture of human adult liver stem cells from patient samples. Currently – with more populations in the enrichment stages – we are characterising already existing stem cell populations, mainly with respect to their differentiation potentials towards mature hepatocytes or cholangiocytes by the means of RT-qPCR and Immunoflourescence. 299 DOSE-DEPENDENT MODULATION OF NF-úB AND miR-21 BY DEOXYCHOLIC ACID DURING APOPTOSIS OF PRIMARY RAT HEPATOCYTES P.M. Rodrigues1 , M.B. Afonso1 , A.L. Sim˜ao1 , D.M.S. Ferreira1 , P.M. Borralho1,2 , C.M.P. Rodrigues1,2 , R.E. Castro1,2 . 1 Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, 2 Department of Biochemistry and Human Biology, Faculty of Pharmacy University of Lisbon, Lisbon, Portugal E-mail: [email protected] Background and Aims: Both deoxycholic acid (DCA), an endogenous bile acid, and miR-21, an oncogenic miRNA, have been implicated in the pathogenesis of liver and gastrointestinal disorders. Interestingly, we have recently shown that DCA inhibits miR-21 expression in hepatocytes. Therefore, we aimed to clarify the mechanisms by which DCA modulates the miR-21 signaling pathway. Methods: Primary rat hepatocytes were incubated with 25–400 mM DCA from 16 to 48 h. miR-21 expression was evaluated by qRT-PCR. Programmed cell death 4 (PDCD4), a miR-21 target, and NF-úB were analysed by immunoblotting. PDCD4 silencing was achieved by transfecting cells with a specific PDCD4 siRNA. NF-úB was inhibited using BAY 11–7085, a specific inhibitor. Cell death, viability and caspase-3 activity were determined by the ApoTox-GloTM Triplex Assay. Results: Our results show that DCA modulates the miR-21/PDCD4 pathway in a dose-dependent manner. While lower doses (25 and 50 mM) exhibited no toxicity and tend to activate this survival pathway, moderate to high doses (>100 mM) significantly inhibited miR-21 (p < 0.05), and activated caspase-3 and apoptosis (p < 0.01). In agreement, PDCD4 expression was significantly increased in the presence of DCA concentrations greater than 100 mM (p < 0.05). Importantly, upon PDCD4 inhibition, DCA-induced caspase-3 and apoptosis were significantly reduced, particularly for the 100 mM concentration. Finally, DCA inhibited NF-úB expression (p < 0.05), in a similar pattern to miR-21 inhibition. Cells incubated with an NF-úB inhibitor also displayed lower miR-21 levels, increased PDCD4 expression and greater cell death (p < 0.05). Conclusions: DCA modulation of the miR-21/PDCD4 pathway correlates with its effects on apoptosis. While low doses of DCA activate survival, moderate to high doses significantly inhibit miR21; PDCD4 is an important functional target. Importantly, DCA appears modulate miR-21 via NF-úB. A better understanding of the mechanisms by which DCA impacts on cell death and survival may allow for the development of new therapeutic tools. Supported by PTDC/SAU-OSM/102099/2008, PTDC/SAU-ORG/ 111930/2009, Pest-OE/SAU/UI4013/2011 and SFRH/BD/88212/2012 from FCT, Lisbon.
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300 IDENTIFICATION OF MATRIX METALLOPROTEASE 10 (MMP10) AS A KEY NEW MEDIATOR OF THE REGENERATIVE RESPONSE OF THE LIVER O. Garcia-Irigoyen1 , M.U. Latasa1 , M.G. Fernandez-Barrena1 , I. Uriarte2 , M. Elizalde1 , R. Urtasun1 , U. Vespasiani-Gentilucci3 , S. Morini3 , S. Carotti3 , J.A. Rodriguez4 , J. Orbe4 , J.A. Paramo4 , J.J. Marin5 , J. Prieto1,2 , C. Berasain1,2 , M.A. Avila1,2 . 1 Division of Hepatology and Gene Therapy, CIMA, Universidad de Navarra, 2 CIBERehd, Clinica Universidad de Navarra, Pamplona, Spain; 3 Medicine, Universita Campus Bio-Medico, Roma, Italy; 4 Cardiovascular Sciences, CIMA, Universidad de Navarra, Pamplona, 5 Physiology and CIBERehd, Universidad de Salamanca, Salamanca, Spain E-mail: [email protected] Background: The wound-healing response triggered after liver injury involves a transitory substitution of damaged hepatic parenchyma by extracellular matrix (ECM) that needs to be removed when liver tissue is regenerated. Matrix metalloproteinases (MMPs) provide important functions during this process, acting as regulatory molecules by the selective proteolytic activation of growth factors and cell surface receptors in addition to their ability to degrade ECM constituents. The role of some MMPs in liver regeneration has been studied. However, little is known about the function of MMP10, also called stromelysin-2, in this process. Recent studies have demonstrated the overexpression of MMP10 in different tumors, and its behaviour as a profibrinolytic factor. We have characterized the expression and relevance of MMP10 in models of liver injury and regeneration. Methods: MMP10 was analysed in different cell lines and in vivo models of liver injury. Acute administration of CCl4 and alfanaphtyl-isothiocyanate (ANIT), partial hepatectomy (PH) and bile duct ligation (BDL) were performed in MMP10+/+ and MMP10−/− mice. Gene expression was analysed by qPCR, western-blotting and immunohistochemistry. Results: MMP10 expression is very low in the healthy liver, but it is significantly up-regulated in all injury and regeneration models tested. The protein was detected in parenchymal cells, biliary epithelium, and inflammatory cells. In the BDL model MMP10 was found in the cytosol and nucleus of hepatocytes and cholangiocytes. The stimulation of cells with ligands of the epidermal growth factor receptor (EGFR) and TGFb, as well as with bile acids and GW4064, an agonist of farnesoid X receptor (FXR), triggers MMP10 expression. In all the models examined the absence of MMP10 caused a significant increase of necrosis and a high mortality after PH. A remarkable intrahepatic accumulation of fibrinogen and fibronectin insoluble complexes was observed in MMP10−/− mice upon liver injury. Conclusions: This is the first description of the expression and function of MMP10 in liver injury and regeneration. We identified MMP10 as a gene regulated by bile acids/FXR and as a target of liver regeneration-related growth factors. MMP10 is essential for correct liver regeneration after an acute injury, MMP10 profibrinolytic activity may be crucial in this process.
Journal of Hepatology 2013 vol. 58 | S63–S227
the progenitor cells can engraft to the liver of severe-combined immuno-deficient (SCID) mice after paracetamol damage. Methods and Results: Cytogenetic analysis confirmed that B-13s carry the Y chromosome thus female SCID mice were dosed with paracetamol to induce liver damage prior to intra-venous injections of B-13 progenitor cells. Immuno-histochemistry, for the pancreatic marker amylase was performed in all tissues to determine if any B-13 cells were present. Clusters of amylase positive cells were found within the portal tracts of the liver but not in other tissues and showed hepatocyte morphology. To confirm the origin of amylase positive cells, fluorescent insitu hybridisation (FISH) was performed to probe for the rat Y chromosome and confirmed the presence of the B-13 cells within the mouse livers. Protein expression for the pancreatic marker confirmed the presence of amylase within the livers positive by immuno-histochemistry. More interestingly RT-PCR for rat specific amylase and CYP2E1 showed that B-13 cells only engrafted to the liver and pancreas and transdifferentiation was only observed within the liver suggesting that within the liver environment their hepatic-phenotype is promoted. Conclusions: These data show that the B-13 cell line is capable of engrafting to the damaged liver and differentiates into a hepatocyte-like morphology. This ability to transdifferentiate in vivo as well as in vitro brings new insights into the cell line and the isolation of a human equivalent would have great clinical potential for the treatment of liver disease. Acknowledgements: Supported by an MRC ITTP Studentship. Reference(s) [1] Wallace K et al, J Cell Sci. 2010 Jun 15; 123(pt12): 2103–10. [2] Wallace K et al, J Cell Sci. 2011 Feb 1; 124(pt3): 405–13.
298 ISOLATION AND CULTIVATION OF PURE HUMAN ADULT LIVER STEM CELL POPULATIONS FROM PATIENT LIVER RESECTATES 1 N. Fekete-Drimusz1 , I. Meder2 , U. Rudrich ¨ , K. Preis1 , R. Gutierrez1 , 1 2 1 1 M.P. Manns , F. Vondran , M. Bock . Department of Gastroenterology, Hepatology and Endocrinology, 2 Clinic for General, Abdominal and Transplant Surgery, Medizinische Hochschule Hannover, Hannover, Germany E-mail: [email protected] Background and Aims: Currently, freshly isolated or thawed human hepatocytes cannot be kept in vitro for reproducible experimentation or therapeutical use: They dedifferentiate rapidly in the absence of significant cell proliferation. Such a rapid loss of function makes it difficult to apply them for cell based therapies in vivo, or pharmacological and toxicological studies in vitro. This explains the need for the isolation and cultivation of liver stem cells from patient samples that can proliferate without signs of senescence and that could be differentiated to a desired state depending on their future application. Methods: Suspensions of liver cells are received from our clinical partners at MHH following a two-step collagenase perfusion treatment of liver biopsies obtained during liver surgery. After having tested a variety of different coating techniques and culture conditions we are now able to successfully enrich and purify populations of adult liver stem cells from patient specific liver cell suspensions using a highly modified ‘plate-and-wait’ method. The cells can be kept continuously in culture or be stored safely frozen in Nitrogen. Results: The populations obtained can be passaged at low ratios (1:3 every 7–10 days) and (so far) show no signs of senescence. They exhibit stunning morphologic plasticity and sensitivity to the slightest changes of culture conditions, strongly indicating an undifferentiated stem cell phenotype. When comparing gene expression levels of our stem cell populations to primary hepatocytes, the most striking difference
Journal of Hepatology 2013 vol. 58 | S63–S227
S125
POSTERS arises in the strong expression of EpCAM, which is upregulated 50fold in the stem cell populations. Expression levels of Albumin and KRT 8/18/7/19 can be rated as intermediate; AFP is barely detectable and basal levels of so-called liver enriched transcription factors (eg HNF1a, 1b, 4a, 6 and more) are largely similar to primary hepatocyte isolates. Conclusion: We have developed a robust method that allows for the enrichment and maintenance culture of human adult liver stem cells from patient samples. Currently – with more populations in the enrichment stages – we are characterising already existing stem cell populations, mainly with respect to their differentiation potentials towards mature hepatocytes or cholangiocytes by the means of RT-qPCR and Immunoflourescence. 299 DOSE-DEPENDENT MODULATION OF NF-úB AND miR-21 BY DEOXYCHOLIC ACID DURING APOPTOSIS OF PRIMARY RAT HEPATOCYTES P.M. Rodrigues1 , M.B. Afonso1 , A.L. Sim˜ao1 , D.M.S. Ferreira1 , P.M. Borralho1,2 , C.M.P. Rodrigues1,2 , R.E. Castro1,2 . 1 Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, 2 Department of Biochemistry and Human Biology, Faculty of Pharmacy University of Lisbon, Lisbon, Portugal E-mail: [email protected] Background and Aims: Both deoxycholic acid (DCA), an endogenous bile acid, and miR-21, an oncogenic miRNA, have been implicated in the pathogenesis of liver and gastrointestinal disorders. Interestingly, we have recently shown that DCA inhibits miR-21 expression in hepatocytes. Therefore, we aimed to clarify the mechanisms by which DCA modulates the miR-21 signaling pathway. Methods: Primary rat hepatocytes were incubated with 25–400 mM DCA from 16 to 48 h. miR-21 expression was evaluated by qRT-PCR. Programmed cell death 4 (PDCD4), a miR-21 target, and NF-úB were analysed by immunoblotting. PDCD4 silencing was achieved by transfecting cells with a specific PDCD4 siRNA. NF-úB was inhibited using BAY 11–7085, a specific inhibitor. Cell death, viability and caspase-3 activity were determined by the ApoTox-GloTM Triplex Assay. Results: Our results show that DCA modulates the miR-21/PDCD4 pathway in a dose-dependent manner. While lower doses (25 and 50 mM) exhibited no toxicity and tend to activate this survival pathway, moderate to high doses (>100 mM) significantly inhibited miR-21 (p < 0.05), and activated caspase-3 and apoptosis (p < 0.01). In agreement, PDCD4 expression was significantly increased in the presence of DCA concentrations greater than 100 mM (p < 0.05). Importantly, upon PDCD4 inhibition, DCA-induced caspase-3 and apoptosis were significantly reduced, particularly for the 100 mM concentration. Finally, DCA inhibited NF-úB expression (p < 0.05), in a similar pattern to miR-21 inhibition. Cells incubated with an NF-úB inhibitor also displayed lower miR-21 levels, increased PDCD4 expression and greater cell death (p < 0.05). Conclusions: DCA modulation of the miR-21/PDCD4 pathway correlates with its effects on apoptosis. While low doses of DCA activate survival, moderate to high doses significantly inhibit miR21; PDCD4 is an important functional target. Importantly, DCA appears modulate miR-21 via NF-úB. A better understanding of the mechanisms by which DCA impacts on cell death and survival may allow for the development of new therapeutic tools. Supported by PTDC/SAU-OSM/102099/2008, PTDC/SAU-ORG/ 111930/2009, Pest-OE/SAU/UI4013/2011 and SFRH/BD/88212/2012 from FCT, Lisbon.
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300 IDENTIFICATION OF MATRIX METALLOPROTEASE 10 (MMP10) AS A KEY NEW MEDIATOR OF THE REGENERATIVE RESPONSE OF THE LIVER O. Garcia-Irigoyen1 , M.U. Latasa1 , M.G. Fernandez-Barrena1 , I. Uriarte2 , M. Elizalde1 , R. Urtasun1 , U. Vespasiani-Gentilucci3 , S. Morini3 , S. Carotti3 , J.A. Rodriguez4 , J. Orbe4 , J.A. Paramo4 , J.J. Marin5 , J. Prieto1,2 , C. Berasain1,2 , M.A. Avila1,2 . 1 Division of Hepatology and Gene Therapy, CIMA, Universidad de Navarra, 2 CIBERehd, Clinica Universidad de Navarra, Pamplona, Spain; 3 Medicine, Universita Campus Bio-Medico, Roma, Italy; 4 Cardiovascular Sciences, CIMA, Universidad de Navarra, Pamplona, 5 Physiology and CIBERehd, Universidad de Salamanca, Salamanca, Spain E-mail: [email protected] Background: The wound-healing response triggered after liver injury involves a transitory substitution of damaged hepatic parenchyma by extracellular matrix (ECM) that needs to be removed when liver tissue is regenerated. Matrix metalloproteinases (MMPs) provide important functions during this process, acting as regulatory molecules by the selective proteolytic activation of growth factors and cell surface receptors in addition to their ability to degrade ECM constituents. The role of some MMPs in liver regeneration has been studied. However, little is known about the function of MMP10, also called stromelysin-2, in this process. Recent studies have demonstrated the overexpression of MMP10 in different tumors, and its behaviour as a profibrinolytic factor. We have characterized the expression and relevance of MMP10 in models of liver injury and regeneration. Methods: MMP10 was analysed in different cell lines and in vivo models of liver injury. Acute administration of CCl4 and alfanaphtyl-isothiocyanate (ANIT), partial hepatectomy (PH) and bile duct ligation (BDL) were performed in MMP10+/+ and MMP10−/− mice. Gene expression was analysed by qPCR, western-blotting and immunohistochemistry. Results: MMP10 expression is very low in the healthy liver, but it is significantly up-regulated in all injury and regeneration models tested. The protein was detected in parenchymal cells, biliary epithelium, and inflammatory cells. In the BDL model MMP10 was found in the cytosol and nucleus of hepatocytes and cholangiocytes. The stimulation of cells with ligands of the epidermal growth factor receptor (EGFR) and TGFb, as well as with bile acids and GW4064, an agonist of farnesoid X receptor (FXR), triggers MMP10 expression. In all the models examined the absence of MMP10 caused a significant increase of necrosis and a high mortality after PH. A remarkable intrahepatic accumulation of fibrinogen and fibronectin insoluble complexes was observed in MMP10−/− mice upon liver injury. Conclusions: This is the first description of the expression and function of MMP10 in liver injury and regeneration. We identified MMP10 as a gene regulated by bile acids/FXR and as a target of liver regeneration-related growth factors. MMP10 is essential for correct liver regeneration after an acute injury, MMP10 profibrinolytic activity may be crucial in this process.
Journal of Hepatology 2013 vol. 58 | S63–S227