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F.N.1 MULTIPOTENT STEM CELLS RESIDE IN HUMAN EXTRAHEPATIC BILE DUCTS (hEHBDs) AND CAN GIVE RISE TO HEPATOCYTES, CHOLANGIOCYTES AND PANCREATIC ISLET CELLS
Abstracts A.I.S.F. Annual Meeting (Rome, February 25th−26th, 2010) / Digestive and Liver Disease 42 Suppl. 1 (2010) S1–S51
Posters, Friday, February 26th, 2010 F.N.1 MULTIPOTENT STEM CELLS RESIDE IN HUMAN EXTRAHEPATIC BILE DUCTS (hEHBDs) AND CAN GIVE RISE TO HEPATOCYTES, CHOLANGIOCYTES AND PANCREATIC ISLET CELLS V. Cardinale1,2 , Y. Wang6 , G. Carpino5 , M. Gatto1,2 , M. Mennini3 , M. Rossi3 , P. Berloco3 , E. Gaudio4 , L.M. Reid6 , D. Alvaro1,2 . 1 Dept. Clinical Medicine, 2 Polo Pontino, 3 Dept. “P. Stefanini”, 4 Dept. Human Anatomy, “Sapienza” University of Rome, Italy; 5 Dept. Health Science, University “Foro Italico” Rome, Italy; 6 UNC School of Medicine, Chapel Hill, NC, USA During embryogenesis, hEHBDs emerge from midgut endoderm with endodermal proliferation resulting in formation of peribiliary glands (PBGs). To investigate hEHBDs for stem cell populations, common hepatic duct, cystic duct, gallbladder and bile duct were obtained from normal human adult livers while hepato-pancreatic ampulla from pancreas. Tissues or isolated cells were characterized by histology, immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, and by RT-PCR for stem/progenitor and mature cell markers. In addition, freshly isolated cells were plated onto plastic and in Kubota’s Medium (KM). Results: Density of the PBGs is significantly higher in hepatopancreatic ampulla, cystic duct and common hepatic duct with respect to bile duct. Aggregates of cells within gall bladder and cells within PBGs were found to contain markers of endodermal precursors (SOX17, SOX9, FOXA2, HNF6) and surface markers (CD326/EpCAM, CD56/NCAM, CD133), also when cultured in vitro on KM. Furthermore, they express low levels of lineage markers of hepatocytes (albumin, transferrin), cholangiocytes (gamma-GT, secretin receptor), and endocrine pancreas (insulin, glucagon). We also observed expression of PDX1 and NGN3 within the nucleus in some cells and perinuclearly in others. hEHBDs cultures yielded cell colonies with several distinct morphologies including one strikingly similar to that of hHSCs (~5−7 mm; high nucleo/cytoplasmic ratio) and with estimated division rates at a division every ~36 hours and with colonies easily maintained for months. Cells isolated from 1 month cultured colonies were transferred to each of several distinct 3D differentiation conditions and yielded cells that lineage restricted to cords of hepatocytes, branching ducts of cholangiocytes, or clusters of pancreatic cells. The preferential location of the cells in PBGs suggests that the glands are possible stem cell niches. Conclusion: We have identified candidate endodermal stem cells and stem cell niches (PBGs), in normal hEHBDs from all age donors, including adults. The hEHBDs stem cells are readily isolated by either immunoselection and/or culture selection strategies, have a strong potential for cell therapy of liver and pancreatic diseases, including diabetes, for regenerative medicine of endodermal organs and with implications for the pathophysiology of cholangiocarcinoma and sclerosing cholangitis.
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F.N.2 PERFORMANCE OF TRANSIENT ELASTOGRAPHY AND NON INVASIVE MARKERS OF LIVER FIBROSIS IN PRIMARY BILIARY CIRRHOSIS N. Cazzagon1 , L. Chemello2 , D. Martines1 , L. Cavalletto2 , V. Baldo3 , R. Lazzari1 , A. Floreani1 . 1 Dept. of Surgical and Gastroenterological Sciences, 2 Dept. of Clinical and Experimental Medicine, 3 Dept. of Hygiene and Public Health, University of Padova, Italy Background: A variety of surrogate markers of liver fibrosis have been proposed over the last years, the majority of whom have been validated in chronic hepatitis C. Transient elastography (TE) is the latest technology advance proposed in this contest, but still understudied in PBC. Aim: To assess: (1) the diagnostic validity of TE in patients with PBC in comparison with a battery of surrogate markers of hepatic fibrosis (APRI, FIB-4, Fibroindex, Forns, AST/ALT Ratio) in identifying fibrosis or cirrhosis in PBC; (2) the correlation between liver stiffness and Mayo score prognostic index. Methods: 110 patients with PBC (100 females and 10 males, mean age 58±12 years, stage I = 16; stage II = 44; stage III = 45; stage IV = 12) were consecutively enrolled. Performance of each marker and of liver stiffness was compared to histological staging evaluated according to Scheuer’s classification at time of liver biopsy (within the last 12 months) and was expressed by sensitivity (Se), specificity (Sp), positive and negative predictive value (PPV, NPV) and area under ROC curve (AUROC). Optimised cut-offs of non invasive markers were obtained through the AUROC analysis. Results: Liver stiffness values ranged between 3.1 and 38.6 kPa (mean 8.9±5.7). Cut-off values were 7.35 for histological stage III and 11.45 for stage IV. The areas under the ROC curve of TE, APRI, FIB-4, Fibroindex, Forns, and AST/ALT ratio were respectively: 0.87, 0.65, 0.63, 0.63, 0.70, 0.55 for stage III and 0.96, 0.89, 0.79, 0.87, 0.88, 0.60 for stage IV. The best performance in identifying fibrosis or cirrhosis was obtained with TE alone, whereas the combination of each surrogate marker plus TE did not improve the area under the ROC curve. TE presented a positive correlation with Mayo score (r = 0.38, p < 0.001). Logistic regression analysis showed that TE (OR = 1.389, 1.142–1.689 95% CI) was significantly associated with advanced fibrosis (p < 0.001). Conclusions: TE is a simple and effective method for assessing liver fibrosis in PBC, whereas the non invasive surrogate markers failed to reveal a satisfactory performance in predicting III−IV staging. F.N.3 PROGENITOR CELL ACTIVATION AND LIVER REPAIR IS ALTERED IN NOTCH2- AND RBP-Jú-DEFECTIVE MICE EXPOSED TO CHOLESTATIC INJURIES R. Fiorotto1,2 , C. Spirli1,2 , R. Scirpo1,3 , T. Gridley4 , S. Huppert5 , B. Torsello6 , C. Ferrero1 , M. Strazzabosco1,2,3 . 1 Dept. of Internal Medicine, Liver Center and Digestive Diseases Section, Yale University, USA; 2 CeLiveR, Ospedali Riuniti di Bergamo, Italy; 3 Depts. of Clinical Medicine and Prevention and 6 of Experimental Medicine, University of Milan-Bicocca, Italy; 4 The Jackson Laboratory, Bar Harbor, USA; 5 Dept of Cell and Dev Biology and Center for Stem Cell Biology, Vanderbilt University Medical Center, USA Background and Aims: Notch signaling controls cell fate, biliary differentiation and tubulogenesis during the development of the intrahepatic biliary tree. Defective Notch signaling causes Alagille Syndrome, a cholestatic cholangiopathy characterized by intrahepatic bile duct paucity. Liver damage repair requires the
Posters, Friday, February 26th, 2010 F.N.1 MULTIPOTENT STEM CELLS RESIDE IN HUMAN EXTRAHEPATIC BILE DUCTS (hEHBDs) AND CAN GIVE RISE TO HEPATOCYTES, CHOLANGIOCYTES AND PANCREATIC ISLET CELLS V. Cardinale1,2 , Y. Wang6 , G. Carpino5 , M. Gatto1,2 , M. Mennini3 , M. Rossi3 , P. Berloco3 , E. Gaudio4 , L.M. Reid6 , D. Alvaro1,2 . 1 Dept. Clinical Medicine, 2 Polo Pontino, 3 Dept. “P. Stefanini”, 4 Dept. Human Anatomy, “Sapienza” University of Rome, Italy; 5 Dept. Health Science, University “Foro Italico” Rome, Italy; 6 UNC School of Medicine, Chapel Hill, NC, USA During embryogenesis, hEHBDs emerge from midgut endoderm with endodermal proliferation resulting in formation of peribiliary glands (PBGs). To investigate hEHBDs for stem cell populations, common hepatic duct, cystic duct, gallbladder and bile duct were obtained from normal human adult livers while hepato-pancreatic ampulla from pancreas. Tissues or isolated cells were characterized by histology, immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, and by RT-PCR for stem/progenitor and mature cell markers. In addition, freshly isolated cells were plated onto plastic and in Kubota’s Medium (KM). Results: Density of the PBGs is significantly higher in hepatopancreatic ampulla, cystic duct and common hepatic duct with respect to bile duct. Aggregates of cells within gall bladder and cells within PBGs were found to contain markers of endodermal precursors (SOX17, SOX9, FOXA2, HNF6) and surface markers (CD326/EpCAM, CD56/NCAM, CD133), also when cultured in vitro on KM. Furthermore, they express low levels of lineage markers of hepatocytes (albumin, transferrin), cholangiocytes (gamma-GT, secretin receptor), and endocrine pancreas (insulin, glucagon). We also observed expression of PDX1 and NGN3 within the nucleus in some cells and perinuclearly in others. hEHBDs cultures yielded cell colonies with several distinct morphologies including one strikingly similar to that of hHSCs (~5−7 mm; high nucleo/cytoplasmic ratio) and with estimated division rates at a division every ~36 hours and with colonies easily maintained for months. Cells isolated from 1 month cultured colonies were transferred to each of several distinct 3D differentiation conditions and yielded cells that lineage restricted to cords of hepatocytes, branching ducts of cholangiocytes, or clusters of pancreatic cells. The preferential location of the cells in PBGs suggests that the glands are possible stem cell niches. Conclusion: We have identified candidate endodermal stem cells and stem cell niches (PBGs), in normal hEHBDs from all age donors, including adults. The hEHBDs stem cells are readily isolated by either immunoselection and/or culture selection strategies, have a strong potential for cell therapy of liver and pancreatic diseases, including diabetes, for regenerative medicine of endodermal organs and with implications for the pathophysiology of cholangiocarcinoma and sclerosing cholangitis.
S33
F.N.2 PERFORMANCE OF TRANSIENT ELASTOGRAPHY AND NON INVASIVE MARKERS OF LIVER FIBROSIS IN PRIMARY BILIARY CIRRHOSIS N. Cazzagon1 , L. Chemello2 , D. Martines1 , L. Cavalletto2 , V. Baldo3 , R. Lazzari1 , A. Floreani1 . 1 Dept. of Surgical and Gastroenterological Sciences, 2 Dept. of Clinical and Experimental Medicine, 3 Dept. of Hygiene and Public Health, University of Padova, Italy Background: A variety of surrogate markers of liver fibrosis have been proposed over the last years, the majority of whom have been validated in chronic hepatitis C. Transient elastography (TE) is the latest technology advance proposed in this contest, but still understudied in PBC. Aim: To assess: (1) the diagnostic validity of TE in patients with PBC in comparison with a battery of surrogate markers of hepatic fibrosis (APRI, FIB-4, Fibroindex, Forns, AST/ALT Ratio) in identifying fibrosis or cirrhosis in PBC; (2) the correlation between liver stiffness and Mayo score prognostic index. Methods: 110 patients with PBC (100 females and 10 males, mean age 58±12 years, stage I = 16; stage II = 44; stage III = 45; stage IV = 12) were consecutively enrolled. Performance of each marker and of liver stiffness was compared to histological staging evaluated according to Scheuer’s classification at time of liver biopsy (within the last 12 months) and was expressed by sensitivity (Se), specificity (Sp), positive and negative predictive value (PPV, NPV) and area under ROC curve (AUROC). Optimised cut-offs of non invasive markers were obtained through the AUROC analysis. Results: Liver stiffness values ranged between 3.1 and 38.6 kPa (mean 8.9±5.7). Cut-off values were 7.35 for histological stage III and 11.45 for stage IV. The areas under the ROC curve of TE, APRI, FIB-4, Fibroindex, Forns, and AST/ALT ratio were respectively: 0.87, 0.65, 0.63, 0.63, 0.70, 0.55 for stage III and 0.96, 0.89, 0.79, 0.87, 0.88, 0.60 for stage IV. The best performance in identifying fibrosis or cirrhosis was obtained with TE alone, whereas the combination of each surrogate marker plus TE did not improve the area under the ROC curve. TE presented a positive correlation with Mayo score (r = 0.38, p < 0.001). Logistic regression analysis showed that TE (OR = 1.389, 1.142–1.689 95% CI) was significantly associated with advanced fibrosis (p < 0.001). Conclusions: TE is a simple and effective method for assessing liver fibrosis in PBC, whereas the non invasive surrogate markers failed to reveal a satisfactory performance in predicting III−IV staging. F.N.3 PROGENITOR CELL ACTIVATION AND LIVER REPAIR IS ALTERED IN NOTCH2- AND RBP-Jú-DEFECTIVE MICE EXPOSED TO CHOLESTATIC INJURIES R. Fiorotto1,2 , C. Spirli1,2 , R. Scirpo1,3 , T. Gridley4 , S. Huppert5 , B. Torsello6 , C. Ferrero1 , M. Strazzabosco1,2,3 . 1 Dept. of Internal Medicine, Liver Center and Digestive Diseases Section, Yale University, USA; 2 CeLiveR, Ospedali Riuniti di Bergamo, Italy; 3 Depts. of Clinical Medicine and Prevention and 6 of Experimental Medicine, University of Milan-Bicocca, Italy; 4 The Jackson Laboratory, Bar Harbor, USA; 5 Dept of Cell and Dev Biology and Center for Stem Cell Biology, Vanderbilt University Medical Center, USA Background and Aims: Notch signaling controls cell fate, biliary differentiation and tubulogenesis during the development of the intrahepatic biliary tree. Defective Notch signaling causes Alagille Syndrome, a cholestatic cholangiopathy characterized by intrahepatic bile duct paucity. Liver damage repair requires the