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Characterization of mesenchymal stem cells-like in human liver
A14
Abstracts / Digestive and Liver Disease 40 (2008) A1–A40
erated the development of CHCCs and increased the number of BM-derived tumors. Conclusions. In our model of CHCC, BM cells could differentiate into OCs and subsequently give rise to liver tumors. The CHCCs expressed G-CSFR and the administration of GCSF accelerated the development of the disease and increased the number of BM-derived liver tumours. doi:10.1016/j.dld.2007.12.047 CHARACTERIZATION OF MESENCHYMAL STEM CELLS-LIKE IN HUMAN LIVER R. Ratti a , N. Saulnier b , F. Colombo c , L. Porretti a , A. Cattaneo a , M.A. Puglisi b , G. Rossi c , G. Piccolo d , B. Foglieni e , D. Prati a,e a
Centro di Medicina Trasfusionale, Terapia Cellulare e Criobiologia, Italy b Ospedale “A. Gemelli”, Roma, Italy c Centro Trapianto Fegato, Italy d Nord Italia Transplant, Fondazione Ospedale Policlinico, Milano, Italy e Ospedale A. Manzoni, Lecco, Italy Background and aim. Mesenchymal stem cells (MSC) are stem cells residing in the connective tissue. So far, MSC are mainly recovered through dissociation of bone marrow and adipose tissue, followed by selection by plastic adhesion. Recent findings suggest the presence of hepatic MSC-like also in liver. However, liver MSC have not been yet well characterized with regards to cultural and immunophenotypic features. The aim of this study was to isolate and characterize MSC from adult human livers rejected from organ transplantation. Materials and methods. After enzymatic digestion, single cell suspensions were seeded into plastic flasks at the density of 7 × 105 cells/cm2 in a MSC specific medium (MSCGM, Lonza, Italy). After 4–5 days of culture, two morphologically different populations were observed: one with spindle shape, the other with cuboidal shape. Analyses were performed at different passages in order to verify if these populations had mesenchymal properties, and to eventually find out differences between the two populations. Cell characterization was performed by flow cytometry (CD14, CD29, CD34, CD45, CD49f, CD73, CD90, CD105, CD117, CD133 and HLA-DR), fluorescence microscopy (␣-fetoprotein, ␣-SMA, albumin, c-met, CD68, CK7, CK18, CK19, fibronectin, Ki67, OV-6 and vimentin) and RT-PCR (CK7, CK18, CK19 and Oct-4). In addition, their capacity to differentiate into adipogenic, osteogenic and hepatic lineages was tested. Results. Analysis revealed a strong positivity for CD29, CD73, CD90, ␣SMA, fibronectin and vimentin for both populations. CD105 was more expressed by spindle-shaped cells (65% vs. 21%), whereas cuboidal cells showed a higher expression of CK7 (90% vs. <1%), CK18 (21% vs. <1%) and Ki67 (22% vs. 4%). At third passage (40 days after initial seeding), both populations showed a spindle-shaped
morphology; Oct-4, a gene commonly expressed by bone marrow MSC, was not expressed. Spindle-shaped cells fully differentiated in osteoblasts but not in adipocytes and hepatic cells. Conclusions. Cuboidal and spindle shape morphology likely represent different maturation steps of the same cell population. Liver cells grown in MSC specific medium express classical MSC antigens and have osteogenic differentiation potential. However, differently from bone marrow-MSC, they did not express Oct-4 and did not differentiate into adipocytes. Liver MSC show typical hepatocyte markers, but we were not able to differentiate them into mature hepatocytes. This study offers novel insights into the liver cell differentiation field. doi:10.1016/j.dld.2007.12.048 HYPERBARIC OXYGEN REDUCES RAT LIVER PRESERVATION INJURY PRESERVED IN A HYPOTERMIC MACHINE PERFUSION F. Giannone a , P. Caraceni a , G. Sgarbi b , A. Baracca b , G. Solaini b , M. Domenicali a , I. Grattagliano a , G. Lenaz b , D. Trer`e c , M. Bernardi a a
Department of Medicina Interna, Cardioangiologia, Epatologia, University of Bologna, Italy b Department of Biochimica, University of Bologna, Italy c Patologia Sperimentale, University of Bologna, Italy Background/aims. Hypothermic machine perfusion (HMP) has been proposed to be superior than the current static liver preservation to prevent cold ischemic injury, but the optimal conditions for HMP are far to be defined. Hyperbaric oxygen may provide the low amount of oxygen which is still required to match the metabolic needs of the liver during cold ischemia. Thus, we aimed to test an innovative HMP which implies the use of hyperbaric oxygen as a new tool to prevent liver preservation injury. Methods. Explanted livers from Sprague-Dawley rats were kept in Celsior solution at 4 ◦ C and preserved for 6, 12 and 24 h according to four different modalities: (1) normobaric static preservation (controls); (2) hyperbaric static preservation at 2 atm; (3) normobaric dynamic preservation, with continuous perfusion at 1 ml/min/g liver; (4) hyperbaric dynamic preservation, with continuous perfusion at 2 atm. After cold preservation, we determined the liver ultrastructure by using electron microscopy, the energy metabolism by measuring the hepatic glycogen content and mitochondrial function (respiratory control ratio, respiratory complex protein content, ATP synthesis rate), and the oxidative damage by assessing the reduced/oxidized glutathione and sulphydril proteins and nitrosothiol concentrations. Results. Compared to controls, after 24 h of cold ischemia, normobaric perfused livers showed improved nuclear and cytosolic ultrastructure, ameliorated glycogen stores and mitochondrial ATP synthesis, but they also suffered a signifi-
Abstracts / Digestive and Liver Disease 40 (2008) A1–A40
erated the development of CHCCs and increased the number of BM-derived tumors. Conclusions. In our model of CHCC, BM cells could differentiate into OCs and subsequently give rise to liver tumors. The CHCCs expressed G-CSFR and the administration of GCSF accelerated the development of the disease and increased the number of BM-derived liver tumours. doi:10.1016/j.dld.2007.12.047 CHARACTERIZATION OF MESENCHYMAL STEM CELLS-LIKE IN HUMAN LIVER R. Ratti a , N. Saulnier b , F. Colombo c , L. Porretti a , A. Cattaneo a , M.A. Puglisi b , G. Rossi c , G. Piccolo d , B. Foglieni e , D. Prati a,e a
Centro di Medicina Trasfusionale, Terapia Cellulare e Criobiologia, Italy b Ospedale “A. Gemelli”, Roma, Italy c Centro Trapianto Fegato, Italy d Nord Italia Transplant, Fondazione Ospedale Policlinico, Milano, Italy e Ospedale A. Manzoni, Lecco, Italy Background and aim. Mesenchymal stem cells (MSC) are stem cells residing in the connective tissue. So far, MSC are mainly recovered through dissociation of bone marrow and adipose tissue, followed by selection by plastic adhesion. Recent findings suggest the presence of hepatic MSC-like also in liver. However, liver MSC have not been yet well characterized with regards to cultural and immunophenotypic features. The aim of this study was to isolate and characterize MSC from adult human livers rejected from organ transplantation. Materials and methods. After enzymatic digestion, single cell suspensions were seeded into plastic flasks at the density of 7 × 105 cells/cm2 in a MSC specific medium (MSCGM, Lonza, Italy). After 4–5 days of culture, two morphologically different populations were observed: one with spindle shape, the other with cuboidal shape. Analyses were performed at different passages in order to verify if these populations had mesenchymal properties, and to eventually find out differences between the two populations. Cell characterization was performed by flow cytometry (CD14, CD29, CD34, CD45, CD49f, CD73, CD90, CD105, CD117, CD133 and HLA-DR), fluorescence microscopy (␣-fetoprotein, ␣-SMA, albumin, c-met, CD68, CK7, CK18, CK19, fibronectin, Ki67, OV-6 and vimentin) and RT-PCR (CK7, CK18, CK19 and Oct-4). In addition, their capacity to differentiate into adipogenic, osteogenic and hepatic lineages was tested. Results. Analysis revealed a strong positivity for CD29, CD73, CD90, ␣SMA, fibronectin and vimentin for both populations. CD105 was more expressed by spindle-shaped cells (65% vs. 21%), whereas cuboidal cells showed a higher expression of CK7 (90% vs. <1%), CK18 (21% vs. <1%) and Ki67 (22% vs. 4%). At third passage (40 days after initial seeding), both populations showed a spindle-shaped
morphology; Oct-4, a gene commonly expressed by bone marrow MSC, was not expressed. Spindle-shaped cells fully differentiated in osteoblasts but not in adipocytes and hepatic cells. Conclusions. Cuboidal and spindle shape morphology likely represent different maturation steps of the same cell population. Liver cells grown in MSC specific medium express classical MSC antigens and have osteogenic differentiation potential. However, differently from bone marrow-MSC, they did not express Oct-4 and did not differentiate into adipocytes. Liver MSC show typical hepatocyte markers, but we were not able to differentiate them into mature hepatocytes. This study offers novel insights into the liver cell differentiation field. doi:10.1016/j.dld.2007.12.048 HYPERBARIC OXYGEN REDUCES RAT LIVER PRESERVATION INJURY PRESERVED IN A HYPOTERMIC MACHINE PERFUSION F. Giannone a , P. Caraceni a , G. Sgarbi b , A. Baracca b , G. Solaini b , M. Domenicali a , I. Grattagliano a , G. Lenaz b , D. Trer`e c , M. Bernardi a a
Department of Medicina Interna, Cardioangiologia, Epatologia, University of Bologna, Italy b Department of Biochimica, University of Bologna, Italy c Patologia Sperimentale, University of Bologna, Italy Background/aims. Hypothermic machine perfusion (HMP) has been proposed to be superior than the current static liver preservation to prevent cold ischemic injury, but the optimal conditions for HMP are far to be defined. Hyperbaric oxygen may provide the low amount of oxygen which is still required to match the metabolic needs of the liver during cold ischemia. Thus, we aimed to test an innovative HMP which implies the use of hyperbaric oxygen as a new tool to prevent liver preservation injury. Methods. Explanted livers from Sprague-Dawley rats were kept in Celsior solution at 4 ◦ C and preserved for 6, 12 and 24 h according to four different modalities: (1) normobaric static preservation (controls); (2) hyperbaric static preservation at 2 atm; (3) normobaric dynamic preservation, with continuous perfusion at 1 ml/min/g liver; (4) hyperbaric dynamic preservation, with continuous perfusion at 2 atm. After cold preservation, we determined the liver ultrastructure by using electron microscopy, the energy metabolism by measuring the hepatic glycogen content and mitochondrial function (respiratory control ratio, respiratory complex protein content, ATP synthesis rate), and the oxidative damage by assessing the reduced/oxidized glutathione and sulphydril proteins and nitrosothiol concentrations. Results. Compared to controls, after 24 h of cold ischemia, normobaric perfused livers showed improved nuclear and cytosolic ultrastructure, ameliorated glycogen stores and mitochondrial ATP synthesis, but they also suffered a signifi-