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371 Human hepatic progenitor cells: Molecular basis for their engraftment
Category 4e." Molecular and Cellular Biology." Gene Transfer/Gene Therapy
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EFFICIENT HEPATOCYTES ENGRAFTMENT IN A NON-HUMAN PRIMATE AS PRECLINICAL MODEL OF CELL T H E R A P Y
I. Dagher I , L. Boudechiche2, J. Branger2, A. Coulomb 3, L. Sentilhes2, M. Hadchouel 2, D. Pariente4, M. Andreoletti5, D. Franco 1, A. Weber 2.
llnserm EMI 00-20 and Department of Su~ery. Hdpital B~l&e, KremlinBieetre, France," 2lnserm EMI 00-20 and University Paris )2I, H@ital Bie~'tre, Kremlin-Bicetre, France; ~Inserm EMI 00-20 and Department of Pathology. H@ital B~cldre, Kremlin-Bicetre, France," 4lnserm EMI 00-20 and Department of Radiology, H@ital Bicgtre, Kremlin-Bicetre, France," SInserm EMI 00-20 and Department of Anestesiology, H@ital B&cl~re, Kremlin-Bicetre, France Cell therapy by transplantation of genetically modified or not hepatocytes is a promising alternative to orthotopic transplantation for the treatment of life-threatening metabolic diseases. However efficient liver repopulation by transplanted hepatocytes were obtained in rodent models in which resident hepatocytes were destroyed or blocked and these models are not transposable to the clinics. Partial venous obstruction is a safe mean to induce liver regeneration in hurnans. The aim of our study was to define an efficient protocol of hepatocyte transplantation in a large animal close to human. Ten Macaca mulatta were used for the study. The hepatic left lateral lobe, representing 20% of the liver was removed for hepatocytes isolation. A catheter was inserted in the inferior rnesenteric vein and ernbolisation of the left portal branch and the right anterior branch was performed with a biological glue, histoacryl. In parallel, isolated hepatocytes were labeled with a fluorescent dye, Hoechst and three animals were transplanted with 400-500million Hoechst-labeled hepatocytes. BrdU was infused through the catheter on day 3, 5 or 7 after embolisation followed or not by transplantation. Resident and transplanted hepatocytes proliferation was assessed on biopsies performed 4 hours after BrdU infusion. Transplanted hepatocytes engraftment in the liver parenchyma were assessed by immunohistochernical analyses. Partial embolisation induced significant hepatocyte proliferation representing 23% of cells in non-ernbolized segments as soon as 3 days after surgery" and 10% of the cells 5 and 7 days after embolisation. Engraftment of transplanted hepatocytes was more efficient than in non-embolized animals. They proliferated and participated to liver regeneration, representing 10.9+0.7% of the monkey liver at 7 days and their number remained stable 15 days after transplantation. Expression o f cennexin 32 between transplanted and resident hepatocytes demonstrated that transplanted cells were integrated within hepatic plates. Liver histology analysed on biopsies performed 9 months after entholization and revealed a normal architecture and an absence of nodular regenerative hyperplasia. These results suggest that partial embolisation is a safe procedure to achieve effective hepatocyte repopulation and that this strategy of cell therapy or ex vivo gene therapy using lentiviral vectors is potentially applicable to patients.
~7i~ HUMAN HEPATIC PROGENITOR CELLS: MOLECULAR BASIS FOR THEIR ENGRAFTMENT
J.E Delgado, L. Sentilhes, S. Mainot, J. Branger, T. Lin, D. MahieuCaputo, A. Weber. INSERM EMI 00-20 and University Paris XI, H@ital
de Bicgtre, Kremlin Bicgtre, France Orthopic transplantation is currently the only treatment to cure hepatic diseases. Hepatocyte transplantation represents an attractive alternative but it is characterized by a limited engraftment of transplanted cells. Moreover, adult human hepatocytes cannot be amplified in vitro and they rapidly loose they differentiated functions. Therefore other sources of cells must be explored. In our laboratory we are exploring the therapeutic potential of hllman foetal hepatic progenitor cells. These cells have been isolated from liver foetuses at 11-13 weeks of gestation and were characterized as bipotent hepatoblasts. They were efficiently transduced by retroviral
137
vectors. After Wansplantation in athymic mice these cells engrafted more efficendy than adult hepatocytes. The aim of this study was to analyze their functionality in terms of ability to display migratory and invasive properties. Human hepatoblats and adult hepatocytes were cultured in presence or absence of different cytokines. ARN were extracted for RT-PCR analysis. Conditioned media were collected and analyzed by zymography for detection of Matrix metalloproteinases 0VIMPs), one of the principal extracellular matrix components that ensure the degradation of adhesion proteins. RT-PCR and zymography analyses showed that MMPs 2 and 9 were expressed at early stages of liver development (11-13 weeks). They were secreted in their inactive form by foetal hepatoblasts in absence of cytokines and in their activated form after 24 and 48 hours of culture in the presence of Hepatocyte Growth Factor (HGF) and Tumor Necrosis Factor alpha (TNFc0. By contrast no activated form was detected in adult hepatocytes. We also showed by immunocytochernistry that TNFc~ induced activation of NFKB transcription factor and its translocation into cell nuclei. This factor induced MMP9 but not MMP2 gene transcription. In vitro experiments using Boyden chambers showed that activation of MMP 2 and 9 was correlated with an increased cell migration and invasion through Matrigel. These results suggest that differences in engraftment ability between adult and foetal hepatocytes are due to different gene regulation involving MMPs, growth factors and adhesion molecules. MMP gene transfer should enhance the engraftment of transplanted hepatoblasts into murine models.
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STRONG H U M O R A L AND CELLULAR IMMUNE R E S P O N S E S ELICITED BY IMMUNIZATION WITH DENDRITIC CELLS T R A N S D U C E D WITH ADENOVIRUS CODING FOR HCV NS3 PROTEIN
J. Encke, C. Eisenbach, C.M. Lupu, E. Evelyn. Department of Internal Medicine IV, University of Heidelberg, Heidelbe~, Germany Background Dendritic cell (DC) vaccination is a promising immunization technique to induce celhtlar and humoral immune responses against a specific antigen espedally in a therapeutic context. We have demonstrated that peptide or protein pulsed DCs induces vigorous B and T cell immune responses in mice model. The potential of DNA-based immunization as for the adenovirus priming and plasmid boosting against HCV NS3 protein in mice has also been studied in our group. However, tile protective iromunity of adenovirus transduced-DCs correlating with clearance of HCV infection in animal models has not been evaluated to date. Aims: Tile object of the study is to establish the dendritic cell-based vaccination approaches by combining two immunization techniques and fiwestigate the protective celhtlar and humoral immune response to HCV specific antigen in different animal models. Methods: Two different delivery strategies were studied by immunizing BALB/c and HLA-A2.1 transgenic mice subcutaneously, inchtding pul sing DC with HCV NS3 protein for 24 hours and transducing DC with adenovirus coding HCV NS3 antigen for 48 hours after removal and purification by magnetic sorting from bone marrow of the same species of mice. 7 days after the last immunization event antibody production against the target HCV NS3 protein and IFN-¥ secretion were evaluated by ELISA and ELISPOZ Results: We found that the dendritic cells transduced with adenovirus for HCV NS3 were capable of generating the significant humoral and T-cell based finmune response (IFN-¥) specific for human NS3 epitope in HLA-A2.1 mice. Comparatively, the vigor of these responses was much stronger than that of dendritic cells pulsed with recombinant NS3 protein. Furthermore, we demonstrated that the previous transduced DC-NS3 immunization does not influence the subsequent use for boosting. Conelusions: Our results dearly indicate that NS3-transduced dendritic cells induce vigorous B and T cell immune responses in HLA.A2.1 transgenic mice, which suggests that this approach has important implications for immune therapy against HCV infection.
•
EFFICIENT HEPATOCYTES ENGRAFTMENT IN A NON-HUMAN PRIMATE AS PRECLINICAL MODEL OF CELL T H E R A P Y
I. Dagher I , L. Boudechiche2, J. Branger2, A. Coulomb 3, L. Sentilhes2, M. Hadchouel 2, D. Pariente4, M. Andreoletti5, D. Franco 1, A. Weber 2.
llnserm EMI 00-20 and Department of Su~ery. Hdpital B~l&e, KremlinBieetre, France," 2lnserm EMI 00-20 and University Paris )2I, H@ital Bie~'tre, Kremlin-Bicetre, France; ~Inserm EMI 00-20 and Department of Pathology. H@ital B~cldre, Kremlin-Bicetre, France," 4lnserm EMI 00-20 and Department of Radiology, H@ital Bicgtre, Kremlin-Bicetre, France," SInserm EMI 00-20 and Department of Anestesiology, H@ital B&cl~re, Kremlin-Bicetre, France Cell therapy by transplantation of genetically modified or not hepatocytes is a promising alternative to orthotopic transplantation for the treatment of life-threatening metabolic diseases. However efficient liver repopulation by transplanted hepatocytes were obtained in rodent models in which resident hepatocytes were destroyed or blocked and these models are not transposable to the clinics. Partial venous obstruction is a safe mean to induce liver regeneration in hurnans. The aim of our study was to define an efficient protocol of hepatocyte transplantation in a large animal close to human. Ten Macaca mulatta were used for the study. The hepatic left lateral lobe, representing 20% of the liver was removed for hepatocytes isolation. A catheter was inserted in the inferior rnesenteric vein and ernbolisation of the left portal branch and the right anterior branch was performed with a biological glue, histoacryl. In parallel, isolated hepatocytes were labeled with a fluorescent dye, Hoechst and three animals were transplanted with 400-500million Hoechst-labeled hepatocytes. BrdU was infused through the catheter on day 3, 5 or 7 after embolisation followed or not by transplantation. Resident and transplanted hepatocytes proliferation was assessed on biopsies performed 4 hours after BrdU infusion. Transplanted hepatocytes engraftment in the liver parenchyma were assessed by immunohistochernical analyses. Partial embolisation induced significant hepatocyte proliferation representing 23% of cells in non-ernbolized segments as soon as 3 days after surgery" and 10% of the cells 5 and 7 days after embolisation. Engraftment of transplanted hepatocytes was more efficient than in non-embolized animals. They proliferated and participated to liver regeneration, representing 10.9+0.7% of the monkey liver at 7 days and their number remained stable 15 days after transplantation. Expression o f cennexin 32 between transplanted and resident hepatocytes demonstrated that transplanted cells were integrated within hepatic plates. Liver histology analysed on biopsies performed 9 months after entholization and revealed a normal architecture and an absence of nodular regenerative hyperplasia. These results suggest that partial embolisation is a safe procedure to achieve effective hepatocyte repopulation and that this strategy of cell therapy or ex vivo gene therapy using lentiviral vectors is potentially applicable to patients.
~7i~ HUMAN HEPATIC PROGENITOR CELLS: MOLECULAR BASIS FOR THEIR ENGRAFTMENT
J.E Delgado, L. Sentilhes, S. Mainot, J. Branger, T. Lin, D. MahieuCaputo, A. Weber. INSERM EMI 00-20 and University Paris XI, H@ital
de Bicgtre, Kremlin Bicgtre, France Orthopic transplantation is currently the only treatment to cure hepatic diseases. Hepatocyte transplantation represents an attractive alternative but it is characterized by a limited engraftment of transplanted cells. Moreover, adult human hepatocytes cannot be amplified in vitro and they rapidly loose they differentiated functions. Therefore other sources of cells must be explored. In our laboratory we are exploring the therapeutic potential of hllman foetal hepatic progenitor cells. These cells have been isolated from liver foetuses at 11-13 weeks of gestation and were characterized as bipotent hepatoblasts. They were efficiently transduced by retroviral
137
vectors. After Wansplantation in athymic mice these cells engrafted more efficendy than adult hepatocytes. The aim of this study was to analyze their functionality in terms of ability to display migratory and invasive properties. Human hepatoblats and adult hepatocytes were cultured in presence or absence of different cytokines. ARN were extracted for RT-PCR analysis. Conditioned media were collected and analyzed by zymography for detection of Matrix metalloproteinases 0VIMPs), one of the principal extracellular matrix components that ensure the degradation of adhesion proteins. RT-PCR and zymography analyses showed that MMPs 2 and 9 were expressed at early stages of liver development (11-13 weeks). They were secreted in their inactive form by foetal hepatoblasts in absence of cytokines and in their activated form after 24 and 48 hours of culture in the presence of Hepatocyte Growth Factor (HGF) and Tumor Necrosis Factor alpha (TNFc0. By contrast no activated form was detected in adult hepatocytes. We also showed by immunocytochernistry that TNFc~ induced activation of NFKB transcription factor and its translocation into cell nuclei. This factor induced MMP9 but not MMP2 gene transcription. In vitro experiments using Boyden chambers showed that activation of MMP 2 and 9 was correlated with an increased cell migration and invasion through Matrigel. These results suggest that differences in engraftment ability between adult and foetal hepatocytes are due to different gene regulation involving MMPs, growth factors and adhesion molecules. MMP gene transfer should enhance the engraftment of transplanted hepatoblasts into murine models.
•2•
STRONG H U M O R A L AND CELLULAR IMMUNE R E S P O N S E S ELICITED BY IMMUNIZATION WITH DENDRITIC CELLS T R A N S D U C E D WITH ADENOVIRUS CODING FOR HCV NS3 PROTEIN
J. Encke, C. Eisenbach, C.M. Lupu, E. Evelyn. Department of Internal Medicine IV, University of Heidelberg, Heidelbe~, Germany Background Dendritic cell (DC) vaccination is a promising immunization technique to induce celhtlar and humoral immune responses against a specific antigen espedally in a therapeutic context. We have demonstrated that peptide or protein pulsed DCs induces vigorous B and T cell immune responses in mice model. The potential of DNA-based immunization as for the adenovirus priming and plasmid boosting against HCV NS3 protein in mice has also been studied in our group. However, tile protective iromunity of adenovirus transduced-DCs correlating with clearance of HCV infection in animal models has not been evaluated to date. Aims: Tile object of the study is to establish the dendritic cell-based vaccination approaches by combining two immunization techniques and fiwestigate the protective celhtlar and humoral immune response to HCV specific antigen in different animal models. Methods: Two different delivery strategies were studied by immunizing BALB/c and HLA-A2.1 transgenic mice subcutaneously, inchtding pul sing DC with HCV NS3 protein for 24 hours and transducing DC with adenovirus coding HCV NS3 antigen for 48 hours after removal and purification by magnetic sorting from bone marrow of the same species of mice. 7 days after the last immunization event antibody production against the target HCV NS3 protein and IFN-¥ secretion were evaluated by ELISA and ELISPOZ Results: We found that the dendritic cells transduced with adenovirus for HCV NS3 were capable of generating the significant humoral and T-cell based finmune response (IFN-¥) specific for human NS3 epitope in HLA-A2.1 mice. Comparatively, the vigor of these responses was much stronger than that of dendritic cells pulsed with recombinant NS3 protein. Furthermore, we demonstrated that the previous transduced DC-NS3 immunization does not influence the subsequent use for boosting. Conelusions: Our results dearly indicate that NS3-transduced dendritic cells induce vigorous B and T cell immune responses in HLA.A2.1 transgenic mice, which suggests that this approach has important implications for immune therapy against HCV infection.