Immortalization of hepatic progenitor cells.

Development of cell therapy-based strategies for the treatment of liver failures and of inherited metabolic diseases has become a necessity because of the limitations of orthotopic liver transplantation, including shortage of donor livers. This shortage limits also the availability for hepatocytes and these terminally differentiated cells cannot be expanded in vitro. Thus, other alternative sources of hepatocytes have to be explored such as hepatic stem cells. Foetal hepatic cells have specific intrinsic properties compared to adult hepatocytes that should overcome some of their limitations. Thus, the availability of in vitro expandable progenitor cells by means of immortalization and without inducing a transformed phenotype and disrupting their differentiation potential would facilitate studies on cell engraftment and differentiation within the hepatic parenchyma. A temporally controlled expression of the immortalizing transgene would also permit to revert the immortalized phenotype prior to cell transplantation. Since characteristics of murine stem cells cannot readily be extrapolated to their human or other primate counterparts, we have immortalized one clone of primate hepatic progenitor cells using a retroviral vector expressing SV40 Large T flanked by lox P sites. These hepatic cells were bipotent, expressing markers of both hepatocytic and biliary lineages. After transplantation into athymic mice, approximately 50% of immortalized cells engrafted, stopped proliferating after a few days and differentiated in adult hepatocytes, suggesting that the hepatic microenvironment plays an important role in such regulations. Upon infection with a retrovirus expressing the CRE recombinase, immortalized cells stopped growing and died, showing that immortalization was dependant on SV40 Large T. These studies suggest new approaches to expand hepatic progenitor cells, analyse their fate in animal models aiming at cell therapy of hepatic diseases.