[Allotransplantation in utero and immortalization of primate fetal hepatocytes]

We are developing cell therapy approaches on non-human primates as a preclinical model for the treatment of hepatic metabolic diseases. In foetuses, the tissues, including liver, are in expansion, which should facilitate hepatocytes engraftment, and the immune system becomes fully mature only after birth. We have set out conditions for isolation of fetal hepatocytes from macaca mulatta at the end of the 2nd trimester of gestation (90-100 days), their cryopreservation and retroviral transduction. Two different routes of administration of hepatocytes were evaluated: the umbilical vein which was deleterious for the foetuses, and the intraparenchymatous injection which was well tolerated by the animals. Administration of hepatocytes into the hepatic parenchyma resulted in microchimerism and allogenic cells were visualized 9 days after transplantation. Another approach has been to immortalize simian foetal hepatocytes using a retroviral vector expressing SV40 Large T flanked by lox sites. A cell line has been established for 2 years, which is not tumorigenic when injected subcutaneously into nude mice and display characteristics of bipotent hepatoblasts, precursors of hepatocytes and biliary cells. After orthotopic transplantation into nude mice via the portal vein, these cells expressed albumin until the sacrifice of the animals (17 days). The next steps will be to define conditions for transplantation of retrovirally transduced fetal primary and/or immortalized hepatocytes into young foetuses (60 days of gestation) and post-natally.