Transplantation of hepatocytes is a promising alternative to liver transplantation for the treatment of severe liver diseases. However, this approach is hampered by the shortage of donor organs and intrinsic limitations of adult hepatocytes. To investigate whether most of the hurdles faced with adult hepatocytes could be surmounted by the use of human fetal hepatoblasts, we have developed a method to isolate, transduce, and cryopreserve hepatoblasts from human livers at an early stage of development (11-13 weeks of gestation). Cells were characterized in vitro for expression of specific markers, and in vivo for their proliferation and differentiation potential after transplantation into athymic mice. Most of the cells (80-90%) harbored a bipotent phenotype, expressing cytokeratins 8/18, albumin, and CK19. They proliferated spontaneously in culture and were efficiently transduced by a beta-galactosidase-expressing retrovirus (90%). After transplantation, cryopreserved cells engrafted into the liver of athymic mice and proliferated, resulting in up to 10% repopulation. Engrafted cells expressed markers of differentiated adult hepatocytes including albumin, alpha1-antitrypsin, cytochrome P450 3A4, and alpha-glutathione-S-transferase. When retrovirally transduced before transplantation they expressed the transgene in vivo. In summary, early human fetal hepatoblasts engraft, proliferate, and mature in athymic mouse liver, without conditioning the donor.

Liver regeneration after partial hepatectomy results primarily from the simple division of mature hepatocytes. However, during embryonic and fetal development or in circumstances under which postnatal hepatocytes are injured, organ regeneration is believed to occur from a compartment of epithelial liver stem or progenitor cells with biliary and hepatocytic bipotentiality. The ability to identify, isolate, and transplant epithelial liver stem cells from fetal liver would greatly facilitate the treatment of hepatic diseases currently requiring orthotopic liver transplantation. Here we report the identification and immortalization by retrovirus-mediated transfer of the simian virus 40 large T antigen gene of primate fetal epithelial liver cells with a dual hepatocytic biliary phenotype. These cells grow indefinitely in vitro and express the liver epithelial cell markers cytokeratins 8͞18, the hepatocyte-specific markers albumin and ␣-fetoprotein, and the biliary-specific markers cytokeratins 7 and 19. Bipotentiality of gene expression was confirmed by clonal analysis initiated from single cells. Endogenous telomerase also is expressed constitutively. After orthotopic transplantation via the portal vein, Ϸ50% of the injected cells integrated into the liver parenchyma of athymic mice without tumorigenicity. Three weeks after transplantation, cells having seeded in the liver parenchyma expressed both albumin and ␣-fetoprotein but had lost expression of cytokeratin 19. These results provide strong evidence for the existence of a bipotent epithelial liver stem cell in nonhuman primates. This unlimited source of donor cells also should enable the establishment of a model of allogenic liver cell transplantation in a large animal closely related to humans and shed light on important questions related to liver organogenesis and differentiation.

Hepatoblasts are bipotent progenitors of both hepatocytes and cholangiocytes. The lack of stable in vitro culture systems for such cells makes it necessary to generate liver progenitor cell lines by means of immortalization. In this study, we describe the long-term behaviour of a clone of simian foetal hepatic progenitor cells immortalized by Simian virus 40 (SV40) large T-antigen (T-Ag) flanked by loxP sites. Immortalization was associated with the reexpression of telomerase activity, which decreased at late passages (population doubling 120) after more than a year in culture. This decrease was concomitant to telomere shortening and karyotypic instability. However, the chromosomes carrying the p53 gene remained intact and long-term immortalized progenitor cells maintained contact inhibition and proliferative properties. They also displayed the features of a normal bipotent phenotype. We constructed a retroviral vector expressing an inducible Cre recombinase and transferred it into the immortalized progenitors. Activation of the Cre recombinase by 4-hydroxy-tamoxifen induced SV40 T-Ag excision, leading to the death of cells expressing Cre recombinase. Immortalized progenitors at late passages stopped growing and eventually disappeared after transplantation into the livers of immunocompromised mice. These cells provide a novel model to study hepatic differentiation and carcinogenesis.

Objective: Because intrauterine transplantation of fetal hepatocytes could become an effective approach for treating severe genetic disorders of the liver, the objective of this study was to demonstrate the feasibility of in utero allotransplantation of fetal hepatocytes in a nonhuman primate model using direct intraparenchymal administration of donor cells. Methods: Fetal primary hepatocytes were isolated from 3 fetal primates (Macaca mulatta) at 89–120 days of gestation, and cryopreserved. When a recipient was available, the cells were thawed and transduced by a β-galactosidase-expressing retrovirus (3 cases) or labelled with a fluorescent dye (4 cases). Hepatocytes were infused directly into the fetal liver under surgical visual control. Engraftment was assessed by surgical liver biopsies taken 8–60 days following transplantation. Results: Six recipients survived until liver biopsy, and 1 died during the surgical procedure. There was no evidence of engraftment in the 3 fetuses that received genetically marked hepatocytes. All 3 monkeys who received 20–25 × 10⁶ hepatocytes from an 89-day-old donor labelled with fluorescent dye had positive liver biopsies 8–11 days following intrauterine transplantation. Conclusions: In utero allotransplantation of fetal hepatocytes is feasible in the nonhuman primate, and direct intraparenchymal administration enables short-term detection of persisting donor hepatocytes.

Reçu le 21 décembre 2000 RÉSUMÉ Nous développons des approches précliniques chez le primate comme modèle de thérapie cellulaire pour le traitement de maladies métaboliques hépatiques. Chez le foetus, les tissus, dont le foie, sont en expan sion, ce qui devrait faciliter la prise de greffe des hépatocytes, et le système immunitaire devient plei nement mature seulement après la naissance. Nous avons défini les conditions d'isolement d'hépatocytes foetaux de macaques à la fin du 2èm e trimestre de ges tation (90-100 jours), ainsi que les conditions de cul ture, de cryopreservation et de transduction rétrovi rale. Nous avons également évalué deux voies d'administration différentes des hépatocytes : la veine ombilicale, qui s'est révélée délétère pour les foetus, et l'injection intraparenchymateuse qui a été parfaite ment tolérée par les animaux. Cette dernière a per mis de mettre en évidence la présence d'hépatocytes allogéniques, c'est-à-dire un microchimérisme 9 jours après transplantation. Nous avons également immor talisé des hépatocytes simiens foetaux en utilisant un vecteur retroviral exprimant grand T de SV40 flan qué de sites lox P. Une lignée cellulaire est maintenant établie depuis 2 ans : elle n'est pas tumorigène après injection sous-cutanée à des souris nude et expriment des marqueurs caractéristiques d'hépatoblastes bipotents, précurseurs des hépatocytes et des cellules des voies biliaires. Après transplantation orthotopique via la veine porte chez des souris nude, ces cellules exprimaient l'albumine jusqu'au moment du sacrifice des animaux (17 jours). Les prochaines étapes seront la mise au point des conditions de transplantation d'hépatocytes transduits primaires et/ou immortalisés chez des foetus plus jeunes (60 jours de gestation) et chez le macaque nouveau-né. SUMMARY In utero allotransplantation and immortalization of non-human primate foetal hépatocytesWe are developing cell therapy approaches on non human primates as a preclinical model for the treat ment 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 intraparenchymous injection which was well tolerated by the animals. Administration of hepatocytes into the hepatic parenchyma resulted in microchimerism and

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