Abstract
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 re-expression 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.
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Acknowledgements
We thank Drs D Mahieu and I Dagher for help in animal surgery, L Cam for helpful discussion and Mrs B Savary for assistance in artwork. This research was supported by the EC, QLK3CT 1999-00364, and grants from Inserm ATC Biothérapies, Association Française contre les Myopathies, the Fondation pour la Recherche Médicale, the Etablissement Français des Greffes, the ‘GIS Maladies Rares’ and the Institut Pasteur. JE Allain was supported by Fondation pour la Recherche Médicale.
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Delgado, JP., Parouchev, A., Allain, JE. et al. Long-term controlled immortalization of a primate hepatic progenitor cell line after Simian virus 40 T-Antigen gene transfer. Oncogene 24, 541–551 (2005). https://doi.org/10.1038/sj.onc.1208089
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DOI: https://doi.org/10.1038/sj.onc.1208089
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