Long-term controlled immortalization of a primate hepatic progenitor cell line after Simian virus 40 T-Antigen gene transfer

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 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.

Development of a conditionally immortalized human pancreatic β cell line

“…Similar approaches were used to produce reversely immortalized human liver sinusoidal endothelial cells and β cells (23,24) and primate hepatic progenitor cell lines (25). However, the first β cell line developed with this approach rapidly lost insulin expression (23).…”

“…The second line, NAKT-15, reported in 2005 (24), was not further studied or distributed despite its promising β cell-specific properties. In addition, upon Cre-mediated SV40 LT deletion in primate hepatic progenitor cell lines, cells exhibited picnotic or fragmented nuclei characteristics of apoptosis and died, which was suggested to be due to p53 accumulation (25). Here, we used excisable lentiviral vectors to generate a stable conditionally immortalized functional human β cell line.…”

Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine

“…Nevertheless, the expression of the transfected large T antigen induced considerable alterations of the karyotype and the mean chromosome number was increased to 60 chromosomes, an effect already described in a variety of SV40 large T immortalized cells. 24,[40][41][42][43] As these alterations might have induced a transformation of the cells, a soft agar cloning assay was performed to check for the ability of the cells to grow anchorage independently. Only a very small percentage (0.65%) of the cells was able to form colonies in soft agar.…”

Cadaveric Hepatocytes Repopulate Diseased Livers: Life After Death

“…3 Immortalization strategies have therefore been applied to primary hepatocytes in an attempt to overcome this problem but have remained unsuccessful. 4 In addition, such an approach might create added complexity to direct utility in the clinic because genetic modification to achieve a proliferative state may result in a failure of contact inhibition and self-limiting proliferation in vivo. Because of the intrinsic problems with primary human hepatocytes several groups have focused on the role that stem cell populations might have in the production of large amounts of human hepatocytes for therapeutic purposes.…”