Conditional immortalization of gunn rat hepatocytes: An ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer

Abstract

Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGR)-mediated endocytosis are being developed to transfer genes for the correction of bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT) deficiency. Ex vivo evaluation of these gene transfer vectors would be facilitated by a cell system that lacks bilirubin-UGT, but expresses differentiated liver functions, including ASGR. We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58). At 33 degrees C, the immortalized hepatocyte clones expressed SV40 large T antigen, synthesized DNA, and doubled in number every 2 to 3 days. At this temperature, differentiated hepatocyte markers, e.g., albumin, ASGR, and androsterone-UGT, were expressed at 5% to 10% of the levels found in primary hepatocytes maintained in culture for 24 hours. Glutathione-S-transferase Yp (GST-Yp), an oncofetal protein, was expressed in these cells at 33 degrees C, but was undetectable in primary hepatocytes. In contrast, when the cells were cultured at 39 degrees C or 37 degrees C, the large T antigen was degraded, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were observed. The expression of albumin, ASGR, and androsterone-UGT, and their corresponding mRNAs, increased to 25% to 40% of the level in primary hepatocytes, whereas GST-Yp expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red-labeled asialoorosomucoid, and binding and degradation of 125I-asialoorosomucoid. After liposome-mediated transfer of a plasmid containing the coding region of human bilirubin-UGT1, driven by the SV40 large T promoter, active human bilirubin-UGT1 was expressed in these cells. The immortalized cells were not tumorigenic after transplantation into severe combined immunodeficiency mice. These conditionally immortalized cells will be useful for ex vivo evaluation of bilirubin-UGT gene transfer vectors.

Stem Cells and Liver Regeneration

“…The only cell lines with documented therapeutic effects in animal models are those lines or primary cultures that were originally derived from primary hepatocytes (eg, Kobayashi et al, 61,62 Gupta and Chowdhury, 63 and Fox et al 64 ). However, studies of primary progenitors are of increasing importance as culture methodology and cell isolation capabilities improve.…”

Strategies for immortalization of primary hepatocytes

“…Moreover, the use of this strategy is not accompanied by the excision of the immortalization gene from the genome and thus could present a potential risk of tumorigenesis [84, 106, 123]. Nevertheless, some conditionally immortalized hepatic cell lines are based on this principle, and these cell lines can be transplanted efficiently in rat models of acute liver failure and chronic hepatic encephalopathy, usually without occurrence of tumorigenicity [88, 90, 102, 118-120, 124, 125]. However, concerns related to tumorigenicity form an important restriction to clinical appreciation of immortalized human hepatocytes [20].…”

“…Importantly, the temperature shift associated with this methodology might induce changes in cellular properties, which can complicate the interpretation of the study outcome. A more sophisticated system based on recombinase regulation is thought to offer a solution for these issues [10, 88, 118, 126, 127].…”

“…Furthermore, these vectors do not provoke harmful immune responses and allow integration of large genes [139]. Retroviral vectors, such as the Mo-MLV-derived vectors, have been frequently used to establish human and rodent hepatic cell lines [2, 9, 19, 84, 87, 102-104, 108, 110, 111, 118, 119, 124, 140, 141]. A major flaw in this system is its inability to transduce non-dividing cells, which makes it unsuitable for non-proliferating cells, including hepatocytes [139, 142].…”

Immortalized Human Hepatic Cell Lines for In Vitro Testing and Research Purposes

“…Since no other temperature-sensitive immortalizing genes have yet been identified, this method is restricted to the use of SV40 Tag (36). Importantly, the temperature shift related to this methodology might induce variations in cellular properties, which can complicate the interpretation of the study outcome (36, 66, 67). More sophisticated systems based upon recombinase or transcriptional regulation are believed to offer a better solution (68).…”