Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

Abstract

Background: Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Methods: Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. Results: After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5-7 days. The remaining transfected hepatocytes persisted for 2-4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. Conclusion: We established a conditional human fetal hepatocytes cell line with mesenchymal characteristics. Thus immortalization of human fetal hepatocytes cell line by telomerase biology offers a great challenge to examine basic biological mechanisms which are directly related to human and best cell source having unlimited population doubling for bioartificial support without any risk of replicative senescence and pathogenic risks. ( J CLIN EXP HEPATOL 2014;4:191-201)

Immortalized common marmoset (Callithrix jacchus) hepatic progenitor cells possess bipotentiality in vitro and in vivo

“…Strong fluorescence signals were detected in mixed liver cells 1 week after three successive infections and EGFP-positive cell clones were sorted with fluorescence-activated cell sorting (FACS) and seeded in six-well plates with further dilution. Notably, EGFP signals became weaker with passaging ( Supplementary Figure S1a), consistent with previous reports 16 . Six clones derived from a mixed population formed colonies and proliferated rapidly after 30 passages, though most cells changed their morphologies during infection and became more like elongated spindle cells (Supplementary Figure S1b).…”

“…Notably, this approach also eliminates the possibility of contaminating cells types such as ESCs or iPSCs, which can result in the possibility of teratoma formation. However, the drawback of this virus infectionbased immortalization method is the influence on cellular morphologies as reported earlier 16 , though spontaneous EMT acquisition during culture can also contribute to the morphological changes. Despite the acquisition of mesenchymal features of MHPCs during immortalization, MHPCs maintain the properties and potency of hepatic progenitor cells, including gene expression profiles and functionality, including epithelial cell surface polarization.…”

Primary-like Human Hepatocytes Genetically Engineered to Obtain Proliferation Competence as a Capable Application for Energy Metabolism Experiments in In Vitro Oncologic Liver Models

“…We strongly suggest the HepaFH3 cell line as a promising tool for analyzing HCC development mechanisms. Immortalized hepatic cell lines have been generated in previous studies from human hepatocytes, e.g., by the transduction of oncogenes including TERT, cyclin D1 or simian virus 40 large t (SV40 lt) antigen, and are a promising tool for hepatology studies [ 57 , 58 ]. Furthermore, hepatic cell lines with hepatoma cell origins, especially the HepaRG cell line, are a widely used tool in hepatology studies [ 59 ].…”

Immortalization of porcine hepatocytes with a α‐1,3‐galactosyltransferase knockout background

“…These immortalized hepatocytes maintain the functions and metabolic characteristics of primary hepatocytes without tumorigenicity. Studies have also proven that these immortalized hepatocytes could be used for the treatment of acute liver failure, clinical research on bioartificial liver, or studies of hepatocyte xenotransplantation …”