Reversibly immortalized hepatic progenitor cell line containing double suicide genes

Fang,Shu‑Yu; Hu,Chao‑Qun; Liu,Meng‑Nan; Tao,Li; Wang,Yi; Gong,Meng‑Jia; He,Yun; He,Tong‑Chuan; Bi,Yang

doi:10.3892/ijmm.2018.3803

Reversibly immortalized hepatic progenitor cell line containing double suicide genes

Authors:
- Shu‑Yu Fang
- Chao‑Qun Hu
- Meng‑Nan Liu
- Li Tao
- Yi Wang
- Meng‑Jia Gong
- Yun He
- Tong‑Chuan He
- Yang Bi
View Affiliations

Affiliations: Department of Pediatric Surgery, Stem Cell Biology and Therapy Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Stem Cell Therapy Engineering Technical Center, The Children's Hospital of Chongqing Medical University, Chongqing 400014, P.R. China
Published online on: August 2, 2018 https://doi.org/10.3892/ijmm.2018.3803
Pages: 1977-1986
Copyright: © Fang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

A large number of functional hepatocytes is required for bioartificial liver therapy. Simian virus 40 T‑antigen (SV40T) has been previously reported to improve the immortalized proliferation of primary hepatocytes to generate a sufficient number of cells; however, these long‑term immortalized hepatocytes may induce further malignant transformation in vivo. In the present study, the SV40T immortalization gene and two suicide genes, herpes simplex virus thymidine kinase (HSV‑tk) and cytosine deaminase (CD), were transducted into primary hepatocytes to construct a novel type of Cre/LoxP‑mediated reversible immortalized hepatocyte line. Polymerase chain reaction analysis and western blotting confirmed that the SV40T, HSV‑tk and CD genes were successfully inserted into hepatic progenitor cells and their expression was controlled by Cre/LoxP recombination. Total removal of SV40T could be achieved via the ganciclovir (GCV)/HSV‑tk suicide system. Cells maintained their biosafety in vivo with CD gene expression and 5‑fluorocytosine (5‑FC) induced cell death. Following transplantation into the carbon tetrachloride (CCl4) model group, the majority of cells had survived after 14 days post‑implantation and a number of the cells had transported into the liver parenchyma. When compared with the CCl4 model group, the transplanted cells repaired the liver biochemical index and pathological structure markedly. Thus, the present study reports a novel reversible immortalized hepatocyte with double suicide genes, which exhibited the cellular phenotype and recovery function of normal liver cells. This method maximally guaranteed the biological safety of immortalized hepatocytes for in vivo application, providing a reliable, safe and ideal cell material for the artificial liver technique.

View Figures

View References

1	Bunchorntavakul C and Reddy KR: Acute Liver Failure. Clin Liver Dis. 21:769–792. 2017. View Article : Google Scholar : PubMed/NCBI
2	Jiang W, Li J, Guo Q, Sun J, Chen C and Shen Z: Liver transplantation for hepatocellular carcinoma. Drug Discov Ther. 9:331–334. 2015. View Article : Google Scholar : PubMed/NCBI
3	Al MI, Abaalkhail FA, Bahili HA, Abdo AH, Elsiesy HA, Al MS, El Sheikh YM, Hegab BS, Kamel YM, AlGoufi TT, et al: Liver transplantation at KFSHRC: Achievement and challenges. Ann Saudi Med. 34:103–106. 2014. View Article : Google Scholar : PubMed/NCBI
4	Pan XP and Li LJ: Advances in cell sources of hepatocytes for bioartificial liver. Hepatobiliary Pancreat Dis Int. 11:594–605. 2012. View Article : Google Scholar : PubMed/NCBI
5	Nibourg GA, Chamuleau RA, van der Hoeven TV, Maas MA, Ruiter AF, Lamers WH, Oude Elferink RP, van Gulik TM and Hoekstra R: Liver progenitor cell line HepaRG differentiated in a bioartificial liver effectively supplies liver support to rats with acute liver failure. PLoS One. 7:e387782012. View Article : Google Scholar : PubMed/NCBI
6	Sakiyama R, Blau BJ and Miki T: Clinical translation of bioartificial liver support systems with human pluripotent stem cell-derived hepatic cells. World J Gastroenterol. 23:1974–1979. 2017. View Article : Google Scholar : PubMed/NCBI
7	Nguyen TH, Mai G, Villiger P, Oberholzer J, Salmon P, Morel P, Bühler L and Trono D: Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes. J Hepatol. 43:1031–1037. 2005. View Article : Google Scholar : PubMed/NCBI
8	Meng FY, Liu L, Yang FH, Li CY, Liu J and Zhou P: Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombination. World J Gastroenterol. 20:13119–13126. 2014. View Article : Google Scholar : PubMed/NCBI
9	Chendeb M, Schneider R, Davidson I and Fadloun A: Selective elimination of long Interspersed element-1 expressing tumour cells by targeted expression of the HSV-TK suicide gene. Oncotarget. 8:38239–38250. 2017. View Article : Google Scholar : PubMed/NCBI
10	Chai LP, Wang ZF, Liang WY, Chen L, Chen D, Wang AX and Zhang ZQ: In vitro and in vivo effect of 5-FC combined gene therapy with TNF-α and CD suicide gene on human laryngeal carcinoma cell line Hep-2. PLoS One. 8:e611362013. View Article : Google Scholar
11	Bi Y, He Y, Huang J, Su Y, Zhu GH, Wang Y, Qiao M, Zhang BQ, Zhang H, Wang Z, et al: Functional characteristics of reversibly immortalized hepatic progenitor cells derived from mouse embryonic liver. Cell Physiol Biochem. 34:1318–1338. 2014. View Article : Google Scholar : PubMed/NCBI
12	Westerman KA and Leboulch P: Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A. 93:8971–8976. 1996. View Article : Google Scholar : PubMed/NCBI
13	Ezzat T, Dhar DK, Malago M and Olde Damink SW: Dynamic tracking of stem cells in an acute liver failure model. World J Gastroenterol. 18:507–516. 2012. View Article : Google Scholar : PubMed/NCBI
14	Hu G, Xu JJ, Deng ZH, Feng J and Jin Y: Supernatant of bone marrow mesenchymal stromal cells induces peripheral blood mononuclear cells possessing mesenchymal features. Int J Biol Sci. 7:364–375. 2011. View Article : Google Scholar : PubMed/NCBI
15	Wei L, Fraser JL, Lu ZY, Hu X and Yu SP: Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats. Neurobiol Dis. 46:635–645. 2012. View Article : Google Scholar : PubMed/NCBI
16	Zhao LF, Pan XP and Li LJ: Key challenges to the development of extracorporeal bioartificial liver support systems. Hepatobiliary Pancreat Dis Int. 11:243–249. 2012. View Article : Google Scholar : PubMed/NCBI
17	van Wenum M, Adam AA, Hakvoort TB, Hendriks EJ, Shevchenko V, van Gulik TM, Chamuleau RA and Hoekstra R: Selecting cells for bioartificial liver devices and the importance of a 3D culture environment: A functional comparison between the HepaRG and C3A cell lines. Int J Biol Sci. 12:964–978. 2016. View Article : Google Scholar : PubMed/NCBI
18	Liu H, You S, Rong Y, Wu Y, Zhu B, Wan Z, Liu W, Mao P and Xin S: Newly established human liver cell line: A potential cell source for the bioartificial liver in the future. Hum Cell. 26:155–161. 2013. View Article : Google Scholar : PubMed/NCBI
19	Yu Y, Wang X and Nyberg SL: Potential and challenges of induced pluripotent stem cells in liver diseases treatment. J Clin Med. 3:997–1017. 2014. View Article : Google Scholar : PubMed/NCBI
20	Nibourg GA, Chamuleau RA, van Gulik TM and Hoekstra R: Proliferative human cell sources applied as biocomponent in bioartificial livers: A review. Expert Opin Biol Ther. 12:905–921. 2012. View Article : Google Scholar : PubMed/NCBI
21	van Wenum M, Chamuleau RA, van Gulik TM, Siliakus A, Seppen J and Hoekstra R: Bioartificial livers in vitro and in vivo: Tailoring biocomponents to the expanding variety of applications. Expert Opin Biol Ther. 14:1745–1760. 2014. View Article : Google Scholar : PubMed/NCBI
22	Ordovás L, Park Y and Verfaillie CM: Stem cells and liver engineering. Biotechnol Adv. 31:1094–1107. 2013. View Article : Google Scholar : PubMed/NCBI
23	Fiers W, Contreras R, Haegemann G, Rogiers R, Van de Voorde A, Van Heuverswyn H, Van Herreweghe J, Volckaert G and Ysebaert M: Complete nucleotide sequence of SV40 DNA. Nature. 273:113–120. 1978. View Article : Google Scholar : PubMed/NCBI
24	Tevethia MJ and Ozer HL: SV40-mediated immortalization. Methods Mol Biol. 165:185–199. 2001.PubMed/NCBI
25	Shay JW, Pereira-Smith OM and Wright WE: A role for both RB and p53 in the regulation of human cellular senescence. Exp Cell Res. 196:33–39. 1991. View Article : Google Scholar : PubMed/NCBI
26	Shahid JM, Iwamuro M, Sasamoto H, Kubota Y, Seita M, Kawamoto H, Nakaji S, Noguchi H, Yamamoto K and Kobayashi N: Establishment of an immortalized porcine liver cell line JSNK-1 with retroviral transduction of SV40T. Cell Transplant. 19:849–856. 2010. View Article : Google Scholar : PubMed/NCBI
27	Prives C: The replication functions of SV40 T antigen are regulated by phosphorylation. Cell. 61:735–738. 1990. View Article : Google Scholar : PubMed/NCBI
28	Matsumura T, Takesue M, Westerman KA, Okitsu T, Sakaguchi M, Fukazawa T, Totsugawa T, Noguchi H, Yamamoto S, Stolz DB, et al: Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT. Transplantation. 77:1357–1365. 2004. View Article : Google Scholar : PubMed/NCBI
29	Ramboer E, De Craene B, De Kock J, Vanhaecke T, Berx G, Rogiers V and Vinken M: Strategies for immortalization of primary hepatocytes. J Hepatol. 61:925–943. 2014. View Article : Google Scholar : PubMed/NCBI
30	Pan X, Wang Y, Yu X, Li J, Zhou N, Du W, Zhang Y, Cao H, Zhu D, Chen Y and Li L: Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes. Int J Med Sci. 12:248–255. 2015. View Article : Google Scholar : PubMed/NCBI
31	Kobayashi N, Noguchi H, Westerman KA, Watanabe T, Matsumura T, Totsugawa T, Fujiwara T, Leboulch P and Tanaka N: Cre/loxP-based reversible immortalization of human hepatocytes. Cell Transplant. 10:383–386. 2001.PubMed/NCBI
32	Kobayashi N, Noguchi H, Totsugawa T, Watanabe T, Matsumura T, Fujiwara T, Miyazaki M, Fukaya K, Namba M and Tanaka N: Insertion of a suicide gene into an immortalized human hepatocyte cell line. Cell Transplant. 10:373–376. 2001.PubMed/NCBI
33	Wang Z, Zhang J, Zeng Y, Sun S, Zhang J, Zhang B, Zhu M, Ouyang R, Ma B, Ye M, et al: Knockout of 4.1B triggers malignant transformation in SV40T-immortalized mouse embryo fibroblast cells. Mol Carcinog. 56:538–549. 2017. View Article : Google Scholar
34	Tátrai P, Szepesi Á, Matula Z, Szigeti A, Buchan G, Mádi A, Uher F and Német K: Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation. Biochem Biophys Res Commun. 422:28–35. 2012. View Article : Google Scholar : PubMed/NCBI
35	Van De Klundert FA and Bloemendal H: SV40 large T antigen-induced inhibition of terminal differentiation of primary skeletal muscle cells is associated with a block in the expression of MyoD and myogenin. Mol Biol Rep. 20:143–148. 1994–1995. View Article : Google Scholar
36	Li M, Chen Y, Bi Y, Jiang W, Luo Q, He Y, Su Y, Liu X, Cui J, Zhang W, et al: Establishment and characterization of the reversibly immortalized mouse fetal heart progenitors. Int J Med Sci. 10:1035–1046. 2013. View Article : Google Scholar : PubMed/NCBI