Research Report
Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 28, 2014; 20(36): 13119-13126
Published online Sep 28, 2014. doi: 10.3748/wjg.v20.i36.13119
Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombination
Fan-Ying Meng, Li Liu, Feng-Hui Yang, Chun-You Li, Jun Liu, Ping Zhou
Fan-Ying Meng, Feng-Hui Yang, Chun-You Li, Jun Liu, Division of Hepato-Biliary-Pancreatic Surgery, Institute of Organ Transplantation, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China
Li Liu, Ping Zhou, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Author contributions: Meng FY and Liu L contributed equally to this work; Zhou P and Liu J proposed the study and contributed equally to this work; Meng FY, Liu L, Yang FH and Li CY performed the research; Meng FY and Liu L collected and analyzed the data; Meng FY and Liu L wrote the manuscript; all authors contributed to the design of the study and interpretation of data, and approved the final version to be published.
Supported by Major Scientific and Technological Project of Shandong Province, No. 201221019; and Cisco Clinical Oncology Research Fund and Bayer Schering Cancer Research Fund, No. Y-B2012-011
Correspondence to: Jun Liu, MD, PhD, Division of Hepato-Biliary-Pancreatic Surgery, Institute of Organ Transplantation, Shandong Provincial Hospital Affiliated to Shandong University, Jingwuwei 7 Road 324, Jinan 250021, Shandong Province, China. 963cat@sohu.com
Telephone: +86-531-68776931 Fax: +86-531-68776931
Received: January 26, 2014
Revised: March 30, 2014
Accepted: June 26, 2014
Published online: September 28, 2014
Abstract

AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.

METHODS: We successfully isolated primary human hepatocytes from surgically resected liver tissue taken from a patient with liver hemangiomas. The freshly isolated cells were then immortalized with retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40T) and hygromycin-resistance genes flanked by paired loxP recombination targets.

RESULTS: The freshly isolated hepatocytes with high viability (85%) were successfully immortalized using retroviral gene transfer of SV40T. SV40T in the immortalized cells was then excised by Cre/loxP site-specific recombination. This cell population exhibited the characteristics of differentiated hepatocytes.

CONCLUSION: We successfully established reversibly immortalized human hepatocytes, which will provide an unlimited supply of cells for practical applications.

Keywords: Hepatocyte, Primary human hepatocytes, Reversible immortalization, Hepatocyte isolation, SV40T

Core tip: It is meaningful to establish reversibly immortalized human hepatocytes which can be economically grown in tissue culture. Toward this goal, we successfully established a method for the reversible immortalization of human hepatocytes using Cre/loxP site-specific recombination, which may offer a good and safe source of hepatocytes for the bioartificial liver (BAL) system in the near future. If a sufficient number of human hepatocytes can be used, these extracorporeal devices will serve as successful “bridge-to-transplant”therapies. With the progress made in bioreactor development, the next-generation BAL system could reach the level of artificial kidney and save more patients.