TIGHTLY REGULATED HUMAN HEPATOCYTES AS A SOURCE OF A BIOARTIFICIAL LIVER

Abstract

Purpose: One of the major limitations for establishing a novel bioartificial liver (BAL) is shortage of healthy human hepatocytes. In an effort to address this issue, we here describe a method to develop human hepatocyle cell lines with tight regulation. Methods: Human hepatocytes were transfected with a plasmid SV3neo containing the simian virus 40 T antigen (SV40T) gene followed by an introduction of the suicide herpes simplex virusthymidine kinase (TK) gene or transduced by a retroviral vector SSR#69 expressing the genes of TK and SV40T intervened by a pair of loxP recombination targets that were excisable by Cre/loxP-site specific recombination. Ganciclovir (GCV) sensitivity, the gene expression of differentiated liver functions, the efficacy of Cre/loxP recombination, and an in-vivo transplantation effect in animals were examined in SV40T-transdcued hepatocytes. Results: OUMS-29/TK, one of SV3neo-transformed hepatocytes, and NKNT-3, one of SSR#69-transduced hepatocytes, grew economically in culture with preserving adequate liver functions and were sensitive to 5 uM GCV. Intrasplenic transplantation of these cells prolonged the survival of 90% hepatectomized rats. A transient expression of Cre recombinase efficiently removed SV40T from NKNT-3 cells. Conclusion: We demonstrate here for the first time an attractive strategy for resolving the problem of donor liver shortage that currently limits the use of primary human hepatocytes for BAL therapy.