Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.
OBJECTIVE
Hepatocyte transplantation and bioartificial liver treatment are attractive alternatives to liver transplantation. The availability of well-characterized human hepatocyte lines facilitates such cell therapies.


METHODS
Human hepatocytes were immortalized with a retroviral vector SSR#197 expressing catalytic subunit of human telomerase reverse transcriptase (hTERT) and enhanced green fluorescent protein (EGFP) cDNAs flanked by a pair of loxP recombination targets. Then, Tamoxifen-dependent Cre recombinase was expressed in SSR#197-immortalized hepatocytes. Cre/LoxP recombination was performed in the established cells by simple exposure to 500 nM Tamoxifen for a week. Then, the reverted population of the cells was recovered by EGFP-negative cell sorting and characterized in vitro and in vivo using a pig model of acute liver failure (ALF) induced by d-galactosamine (0.5 g/kg) injection.


RESULTS
A human hepatocyte cell line 16T-3 was established. Reverted 16-T3 cells showed the increased expression of hepatic markers in association with enhanced levels of transcriptional factors. Compared to normal human hepatocytes, albumin production and lidocaine-metabolizing activities of reverted 16-T3 cells were 0.32 and 0.50-fold, respectively. Transplantation of reverted 16T-3 cells significantly prolonged the survival of ALF pigs.


CONCLUSIONS
Here we demonstrate the usefulness of Cre/LoxP -mediated reversible immortalization of human hepatocytes with Tamoxifen-mediated self-recombination.
Totsugawa
Toshinori
T
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.
Yong
Chen
C
Rivas-Carrillo
Jorge David
JD
Soto-Gutierrez
Alejandro
A
Navarro-Alvarez
Nalú
N
Noguchi
Hirofumi
H
Okitsu
Teru
T
Westerman
Karen A
KA
Kohara
Michinori
M
Reth
Michael
M
Tanaka
Noriaki
N
Leboulch
Philippe
P
Kobayashi
Naoya
N
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002461:Cell Line, Transformed / Q000737:chemistry / Q000166:cytology / Q000637:transplantation*; D022781:Hepatocytes / Q000737:chemistry / Q000187:drug effects / Q000637:transplantation*; D006801:Humans; D019426:Integrases / Q000235:genetics; D017093:Liver Failure / Q000473:pathology / Q000601:surgery*; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D034421:Sus scrofa; D013629:Tamoxifen / Q000494:pharmacology; D016896:Treatment Outcome
D015415:Biomarkers; D013629:Tamoxifen; C045073:Cre recombinase; D019426:Integrases