Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers.
BACKGROUND
Maintenance of liver-specific functions has been shown to be stabilized by co-cultivation of hepatocytes with hepatic stellate cells (HSC). Because the limited lifespan of human HSC is a major hurdle to their use, the authors report here the amplification of human HSC populations in vitro by retroviral transfer of human telomerase reverse transcriptase (hTERT).


METHODS
Human HSC strain LI 90 cells were transduced with a retroviral vector SSR#197 expressing hTERT and green fluorescent protein (GFP) cDNA flanked by a pair of loxP. TWNT-1, one of SSR#197-immortalized HSC, was characterized. Differentiated liver functions were evaluated in an immortalized human hepatocyte NKNT-3-TWNT-1 co-culture system.


RESULTS
TWNT-1 cells showed differential functions of HSC, including uptake of acetylated low-density lipoproteins and synthesis of collagen type I and hepatocyte growth factor. Efficient excision of the retrovirally transferred hTERT and GFP cDNAs was achieved by TAT-mediated expression of the Cre recombinase and subsequent GFP-negative cell sorting. When co-cultured with TWNT-1 cells, NKNT-3 increased protein expression of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9 and urea synthesis.


CONCLUSIONS
TWNT-1 cells could be valuable in the study of integrated liver functions and contribute to the optimization of liver cell therapies and bioartificial livers.
Watanabe
Takamasa
T
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Shibata
Norikuni
N
Westerman
Karen A
KA
Okitsu
Teru
T
Allain
Jean E
JE
Sakaguchi
Masakiyo
M
Totsugawa
Toshinori
T
Maruyama
Masanobu
M
Matsumura
Toshihisa
T
Noguchi
Hirofumi
H
Yamamoto
Shinichiro
S
Hikida
Masaki
M
Ohmori
Akira
A
Reth
Michael
M
Weber
Anne
A
Tanaka
Noriaki
N
Leboulch
Philippe
P
Kobayashi
Naoya
N
D000107:Acetylation; D000199:Actins / Q000235:genetics; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D002232:Carbocyanines; D002455:Cell Division; D002461:Cell Line, Transformed; D008078:Cholesterol, LDL / Q000493:pharmacokinetics; D018920:Coculture Techniques; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004268:DNA-Binding Proteins; D004705:Endocytosis; D005260:Female; D005456:Fluorescent Dyes; D015870:Gene Expression; D015696:Gene Products, tat / Q000235:genetics; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D019426:Integrases / Q000235:genetics; D019164:Liver, Artificial; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D016615:Telomere / Q000378:metabolism; D014508:Urea / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C035005:3,3'-dihexadecylindocarbocyanine; D000199:Actins; D002232:Carbocyanines; D008078:Cholesterol, LDL; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D015696:Gene Products, tat; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D014508:Urea; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D020797:Receptor, Platelet-Derived Growth Factor beta; C045073:Cre recombinase; D019426:Integrases; D019098:Telomerase; D001616:beta-Galactosidase