title: Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers.
abstract: 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.
authors.affiliation: Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
authors.forename: Takamasa
authors.initials: T
authors.lastname: Watanabe
authors.forename: Norikuni
authors.initials: N
authors.lastname: Shibata
authors.forename: Karen A
authors.initials: KA
authors.lastname: Westerman
authors.forename: Teru
authors.initials: T
authors.lastname: Okitsu
authors.forename: Jean E
authors.initials: JE
authors.lastname: Allain
authors.forename: Masakiyo
authors.initials: M
authors.lastname: Sakaguchi
authors.forename: Toshinori
authors.initials: T
authors.lastname: Totsugawa
authors.forename: Masanobu
authors.initials: M
authors.lastname: Maruyama
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Matsumura
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Noguchi
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Masaki
authors.initials: M
authors.lastname: Hikida
authors.forename: Akira
authors.initials: A
authors.lastname: Ohmori
authors.forename: Michael
authors.initials: M
authors.lastname: Reth
authors.forename: Anne
authors.initials: A
authors.lastname: Weber
authors.forename: Noriaki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Philippe
authors.initials: P
authors.lastname: Leboulch
authors.forename: Naoya
authors.initials: N
authors.lastname: Kobayashi
mesh_terms: 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
chemical_list: 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