Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers

Takamasa Watanabe, Norikuni Shibata, Karen A. Westerman, Teru Okitsu, Jean E. Allain, Masakiyo Sakaguchi, Toshinori Totsugawa, Masanobu Maruyama, Toshihisa Matsumura, Hirofumi Noguchi, Shinichiro Yamamoto, Masaki Hikida, Akira Ohmori, Michael Reth, Anne Weber, Noriaki Tanaka, Philippe Leboulch, Naoya Kobayashi

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)1873-1880
Number of pages8
JournalTransplantation
Volume75
Issue number11
DOIs
Publication statusPublished - Jun 15 2003

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Artificial Liver
Hepatic Stellate Cells
Green Fluorescent Proteins
Liver
Hepatocytes
Complementary DNA
Hepatocyte Growth Factor
Cell- and Tissue-Based Therapy
Coculture Techniques
Collagen Type I
Cytochrome P-450 Enzyme System
Isoenzymes
Urea
Proteins
Maintenance
Population
human TERT protein

ASJC Scopus subject areas

  • Transplantation
  • Immunology

Cite this

Watanabe, T., Shibata, N., Westerman, K. A., Okitsu, T., Allain, J. E., Sakaguchi, M., ... Kobayashi, N. (2003). Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers. Transplantation, 75(11), 1873-1880. https://doi.org/10.1097/01.TP.0000064621.50907.A6

Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers. / Watanabe, Takamasa; Shibata, Norikuni; Westerman, Karen A.; Okitsu, Teru; Allain, Jean E.; Sakaguchi, Masakiyo; Totsugawa, Toshinori; Maruyama, Masanobu; Matsumura, Toshihisa; Noguchi, Hirofumi; Yamamoto, Shinichiro; Hikida, Masaki; Ohmori, Akira; Reth, Michael; Weber, Anne; Tanaka, Noriaki; Leboulch, Philippe; Kobayashi, Naoya.

In: Transplantation, Vol. 75, No. 11, 15.06.2003, p. 1873-1880.

Research output: Contribution to journalArticle

Watanabe, T, Shibata, N, Westerman, KA, Okitsu, T, Allain, JE, Sakaguchi, M, Totsugawa, T, Maruyama, M, Matsumura, T, Noguchi, H, Yamamoto, S, Hikida, M, Ohmori, A, Reth, M, Weber, A, Tanaka, N, Leboulch, P & Kobayashi, N 2003, 'Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers', Transplantation, vol. 75, no. 11, pp. 1873-1880. https://doi.org/10.1097/01.TP.0000064621.50907.A6
Watanabe, Takamasa ; Shibata, Norikuni ; Westerman, Karen A. ; Okitsu, Teru ; Allain, Jean E. ; Sakaguchi, Masakiyo ; Totsugawa, Toshinori ; Maruyama, Masanobu ; Matsumura, Toshihisa ; Noguchi, Hirofumi ; Yamamoto, Shinichiro ; Hikida, Masaki ; Ohmori, Akira ; Reth, Michael ; Weber, Anne ; Tanaka, Noriaki ; Leboulch, Philippe ; Kobayashi, Naoya. / Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers. In: Transplantation. 2003 ; Vol. 75, No. 11. pp. 1873-1880.
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AU - Shibata, Norikuni

AU - Westerman, Karen A.

AU - Okitsu, Teru

AU - Allain, Jean E.

AU - Sakaguchi, Masakiyo

AU - Totsugawa, Toshinori

AU - Maruyama, Masanobu

AU - Matsumura, Toshihisa

AU - Noguchi, Hirofumi

AU - Yamamoto, Shinichiro

AU - Hikida, Masaki

AU - Ohmori, Akira

AU - Reth, Michael

AU - Weber, Anne

AU - Tanaka, Noriaki

AU - Leboulch, Philippe

AU - Kobayashi, Naoya

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N2 - 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.

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