Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT

Masanobu Maruyama, Naoya Kobayashi, Karen A. Westerman, Masakiyo Sakaguchi, Jean E. Allain, Toshinori Totsugawa, Teru Okitsu, Takuya Fukazawa, Anne Weber, Donna B. Stolz, Philippe Leboulch, Noriaki Tanaka

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

Background. Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT). Methods. SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS). Results. One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel. Conclusion. We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

Original languageEnglish
Pages (from-to)446-451
Number of pages6
JournalTransplantation
Volume77
Issue number3
DOIs
Publication statusPublished - Feb 15 2004

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Simian virus 40
Cell Line
Lipopolysaccharides
Keratin-7
Keratin-19
Liver
Cell- and Tissue-Based Therapy
Green Fluorescent Proteins
Bile Ducts
Transfection
Interleukin-6
Spleen
Complementary DNA
Clone Cells
Gene Expression
human TERT protein
Research
matrigel
Therapeutics

ASJC Scopus subject areas

  • Transplantation
  • Immunology

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Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT. / Maruyama, Masanobu; Kobayashi, Naoya; Westerman, Karen A.; Sakaguchi, Masakiyo; Allain, Jean E.; Totsugawa, Toshinori; Okitsu, Teru; Fukazawa, Takuya; Weber, Anne; Stolz, Donna B.; Leboulch, Philippe; Tanaka, Noriaki.

In: Transplantation, Vol. 77, No. 3, 15.02.2004, p. 446-451.

Research output: Contribution to journalArticle

Maruyama, M, Kobayashi, N, Westerman, KA, Sakaguchi, M, Allain, JE, Totsugawa, T, Okitsu, T, Fukazawa, T, Weber, A, Stolz, DB, Leboulch, P & Tanaka, N 2004, 'Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT', Transplantation, vol. 77, no. 3, pp. 446-451. https://doi.org/10.1097/01.TP.0000110292.73873.25
Maruyama, Masanobu ; Kobayashi, Naoya ; Westerman, Karen A. ; Sakaguchi, Masakiyo ; Allain, Jean E. ; Totsugawa, Toshinori ; Okitsu, Teru ; Fukazawa, Takuya ; Weber, Anne ; Stolz, Donna B. ; Leboulch, Philippe ; Tanaka, Noriaki. / Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT. In: Transplantation. 2004 ; Vol. 77, No. 3. pp. 446-451.
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AU - Allain, Jean E.

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N2 - Background. Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT). Methods. SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS). Results. One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel. Conclusion. We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

AB - Background. Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT). Methods. SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS). Results. One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel. Conclusion. We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

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