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

• Published in: Transplantation Vol. 77; no. 3; pp. 446 - 451
• Main Authors: MARUYAMA, Masanobu, KOBAYASHI, Naoya, LEBOULCH, Philippe, TANAKA, Noriaki, WESTERMAN, Karen A, SAKAGUCHI, Masakiyo, ALLAIN, Jean E, TOTSUGAWA, Toshinori, OKITSU, Teru, FUKAZAWA, Takuya, WEBER, Anne, STOLZ, Donna B
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD Lippincott 15-02-2004
• Subjects: Animals; Antigens, Polyomavirus Transforming - genetics; Biocompatible Materials; Biological and medical sciences; Biomarkers - analysis; Cell Differentiation; Cell Line, Transformed; Cell Transplantation; Collagen; Combined surgery. Multiple transplantations; DNA-Binding Proteins; Drug Combinations; Humans; Interleukin-6 - biosynthesis; Laminin; Lipopolysaccharides - pharmacology; Liver - cytology; Liver - drug effects; Liver - metabolism; Medical sciences; Mice; Mice, SCID; Microscopy, Electron, Scanning; Proteoglycans; Simian virus 40; Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases; Telomerase - genetics; Telomerase - metabolism; Transfection; Human; Cell line; Treatment; Surgery; Established cell line; Graft; Transplantation; Cell immortalization; Cell
• ISSN: 0041-1337; 1534-6080
• DOI: 10.1097/01.TP.0000110292.73873.25

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).
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).
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.
We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.