Establishment of an Immortalized Human Hepatic Stellate Cell Line to Develop Antifibrotic Therapies

Author:

Shibata Norikuni1,Watanabe Takamasa2,Okitsu Teru2,Sakaguchi Masakiyo2,Takesue Michihiko1,Kunieda Takemi1,Omoto Kenji1,Yamamoto Shinichiro1,Tanaka Noriaki2,Kobayashi Naoya2

Affiliation:

1. Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0114 Japan

2. Department of Surgery and ‡Department of Cell Biology, Okayama University Graduate School of Medicine and Dentistry, 2–5-1 Shikata-cho, Okayama 700-8558, Japan

Abstract

Because human hepatic stellate cells (HSCs) perform a crucial role in the progress of hepatic fibrosis, it is of great value to establish an immortalized human cell line that exhibits HSC characteristics and grows well in tissue cultures for the development of antifibrotic therapies. Thus, we engineered an immortalized human hepatic stellate cell (HSC) line TWNT-4 by retrovirally inducing human telomerase reverse transcriptase (hTERT) into LI 90 cells established from a human liver mesenchymal tumor. Parental LI 90 entered replicative senescence, whereas TWNT-4 showed telomerase activity and proliferated for more than population doubling level (PDL) 200 without any crisis. TWNT-4 expressed platelet-derived growth factor-β receptor (PDGF-βR), α-smooth muscle actin (α-SMA), and type I collagen (α1) and was considered to be an activated form of HSCs. Treatment of TWNT-4 cells with either 100 U/ml of IFN-γ or 1 ng/ml of rapamycin (Rapa) for 14 days led to lower expression of type I collagen (α1) at RNA and protein levels. Exposure of TWNT-4 cells to both of IFN-γ (10 U/ml) and Rapa (0.1 ng/ml) for 14 days effectively decreased the expression of type I collagen (α1), PDGF-βR, and α-SMA expression and suppressed TGF-β1 secretion of TWNT-4 cells. We successfully induced apoptosis by transducing TNF-related apoptosis-inducing ligand (TRAIL) into TWNT-4 cells using adenovirus vectors Ad/GT-TRAIL and Ad/PGK-GV-17. These findings suggested that immortalized activated HSC line TWNT-4 would be a useful means to develop antifibrotic therapies.

Publisher

SAGE Publications

Subject

Transplantation,Cell Biology,Biomedical Engineering

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