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

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.

Long-Term Treatment of Bile Duct-Ligated Rats with Rapamycin (Sirolimus) Significantly Attenuates Liver Fibrosis: Analysis of the Underlying Mechanisms

“…The reduced expression of TGF-␤1 mRNA in rapamycin-treated animals was paralleled by only a minor decrease in TGF-␤1 plasma levels; this is due to the predominantly auto-and paracrine action of TGF-␤1. Our results are in line with a suppression of TGF-␤1 secretion in an immortalized human HSC line (Shibata et al, 2003) and the decreased TGF-␤1 mRNA levels in the liver of rapamycin-treated rats (Zhu et al, 1999). However, our results differ from the findings of other authors who described an increase in TGF-␤1 mRNA and/or plasma levels by rapamycin in the plasma of human kidney transplant recipients (Khanna et al, 2002), human lymphocytes (Khanna, 2000), transformed and nontransformed cultured cells (Law et al, 2002), rat kidney (Shihab et al, 2004), and prostate cancer cells (van der Poel, 2004).…”

Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

“…5F). HGF has been shown to have a pro-apoptotic on liver myofibroblasts [16], which we have reproduced in vitro (suppl. Fig.…”

“…Immortalized human SCs were derived as previously reported [16]. Primary rat SCs were isolated from 150-200 g female Lewis rats using a two-step step collagenase perfusion [17] followed by a Percoll density gradient separation as previously described [18].…”

Targeting liver fibrosis: Strategies for development and validation of antifibrotic therapies

“…22 Several HSC lines have been generated and characterized, such as clones of spontaneously immortalized rat HSC isolated from CCL 4 -fibrotic liver, 23 human LX-1 and LX-2 HSC immortalized by SV40 T-antigen, 24 or TWNT, a human HSC line generated by retroviral transfection of the telomerase reverse transcriptase gene. 25 Despite their limitations, these cell lines are valuable and cost-efficient tools in early drug discovery.…”