Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis

Abstract

Background: Hepatic stellate cells (HSCs) are a major fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. With increasing interest in developing antifibrotic therapies, there is a need for cell lines that preserve the in vivo phenotype of human HSCs to elucidate pathways of human hepatic fibrosis. We established and characterised two human HSC cell lines termed LX-1 and LX-2, and compared their features with those of primary human stellate cells. Methods and results: LX-1 and LX-2 were generated by either SV40 T antigen immortalisation (LX-1) or spontaneous immortalisation in low serum conditions (LX-2). Both lines express a smooth muscle actin, vimentin, and glial fibrillary acid protein, as visualised by immunocytochemistry. Similar to primary HSCs, both lines express key receptors regulating hepatic fibrosis, including platelet derived growth factor receptor b (bPDGF-R), obese receptor long form (Ob-R L ), and discoidin domain receptor 2 (DDR2), and also proteins involved in matrix remodelling; matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-2, and MT1-MMP, as determined by western analyses. LX-2 have reduced expression of TIMP-1. LX-2, but not LX-1, proliferate in response to PDGF. Both lines express mRNAs for a1(I) procollagen and HSP47. Transforming growth factor b1 stimulation increased their a1(I) procollagen mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction. LX-2, but not LX-1, cells are highly transfectable. Both lines had a retinoid phenotype typical of stellate cells. Microarray analyses showed strong similarity in gene expression between primary HSCs and either LX-1 (98.4%) or LX-2 (98.7%), with expression of multiple neuronal genes. Conclusions: LX-1 and LX-2 human HSC lines provide valuable new tools in the study of liver disease. Both lines retain key features of HSCs. Two unique advantages of LX-2 are their viability in serum free media and high transfectability.

Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis

“…However, native ChIP (NChIP), which favours the detection of stable and direct protein: DNA interactions, failed to detect MeCP2 interaction indicating that MeCP2 associates with the promoter in a relatively unstable manner or alternatively requires proteinprotein interactions retained in the XChIP assay (data not shown). To determine a role for MeCP2 as a regulator of IkBa expression, the LX-2 human HSC-derived myofibroblast cell line, 25 Figure 3C. Use of paired t-test revealed difference between control activated and 5-azadC treated rHSC to be significant with P ¼ 0.0024 for TIMP1 (represented by two stars) and P ¼ 0.029 for collagen1 (represented by one star).…”

“…Cultured HSC loose their vitamin A, enter the cell cycle and display de novo expression of a wide variety of profibrogenic and proinflammatory molecules that are characteristic of HM and which are described in detail elsewehere. 7,[12][13] Rat HSC and the LX2 human stellate cell line 25 were cultured on plastic in Dulbecco's modified Eagle's medium (DMEM), supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 16% or 10% fetal calf serum, respectively. Cell cultures were maintained at 371C at an atmosphere of 5% CO 2 .…”

Expression of mediators of purinergic signaling in human liver cell lines

“…Cell lines and culture conditions LX-2 [33] [38] (kindly provided by Dr. Doug Jefferson, Tufts University School of Medicine, Boston, MA) were cultured, as previously described [38], in a mixture (1:1, volume/volume) of DMEM and DMEM/F12 media containing 10 % FBS, 1 % antibiotics, 1 % adenine, 0.125 % insulin, 0.1 % epinephrine, 0.1 % triiodothyronine-transferrin, 0.33 % epidermal growth factor, and 0.267 % hydrocortisone. All cell cultures were maintained at 37°C with a 5.0 % CO 2 , humidified environment, and passaged every 2-3 days.…”

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.…”