Established Hepatic Stellate Cell Lines in Hepatology Research

Hepatic fibrosis is the result of an imbalance between production and degradation of extracellular matrix. It can be caused by various causes, such as chronic viral hepatitis infection, alcohol abuse, genetic disorders, drug intoxication, cholestasis, metabolic disorders, parasitic infections, and cryptogenic causes [1]. The most relevant profibrogenic cells operating as cellular key drivers in the pathogenesis of liver fibrosis are hepatic stellate cells (HSCs). In the normal liver, HSC reside in the perisinusoidal space between hepatocytes and the sinusoids and exhibit a quiescent phenotype with the main function of storing vitamin A. During chronic hepatic disease, HSC progressively lose their intracellular vitamin A content, and become activated and transdifferentiate into fibrogenic myofibroblasts (MFBs) that drive the fibrogenic process by expressing large quantities of extracellular matrix compounds such as collagens. The activation process is further marked by increased expression of α-smooth muscle actin (α-SMA) and other typical markers including collagen type I (Col I), platelet-derived growth factor receptor type β (PDGFRβ), Vimentin, cysteine- and glycine-rich protein 2 (CSRP2), Fibulin, tissue inhibitor of metalloproteinase 1 (TIMP1), secreted protein acidic and rich in cysteine (SPARC), transgelin (TAGLN), and many others [1]. Much of our current knowledge of HSC/MFB biology has been gained through primary culture studies. When HSC are cultured on the uncoated plastic, they undergo a spontaneous activation process that strongly resembles those observed in the injured liver. However, the isolation and purification of highly pure primary HSC is rather complex and the limited supply and ever-increasing demand for these cells, combined with the additional tightening of formal standards in animal welfare policy and other ethical issues, make working with these cells increasingly difficult [2]. Moreover, challenges resulting from the demand to implement the 3R principle of “Replacement”, “Reduction”, and “Refinement” originally proposed by Russell and Burch in 1959 are additional hurdles, which oppose the use of primary cells in biomedical research [3]. Therefore, scientists have generated more than 30 infinite HSC cell lines originating from mouse, rat, or human HSC (Table 1). All these cell lines have specific cellular and morphological characteristics that are similar to those of primary HSC/MFB. They grow as adherent cells, express typical HSC/MFB markers, respond and transmit profibrogenic signals, and have the capacity to take up and store vitamin A. Therefore, these established HSC lines have been already important tools in investigations studying diverse aspects of HSC/MFB biology. Particularly, several of these lines have been extensively used in studies investigating aspects of profibrogenic signaling, extracellular matrix synthesis, retinoid metabolism, cellular toxicity, proliferation control, cell adhesion, and cell migration. In particular, murine cell line GRX [10,11], rat cell line HSC-T6 [5,12], different derivatives of rat cell line CFSC [4,13,14], and human LX-2 cell line [15,16] are used in many laboratories worldwide (cf. Table 1). The phenotype of most of these continuous growing lines determined by their overall biochemical and morphological characteristics suggests that most of these lines have to be classified as “activated HSC” or “fully transdifferentiated MFB” (Figure 1).Although some of these cell lines can partly revert into the “activated” phenotype by treatment with high concentrations of retinol, fatty acids, latency-associated peptide, or by culturing on special basement membrane-like matrix components, none is capable to fully acquire back a quiescent cell state. In contrast to these immortalized cell lines, it has been shown that primary MFB isolated from human cirrhotic livers changed to an α-SMA negative, lipid droplet-containing quiescent morphology when re-cultured on a basement membrane-like substrate (i.e., Matrigel) [17]. Moreover, this treatment further reduced the expression of Col I, while the expression of matrix metalloproteinase-1 (MMP-1) was induced, which underpins the reversal to an activated phenotype. Another study previously demonstrated that embedding the cells in a three-dimensionally distributed extracellular matrix prepared of rat tail type I collagen is suitable to revert their morphology, proliferation rate and functions [18]. Each cell line has some characteristics that can be used for cell authentication. The most important one is the short tandem repeat (STR) profile that is established for some continuous growing HSC lines (e.g., GRX, Col-GFP HSC, HSC-T6, CFSC-2G, LX-2), which allows to confirm cell line identity and to identify misidentification or cross-contamination. In addition, the detection of typical HSC/MFB markers such as α-SMA, Col I, desmin, and glial fibrilliary acidic protein (GFAP) might be useful for identification. Other cell lines express the green fluorescent protein (GFP), Simian virus 40 large T-antigen (SV40T), or human telomerase reverse transcriptase (hTERT) that were stably introduced into these cells during the immortalization process. Moreover, some HSC lines have special growth properties, morphologies, or other characteristic features that are useful for cell identification. Nowadays, several of these HSC cell lines have become in particular attractive experimental tools in studies testing compounds for their anti-fibrotic activity. Palmic acid for instance induced growth arrest and decreased α-SMA and Col I expression in the rat HSC line PAV-1 [19]. Likewise, glycyrrhizic acid attenuated liver fibrosis in human cell line LX-2 and carbon tetrachloride (CCl4)-induced liver fibrosis, showing that results obtained in continuous growing HSC lines can provide valuable hints for the *in vivo* situation [20]. Anti-fibrotic effects of extracellular vesicles derived from tea leaves were also reported for both HSC line LX-2 and a mouse model of CCl4-induced liver fibrosis [21]. In the same models, a derivative of ferulic acid termed FA11 showed excellent anti-fibrotic activity as assessed by reduced HSC activation and alleviated collagen expression [22]. Moreover, the anti-parasitic drug Ivermectin was recently shown to block activation in cultured CFSC cells and in mouse repeatedly subjected to CCl4 [23]. In all these and many other studies, the efficacy of the drugs was majorly measured by their activity to suppress expression of genes associated with HSC activation and/or transdifferentiation (e.g., α-SMA, Col I), which underpins the key function of HSC in promoting fibrosis in response to liver injury. Unfortunately, most of the results of these preclinical studies were not translated to the clinic yet [24]. Nevertheless, all these *in vitro* studies conducted in established HSC lines show that anti-fibrotic acting agents are majorly effective in counteracting intracellular reactive oxygen species (ROS) formation, pro-inflammatory and pro-fibrotic signaling, and extracellular matrix generation. Therefore, these studies provide the basis to develop effective drugs for treatment of human liver disease.