Genetic and Molecular Characterization of the Immortalized Murine Hepatic Stellate Cell Line GRX

Abstract

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.

Genetic Characterization of Rat Hepatic Stellate Cell Line HSC-T6 for In Vitro Cell Line Authentication

“…Moreover, the antiviral response protein bone marrow stromal cell antigen 2, also known as tetherin, that was recently also found in murine HSC line GRX [ 8 ], was expressed in large quantities in HSC-T6 cells.…”

“…They were established from mouse, rat, and human. However, for only two of them are STR profiles available, namely the murine cell line GRX [ 8 ] and the human cell line LX-2 [ 7 ], while none of the rat lines was genetically characterized yet.…”

“…High quality RNA from HSC-T6 cells was isolated by an established procedure using CsCl density gradient centrifugation [ 8 ]. In brief, confluent cells were lysed and homogenized in a guanidine thiocyanate-containing buffer.…”

“…Reverse transcription of RNA isolated from HSC-T6 cells was conducted according to standard procedures [ 8 ]. All primers used in the study are given in Table S2 .…”

“…Similarly, alteration in the karyotype including changes in the chromosomal modal number or characteristic changes to chromosome structure (deletion, inversions, and translocation) may create typical marker chromosomes by which a cell line can be identified. To date, however, genetic details have only been reported for two continuous stellate cell lines, namely human line LX-2 and murine cell line GRX [ 7 , 8 ], while none of the established rat HSC lines has been genetically characterized yet.…”