Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication

Abstract

Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).

Genetic Characterization of Rat Hepatic Stellate Cell Line PAV-1

“…To establish a characteristic STR profile of PAV-1 cells, we genotyped 31 established polymorphic markers that we previously used to profile two other rat HSC lines, namely HSC-T6 [ 19 ] and CFSC-2G [ 20 ]. The sum of generated STR profiles for PAV-1 cells revealed a unique pattern of allele sizes for the 31 markers that was different from those of CFSC-2G and HSC-T6 that we have recently established ( Table 6 , Figure S5 ).…”

“…Detection of chromosome rearrangements in PAV-1 cells was accomplished using a commercially available rat SKY probe (Applied Spectral Imaging Inc., Carlsbad, CA, USA) and published protocols [ 19 , 20 , 27 ]. For silver (Ag)-staining of the nucleolus organizer regions (NORs), slides with metaphase chromosomes were stained with AgNO 3 according to the method of Goodpasture and Bloom [ 28 ].…”

“…In previous studies, we identified several genetic characteristics and STR profiles for several HSC lines, including human LX-2 [ 18 ], rat cell lines HSC-T6 and CFSC-2G [ 19 , 20 ], and mouse HSC cell lines GRX [ 21 ] and Col-GFP HSC [ 22 ]. Now we have extended our studies and performed karyotype analysis and multicolor spectral karyotyping (SKY) in the rat HSC line PAV-1, which was isolated by amplification of a colony obtained via the spontaneous immortalization of primary HSC isolated from an over 8-month-old male Wistar rat [ 23 ].…”

Established Hepatic Stellate Cell Lines in Hepatology Research

“…Certainly, these suggestions are only rough indications that need to be independently confirmed before conducting extensive studies. Expression data for HSC-T6 and CFSC-2G were taken from previous studies [4,5], while data for LX-2 and PAV-1 was taken from unpublished data.…”

“…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).…”