Characterization of the novel spontaneously immortalized rat Müller cell line SIRMu-1

Abstract

Müller cells (MCs) play a crucial role in the retina, and cultured MC lines are an important tool with which to study MC function. Transformed MC lines have been widely used; however, the transformation process can also lead to unwanted changes compared to the primary cells from which they were derived. To provide an alternative experimental tool, a monoclonal novel spontaneously immortalized rat Müller cell line, SIRMu-1, was derived from primary rat MCs and characterized. Immunofluorescence, western blotting and RNA-sequencing demonstrate that the SIRMu-1 cell line retains similar characteristics to cultured primary MCs in terms of expression of the MC markers cellular retinaldehyde-binding protein, glutamine synthetase, S100, vimentin and glial fibrillary acidic protein at both the mRNA and protein levels.Both the cellular morphology and overall transcriptome of the SIRMu-1 cells are more similar to primary rat MCs than the commonly used rMC-1 cells, a well-described, transformed rat MC line. Furthermore, SIRMu-1 cells proliferate rapidly, have an effectively indefinite life span and a high transfection efficiency. The expression of Y chromosome specific genes confirmed that the SIRMu-1 cells are derived from male MCs. Thus, the SIRMu-1 cell line represents a valuable experimental tool to study roles of MCs in both physiologic and pathologic states. This article is a preprint. List of abbreviations CMF-HBSS, calcium-and magnesium-free Hank's balanced salt solution cpm, count per million reads CRALBP, cellular retinaldehyde-binding protein DAPI, 4',6-diamidino-2-phenylindole dihydrochloride DEGs, differentially expressed genes FBS, fetal bovine serum FDR, false discovery rate GFAP, glial fibrillary acidic protein GO, gene ontology GS, glutamine synthetase HBSS, Hank's balanced salt solution HPV, human papillomavirus MCs, Müller cells MEM, minimal essential medium mRNA-seq, mRNA sequencing NG2, neuron glial antigen-2 PBS-HS, horse serum in phosphate-buffered saline PEI, polyethylenimine RPE, retinal pigmented epithelial RPKM, reads per kilobase per million SIRMu, spontaneously immortalized rat Müller cells SIRMu-1, spontaneously immortalized rat Müller cell line SV40, simian virus 40 This article is a preprint.

Optimization of a Method to Isolate and Culture Adult Porcine, Rats and Mice Müller Glia in Order to Study Retinal Diseases

“…Consequently, the majority of studies focusing on these cells have used permanent cell lines obtained through the immortalization of primary cells with viral oncogenes. Various Müller cell lines have been reported in the literature, including the rMC-1 Müller cell line from the adult rat retina (Sarthy et al, 1998), the first permanent Müller cell line, as well as the TR-MUL cell line (Tomi et al, 2003), the spontaneously immortalized human Müller cell line (MIO-M1) (Limb et al, 2002) and novel spontaneously immortalized rat Müller cell line, SIRMu-1 (Kittipassorn et al, 2019). Cell lines can change genetically over multiple passages, leading to phenotypic differences that leads to potentially different experimental results.…”

VEGF Upregulates EGFR Expression to Stimulate Chemotactic Behaviors in the rMC-1 Model of Müller Glia

“…Further, the published literature illustrates that both the levels of VEGF are upregulated and levels of pigment epithelium-derived factor (PDEF) are downregulated in rMC-1 cells, to correlate with the expression levels and patterns in their in vivo counterparts in diabetic retina [ 52 ]. Recent studies have also compared the gene expression of more than 9000 genes in rMC-1 cells to demonstrate that approximately 91% of the genes evaluated were not differentially expressed compared to primary MG. That is, a 91% alike genotype was recorded between rMC-1 cells and primary MG [ 53 ]. In addition, the use of cultured cell models has been widely accepted for the study of cell behaviors within controlled environments, such as microfluidic devices [ 32 , 33 ].…”

RNA sequencing data of cultured primary rat Müller cells, the spontaneously immortalized rat Müller cell line, SIRMu-1, and the SV40-transformed rat Müller cell line, rMC-1

“…Primary rat MC cultures were prepared as described previously [1]. Briefly, mixed retinal cultures, consisting of retinal neurons and glial cells, were generated from 2 to 4 day post-natal Sprague-Dawley rat pups.…”

“…The data presented here are mRNA sequencing analyses of 3 populations of cultured rat MCs: primary cells, the novel spontaneously immortalized rat MC line, SIRMu-1 [1], and the SV40-transformed rat MC line, rMC-1 [2]. RNA samples were extracted from 4 biological replicates of primary MCs, 5 biological replicates of SIRMu-1 cells, and 3 biological replicates of rMC-1 cells.…”

“…The data relating to this article are stored in NCBI Gene Expression Omnibus [3] (GEO ID: GSE123161), including: fastq files containing raw sequencing reads (one per sample, 12 in total); csv files containing numbers of reads per gene (un-normalized and unfiltered, one per sample, 12 in total); a tsv file containing TMM-normalized log2 counts per million reads for all 12 samples. Please refer to the related research article [1] for details on data processing.…”