Characterization of a Spontaneously Immortalized Murine Müller Glial Cell Line QMMuC-1

Augustine, Josy; Pavlou, Sofia; O’Hare, Michael; Harkin, Kevin; Stitt, Alan W.; Curtis, Tim M.; Xu, Heping; Chen, Mei

doi:10.1167/iovs.17-23293

Cited by 13 publications

(19 citation statements)

References 32 publications

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“…Immortalized rat MC lines generated via transformation, such as rMC-1 (Sarthy et al, 1998), RMC HPV-16 E6/E7 (Roque et al, 1997) and TR-MUL5 (Tomi et al, 2003), have been used extensively for the analysis of MC function. Similar to the spontaneously immortalized human MC line MIO-M1 (Limb et al, 2002) and the spontaneously immortalized murine MC line QMMuC-1 (Augustine et al, 2018) serving as an alternative model for studying human and mouse MCs, respectively, the spontaneously immortalized SIRMu-1 cells described here provide an alternative system for investigating MC function in rat cells not transformed with viral oncogenes. Although the mechanism of spontaneous immortalization remains to be ascertained, the SIRMu-1 cells appear capable of proliferating indefinitely.…”

Section: Discussionmentioning

confidence: 95%

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

Kittipassorn

Haydinger

Wood

et al. 2019

Experimental Eye Research

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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.

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Section: Discussionmentioning

confidence: 95%

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

Kittipassorn

Haydinger

Wood

et al. 2019

Experimental Eye Research

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“…The blot was performed using 15 to 20 µg of protein according to previously described methods. 23 Primary and secondary antibodies used are detailed in Table 3 . Membranes were visualized with enhanced chemiluminescence (Clarity Western ECL Blotting Substrates; Bio-Rad Laboratories) and bands detected using Syngene G-Box imaging system (Syngene, Cambridge, UK).…”

Section: Methodsmentioning

confidence: 99%

“…QMMuC-1, an immortalized murine Müller cell line 23 and the human Müller cell line Moorfields Institute of Ophthalmology-Müller 1 (MIO-M1) (courtesy of Dr. G.A. Limb, UCL Institute of Ophthalmology, London, UK) 24 were cultured in Dulbecco's Modified Eagle Media (Thermo Fisher Scientific, Waltham, MA; Catalog# 41965039) supplemented with 10% fetal calf serum.…”

Section: Cell Culture and Treatmentsmentioning

confidence: 99%

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VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

Llorián-Salvador

Barabás

Byrne

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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Müller glia are important in retinal health and disease and are a major source of retinal VEGF-A. Of the different VEGF family members, the role of VEGF-A in retinal health and disease has been studied extensively. The potential contribution of other VEGF family members to retinal pathophysiology, however, remains poorly defined. This study aimed to understand the role of VEGF-B in Müller cell pathophysiology. METHODS. The expression of different VEGFs and their receptors in human MIO-M1 and mouse QMMuC-1 Müller cell lines and primary murine Müller cells was examined by RT-PCR, ELISA, and Western blot. The effect of recombinant VEGF-B or VEGF-B neutralization on Müller cell viability and survival under normal, hypoxic, and oxidative (4-hydroxynonenal [4-HNE]) conditions was evaluated by Alamar Blue, Yo-Pro uptake, and immunocytochemistry. The expression of glial fibrillary acidic protein, aquaporin-4, inward rectifying K + channel subtype 4.1, glutamine synthetase, and transient receptor potential vanilloid 4 under different treatment conditions was examined by RT-PCR, immunocytochemistry, and Western blot. Transient receptor potential vanilloid 4 channel activity was assessed using a Fura-2-based calcium assay. RESULTS. VEGF-B was expressed in Müller cells at the highest levels compared with other members of the VEGF family. VEGF-B neutralization did not affect Müller cell viability or functionality under normal conditions, but enhanced hypoxia-or 4-HNE-induced Müller cell death and decreased inward rectifying K + channel subtype 4.1 and aquaporin-4 expression. Recombinant VEGF-B restored Müller cell glutamine synthetase expression under hypoxic conditions and protected Müller cells from 4-HNE-induced damage by normalizing transient receptor potential vanilloid 4 channel expression and activity. CONCLUSIONS. Autocrine production of VEGF-B protects Müller cells under pathologic conditions.

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“…241 To date, retinal cell lines have not been widely used to investigate purinergic signaling. However, several immortalized retinal cell lines have been described, such as those derived from ganglion cells, 242 photoreceptors, 243,244 Muller glia cells, [245][246][247][248][249][250][251] astrocytes, 252 endothelial cells, 253,254 pericytes, 255 and RPE. 256 The photoreceptor derived 661 W cell line is a mouse photoreceptor-derived cell line, obtained from a retinal tumor formed in a transgenic mouse expressing SV40 large T-antigen under the control of the human interphotoreceptor retinoid-binding protein promoter.…”

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confidence: 99%

The purinergic P2X7 receptor as a potential drug target to combat neuroinflammation in neurodegenerative diseases

Calzaferri

Ruiz-Ruiz

Diego

et al. 2020

Medicinal Research Reviews

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Neurodegenerative diseases (NDDs) represent a huge social burden, particularly in Alzheimer's disease (AD) in which all proposed treatments investigated in murine models have failed during clinical trials (CTs). Thus, novel therapeutic strategies remain crucial. Neuroinflammation is a common pathogenic feature of NDDs. As purinergic P2X7 receptors (P2X7Rs) are gatekeepers of inflammation, they could be developed as drug targets for NDDs. Herein, we review this challenging hypothesis and comment on the numerous studies that have investigated P2X7Rs, emphasizing their molecular structure and functions, as well as their role in inflammation. Then, we elaborate on research undertaken in the field of medicinal chemistry to determine potential P2X7R antagonists. Subsequently, we review the state of neuroinflammation and P2X7R expression in the brain, in animal models and patients suffering from AD, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, and retinal degeneration. Next, we summarize the in vivo studies testing the hypothesis that by

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Characterization of a Spontaneously Immortalized Murine Müller Glial Cell Line QMMuC-1

Cited by 13 publications

References 32 publications

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

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

VEGF-B Is an Autocrine Gliotrophic Factor for Müller Cells under Pathologic Conditions

The purinergic P2X7 receptor as a potential drug target to combat neuroinflammation in neurodegenerative diseases

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