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Nicolas Cuenca, Violeta Gomez-Vicente, Ana Flores, Pedro Lax, Celia Murciano, Alberto Yañez, Maria Luisa Gil, Daniel Gonzalbo, Victoria Maneu; Characterization of a new retinal cell line (MU-PH1) expressing stem cell markers from adult mouse. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2222.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize a novel cell line, derived from retinal progenitors, which could serve as an in vitro model for the study of photoreceptor degenerative mechanisms and to evaluate the efficacy of neuroprotective compounds.
A Müller-derived cell line (MU-PH1) was isolated from adult C57BL/6 mouse retina. RT-PCR, immunoblot and immunofluorescence analyses were performed to characterize these cells. Calcium imaging allowed the determination of light-responsiveness. To establish a model for the screening of potential neuroprotective drugs, apoptosis was induced with cytotoxic agents and measured using XTT and crystal violet viability assays.
We report the isolation and characterization of a novel, spontaneously immortalized, Müller-derived cell line (MU-PH1). These cells demonstrated a glial-like morphology and were able to form neurospheres under the appropriate culture conditions. RT-PCR, immunoblot and immunofluorescence techniques showed that MU-PH1 cells expressed the Müller cell markers vimentin, S-100 and glutamine synthetase, and several neural and stem cell markers (nestin, Abcg2, alpha-tubulin, beta-III-tubulin and Ascl1). The purity of the culture was confirmed, as the expression of other non-neuronal populations’ markers such as CRALBP (retinal pigment epithelial cells), GFAP (astrocytes) or CD31 (endothelium) was undetectable by RT-PCR. The presence of CD11b immunopositive cells (microglia) was also excluded by immunomagnetic purification. Interestingly, MU-PH1 cells expressed the photoreceptor markers rhodopsin, recoverin, transducin, melanopsin and blue and red/green opsins. Stimulation with 480 nm light evoked slow and fast transient calcium responses in most of the cells tested. MU-PH1s were sensitive to oxidative stress induced by sodium nitroprusside or oligomycin/rotenone and cell death was prevented by anti-apoptotic and antioxidant compounds such as tauroursodeoxycholate and N-acetylcysteine.
Availability of this mouse cell line could be of great interest as a photoreceptor model and will facilitate studies aimed to a better understanding of retinal Müller cell biology, as well as a convenient model for the screening and development of new therapeutic drugs.
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