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Silke Becker, Shweta Singhal, Hari Jayaram, Lauren M. James, G. Astrid Limb; Differentiation of Human Müller Stem Cells Towards a Retinal Ganglion Cell Phenotype. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2235.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is one of the leading causes of blindness in the developed world, characterized by the loss of retinal ganglion cells (RGC). In patients with end stage glaucoma, one of the strategies to conserve or restore vision is the replacement of damaged RGC. Human Müller stem cells (hMSC) from the adult retina constitute a potential source for RGC replacement. The present study investigated whether Notch-1 inhibition, which has been shown to induce differentiation of hMSC into RGC, increases hMSC expression of functional nicotinic acetylcholine receptors (nAChR), indicative of a ganglion cell phenotype.
The hMSC line MIO-M1 was used in the present investigation. Cells were divided into three treatment groups, which received either no treatment (NM) or which were cultured on matrigel-coated surfaces with FGF-2 in the absence (MF) or presence of the Notch inhibitor DAPT (MFD) for 7 days. Total RNA was extracted, reverse transcribed and PCR was performed using specific primers for nAChR alpha subunits 1-4, 6 and 7 and beta-actin. PCR products were analysed by gel electrophoresis and quantified by densitometry. For calcium imaging MIO-M1 cells were grown on LAB-TEK chambered coverglasses, loaded with Fura Red-AM and the intracellular calcium concentration was estimated by fluorescence microscopy.
Using RT-PCR we showed that RNA expression for alpha4 and alpha6 nAChR subunits was significantly increased by Notch-1 inhibition. In addition RGC committed precursors, but not undifferentiated MIO-M1 cells, responded to stimulation with nicotine with a substantial rise in the intracellular calcium concentration, which was strongly inhibited by the alpha4 beta2 and alpha6 beta2 nicotinic receptor antagonist methyllycaconitine.
Our findings indicate that differentiation of MIO-M1 cells by Notch-1 inhibition leads to acquisition of a functional RGC phenotype. The present study provides an important insight into the neural progenicity and differentiation of MIO-M1 cells, which may prove to be an important tool for RGC transplantation to repair retinal function.
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