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I.S. Lin, R.–K. Tsai; NeuroD Inducing Rosette Formation in Cultured Pigmented Ciliary Margin Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2798.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate if pigmented ciliary margin (PCM) cells can develop neurospheres with adeno–associated virus (AAV) transducing neuroD gene.
Human PCM cells were acquired from donor and harvested. NeuroD cDNA was constructed by RT–PCR and cloned. Then AAV carrying neuroD gene was constructed and applied on PCM cells. After the infection, western blot for neuroD, morphology examination, and immunocytochemistry utilizing different kinds of retinal cell markers were conducted.
NeuroD expression can be demonstrated in PCM cells as early as on the 4th–5th day after infection with AAV. Morphology change can be observed. By one week, significant neurospheres formation is noted. Then rosette formation develops. Immunocytochemistry of the rosettes reveals positive expression of rhodopsin, green opsin, blue opsin, calbindin, CHX10–NT, GFAP, and syntaxin, etc.. Such neurospheres can last for one month at least without need of other neurotrophines.
With AAV transducing neuroD gene, PCM cells can develop neurospheres and form rosettes, which reveal various retinal neuron markers.
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