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P.–K. Lin, J.–H. Liu, Y.–C. Chen; Morphology and Phenotype Presentation of Transdifferentiated Human Retinal Pigment Epithelial Cells by Adeno–Associated Virus Carrying Ciliary Neurotrophic Factor . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2771.
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© ARVO (1962-2015); The Authors (2016-present)
To transdifferentiate human retinal pigment epithelium (RPE) into retinal neurons with ciliary neurotrophic factor (CNTF) and demonstrate the morphology.
ARPE–19 cell line was purchased and incubated. CNTF cDNA was constructed with RT–PCR using fetal brain as template. CNTF cDNA was further cloned and integrated to adeno–associated virus (AAV) cassette with CMV promoter. The replication defective AAV–CNTF then was added to ARPE–19 culture. Western blot analysis was performed to verify proper CNTF manifestation from cultured RPE cells. Morphology and retinal markers investigation were done to identify the transdifferentiation.
After adding AAV–CNTF to the ARPE–19 culture, CNTF expression can be demonstrated on the 3rd day after the infection. By the 5th day after the infection, neurospheres formation with pigment loss is noted. Gradually, rosettes formed. With proper culture, lamina formation can be shown. The immunocytochemistry reveals positive expression of PKC, rhodopsin, calbindin, GFAP, and syntaxin, etc..
ARPE–19 cells can be transdifferentiated into retinal neurons with AAV transducing CNTF. Neurosphere, rosette, and lamina formation were noted. Outer retinal markers were also shown. The therapeutic potential of such transdifferentiation may be promising.
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