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M. A. Fields, L. Vickers, H. Cai, J. Gong, S. Tsang, L. Del Priore; Ex Vivo Gene Expression of Retinal Differentiation Transcription Factors Induces a Photoreceptor Phenotype in Mouse Neural and Embryonic Stem Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2661.
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© ARVO (1962-2015); The Authors (2016-present)
Embryonic stem cell (ESC) transplantation is a promising therapeutic approach for the replacement of degenerated retinal cells in patients with age-related macular degeneration (AMD) and other retinal degenerations. Previously we have used microarray analysis to identify key transcription factors in murine retinal development. Herein we perform ex vivo gene therapy to express these transcription factors in ESC to induce their differentiation into photoreceptors.
Retinal samples were collected from E11 to adult murine eyes and developmental transcription factors expressed at different development time points were identified by microarray analysis. cDNA of identified transcription factors [namely cone-rod homeobox (Crx), orthodenticle homeobox 2 (Otx2), neural retina leucine zipper (Nrl), neurogenic differentiation 1 (NeuroD1) and neurogenic differentiation 4 (NeuroD4)], were cloned into the pCDH-EF1-MCST2A expression vector containing a red fluorescent protein (RFP) or green fluorescent protein (GFP) reporter gene. Mouse neural stem cells or mouse ESD3 cells were plated on laminin cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS, β-mercaptoethanol and retinoic acid. Purified DNA was transfected into mouse neural stem cells or mouse ESD3 cells. Various neural marker antibodies and fluorescence microscopy were used to identify expression of neural/photoreceptor markers.
Transfection with Crx and NeuroD1 induced phenotypical changes in embryonic stem cells and neural stem cells demonstrated by positive immunofluorescence staining for neural markers β-tubulin III, NF-200 and photoreceptor markers Nrl, Crx, and recoverin. Conversely, we did not observe similar effects from transfection of Otx2 or Nrl into neural stem or ESD3 cells.
Microarray analysis reveals high expression levels of transcriptions factors involved in mouse retinal development, consistent with the previous reports which suggest that the time between embryonic day 18 and postnatal day 5 appears to be critical for photoreceptor differentiation in the neural retina. Ex vivo gene expression of selected genes into mouse neural stem cells and mouse embryonic stem cells direct stem cells to differentiate into a neural/photoreceptor phenotype.
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