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Mark A. Fields, Laura Vickers, Hui Cai, Jie Gong, Stephen Tsang, Lucian V. Del Priore; Expression of Transcription Factors Involved in Murine Stem Cell Differentiation Induce Photoreceptor Phenotypes in Mouse Embryonic Stem Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5990. doi: https://doi.org/.
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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. Previously we have used microarray analysis to identify key transcription factors in murine retinal development. Herein we perform ex vivo gene therapy using a lentiviral delivery system to express the transcription factor NeuroD1 in ESCs to induce their differentiation into photoreceptors.
Retinal samples were collected from E11, E18, P5, P8 and older adult murine eyes and the transcription factors expressed at different developmental time points were identified by microarray analysis. DNA of key transcription factors (in particular NeuroD1) were cloned into the pCDH-EF1-MCST2A expression vector (System Biosciences, Mountain View, CA) containing a red fluorescent protein (RFP) or green fluorescent protein (GFP) reporter gene. The cloned NeuroD1-RFP lentivirus was prepared using a lentiviral packaging system (System Biosciences). Mouse embryonic stem cells ES-D3 (ATCC, Manassas, VA) were plated into a six-well culture dish containing a feeder layer of Mitomycin C-treated CF-1 mouse embryonic fibroblasts cultured at 37°C, 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and β-mercaptoethanol. Cultured ES-D3 cells were infected with the NeuroD1-RFP and isolated by fluorescent activated cell sorting (FACS) using a BD Aria II sorter (BD Biosciences, San Jose, CA). Cells expressing NeuroD1-RFP were plated on laminin-coated dishes and cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS and β-mercaptoethanol. Various antibodies and fluorescence microscopy were used to identify expression of neural/photoreceptor markers and NeuroD1 protein expression.
RFP-positive cells were confirmed by immunostaining for NeuroD1; RFP-positive cells were isolated using FACS. Transduction with NeuroD1 induced phenotypical changes in ES-D3 cells and positive immunofluorescence staining for neural markers β-tubulin III. Conversely, we did not observe similar staining from ES-D3 cells without transduction cultured on laminin plated controls.
Microarray analysis reveals high expression levels of transcriptions factors involved in mouse retinal development, such as NeuroD1. Ex vivo gene expression by a lentiviral delivery system of a single gene such as NeuroD1 into mouse ESCs direct their differentiation towards a neural but not photoreceptor phenotype.
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