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J. A. Cooke, L. A. Crompton, J. B. Uney, A. D. Dick, M. A. Caldwell; The Generation of Photoreceptors from Embryonic Stem Cells: Towards a Transplant Model. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5141.
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Our aim is to differentiate photoreceptor cells from embryonic stem (ES) cells. We have differentiated mouse ES cells into neural progenitor cells (NPCs) using an in vitro selection method. Following this step we have further differentiated the ES cell-derived NPCs into photoreceptors by over-expressing the transcription factor, Cone-rod homeobox (Crx) using a lentiviral vector.
Mouse ES cells were cultured as embryoid bodies for four days followed by an eight day Nestin selection protocol. The Nestin-positive ES-derived NPCs were then cultured for a further 6-10 days in the presence of FGF-2 to expand the population of cells. The full-length mouse Crx gene was cloned into a lentivirus containing an internal ribosomal entry site and green fluorescent protein as a reporter gene. The ES-derived NPCs were transduced with the Crx-containing lentivirus to further differentiate the cells into photoreceptors.
Mouse ES cells were successfully differentiated into a population of neural progenitor cells using selection media. These cells were characterised by RT-PCR and immunocytochemistry and were shown to express neural progenitor markers including Nestin, Sox2, and Pax6. The ES-NPCs were then further differentiated into putative photoreceptor cells by over-expressing the transrciption factor, Crx.
We have developed a reliable method to produce neural progenitor cells from mouse ES cells. These cells are expandable in the presence of FGF-2 and can be further manipulated into a neural retinal fate. This study provides us with a population of cells that can be used as a cell replacement therapy in a model of retina degeneration.
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