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Jorn Lakowski, Yating Han, Anai A. Gonzalez-Cordero, Emma West, Robin R. Ali, Rachael A. Pearson, Jane C. Sowden; Towards Photoreceptor Replacement Therapy: Identifying The Cell Surface Marker Profile Of Photoreceptor Precursors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2695.
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© ARVO (1962-2015); The Authors (2016-present)
Loss of photoreceptors due to retinal degeneration is a major cause of blindness in the developed world. While no effective treatment is currently available, cell replacement therapy, using in vitro generated photoreceptor precursor cells, may be a feasible future treatment. Safety and efficacy of this approach depends on stringent selection and purification of optimal donor cells from stem cell cultures. As genetic labeling of donor cells is not desirable for therapy, we developed cell selection strategies using cell surface antigens. Previously, we demonstrated that CD73/CD24 double positive rod precursors derived from the developing retina integrate efficiently into the normal and diseased mouse eye after sub-retinal transplantation. Here, we developed a cell selection protocol for the isolation of correctly staged rod precursors from differentiated mouse embryonic stem cell (mESC) cultures. In addition, we assessed the deployment of the same cell surface markers for human cells.
The developmental expression profiles of 34 highly expressed cell surface antigens identified by microarray analysis of Nrl.GFP expressing rod precursors were analysed using semi-quantitative PCR and flow cytometry. Markers for positive and negative selection of post-mitotic, yet immature, rod precursors were tested on postnatal mouse retinal cells and differentiated mESCs. Cell surface marker expression in human retinal cells was established using immunohistochemistry and flow-cytometry.
A panel of 8 cell surface markers was identified that in combination facilitates the isolation of post-mitotic rod precursor from differentiated mESCs. CD73, CD24, CD133 and CD47 were transiently co-expressed during retinal development and were useful for the positive selection of rod precursors from the developing retina and differentiated mESC. Removal of retinal progenitors and other retinal cell types was accomplished by negative selection via CD15, CD117, CD44 and CD138 expression.
Correctly staged photoreceptor precursors for the purpose of retinal therapy can be isolated from complex mixtures of cells, such as differentiated mESC, via cell surface marker expression. In addition to positive selection of donor cells, removal of undesirable or mitotically active retinal cells can be accomplished through cell surface marker-mediated cell depletion.
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