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M. Mandai, H. Ikeda, K. Iseki, C. Ishigami, Z.-B. Jin, M. Takahashi; Efficient Generation of Photoreceptors From Mouse Es Cells by Negative Selection Using Lectins to Enrich Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1280.
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© ARVO (1962-2015); The Authors (2016-present)
In view of transplantation therapy for the diseases with photoreceptor degeneration, we developed a protocol to obtain Rx positive retinal progenitor cells using SFEB/DSFA method from mouse ES cells. We also reported that purification of Rx-GFP positive cells followed by a notch inhibitor treatment significantly enhanced the differentiation of photoreceptor cells. In order to establish a more generalized protocol, we aimed to establish a method to enrich Rx positive cells for the subsequent efficient generation of ES-derived photoreceptor cells
Using GFP knocked in ES cell line at Rx lucus, we screened the staining pattern of 20 lectins against Rx-GFP positive cells. Then with the selected combination of lectins, efficiency of Rx enrichment was tested. The characteristics of lectin unbound population followed by negative selection was also investigated by immunohistochemistry and gene expressions compaired to lectin bound population. The efficiency of photoreceptor differentiation in these lectin unbound population was also studied.
We could consistently double the percentage of Rx positive cells by the negative selection using the specific combination of lectins. Lectin unbound population contained significantly lower amount of oct3/4 positive cells and the gene expressions of brachiury and HNF4 by lectin unbound population was signigicantly lower than those by lectin bound population. A notch inhibitor DAPT significantly increased the crx positive photoreceptor precursor cells in lectin unbound population, and these cells differentiated to rhodopsin positive cells.
We could establish a protocol to obtain ES-derived photoreceptor cells more efficiently using a negative selection with lectins to enrich retinal progenitor cells as a part of the procedure.
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