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Q. Li, B. Coles, D. van der Kooy, R. McInnes; High Enrichment and Molecular Characterization of Adult Mammalian Retinal Stem Cells (RSCs) . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5413.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:We have previously shown that the pigmented portion of ciliary epithelium in the adult mammalian eye harbors mitotically quiescent RSCs, which are capable of self–renewal and differentiating into retinal neurons when assayed in vitro. We aim to purify the RSCs in order to define their ‘molecular signature’, to compare their signature to that of multipotent retinal progenitors, and to identify key molecules that may regulate the maintenance and proliferation of RSCs during development. Methods:We have taken a FACS–based approach; combining surface markers, dye efflux and an intracellular reporter gene system to enrich RSCs. Cells from ciliary margin of adult mouse eyes were dissected, dissociated and expanded in vitro. Both primary dissected and in vitro expanded cells were systematically tested for candidate surface markers, dye efflux and reporter gene expression (from nestin–GFP transgenic mouse) by flow cytometry. Stem cell characteristics (self–renewal and multipotency) of the enriched cell populations were tested by in vitro sphere and in vitro differentiation assays. RNA was isolated from both enriched and unenriched cells, as well as from E10.5 mouse embryos. Microarray analysis of gene expression was performed using the Affymetrix system. Results: Several markers for negative depletion were identified for segregating primary dissected cells, including CD31, CD34, CD36, Sca1, CD51, CD57, and CD59. Selection using a combination of all these markers represented >60% of total cells, and at least 2–fold enrichment. Moreover, a "side population" (SP), of 2–5% of the total cell population isolated from the ciliary margin, was present; this population was at least 5–fold enriched for RSCs (yielding ∼ 1 RSCs per 100 total cells). Combining surface markers and SP sorting, a >10 fold enrichment was achieved. While primary cells do not express nestin, it is activated in retinal sphere cells. By selecting GFP+ cells from in vitro expanded sphere cells derived from a nestin–GFP transgenic mouse, >10 fold enrichment was achieved (yielding at least 1 RSCs per 20–30 total cells). Conclusions:We have achieved moderately high enrichment for RSCs from both primary ciliary margin cells and in vitro expanded cells. Gene expression profiling is being performed using RNA isolated from enriched RSCs and un–enriched cells, as well as from retinal progenitors (E10.5) by microarray analysis. This work will identify molecular marker(s) that will further enrich the RSC fraction, and that may also participate in retinal development.
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