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Brian G. Ballios, Laura Clarke, Brenda L. Coles, Molly S. Shoichet, Derek van der Kooy; The Adult Retinal Stem Cell Is A Rare Cell In The Ciliary Epithelium Whose Progeny Can Differentiate Into Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5892.
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© ARVO (1962-2015); The Authors (2016-present)
The clonal derivation of adult retinal stem cells (RSCs) has been reported in mouse (Tropepe et al. Science, 2000) and human (Coles et al. PNAS, 2004). Recent arguments in the literature against the "stemness of the RSC" include: (1) the equal ability of all ciliary epithelial (CE) cells to undergo in vitro clonal expansion and transdifferentiation to produce mature retinal cell types, rather than a rare pigmented stem cell population (Cicero et al. PNAS, 2009) (2) poor retinal differentiation, with unconvincing photoreceptor differentiation (Cicero et al. PNAS, 2009; Gualdoni et al. Stem Cells, 2010). Here, we outline new evidence in support of the adult RSC as a bona fide stem cell population.
We use cell sorting of dissociated CE cells on the basis of size (forward-scatter) and pigmentation (side-scatter), as well as fluorescently-labeled P-cadherin expression. Differentiation of clonally-derived spheres from Nrl.gfp animals is performed on laminin-coated plates for 40 days in vitro, using combinations of small molecules to direct differentiation (taurine, retinoic acid, sonic hedgehog, sodium butyrate, FGF2). Specific expression of mature photoreceptor markers (Rhodopsin, Crx) is confirmed by immunocytochemistry and Q-PCR.
The RSC population can be enriched from CE cells on the basis of medium-size and high pigmentation (13-fold enrichment, n=3) and low P-cadherin expression (7.7-fold enrichment, n=3). Furthermore, clonally-derived RSC progeny can be directed towards rod photoreceptor differentiation with high efficiency (90-95%) using exogenous culture additives, including combinations of taurine and retinoic acid. This represents a significant advancement over previous rates of differentiation (10%) in serum. The expression of post-mitotic rod genes (Nrl and rhodospin) shows a similar temporal profile to normal mouse rod photoreceptor development, and reveals an instructive differentiation mechanism, rather than a selective survival of post-mitotic rods.
Our use of exogenous factors to direct differentiation avoids genomic manipulation, and eliminates the risk of insertional mutagenesis. These RSC-derived photoreceptors may prove useful for transplantation to replace photoreceptors damaged by retinal diseases such as age-related macular degeneration or retinitis pigmentosa.
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