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B.V. Castillo; Isolation and Culture of Adult Human Retinal Stem Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1020.
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Purpose: Retinal stem cells have been recently identified in adult mammalian eyes and were present in the pigmented epithelium of the ciliary body, a homologous structure of the ciliary marginal zone found in fishes and amphibians. The purpose of this study is to isolate retinal stem cells from an adult human eye. Methods: A globe from a 47 y.o. female donor was obtained from the Midwest Eye Bank. The anterior segment was separated and the lens removed. The tissue was treated with 2% dispase for 90 minutes at 37oC. The pigment epithelium was stripped from the ciliary processes and was collected in HBSS. The sheets of epithelium were dissociated with 0.25% trypsin and grown in serum-free defined medium (DMEM/F12 + N2+FGF) at a density of 1 x 104 per ml. After 3 days, the suspended cells were collected and grown in laminin coated chamber slides with defined medium + 1% FBS. The cells were fixed with 4% paraformaldehyde on day 4 in preparation for fluorescence immunocytochemistry. Results: The pigment epithelium was peeled off easily after treatment with dispase with a yield of 4 x 105 cells. In serum-free defined medium, the cells remained pigmented but grew mostly as suspended cell clusters similar to the findings of Tropepe et al. The presence of laminin and 1% FBS stimulated adhesion and dispersion of the previously clustered cells. Immunocytochemistry showed anti-nestin immunoreactivity in a focal population of cells. Some were also positive for NSE. The majority of the cells stained positive for vimentin. Conclusions: Retinal stem cells are present in adult human eyes and can be grown in vitro. The results of this study has potential application in treatment of retinal degenerative disorders.
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