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A. Usui, S. Shimmura, Y. Matsuzaki, H. Okano, K. Tsubota; Immature Side Population (SP) Cells Isolated from Corneal Limbal Tissue . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1356.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To demonstrate the presence of immature side population (SP) cells in limbal epithelium. Methods: SP cells, first identified in bone marrow and subsequently in other organs, are an immature fraction of cells that contain pluripotent stem cells. SP cells were defined as Hoechst 33342-excluding cells by fluorescence-activated cell sorting analysis (FACS). Corneo-scleral rims were prepared from enucleated rabbit eyes. Limbal tissue was treated with 50U/ml Dispase II, followed by 0.2% collagenase and 0.25% Trypsin-0.02% EDTA. Single cells were collected and suspended with or without reserpine, an inhibitor of the multi drug resistance (MDR) gene. Reserpine-treated and untreated cells were incubated with Hoechst 33342 dye, and analyzed and sorted by FACS. Sorted cells were further suspended in culture medium with supplements, and incubated at 37 C. All animal experiments adhered to the ARVO statement for the use of animals in ophthalmic and vision research. Results: A typical SP profile was recognized in single cell suspension of corneal limbal cells. Fluorescence was blocked by the addition of reserpine, confirming the presence of SP cells. In vitro culture demonstrated that cultured SP cells adhered to the dish, and expanded into both epithelial cell-like and fibroblast-like morphology after 2 days. Conclusions: SP cells were shown to exist in the corneal limbus. This fraction of cells may be used to further identify and characterize corneal epithelial stem cells.
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