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H. Miyashita, S. Shimmura, Y. Matsuzaki, K. Higa, H. Okano, J. Shimazaki, K. Tsubota; Characterization of Human Limbal Side Population (SP) Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2085.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the molecular marker expression profile and colony forming efficiency of limbal epithelial SP cells. Methods: Four to ten limbal segments from human eyebank corneas were treated enzymatically to obtain single cell suspensions. Cells were incubated with Hoechst 33342 solution (4 ∼ 5 µg / ml) with or without reserpine at 37 °C for 60 min, stained with propidium iodide (PI), and analyzed by flow cytometry. Dead (PI +) cells were gated out. SP cells were defined as reserpine sensitive Hoechst blue (low) and red (low) population. Cytospin samples of SP cells and main population (MP) cells were stained with anti–keratin 19, anti–keratin 3, anti–vimentin, and anti–MART1 (melanocyte marker) antibodies. Unsorted limbal cells were used as control. SP and MP cells were cultured with mitomycin C treated 3T3 cells to observe colony formation. Results: Percentage of SP cells in viable (PI–) cells ranged from 0.01 % to 0.92 % (average=0.25 % ± 0.32 %, n= 10). K19, K3, vimentin, and MART1 positive cells were 52.8 %, 10.8 %, 19.3 %, and 6.4 % in SP cells, 53.8 %, 26.6 %, 8.5 %, and 5.0 % in MP cells, and 44.1%, 44.6 %, 9.7 %, and 2.0 % in unsorted limbal cells, respectively. Colony forming efficiency (CFE) of SP and MP cells was 2.7 % and 2.0 %, respectively. Conclusions: SP fraction of limbal epithelial cells contained fewer K3 (+) cells and higher levels of vimentin (+) cells compared with the MP fraction, however, there was no difference observed in CFE.
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