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J.M. Wolosin, M. Zhou, R.M. Lavker; Ocular surface side population cells are slow cycling in vivo. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3772.
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Purpose:The limbal (Li) and conjuctival (Cnj) epithelia (E) contain a small population of cells that, when analyzed by flow cytometry (Wolosin etal in Progress in Stem Cell Research, Nova Science Publishers. in press) display the side population (SP) phenotype (Goodell etal, J Exp Med. 183:1797, 1996). This phenotype reflects the ability of cells to prevent the accumulation of the DNA–binding dye Hoechst 33342 (Zhou etal, Nat Med. 7:1028, 2001). SP cells have been shown in several organs to belong to the stem cell (SC) pool. Slow cycling is one of the best–known features of ocular surface SCs (Lavker and Sun PNAS. 97:13473, 2000). Accordingly, this study examined the rate of cells proliferation of SP cells in vivo during the two weeks prior to cell isolation. Methods:Alzet mini pumps (3) were implanted subcutaneously into rabbits (3 kg) calibrated to deliver ∼ 24 mg BrdU (a thymidine analog) per day. On day 5 after implantation, one eye of each animal was treated topically with petrolatum containing 0.1 % phorbol myristate acetate (PMA), an enhancer of proliferation for stem and transient amplifying epithelial cells. Rabbits were sacrificed after 13 days of continuous BrdU infusion. Small sections of ocular surface tissue were fixed and processed for BrdU immuno–histology to determine a basal cell labeling index (LI; in %). Single cell suspensions of CnjE and LiE cells were incubated with Hoechst 33342 and sorted into SP and non–SP cells by flow cytometry. The sorted cells were plated on polylysine–coated glass coverslips and the LI of cells that adhered to the substratum within 16 hr was determined by BrdU immunofluorescence. Results:The 13 day LI for the basal cells in the limbal and conjunctival domains were respectively, 52 %, and 82 %, for the untreated eye and 71 % and 89 % for the PMA–exposed tissues. The respective LIs (n = 2) for the adhering CnjE SP and non–SP domains were 11 % and 54 % for the untreated and 31 % and 93 % for the PMA treated tissues. Similarly, a very low LI was also observed in the LiE SP cells. Conclusions:Rabbit ocular surface SP cells in adult rabbit are near quiescent in resting conditions but be stimulated to proliferate by exposure to PMA, features consistent with a stem cell phenotype.
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