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MT Budak, JM Wolosin; Effect of Phorbol Ester on Clone Initiation by Corneal and Limbal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1640.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Stem (St) and transient amplifying (TA) cells, represent sequential stages of the orderly growth and differentiation process in the limbo-corneal (LC) epithelium. Both St and TA cells are able to proliferate by clonal initiation and form similar colonies. Yet, in vivo, they display distinct responses to topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and protein kinase C activator. Only St cells, which are present exclusively in the limbus, show an enhanced proliferative response. On the progenitor cells TPA acts as a promoter of differentiation. The purpose of this study was to determine whether TPA has a differential effect on clone initiation and progression by freshly dissociated populations of the St cell-rich limbal and St cell-free peripheral corneal cells. Methods: Cells were obtained by sequential tissue treatment with Dispase and trypsin, seeded (10/cm2) over mitomycin treated 3T3's (1,500/cm2) and cultured in DMEM/F12-20% FBS. After 12 to 14 days, colony formation index (CFI; colonies/100 cells) was determined. TPA was introduced for 24 to 72 hr at, or at various times after, seeding. Results: CFI for the cells of the limbus and corneal periphery (CPe; the 1 mm annulus adjacent to the limbus), where 8.9 (average for n = 11) and 3.5 (n = 7), respectively. Introduction of 100 nM TPA at t= 6 hr for 24 hr decreased limbal cell CFI to 38% of control (n = 6) but completely abolished the CFI of CPe cells (n = 7). Applied at 20 nM for 72 hr, TPA reduced Li CFI by < 10% but caused a 75% reduction of CPe cell CFI (n = 2). The inhibitory effect of the 24 hr TPA exposure on the Li cells was essentially independent from the time of application, whehter at seeding or up to day 4 of culture. Extending the exposure to 100 nM TPA for up to the first 72 hr post-seeding had also minimal effect on the extent of limbal cell CFI inhibition (n = 4). On the other hand, introducing TPA for 72 hr starting on day 4 abolished all limbal CFI (n = 2). Microscopic inspection of these latter cells indicated arrested proliferation and morphological changes consistent with differentiation.Conclusion: TPA inhibits clonal initiation of all corneal cells and the majority of limbal cells. It is proposed that the clonogenic limbal cells displaying TPA resistance are stem cells and/or their early progeny. In culture stem cells phenotype and hence, resistance to TPA, are gradually lost.
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