The immediate objective of this application is the isolation and characterization of stem cells in the closely related limbo-corneal and conjunctive epithelial lineages. The new knowledge from these studies will provide the fundamental information needed to develop strategies for clinical reconstruction of corneal tissues. Recent studies indicate that a large variety of somatic stem cells express the multidrug resistance transporter (MDR) ATP binding Cassette (ABC) transporter, ABCG2/BCRP. The activity of this transporter causes ceils to appear in flow cytometry blue vs. red fluorescence emission plots as a characteristically isolated group of cells positioned to the left (blue) side of the normal blue/red ratio. These cells have been termed side population (SP) cells. Accordingly both the ABCG2 + and SP phenotypes have been proposed to be general features of stem cells. We have recently identified ABCG2 in the human limbal and conjunctival epithelia and isolated SP cells from these tissues in human and rabbit by fluorescence activated cell sorter. The isolated ceils display multiple features associated with the epithelial stem cell phenotype including, a) an extremely low intracellular 'granularity'and a small size;b) a quiescent phenotype in vivo;c) a selective resiliency to induced differentiation by phorbol esters in vitro;and d) slow recruitment to proliferation when set in culture. We propose now to characterize these cells in detail from several biological and biochemical perspectives. We will a) examine whether SP or ABCG2 + cells exhibit the most recognizable features of epithelial stem cells including slow cycling and long term proliferative capacity;b) determine the complexity and diversity of the stem/precursor cell population;and c) identify ocular surface SP cell associated genes. Completion of these initial studies will provide critical new information on the features of the ocular precursor cell pool.
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