OCULAR SURFACE EPITHELIAL PRECURSORS

描述（由申请人提供）：本申请的直接目的是在密切相关的角膜边缘和结膜上皮谱系中分离和表征干细胞。这些研究的新知识将为制定临床角膜组织重建策略提供必要的基础信息。近年来的研究表明，多种体细胞干细胞表达多药耐药转运体（MDR） ATP结合盒体（ABC）转运体ABCG2/BCRP。这种转运体的活性导致细胞在流式细胞术中出现蓝色和红色荧光发射图，作为一组典型的分离细胞，位于正常蓝/红比值的左侧（蓝色）。这些细胞被称为侧群（SP）细胞。因此，ABCG2 +和SP表型都被认为是干细胞的一般特征。我们最近在人角膜缘和结膜上皮中发现了ABCG2，并利用荧光激活细胞分选仪从人和兔的这些组织中分离出SP细胞。分离的细胞显示出与上皮干细胞表型相关的多种特征，包括：a)细胞内“粒度”极低且体积小；B)体内静态表型；C)对酚酯诱导的体外分化具有选择性弹性；d)在培养环境中，细胞增殖缓慢。我们现在建议从几个生物学和生物化学的角度来详细描述这些细胞。我们将a)检查SP或ABCG2 +细胞是否表现出上皮干细胞最明显的特征，包括慢循环和长期增殖能力；B)确定干细胞/前体细胞群体的复杂性和多样性；c)鉴定眼表SP细胞相关基因。这些初步研究的完成将为眼前体细胞库的特征提供重要的新信息。

DESCRIPTION (provided by applicant): 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.