OCULAR SURFACE EPITHELIAL PRECURSORS

描述（由申请人提供）：本申请的直接目的是分离和表征密切相关的角膜缘-角膜和结膜上皮谱系中的干细胞。这些研究的新知识将为制定角膜组织临床重建策略提供所需的基本信息。最近的研究表明，多种体干细胞表达多药耐药转运蛋白（MDR）ATP结合盒（ABC）转运蛋白ABCG 2/BCRP。该转运蛋白的活性导致细胞在流式细胞术蓝色与红色荧光发射图中显示为位于正常蓝色/红色比率的左侧（蓝色）的特征性分离的细胞组。这些细胞被称为侧群（SP）细胞。因此，ABCG 2+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鉴定了ABCG 2，并通过荧光激活细胞分选仪从人和兔的这些组织中分离了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）极低的细胞内“粒度”和小尺寸; B）体内静止表型; c）体外对佛波醇酯诱导的分化的选择性弹性;和d）当置于培养物中时，缓慢募集增殖。我们现在建议从几个生物学和生物化学的角度详细描述这些细胞。我们将a）检查SP或ABCG 2+细胞是否表现出上皮干细胞的最可识别的特征，包括缓慢的循环和长期增殖能力; B）确定干细胞/前体细胞群的复杂性和多样性;和c）鉴定眼表面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.