OCULAR SURFACE EPITHELIAL PRECURSORS

描述(由申请人提供)：本申请的直接目标是分离和鉴定密切相关的角膜缘和结膜上皮细胞系中的干细胞。来自这些研究的新知识将为制定临床重建角膜组织的策略提供必要的基本信息。最近的研究表明，许多体细胞都表达多药耐药转运蛋白(MDR)三磷酸腺苷结合盒(ABC)转运蛋白ABCG2/BCRP。这种转运蛋白的活性使细胞在流式细胞仪蓝色和红色荧光发射曲线图中显示为一组典型的孤立的细胞，位于正常蓝/红比率的左侧(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。我们最近在人的角膜缘和结膜上皮细胞中发现了ABCG2，并用荧光激活细胞分选仪从人和兔的这些组织中分离出了SP细胞。分离的细胞表现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)体内静止的表型；c)在体外对佛波酯诱导分化的选择性弹性；以及d)在培养时缓慢的募集到增殖。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)检测SP或ABCG2+细胞是否表现出上皮干细胞最明显的特征，包括缓慢的周期和长期的增殖能力；b)确定干细胞/前体细胞群体的复杂性和多样性；以及c)识别眼表SP细胞相关基因。这些初步研究的完成将提供有关眼前体细胞库特征的关键新信息。

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.