OCULAR SURFACE STEM CELL GENES

描述（由申请人提供）：眼表内膜、结膜和角膜边缘上皮的稳态更新和再生最终依赖于一小群终生自我复制的干细胞。这些干细胞在存活状态下的分离将为它们在眼部疾病的治疗或眼部发育和修复机制的基础研究中提供新的场所。活细胞的分离通常依赖于鉴定质膜表面表位来“标记”这些细胞，而不破坏或修改它们，以便通过分选或筛选进行分离。分离眼表干细胞的一个主要障碍是缺乏已知的干细胞标记物或对识别和分离有用的特性。在这种情况下，最近的实验证据表明，许多体细胞可能具有共同的解毒机制，该机制由一种特定的多药耐药转运体ABCG2/BCRP/MTX的过度表达所支撑。当富含abc_2的细胞与DNA结合染料Hoechst-33343 （Ho333）一起孵育，并使用紫外激发源进行荧光激活细胞分选（FACS）分析时，高效的Ho333外排导致荧光光谱移位。这种移位导致细胞在发射图中显示为正常蓝/红比值左侧（蓝色）的典型孤立细胞群。我们现在已经证实这种蛋白存在于眼表上皮中，并建立了分离SP细胞的有效方法。这些细胞符合上皮干细胞表型的主要已知或预期特征，包括：a)它们明显缺乏细胞内“粒度”和小尺寸；B)长时间暴露于佛波酯后启动克隆增殖的独特能力；C)在培养环境中缓慢的增殖。本应用程序的目的是利用从农场哺乳动物的眼睛中分离足够数量的这些细胞的可能性，以便鉴定在眼表干细胞中差异表达的基因家族，以及那些表达标志着分化开始的基因。鉴于我们目前对干细胞的了解非常有限，在解开这些特权细胞功能背后的过程的漫长道路上，获得基因库是必不可少的，影响很大的一步。

DESCRIPTION (provided by applicant): Homeostatic renewal and regeneration of the ocular surface linings, the conjunctival, and limbo-corneal epithelia ultimately depends on a small population of stem cells that self replicate throughout life. Isolation of these stems cells in the viable state will provide new venues to use them in the treatment of ocular disease, or in fundamental studies on mechanisms of ocular development and repair. Isolation of cells in viable form is generally dependent on identification of plasma membrane surface epitopes to 'mark' these cells without damaging or modifying them for isolation by sorting or panning. A major impediment to isolation of ocular surface stem cells is the absence of known stem cell markers or properties useful for their recognition and isolation. In this context, recent experimental evidence suggests that many somatic stem cells may share a common detoxification mechanisms underpinned by over-expression of a particular multidrug resistance transporter, known as ABCG2/BCRP/MTX. When ABCG2-rich cells are incubated with the DNA binding dye Hoechst-33343 (Ho333) and analyzed by fluorescence activated cell sorting (FACS) using a UV excitation source, highly efficient Ho333-efflux results in a fluorescence spectral shift. This shift causes cells to appear in emission plots as a characteristically isolated group of cells to the left (blue) side of the normal blue/red ratio. We have now confirmed the presence of this protein in the ocular surface epithelia and have established effective ways to isolate SP cells. These cells comply with the main known or expected features of the epithelial stem cell phenotype including, a) their remarkable lack of intracellular 'granularity' and a small size; b) unique ability to initiate clonogenic proliferation following prolonged exposure to phorbol ester; c) slow recruitment to proliferation when set in culture. The objective of this application is to exploit the possibility of isolating sufficient amounts of these cells from the ocular of farm mammals to allow the identification of the family of genes that are differentially expressed in the ocular surface stem cells, as well as those genes whose expression hallmark the start of differentiation. Given our current very limited understanding of stem cells, acquisition of a gene library is an essential, high impact step in the long path to unravel the process underlying the functionality of these privileged cells.