OCULAR SURFACE STEM CELL GENES

描述（由申请人提供）：眼表衬里、结膜和角膜缘上皮的稳态更新和再生最终取决于在整个生命过程中自我复制的少量干细胞。 分离这些活状态的干细胞将为将其用于眼部疾病的治疗或用于眼部发育和修复机制的基础研究提供新的途径。活形式的细胞的分离通常依赖于质膜表面表位的鉴定以“标记”这些细胞而不损伤或修饰它们以通过分选或淘选进行分离。分离眼表面干细胞的主要障碍是缺乏已知的干细胞标志物或对其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，许多体干细胞可能有一个共同的解毒机制，由一个特定的多药耐药转运蛋白，称为ABCG 2/BCRP/MTX的过度表达支持。当ABCG 2富集细胞与DNA结合染料Hoechst-33343（Ho 333）一起孵育并使用UV激发源通过荧光激活细胞分选（FACS）进行分析时，高效Ho 333流出导致荧光光谱偏移。这种偏移导致细胞在发射图中显示为正常蓝/红比率左侧（蓝）的一组特征性孤立细胞。我们现在已经证实了这种蛋白存在于眼表上皮细胞，并建立了有效的方法来分离SP细胞。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）它们显著缺乏细胞内“粒度”和小尺寸; B）在长期暴露于佛波酯后启动克隆增殖的独特能力; c）当置于培养物中时，增殖的缓慢募集。本申请的目的是利用从农场哺乳动物的眼分离足够量的这些细胞的可能性，以允许鉴定在眼表面干细胞中差异表达的基因家族，以及其表达标志分化开始的那些基因。鉴于我们目前对干细胞的了解非常有限，获得基因文库是解开这些特权细胞功能背后过程的漫长道路上必不可少的，高影响力的步骤。

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.