OCULAR SURFACE STEM CELL GENES

描述(申请人提供)：眼表衬里、结膜和角膜缘上皮的动态平衡更新和再生最终依赖于在一生中自我复制的一小部分干细胞。这些处于存活状态的干细胞的分离将为它们用于眼部疾病的治疗或眼部发育和修复机制的基础研究提供新的场所。活细胞的分离通常依赖于质膜表面表位的鉴定，以便在不破坏或修改这些细胞的情况下，通过分选或淘洗来分离这些细胞。眼表干细胞分离的一个主要障碍是缺乏识别和分离它们的已知干细胞标记或特性。在这种背景下，最近的实验证据表明，许多体细胞可能分享一个共同的解毒机制，这是由一种特定的多药耐药转运蛋白ABCG2/BCRP/MTX的过度表达支撑的。当富含ABCG2的细胞与DNA结合染料Hoechst-33343(Ho333)孵育并使用紫外光激发源荧光激活细胞分选进行分析时，高效的Ho333-外排导致荧光光谱漂移。这种变化导致细胞在发射曲线图中显示为正常蓝/红比率左侧(蓝色)的一组特征隔离的细胞。我们现在已经证实了这种蛋白在眼表上皮细胞中的存在，并建立了分离SP细胞的有效方法。这些细胞符合已知或预期的上皮干细胞表型的主要特征，包括：a)它们显著缺乏细胞内的‘粒度’和小尺寸；b)在长期接触佛波酯后启动克隆性增殖的独特能力；c)在培养时缓慢的募集到增殖。这项应用的目的是探索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，以便识别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，在揭开这些特权细胞潜在功能的漫长道路上，获得基因库是至关重要的、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.