Cornea Epithelial Stem Cell Isolation for Bioengineering

描述(申请人提供)：角膜上皮干细胞疾病或功能障碍，称为角膜缘干细胞缺乏症，是世界上最常见的致盲眼病之一。直到过去十年，角膜缘移植用于这种疾病的表面重建的潜力被意识到之前，没有有效的治疗方法来治疗这些疾病。角膜缘移植的成功归功于角膜上皮干细胞的愈合潜力，而角膜上皮干细胞只是移植到这些移植物中的细胞混合物中的一小部分。从小的角膜缘活检组织中分离出纯净的角膜上皮干细胞，在培养中扩增，并用于再生具有正常表型和再生能力的角膜表面的能力，将是该领域的重大进展。但到目前为止，还没有鉴定出角膜上皮干细胞的特异性标志物。角膜上皮干细胞的分离尚未实现。我们的初步研究提供了令人鼓舞的结果，支持我们的假设，即新的分离方法和建议的非眼组织干细胞和基底细胞标记物可以用于分离纯净的角膜上皮干细胞群体。提出的两个具体目标将实现长期目标。目标1将创造新的方法来分离角膜上皮干细胞，基于其独特的性质和分子标记，用于角膜组织工程。三种分离策略将被结合使用：1)基于细胞分化和扩大的大小之间的相关性进行大小分类；2)基于其较高的β1整合素表达而通过它们与细胞外基质的快速黏附来进行浓缩；3)使用重要的DNA结合染料Hoechst 33342通过流式细胞术来纯化作为辅助群体的干细胞。目的2将利用先进的基因阵列/微阵列技术，通过鉴定纯化的干细胞群体中的基因表达模式，寻找新的角膜上皮干细胞特异性标记物。随着上述目标的实现，将首次实现角膜上皮干细胞的纯种群，这将使角膜上皮干细胞的概念成为现实，并将干细胞研究和临床应用带入一个新的时代。这项工作将对成人干细胞的概念具有重要的科学意义和高度的影响，不仅对角膜和眼表，而且对其他组织也是如此。

DESCRIPTION (provided by applicant): Corneal epithelial stem cell disease or dysfunction, known as limbal stem cell deficiency, is among the most common blinding eye conditions worldwide. There was no effective therapy for these conditions until the potential of limbal transplantation for surface reconstruction of eyes with this condition was realized in the past decade. The success of limbal transplantation has been attributed to the healing potential of the corneal epithelial stem cells that are only a small subpopulation contained within the mixture of cells transplanted in these grafts. The ability to isolate a pure population of corneal epithelial stem cells from small limbal biopsies, expand them in culture and use them for regenerating a corneal surface of normal phenotype and regenerative capacity would represent a major advance in this field. But to date, no specific markers for corneal epithelial stem cells have been identified. Isolation of corneal epithelial stem cells has not been achieved. Our preliminary studies have provided encouraging results in support of our hypothesis that the novel isolation methods and proposed markers for stem cells and basal cells in non-ocular tissues can be utilized to isolate a pure population of corneal epithelial stem cells. Two proposed Specific Aims will realize the long-term objectives. Aim 1 will create novel approaches to isolate corneal epithelial stem cells based on their unique properties and molecular markers for use in corneal tissue engineering. Three isolating strategies in combination will be used: 1) Size sorting based on the correlation between cell differentiation and enlarged size; 2) Enrichment by their rapid adherence to extracellular matrix based on their higher-expression of beta1integrin; 3) Purification of stem cells as a side population by flow cytometry using a vital DNA binding dye, Hoechst 33342. Aim 2 will search for new specific markers for corneal epithelial stem cells by characterizing gene expression patterns in the purified populations of stem cells using advanced gene array/microarray techniques. With the proposed Aims accomplished, pure populations of corneal epithelial stem cells will be available for the first time, which will make the corneal epithelial stem cell concept become reality and bring stem cell research and clinical application into a new age. This work will have important scientific significance and high impact on the adult stem cell concept, not only for cornea and ocular surface, but also for other tissues.