Ocular surface epithelial precursors

描述（由申请人提供）：少量干细胞维持角膜边缘和结膜谱系。在角膜边缘系统中，干细胞优先定位于角膜边缘，整体或区域性损伤会产生上皮性角膜边缘干细胞缺乏综合征。近年来，各种外科干细胞移植方法已被应用于重建角膜缘干细胞生态位以恢复视力。我们研究的长期目标是a)了解眼表干细胞，特别是眼缘干细胞的功能性质；b)将这一关键知识应用于优化体外角膜缘细胞群扩增，以改进基于富含干细胞的细胞群移植的表面重建程序。在这项资助的初始资助期间，我们对结膜和角膜缘上皮的小干细胞群进行了表征，称为侧群（SPs）。我们的研究表明，这些细胞来源于体内处于缓慢循环状态的细胞，并在体外显示出干细胞的其他几个特征。基于这一时期的发现，我们现在提出，在Specific ai1中，为了验证表达CD62E或排除有丝分裂跟踪器深红色的细胞群构成异质干细胞/前体缘和结膜上皮细胞群的额外组成部分的假设，在Specific ai2中，为了验证观察到能够影响基因转录的几个基因的过度表达的假设，这些基因通常被称为主基因或细胞命运基因(PAX6, MEIS1, SIX1, MSX1，HES1 ID1，即控制MYC活性的MXD1/MAX系统)参与了角膜缘上皮细胞的干细胞性。在Aim 1中与干细胞/前体细胞区室相关的细胞群的比较表征中获得的知识，结合特异性Aim 2中与干细胞/前体细胞状态保存或丧失相关的基因表达和信号通路的鉴定，将为改进在体外角膜缘细胞扩增过程中最大化干细胞/前体细胞产量的策略提供有力的基础，或者，通过表型逆转促进干细胞从一般增殖（基底）上皮细胞中拯救出来，从而有利于眼表重建手术。具体目标3旨在应用在前两个具体目标中获得的知识和专业知识，对新分离的角膜缘上皮细胞进行遗传和/药理学操作，以实现这些细胞的有效扩增，同时保持其在体内角膜上皮功能恢复的能力。

DESCRIPTION (provided by applicant): Small populations of stem cells sustain the limbal-corneal and the conjunctival lineages. In the limbal-corneal system, where stem cells localize preferentially to the limbus, total or zonal damage produces epithelial limbal stem cell deficiency syndromes. In recent years, a variety of surgical stem cell transplant methodologies have been applied to reestablish the limbal stem cell niche for vision restoration. The long term goals of our studies are a) understand the functional nature of ocular surface stem cells, in particular those of the limbus; and b) apply this critical knowledge for the optimization of ex vivo limbal cell population expansion to improve surface reconstructive procedures based on transplantation of stem cell-rich cell populations. During the initial funding period of this grant we characterized small stem cell cohorts known as side populations (SPs) from both conjunctival and limbal epithelia. Our studies showed that these cells derive from cells that have been in the slow cycling state in vivo and to display several other features recognized for stem cells in vitro. Based on finding during this period we now propose, in Specific Aim1 to test the hypothesis that cell cohorts expressing CD62E or excluding mitotrakcker deep red constitute additional component of an heterogeneous stem/precursor limbal and conjunctival epithelial cell population and in Specific Aim2 to test the hypothesis that observed overexpressions of several genes capable of affecting gene transcription globally and usually referred as master or cell fate genes (PAX6, MEIS1, SIX1, MSX1, HES1 ID1, the MXD1/MAX system controlling MYC activity) contribute to cell stemness in the limbal epithelium. The knowledge acquired in the comparative characterization of the cell cohorts associated with the stem/precursor cell compartment in Aim 1, combined with the identification of gene expressions and signal pathways that are associated with preservation or loss of the stem/precursor cell state in Specific Aim 2, will provide the cogent basis to improve strategies for either maximization of stem/precursor cell yields during limbal cells expansion in vitro, or alternatively, facilitate stem cell rescue from generic proliferative (basal) epithelial cells through reversion of phenotype for the benefit of reconstructive ocular surface procedures. Specific aim 3 seeks to apply the knowledge and expertise acquired in the two previous specific aims for the genetic and/pharmacological manipulation of freshly isolated limbal epithelial cells to attain effective expansion of these cells while maintaining their capacity for corneal epithelial function recovery in vivo.