Cell and Molecular Biology of Cornea Epithelial Stem Cells

描述（由申请人提供）：角膜，我们的“世界之窗”，是受到伤害，感染和其他疾病造成的损害。正在开发通过角膜上皮干细胞移植来治疗这些角膜的方法。一个主要的医学挑战在于能够分离足够数量的这些细胞用于培养、扩增和移植。我们仍然缺乏对它们的增殖、迁移行为和识别这些细胞的精确标记的基本了解。我们也不知道是什么因素调节这些行为，而这对于增强角膜上皮干细胞的治疗潜力至关重要。最近的证据表明，皮肤神经可能是上皮干细胞龛的关键组成部分。非洲爪蟾是一种成熟的脊椎动物模型，在细胞和分子生物学研究中具有明显的优势。成熟青蛙角膜的发育和形态基本上与人类相同。我们开发了工具，使非洲爪蟾成为研究这些细胞生物学的优秀系统。我们的中心假设是，角膜缘干细胞和它们的增殖后代表现出特定的分裂模式，并表达特定的基因组合，这将使人们能够区分这些细胞。此外，这些细胞对损伤释放的信号作出反应，以进行增加的增殖，能够迁移到伤口部位，最后，角膜的神经代表了调节这些细胞行为的上皮干细胞生态位的关键组成部分。该提案有三个具体目标来解决这些假设：I）确定正常增殖模式和基因表达谱以区分这些干细胞及其后代，并检查多能性基因在调节这些细胞行为中的作用。II）确定这些细胞如何对损伤做出反应，以及它们是否在稳态和修复期间进行主动迁移。III）。确定角膜的皮肤神经是否在支持这些增殖细胞中起关键作用。为了实现这些目标，我们开发了新的工具，可以在活体动物中长时间直接连续观察这些细胞。我们还开发了爪蟾模型来研究角膜伤口修复和角膜缘干细胞缺乏症（LSCD）。最后，我们可以非常精确地结扎角膜神经，以测试它们在支持这些细胞中的作用。这项研究在非洲爪蟾中使用高通量转基因方法以允许实时观察活角膜上皮干细胞方面具有创新性，其中我们可以表征识别这些细胞的基因表达（标记物）的特定模式。这项研究还将确定角膜神经是否代表支持这些细胞的干细胞生态位的关键组成部分。在美国，每年有数万名患者患上神经营养性角膜炎、角膜营养不良、LSCD和其他需要角膜移植的眼部疾病（约40，000/年）。这项研究意义重大，因为它将开发可用于治疗这些疾病和角膜损伤的工具和方法。

DESCRIPTION (provided by applicant): The cornea, our "window to the world," is subject to damage caused by injury, infection and other diseases. Approaches are being developed to treat these corneas by transplantation of cornea epithelial stem cells. A major medical challenge lies in being able to isolate sufficient quantities of these cells for culture, expansion, and transplantation. We still lack a basic understanding of their proliferation, migratory behaviors and precise markers to identify these cells. We also do not know what factors regulate these behaviors, which is essential for enhancing the therapeutic potential of cornea epithelial stem cells. Recent evidence suggests that cutaneous nerves may be a key component of the epithelial stem cell niche. The frog Xenopus is an established vertebrate model with distinct advantages for studies of cell and molecular biology. The development and morphology of the mature frog cornea is essentially identical to that of humans. We developed tools that make Xenopus an excellent system to study the biology of these cells. Our central hypotheses are that limbal stem cells and their proliferative progeny exhibit specific cleavage patterns and express specific combinations of genes that will allow one to distinguish these cells. Further, these cells respond to cues released by damage to undergo increased proliferation, being able to migrate to wound sites, and finally that nerves serving the cornea represent a critical component of the epithelial stem cell niche that regulates the behavior of these cells. This proposal has three specific aims to address these hypotheses: I) determine the normal proliferative patterns and gene expression profiles to distinguish these stem cells and their progeny, and examine the role of pluripotency genes in regulating the behavior of these cells. II) Determine how these cells respond to damage and whether they undergo active migration during homeostasis and repair. III). Determine if cutaneous nerves of the cornea play a key role in supporting these proliferative cells. To address these aims we developed new tools that permit direct, continuous visualization of these cells in live animals over prolonged periods of time. We have also developed Xenopus models to study cornea wound repair and limbal stem cell deficiency (LSCD). Finally, we can ligate the corneal nerves with tremendous precision to test their role in supporting these cells. This research is innovative in its use of high-throughpu transgenic approaches in Xenopus to permit real-time observations of live cornea epithelial stem cells in which we can characterize specific patterns of gene expression (markers) that identify these cells. This study will also determine if corneal nerves represent a critical component of the stem cell niche that supports these cells. Tens of thousands of patients develop neurotrophic keratitis, forms of corneal dystrophy, LSCD, and other eye conditions annually in the U.S. that require corneal transplants (about 40,000/year). The proposed research is significant, as it will develop tools and approaches that can be used to treat these diseases and injuries of the cornea.