Stem Cells for Corneal Engineering

描述（由申请人提供）：角膜失明影响全球数百万人，通常只能通过移植捐赠的组织来治疗。在世界上许多地区，供体组织不可用;在美国，用于移植的角膜供应受到越来越多的眼睛进行屈光手术的威胁。该项目的长期目标是通过开发基于干细胞的疗法来解决角膜失明问题。该项目已经鉴定和表征了成人角膜基质干细胞（CSSC），其可以在培养中扩增，同时保留采用角膜细胞表型和精心制作丰富的基质样结缔组织的能力。当CSSC在聚（酯氨基甲酸酯）脲（PEUU）（一种可生物降解的聚合物）的对齐的纤维素纳米纤维的支架上培养时，它们分泌具有与正常角膜基质相似的对齐胶原的结缔组织。当将CSSC注射到Lumican敲除小鼠的基质中时，基质中的胶原变得更加组织化，并且角膜透明度增加。重要的是，人CSSC在小鼠中表现出免疫豁免，在没有免疫排斥的情况下保持存活数月。新的提案将测试直接来自这些令人兴奋的发现的特定假设。目的1通过测试以下预测记录了CSSC的组织再生过程：（a）CSSC首先通过时间调节特定基质金属蛋白酶组的表达来去除现有组织;（B）在该去除过程之后，CSSC通过沉积新的结缔组织来重建基质组织;以及（c）该再生过程可以重塑与导致大多数人角膜失明的瘢痕组织类似的瘢痕组织。目的2：将CSSC与PEUU支架结合，制备新型生物工程组织，并验证以下预测：（a）PEUU支架上的CSSC单层可以整合到小鼠角膜移植物中，并作为透明基质组织发挥作用;（B）CSSC可以整合到具有角膜基质厚度和强度的多层PEUU支架中;和（c）在使用深前板层角膜移植术移植到兔角膜中后，多层构建体可以在功能上替代基质。这项研究的科学影响将证明两种新的基于干细胞的角膜瘢痕治疗方法的有效性，表明每种方法都准备好进行临床试验。 公共卫生相关性：角膜失明是一个全球性的健康问题。对于许多患有这种疾病的人来说，没有恢复视力的选择。该项目将使用成人干细胞开发治疗角膜瘢痕的方法，这是失明的主要原因。这些干细胞可以大量产生，是安全的，并引起组织排斥。这些疗法的成功开发可能会为许多人恢复视力提供新的机会。

DESCRIPTION (provided by applicant): Corneal blindness affects millions of individuals world-wide and usually can only be treated by transplantation of donated tissue. In many regions of the world donor tissue is not available; in the US, the supply of corneas for transplant is threatened by increasing numbers of eyes with refractive surgeries. The long-term goal of this project is to address corneal blindness by developing therapies based on stem cells. This project has already identified and characterized adult human corneal stromal stem cells (CSSC), which can be expanded in culture while retaining the ability to adopt a keratocyte phenotype and to elaborate abundant stroma-like connective tissue. When CSSC were cultured on a scaffolding of aligned paralel nanofibers of poly(ester urethane)urea (PEUU), a biodegradable polymer, they secreted connective tissue with aligned collagen similar to that of normal corneal stroma. When CSSC were injected into stromas of lumican knockout mice, collagen in the stroma became more organized and corneal transparency was increased. Importantly, human CSSC exhibited an immune privilege in mice, remaining viable for months without immune rejection. The new proposal will test specific hypotheses emanating directly from these exciting findings. Aim 1 documents the process of tissue regeneration by CSSC by testing the predictions that (a) CSSC first remove existing tissue by temporally regulated expression of a specific group of matrix metalloproteinases; (b) after this removal process, CSSC rebuild stromal tissue by deposition of new connective tissue; and (c) this regeneration process can remodel scar tissue similar to that causing most human corneal blindness. Aim 2 combines CSSC with PEUU scaffolding to produce a novel bioengineered tissue and tests the predictions that (a) CSSC monolayers on PEUU nanofiber scaffolding can integrate and function as transparent stromal tissue in mouse corneal transplants; (b) CSSC can be incorporated into multilayered PEUU scaffolding with the thickness and strength of a corneal stroma; and (c) the multilayered constructs can functionally replace stroma after transplantation into rabbit corneas using deep anterior lamellar keratoplasty. The scientific impact of this study will be a demonstration of effectiveness of two novel stem-cell based therapeutic approaches for corneal scarring, indicating a readiness for translation of each to clinical trials. PUBLIC HEALTH RELEVANCE: Corneal blindness is a health problem world-wide. For many individuals with this condition there is no option for restoration of vision. This project will use adult human stem cells to develop therapy for corneal scarring, a major source of blindness. These stem cells can be produced in large numbers, are safe, and do cause tissue rejection. Successful development of these therapies may open new opportunity to restore vision to large numbers of people.