Stem Cells for Corneal Engineering

 描述（由申请人提供）：角膜混浊是全球数百万人失明的原因。使用尸体角膜的穿透性角膜移植术（PK）非常成功，但捐献的组织供应有限，随着时间的推移，PK移植物的并发症和失败率很高。开发更好的治疗角膜混浊是世界卫生保健的需要，激发了这个项目。在该项目的10年中，我们已经鉴定了来自人角膜基质（CSSC）的干细胞，并表明这些细胞（a）分化为角膜细胞，（B）在体外产生角膜基质组织，和（c）在体内防止小鼠模型中角膜瘢痕的形成。该项目的长期目标是开发基于干细胞的治疗应用，可以治疗或逆转角膜瘢痕形成，减少对PK的需求。在项目的更新中，我们将通过三个具体目标来实现这一目标。CSSC表达N-cadherin（NCad），其他基质细胞不表达NCad。目的1将检验NCad鉴定具有角膜细胞分化潜能的细胞群并提供用于分离具有高潜能实现角膜再生的细胞的细胞表面标记物的假设。我们已经发现CSSC可以分泌具有与角膜基质相似的排列的胶原和蛋白多糖的结缔组织。目的2将测试的作用，微观结构的拓扑结构和刚度的固体基板在指导该CSSC分泌的基质的组成和组织。目的还将测试胶原交联酶LOXL 2和TGM 2在增加CSSC精心制作的基质的强度和刚度中的作用。该目标将通过堆叠工程化组织片来组装多层结构。这些实验将提供关于3D环境如何指导透明基质组织的分泌和组织的见解。我们最近已经表明，CSSC滴注在愈合的角膜伤口防止瘢痕形成和重建消融组织与基质与天然角膜难以区分。目的3研究CSSC如何诱导组织再生。CSSC减少嗜中性粒细胞的浸润，并诱导TGF β 3的存在，TGF β 3是一种细胞因子，可诱导无瘢痕伤口愈合。这一目的将检验CSSC通过分泌TSG-6蛋白减少嗜中性粒细胞浸润并影响巨噬细胞采用通过分泌TGF β 3刺激组织再生的替代活化（M2）表型的假设。这些研究的影响将是识别控制角膜基质组织基质合成和组织的新分子机制。特别具有潜在意义的是鉴定诱导天然哺乳动物组织再生的机制。CSSC的临床应用目标也将取得进展。将产生更厚、更强、更有组织的组织结构，增加用于板层移植物的潜力。鉴定与CSSC的免疫抑制和再生潜力相关的特性将提高这些细胞在基于细胞的治疗中的应用潜力。

 DESCRIPTION (provided by applicant): Corneal opacity is a source of blindness for millions worldwide. Penetrating keratoplasty (PK) using cadaveric corneas is highly successful, but donated tissue is in limited supply and over time PK grafts are subject to a significant rate of complications and failure. Developing better therapy for corneal opacity is world health-care need that has inspired this project. In the 10 years of this project we have identified stem cells from human corneal stroma (CSSC) and showed that these cells (a) differentiate to keratocytes, (b) produce corneal stromal tissue in vitro, and (c) prevent formation of corneal scars in mouse models in vivo. The long-term goal of the project is develop stem cell-based therapeutic applications that can treat or reverse corneal scarring reducing the need for PK. In the renewal of the project we will work toward this goal with three specific aims. CSSC but no other stromal cells express N-cadherin (NCad). Aim 1 will test the hypothesis that NCad identifies a population of cells with keratocyte differentiation potential and provides a cell surface marker useful for isolating cells with high potential to effect corneal regeneration. We have found that CSSC can secrete connective tissue with aligned collagen and proteoglycans similar to corneal stroma. Aim 2 will test the role of microstructure topology and stiffness of solid substrates in directing the composition and organization of this CSSC-secreted matrix. The aim will also test the role of collagen crosslinking enzymes LOXL2 and TGM2 in increasing the strength and stiffness of the matrix elaborated by CSSC. This aim will assemble multilamellar constructs by stacking of the engineered tissue sheets. These experiments will provide insights on how the 3D environment directs secretion and organization of transparent stromal tissue. We have recently shown that CSSC instilled in healing corneal wounds prevent scarring and reconstitute ablated tissue with matrix indistinguishable from native cornea. Aim 3 will investigate how CSSC induce this tissue regeneration. CSSC reduce infiltration of neutrophils and induce the presence of TGFß3, a cytokine attributed to induce scarless wound healing. This aim will test the hypothesis that via secretion of TSG-6 protein, CSSC reduce neutrophil infiltration and influence macrophages to adopt an alternative activated (M2) phenotype stimulating tissue regeneration by secretion of TGFß3. The impact of these studies will be identification of novel molecular mechanisms controlling corneal stromal tissue matrix synthesis and organization. Of particular potential significance is identification of a mechanism to induce regeneration of native mammalian tissue. There will also be progress toward the goal of clinical use of CSSC. Thicker, stronger, and more organized tissue constructs will be produced with increased potential for use in lamellar grafts. Identification of properties linked to immunosuppressive and regenerative potential for CSSC will improve potential for use of these cells in cell-based therapy.