Bioengineering Cornea with Autologous Stem Cells

 描述（由申请人提供）：该多PI项目结合了两个具有不同专业领域的研究小组的努力，以解决开发生物工程角膜基质和内皮组织的长期目标，为角膜失明患者提供治疗。 这些组织将从成体干细胞中进行生物工程改造，成体干细胞可以从待治疗的个体中获得。 将这些细胞组织成组织将由天然细胞外基质蛋白构建的支架引导，使用仿生表面诱导组装过程制造。 匹兹堡大学的Funderburgh实验室将从捐赠的人类角膜缘基质中获得干细胞。 他们对这些角膜基质干细胞（CSSC）的广泛研究表明，它们分化为基质角膜细胞和角膜内皮细胞，这些组织负责大多数角膜混浊。 最近，我们证明了CSSC可以从活检样品中获得，从而提供了产生自体生物工程组织的机会。 卡内基梅隆大学的Feinberg实验室开发了一种组装天然细胞外基质蛋白的新方法，以产生具有定义的3D结构的组织样支架。 目标1将集中于组装生物工程基质，其中该项目将阐明由支架提供的空间和生化线索以及可溶性线索（缺氧，TGF β 3，FGF 2）在从CSSC产生基质样组织中的作用。 组织的各个层将被堆叠以形成类似于角膜基质的多层3-D组织。 将检查这些实验中产生的组织的组成、结构、生物力学性质以及在体内兔模型中充当基质的功能替代物的能力。 目的2：将CSSC培养成球状，然后转移到含有基底膜蛋白的基质中，形成多边形单层，并表达角膜内皮细胞的典型基因，从而将角膜内皮细胞生物工程化。 使用表面引发的组装，Feinberg小组开发了一种生物工程等效的Descemet膜，可以增加培养的角膜内皮细胞的分化，扩增和功能。 角膜内皮层将由CSSC在这些膜上产生，并测试基因表达模式、细胞密度、细胞形态和内皮泵功能。 构建体的功能将在长期兔体内模型中得到证实。 该项目将使用创新的实验方法，对不溶性细胞外微环境如何指导角膜组织的形成和功能进行新的理解。 重要的是，在研究期间开发的组织有可能进入临床相关研究，为角膜失明的古老问题提供新的治疗方法。

 DESCRIPTION (provided by applicant): This Multi-PI project combines the efforts of two research groups with different areas of expertise to address the long-term goal of developing bioengineered corneal stroma and endothelial tissues to provide therapy for individuals with corneal blindness. These tissues will be bioengineered from adult stem cells, which can be obtained from the individuals to be treated. Organization of these cells into tissues will be guided by scaffolds constructed of native extracellular matrix proteins, fabricated using a biomimetic, surface-induced assembly process. The Funderburgh Lab at the University of Pittsburgh will obtain stem cells from limbal stroma of donated human corneas. Their extensive work with these corneal stromal stem cells (CSSC) shows that they differentiate to stromal keratocytes and to corneal endothelial cells, tissues responsible for most corneal opacity. Recently we demonstrated that CSSC could be obtained from biopsy samples, presenting the opportunity to generate autologous bioengineered tissues. The Feinberg Lab at Carnegie Melon University has developed a novel approach of assembling native extracellular matrix proteins to produce tissue-like scaffolding with defined 3-D architecture. Aim 1 will focus on assembling bioengineered stroma in which the project will elucidate spatial and biochemical cues provided by the scaffolding and the role of soluble cues (hypoxia, TGFß3, FGF2) in generating a stroma-like tissue from CSSC. Individual layers of tissue will be stacked to form multilamellar 3-D tissue similar to that of the corneal stroma. Tissues produced in these experiments will be examined for composition, structure, biomechanical properties, and for the ability to act as a functional substitute for stroma in an in vivo rabbit model. Aim 2 will bioengineer corneal endothelium, based on our observation that CSSC cultured as spheres and then transferred to substratum containing basement membrane proteins form polygonal monolayers and express genes typical of corneal endothelium. Using surface-initiated assembly, the Feinberg group has developed a bioengineered equivalent of Descemet's membrane that increases differentiation, expansion, and function of cultured corneal endothelial cells. Corneal endothelial layers will be generated on these membranes from CSSC, and tested as to gene expression patterns, cell density, cell morphology, and endothelial pump function. Functionality of the constructs will be demonstrated in long-term rabbit models in vivo. This project will use innovative experimental approaches to develop a new understanding of how the insoluble extracellular microenvironment guides formation and function of corneal tissues. Importantly, tissues developed during the study hold the potential of being advanced into clinically relevant studies to provide a novel therapeutic approach to the age-old problem of corneal blindness.