Bioengineering Cornea with Autologous Stem Cells

自体干细胞生物工程角膜

• 批准号: 9113712
• 负责人: JAMES L FUNDERBURGH
• 金额: $ 38.71万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9113712
• 关键词: 3-Dimensional; Address; Age; Allogenic; Architecture; Area; Autologous; Basement membrane; Biochemical; Biomechanics; Biomedical Engineering; Biomimetics; Biopsy; Biopsy Specimen; Blindness; Cations; Cell Culture Techniques; Cell Density; Cells; Cellular Morphology; Clinical; Collagen; Cornea; Corneal Endothelium; Corneal Opacity; Corneal Stroma; Cues; Data; Descemet' s membrane; Development; Dimensions; Electrical Resistance; Endothelial Cells; Engineering; Extracellular Matrix; Extracellular Matrix Proteins; FGF2 gene; Failure; Future; Gene Expression Profile; Generations; Genes; Goals; Graft Rejection; Human; Hypoxia; Immune; In Vitro; Individual; Ion Transport; Keratoplasty; Melons; Membrane; Membrane Proteins; Modeling; Molecular; Nanostructures; Oryctolagus cuniculus; Outcomes Research; Patients; Penetrating Keratoplasty; Performance; Peripheral; Process; Property; Proteins; Psyche structure; Publications; Publishing; Pump; Research; Research Project Grants; Retrospective Studies; Role; Shapes; Source; Stem cells; Structure; Surface; Techniques; Testing; Therapeutic; Thick; Tissue Donors; Tissue Engineering; Tissues; Translations; Transplant Recipients; Transplantation; Transplanted tissue; Universities; Validation; Vision; Visually Impaired Persons; Work; adult stem cell; base; clinically relevant; extracellular; flexibility; high risk; immunogenic; in vivo; in vivo Model; innovation; limbal; monolayer; nanostructured; novel strategies; novel therapeutic intervention; public health relevance; research study; response; scaffold; stem cell biology; stem cell differentiation

 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.

 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，以实现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。 这些组织将由成体干细胞进行生物工程改造，这些细胞可以从待治疗的个体中获得。 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，该支架是使用仿生、表面诱导的组装过程制造的。 匹兹堡大学芬德堡实验室将从捐赠的人类角膜的角膜缘基质中获取干细胞。 他们对这些角膜基质干细胞（CSSC）的广泛研究表明，它们可以分化为基质角膜细胞和角膜内皮细胞，这是造成大多数角膜混浊的组织。 最近，我们证明可以从活检样本中获得 CSSC，这为生成自体生物工程组织提供了机会。 卡内基梅隆大学 Feinberg 实验室开发了一种组装天然细胞外基质蛋白的新方法，以生产具有明确 3D 结构的组织样支架。 目标 1 将侧重于组装生物工程基质，其中该项目将阐明支架提供的空间和生化线索以及可溶性线索（缺氧、TGFβ3、FGF2）在从 CSSC 生成基质样组织中的作用。 各层组织将堆叠起来形成类似于角膜基质的多层 3D 组织。 将检查这些实验中产生的组织的组成、结构、生物力学特性，以及在体内兔模型中作为基质功能替代品的能力。 目标 2 将生物工程角膜内皮，基于我们的观察，CSSC 培养为球体，然后转移到含有基底膜蛋白的培养基中，形成多边形单层并表达角膜内皮的典型基因。 Feinberg 小组利用表面引发的组装技术，开发出一种生物工程的后弹力层膜，可增强培养的角膜内皮细胞的分化、扩张和功能。 CSSC 将在这些膜上生成角膜内皮层，并测试基因表达模式、细胞密度、细胞形态和内皮泵功能。 该构建体的功能将在长期兔体内模型中得到证明。 该项目将采用创新的实验方法，对不溶性细胞外微环境如何引导角膜组织的形成和功能产生新的认识。 重要的是，研究期间开发的组织有可能进入临床相关研究，为角膜失明这一古老问题提供一种新的治疗方法。