hiPSC-derived tissue mimetics of the retina blood barrier

hiPSC 衍生的视网膜血屏障组织模拟物

• 批准号: 10080730
• 负责人: Danielle S. Benoit
• 金额: $ 22.41万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10080730
• 关键词: Address; Adult; Age related macular degeneration; Animal Model; Basement membrane; Biocompatible Materials; Blindness; Blood Vessels; Blood-Retinal Barrier; Bruch' s basal membrane structure; Cell Culture Techniques; Cell Differentiation process; Cells; Characteristics; Coculture Techniques; Collagen; Complex; Cues; Data; Deposition; Development; Diffusion; Disease; Disease model; Drug Screening; Elastin; Endothelial Cells; Engineering; Environment; Exposure to; Eye; Eye diseases; Fibronectins; Functional disorder; Future; Genetic; Gold; Growth Factor; Human; Hydrogels; In Vitro; Individual; Laminin; Matrix Metalloproteinases; Membrane; Mesenchymal; Mesenchymal Stem Cells; Metabolic; Modeling; Mus; Nutrient; Ocular Physiology; Pathology; Patients; Phagocytosis; Pharmacotherapy; Physiological; Pigment Epithelium; Pigmentation physiologic function; Pigments; Play; Protocols documentation; Public Health; Research; Resistance; Retinal Degeneration; Role; Structure; Structure of retinal pigment epithelium; Study models; TIMP3 gene; Testing; Tight Junctions; Tissue Engineering; Tissue Model; Tissue Transplantation; Tissues; VEGFA gene; Vitamin A; Work; arginyl-glycyl-aspartyl-serine; cell type; endothelial stem cell; ethylene glycol; flexibility; fluorescein isothiocyanate dextran; human fetal retinal pigment epithelial cell; improved; in vitro Model; in vivo; induced pluripotent stem cell; induced pluripotent stem cell technology; macromolecule; macula; mimetics; model development; mouse model; multidisciplinary; novel therapeutics; photoreceptor cell outer segment; retinal progenitor cell; stem cell derived tissues; therapeutic development; wasting

The retinal pigment epithelium (RPE)-Bruch’s Membrane (BrM)-choriocapillaris (CC) complex is a highly selective diffusional barrier for nutrients and metabolic wastes in the eye. Dysfunctions in the RPE-BrM-CC complex underlies eye pathologies such as age-related macular degeneration (AMD), the leading cause of adult blindness in the US. Within the RPE-BrM-CC, BrM is a multilayered tissue that divides RPE and CC and is the first structure to show anomalies in AMD, yet it is not clear whether RPE and/or CC dysfunction initiates BrM alterations. Murine models fail to recapitulate AMD since mice lack macula. The inability to use animal models or recreate a functional RPE-BrM-CC tissue complex limits our ability to investigate crucial aspects of eye diseases, including AMD, where the integrity of the entire tissue is compromised. Therefore, we hypothesize that by using developmentally-inspired cues, a functional tissue mimetic can be developed for ex vivo study. Specifically, we will exploit human induced pluripotent stem cell (hiPSC) technology and tissue engineering to establish functional tissue mimetics. Because of their modularity, hiPSC-derived in vitro models allow the flexibility to study the role individual cell type(s) and intercellular interaction in disease pathophysiology. Our strong preliminary data demonstrate that hiPSC-RPE seeded onto RGDS-functionalized poly(ethylene glycol) (PEG) hydrogels are stable and become pigmented over 2-3 weeks. RPE layers deposit basement membrane, composed of some BrM components. Furthermore, CC-like vasculature, complete with Col6-postiive basement membrane, can be developed from hiPSC-endothelial cells (EC) and mesenchymal stem cells (MSC) entrapped within the PEG hydrogels underlying RPE. However, our current tissue mimetic lacks structurally complete BrM, which limits overall tissue mimetic structure and function. During development, RPE cells differentiate and become pigmented with support from underlying mesenchymal matrix and soluble cues. Then the CC layer develops while the mature BrM is deposited by coordinated interplay of the RPE and CC. Thus, to improve the mimetic, our aims are to 1) exploit developmentally-inspired cues to further enhance BrM structural development. In Aim 1a, the temporal introduction of the CC tissue mimetics after hydrogel- seeded RPE layer maturation/pigmentation and in Aim 1b, soluble mesenchymal cues via co-culture, will be explored to further promote BrM development. Aim 2 will assess the RPE-BrM-CC mimetic function as compared to in vivo levels and gold standard RPE culture models. Our expertise with engineering biomaterials to regulate the cell environment (Benoit) and eye physiology and hiPSC-derived RPE, MSC, and EC cell types is crucial to developing RPE-BrM-CC tissue mimetics using patient-derived cells. Successful completion of these aims would be a significant step towards ocular disease modeling and subsequent development of novel drug therapies for several retinal degenerative diseases, including age-related macular degeneration (AMD). !

视网膜色素上皮(RPE)-Bruch膜(BRM)-脉络膜毛细血管(CC)复合体是一种高度 营养物质和代谢废物在眼睛中的选择性扩散屏障。RPE-BRM-CC的功能障碍 复杂的眼部病理，如老年性黄斑变性(AMD)，是 在美国，成人失明。在RPE-BRM-CC内，BRM是一个多层组织，将RPE和CC分开， 是第一个显示AMD异常的结构，但尚不清楚RPE和/或CC功能障碍是否启动 BRM更改。小鼠模型不能概括AMD，因为小鼠没有斑点。不能使用动物 模型或重建功能性RPE-BRM-CC组织复合体限制了我们研究关键方面的能力 眼科疾病，包括AMD，整个组织的完整性受到损害。因此，我们 假设通过使用受发育启发的线索，可以为EX开发出一种功能组织模拟物 活体研究。具体地说，我们将利用人类诱导多能干细胞(HiPSC)技术和组织 工程学以建立功能组织模拟学。由于它们的模块化，hPSC衍生的体外模型 允许灵活地研究单个细胞类型(S)和细胞间相互作用在疾病中的作用 病理生理学。我们强有力的初步数据表明，HiPSC-RPE种在RGDS上是功能化的 聚乙二醇(PEG)水凝胶是稳定的，在2-3周内变得有色。RPE层沉积 基底膜，由一些BRM组分组成。此外，CC样血管形成，完整的 Col6阳性基底膜可由HPSC-内皮细胞(EC)和间充质形成 干细胞(MSC)被包裹在RPE下面的聚乙二醇水凝胶中。然而，我们目前的组织模拟物 缺乏结构完整的BRM，这限制了整体组织的模拟结构和功能。在开发过程中， RPE细胞在底层间充质基质的支持下分化并着色，可溶 暗示。然后，通过RPE和RPE的协调相互作用，在成熟的BRM沉积的同时发展CC层 CC.因此，为了提高模仿性，我们的目标是1)利用受发展启发的线索来进一步增强 BRM结构发展。在目标1a中，水凝胶后CC组织模拟物的时间介绍- 种子RPE层成熟/着色和在目标1b中，通过共培养的可溶间充质线索将被 探索进一步推动BRM发展。目标2将评估RPE-BRM-CC模拟功能为 与活体水平和黄金标准RPE培养模型进行比较。我们在工程生物材料方面的专业知识 调节细胞环境(Benoit)、眼生理和HiPSC衍生的RPE、MSC和EC细胞类型 对于使用患者来源的细胞开发RPE-BRM-CC组织模拟物至关重要。成功完成 这些目标将是朝着眼病建模和随后的小说发展迈出的重要一步 治疗多种视网膜退行性疾病的药物疗法，包括老年性黄斑变性(AMD)。 好了！