hiPSC-derived tissue mimetics of the retina blood barrier

视网膜色素上皮（RPE）-Bruch膜（BrM）-脉络膜毛细血管（CC）复合体是高度依赖于视网膜色素上皮细胞（RPE）的一种细胞因子。 眼内营养物质和代谢废物的选择性扩散屏障。RPE-BrM-CC功能障碍 老年性黄斑变性（AMD）是老年性黄斑变性的主要原因。 美国的成人失明在RPE-BrM-CC内，BrM是将RPE和CC分开的多层组织， 是AMD中显示异常的第一个结构，但尚不清楚RPE和/或CC功能障碍是否引发AMD。 BRM改变。小鼠模型不能概括AMD，因为小鼠缺乏黄斑。无法使用动物 模型或重建功能性RPE-BrM-CC组织复合体限制了我们研究RPE-BrM-CC关键方面的能力。 包括AMD在内的眼部疾病，其中整个组织的完整性受到损害。所以我们 假设通过使用发育启发的线索，可以开发功能性组织模拟物， 体内研究具体来说，我们将利用人类诱导多能干细胞（hiPSC）技术和组织 工程化以建立功能性组织模拟物。由于其模块性，hiPSC衍生的体外模型 允许灵活地研究单个细胞类型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，接种到RGDS官能化的 聚（乙二醇）（PEG）水凝胶是稳定的，并在2-3周内着色。RPE层存款 基底膜，由一些BrM成分组成。此外，CC样脉管系统， Col 6-阳性基底膜，可以从hiPSC-内皮细胞（EC）和间充质细胞（MSC）发育而来。 干细胞（MSC）包埋在RPE下面的PEG水凝胶内。然而，我们目前的组织模拟 缺乏结构完整的BrM，这限制了整体组织模拟结构和功能。在开发过程中， RPE细胞分化并在下面的间充质基质和可溶性基质的支持下着色。 线索然后，CC层发展，而成熟的BrM通过RPE和 CC.因此，为了改善模仿，我们的目标是1）利用发展启发的线索，以进一步提高 BRM结构发展。在目标1a中，在水凝胶-凝胶注射后暂时引入CC组织模拟物。 接种的RPE层成熟/色素沉着和目标1b中，通过共培养的可溶性间充质线索，将被 探索进一步促进BRM的发展。目标2将评估RPE-BrM-CC模拟功能， 与体内水平和金标准RPE培养模型相比。我们在工程生物材料方面的专业知识 调节细胞环境（Benoit）和眼生理学以及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 这些目标将是朝向眼部疾病建模和随后开发新的治疗方法的重要步骤。 药物治疗几种视网膜变性疾病，包括年龄相关性黄斑变性（AMD）。 !

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). !