Using hiPSCs to develop physiologically-relevant outer retina tissue mimetics

外血视网膜屏障（oBRB）由视网膜色素上皮（RPE）细胞和下层视网膜色素上皮（RPE）细胞组成。 有孔脉络膜毛细血管（CC）与血液供应接口。RPE-CC复合函数 协同地支持对视力至关重要的感光细胞健康。一致的，视网膜色素上皮的功能障碍- CC导致近视性眼病中的视网膜变性，包括年龄相关性黄斑变性（AMD）， 是美国50岁以上成年人不可逆失明的最大原因。然而，缺乏在 忠实地再现RPE-CC复合物的体外组织模拟物显著损害了对RPE-CC复合物的研究。 obRB的正常和患病生理。RPE-CC组织模拟物开发的主要挑战 是我们对人类视网膜发生的有限理解。这尤其与CC层相关，在CC层中， 大多数推论是从胚胎人视网膜的组织学研究中得出的。人诱导多能 干细胞（hiPSC）提供了开发体外oBRB模型的独特平台。事实上，有几项研究 现在显示，与RPE-CC复合物相关的特定细胞类型，包括RPE、内皮细胞（EC）和 间充质干细胞（MSC）可以从hiPSC分化。此外，我们最近开发了 一种原始的RPE-CC组织模拟物，通过利用聚（乙二醇）（PEG）水凝胶基 工程化ECM（eECM）和hiPSC衍生的靶细胞，以模拟RPE、EC和 间充质RPE-CC组织模拟物能够重现体内的重要生理特征 RPE-CC复合物，如CC样有孔血管，以前在体外难以实现。虽然 该模型提供了生理RPE-CC发展的框架，目前它有几个局限性， 包括未优化的eECM生化和生物物理线索，缺乏发育指导的颞叶细胞， 细胞提示，以及缺乏血管灌注，导致组织模型在体内不能完全重现 结构（例如，明确的Bruch膜样ECM和CC空间血管结构）和功能（例如， 营养物质运输和大分子扩散）。在这个建议中，我们假设，更好地了解 eECM要求（目标1），ii）纳入时间发育线索（目标2），以及整合 血管灌注（目的3）将促进模块化、空间相关和功能性RPE-CC的发展 组织模拟物。最终，生理和模块化人类外视网膜（RPE-CC）的发展 组织模拟物将对随后的疾病建模、药物筛选和 移植研究。

The outer blood retina barrier (oBRB) comprises of the retinal pigment epithelium (RPE) cells and underlying fenestrated choriocapillaris (CC) that interfaces with the blood supply. The RPE-CC complex functions synergistically to support photoreceptor cell health that is critical for vision. Consistently, dysfunction of the RPE- CC leads to retinal degeneration in myraid eye diseases, including age-related macular degeneration (AMD), the single biggest cause of irreversible blindness in adults > 50 years of age in the US. However, the lack of in vitro tissue mimetics that faithfully recapitulate the RPE-CC complex has significantly impaired the study of normal and diseased physiology of the oBRB. A major challenge for the development of RPE-CC tissue mimetics is our limited understanding of human retinogenesis. This is especially relevant to the CC layer in which the majority of inferences are drawn from histological studies of embryonic human retina. Human induced pluripotent stem cells (hiPSCs) provide a unique platform to develop in vitro oBRB models. Indeed, several studies have now shown that specific cell types relevant to the RPE-CC complex, including RPE, endothelial cells (ECs) and mesenchymal stem cells (MSCs) can be differentiated from hiPSCs. Furthermore, we have recently developed a primitive RPE-CC tissue mimetic by exploiting the versatility of poly(ethylene glycol)(PEG) hydrogel-based engineered ECM (eECM) and hiPSC-derived target cells to emulate the spatial organization of RPE, ECs, and mesenchyme. The RPE-CC tissue mimetic is able to recapitulate important physiological features of the in vivo RPE-CC complex, such as CC-like fenestrated vasculature, that had previously been elusive in vitro. Although this model provides a framework for physiological RPE-CC development, it currently has several limitations, including unoptimized eECM biochemical and biophysical cues, lack of developmentally-instructed temporal cell- cell cues, and absence of vascular perfusion, resulting in a tissue model that does not fully recapitulate in vivo structure (e.g., well-defined Bruch’s membrane-like ECM and CC spatial angioarchitecture) and function (e.g., nutrient transport and macromolecular diffusion). In this proposal, we hypothesize that better understanding of the eECM requirements (Aim 1), ii) incorporation of temporal developmental cues (Aim 2), and integration of vascular perfusion, (Aim 3) will promote development of modular, spatially relevant, and functional RPE-CC tissue mimetic(s). Ultimately, the development of a physiological and modular human outer retina (RPE-CC) tissue mimetic will have important implications for subsequent disease modeling, drug screening, and transplantation studies.