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 有窗孔的脉络膜毛细血管(CC)，与血液供应相连。RPE-CC复合函数 协同支持对视力至关重要的感光细胞健康。一如既往地，RPE功能障碍- 在包括老年性黄斑变性(AMD)在内的眼科疾病中，CC会导致视网膜变性， 在美国，导致成年人失明的最大原因是50岁。然而，In的缺乏 忠实地概括RPE-CC复合体的体外组织模拟显著损害了对 OBRB的正常和患病的生理学。RPE-CC组织模拟学发展面临的主要挑战 是我们对人类视网膜发生的有限理解。这与CC层尤其相关，在CC层中 大多数推论来自于对胚胎人类视网膜的组织学研究。人类诱导多能性 干细胞(HiPSCs)为体外建立人卵巢癌模型提供了独特的平台。事实上，有几项研究已经 现在表明，与RPE-CC复合体相关的特定细胞类型，包括RPE、内皮细胞(ECs)和 间充质干细胞(MSCs)可以从HiPSCs分化而来。此外，我们最近开发了一种 利用聚乙二醇基水凝胶的通用性开发原始的RPE-CC组织模拟物 工程化ECM(EECM)和HiPSC派生的目标细胞，以模拟RPE、ECs和 间充质的我。RPE-CC组织模拟物能够概括体内的重要生理特征 RPE-CC复合体，如CC样有窗的维管系统，以前在体外是难以捉摸的。虽然 这个模型为生理性RPE-CC的发展提供了一个框架，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- 细胞信号，以及缺乏血管灌流，导致组织模型不能在体内完全概括 结构(例如，明确定义的Bruch膜状ECM和CC空间血管结构)和功能(例如， 养分运输和大分子扩散)。在这项提案中，我们假设更好地理解 EECM要求(目标1)，二)纳入时间发展线索(目标2)，以及 血管灌注，(目标3)将促进模块化、空间相关和功能性RPE-CC的发展 组织模拟(S)。最终，一个生理的、模块化的人类外视网膜(RPE-CC)的发展 组织模拟将对后续的疾病建模、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.