A 3D vascularized islet biomimetic to model type 1 diabetes

项目总结/摘要 1型糖尿病（T1 D）是一种自身免疫性疾病，被认为是由免疫介导的对糖尿病细胞的破坏引起的。 胰岛中产生胰岛素的β细胞。研究β细胞破坏的机制， 患有T1 D的人一直具有挑战性，因为大多数重要的免疫学事件发生在 诊断.此外，虽然啮齿动物模型在定义T1 D病因学的某些方面提供了信息， 啮齿动物和人类胰腺在胰岛结构方面存在根本差异， 血管，以及啮齿动物和人类免疫系统之间。此外，人的重要方面 T1 D病理学在啮齿动物模型中未复制。因此，为了充分了解人类T1 D 在病理生理学中，开发一个人类模型至关重要，在这个模型中，参与疾病的所有细胞的相互作用 过程（例如，β细胞、内皮细胞（EC）、先天性和适应性免疫细胞）可以在以下背景下进行研究： 正常的胰岛结构，包括脉管系统、基质细胞和天然胰岛基质。过去三年 通过NIH“人类胰岛仿生学联盟”，我们的团队（共同PI桑德：人类诱导多能 干细胞（hiPSC）和糖尿病;共同PI休斯：血管生物学和生物工程学;共同I Christman 生物材料和组织工程; co-I乔治微流体和运输）已经开发了一种基于微流体的 一个平台，其中原代人胰岛或hiPSC衍生的胰岛样簇由灌注的胰岛细胞网络支持， 人体微血管我们的3D血管化胰岛微器官（VMO-I）平台允许生理， 微血管介导的营养物、疾病相关刺激物或免疫细胞向胰岛的递送。我们提出 利用我们的VMO-I平台的独特功能来模拟胰岛中发生的细胞间相互作用， 在T1 D发病过程中的小生境，即免疫细胞外渗，组织渗透和迁移，以及 β-细胞杀伤。对于这些研究，Co-I Teyton将提供T1 D免疫学方面的专业知识。我们建议雇用两名 不同的体外模型：第一个是在UG 3阶段开发的，是非自体的，由原发性 从原代EC获得人胰岛和脉管系统。在这里，我们将介绍同种异体淋巴细胞（Aim G1） 或胰岛供体匹配的β细胞反应性T细胞克隆（Aim G2），以建立用于T细胞模型化的参数。 外渗和T细胞介导的β细胞杀伤。我们还将努力实现生成VMO-I模型的目标 完全源自hiPSC（Aim G3）。第二个模型是在UH 3阶段开发的，将是完全自体的， 包含源自T1 D患者hiPSC的β细胞、脉管系统和基质细胞，其将与 从同一患者的血液中分离的自身反应性T细胞。通过结合实时传感器和实时成像 通过分子和生化分析，我们将使用这些模型来研究胰岛细胞如何对T1 D做出反应， 相关的应激源，如促炎细胞因子、高血糖和缺氧，免疫细胞和β- 细胞相互作用，以及β细胞如何被杀死。最后，我们将证明该平台可用于评估 候选疗法的有效性，长期目标是利用该平台筛选新的治疗方法。

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is an autoimmune disease thought to be caused by immune-mediated destruction of the insulin-producing β-cells in the pancreatic islets. Studying the mechanisms that underlie β-cell destruction in humans with T1D has been challenging because most of the important immunological events occur before diagnosis. Furthermore, while rodent models have been informative in defining some aspects of T1D etiology, there are fundamental differences between the rodent and human pancreas with respect to islet architecture and vasculature, as well as between rodent and human immune systems. Additionally, important aspects of human T1D pathology are not replicated in the rodent models. Therefore, to fully understand human T1D pathophysiology, it is critical to develop a human model, where the interactions of all cells involved in the disease process (e.g. β-cells, endothelial cells (EC), innate and adaptive immune cells) can be studied in the context of normal islet architecture, including vasculature, stromal cells, and native islet matrix. Over the past three years through the NIH “Consortium on Human Islet Biomimetics”, our team (co-PI Sander: human induced pluripotent stem cells (hiPSC) and diabetes; co-PI Hughes: vascular biology and bioengineering; co-I Christman biomaterials and tissue engineering; co-I George microfluidics and transport) has developed a microfluidic-based platform in which primary human islets or hiPSC-derived islet-like clusters are supported by a network of perfused human microvessels. Our 3D vascularized islet micro-organ (VMO-I) platform allows for physiologic, microvessel-mediated delivery of nutrients, disease-relevant stimuli, or immune cells to the islets. We propose to leverage the unique features of our VMO-I platform to model the cell-cell interactions that occur in the islet niche during T1D pathogenesis, namely immune cell extravasation, tissue penetration, and migration as well as β-cell killing. For these studies co-I Teyton will provide expertise in T1D immunology. We propose to employ two distinct in vitro models: The first, developed in the UG3 phase, is non-autologous and comprised of primary human islets and vasculature from primary EC. Here, we will introduce either allogeneic lymphocytes (Aim G1) or islet donor-matched β-cell-reactive T cell clones (Aim G2) to establish parameters for modeling T cell extravasation and T cell-mediated β-cell killing. We will also work towards the goal of generating a VMO-I model entirely derived from hiPSC (Aim G3). The second model, developed in the UH3 phase, will be fully autologous, comprising β-cells, vasculature, and stromal cells derived from T1D patient hiPSC, which will be combined with autoreactive T cells isolated from blood of the same patient. By combining live-sensors and real-time imaging with molecular and biochemical assays, we will use these models to study how cells in the islet respond to T1D- relevant stressors, such as pro-inflammatory cytokines, hyperglycemia, and hypoxia, how immune cells and β- cells interact, and how β-cells are killed. Finally, we will demonstrate that the platform can be used to assess candidate therapies for efficacy with the long-term goal to utilize the platform to screen for new therapeutics.