Collaborative Research: Engineer a functional 3D vascularized islet organoid from pluripotent stem cells

胰岛是产生和释放多种激素（主要是胰岛素）到血液中的微型器官。胰岛健康和功能的恶化导致糖尿病，已成为世界性的流行病。胰岛移植是一种很有前景的糖尿病治疗方法——将捐赠者分离的胰岛移植到患者体内。由于缺乏足够的胰岛供体，目前的重点是在实验室中从人类多能干细胞（hPSC）中产生胰岛。胰岛主要由产生激素的细胞组成，这些细胞与密集的毛细血管网络交织在一起，以有效地运输释放的激素。除了营养输送外，胰岛血管系统在胰岛发育和功能中起着至关重要的作用。因此，当从造血干细胞中提取胰岛类器官时，在胰岛内血管系统的工程设计上将有多种好处，这是本项目的目标。这一目标将通过整合多种新技术来实现，包括将胰腺细胞聚集成球体和包含从脂肪组织获得的微血管碎片（MFs）以增强血管化。再生类器官最重要的影响将是在糖尿病的细胞治疗中。一个更容易实现的目标是使用功能性胰岛类器官作为测试糖尿病药物化合物的功效和毒性的体外模型。代表匹兹堡大学、亚利桑那州立大学和路易斯维尔大学的跨学科教师团队将利用这个项目的多学科方法来培训研究生和本科生，并扩大外展项目，为不同的学生群体增加机会。该团队将制定一项联合暑期实习计划，两所大学的少数族裔学生将在另外两所大学实习，从而增加合作机会和学生培训。该合作项目的目标是从人类多能干细胞（hPSCs）中设计体外血管化胰岛类器官。造血干细胞的自组织将被设计成具有生理胰岛血管网络和内分泌功能的异质三维（3D）结构。该团队已经开发出一种新的水凝胶系统，通过hPSC衍生的胰腺祖细胞的自组织，密切模仿3D胰岛生理学。水凝胶平台可以精确控制3D培养配置，并允许多细胞聚集。这是对现状的实质性背离，在现状中，hPSCs在搅拌悬浮液中随机聚集，导致不同大小和表型的不受控制的聚集体。体外血管化将通过在工程三维细胞结构中加入分离的脂肪来源的微血管片段来进行工程。这些微碎片保留了血管生成所必需的内皮细胞、血管基质成分和支持细胞。这种血管网络的预嵌入和发展是一种实质性的背离，在目前的现状中，微血管网络的形成依赖于体内植入和宿主血管整合。该项目可能是第一次尝试从造血干细胞中产生体外血管化胰岛类器官。该研究计划围绕三个目标进行：1)确定诱导hPSC衍生细胞聚集的培养条件；2)在胰岛类器官内诱导胰岛特异性微血管网络；3)在体外和体内免疫功能低下小鼠模型中诱导和表征血管化类器官成熟的胰岛功能（内分泌表型和葡萄糖反应性胰岛素产生）。假设类器官内胰岛微环境的充分繁殖将诱导脂肪源性微血管的胰岛特异性血管特征和表型。这种再生的胰岛类器官将与胰腺组织和器官工程直接相关，并且所开发的方法具有改变组织工程领域的潜力。

PIs: Banerjee, Ipsita / Rege, Kaushal / Hoying, JamesProposal Numbers: 1706674 / 1706268 / 1706742Pancreatic islets are micro-organs that produce and release multiple hormones, primarily insulin, into the blood stream. Deterioration of islet health and function results in diabetes, which has become an epidemic healthcare problem worldwide. A promising treatment of diabetes lies in islet transplantation--where isolated islets from donors are transplanted into the patient. In the absence of sufficient islet donors, the current focus is on generating islets in the laboratory from human pluripotent stem cells (hPSC). Pancreatic islets primarily consist of hormone producing cells interlaced with a dense network of capillaries for efficient transport of released hormones. In addition to nutrient delivery, the islet vasculature plays a critical role in islet development and function. Hence, when deriving islet-organoids from hPSCs, there will be multiple benefits in engineering the intra-islet vasculature, which is the objective of this project. This objective will be achieved by integration of multiple novel techniques, including the aggregation of pancreatic cells into spheroids and the inclusion of microvessel fragments (MFs) obtained from adipose (fat) tissue to enhance vascularization. The most significant impact of the regenerative organoids will be in cell therapy for diabetes. An even more achievable goal is the use of functional islet organoids as an in-vitro model for testing the efficacy and toxicity of drug compounds for diabetes. The interdisciplinary faculty team, representing the University of Pittsburgh, Arizona State University and the University of Louisville, will leverage the multidisciplinary approach of this project to train students at the graduate and undergraduate levels and to broaden outreach programs to increase opportunities for a diverse population of students. The team will develop a joint summer internship program, where minority students from each institution will intern in the other two Universities, thereby enhancing collaborative opportunities as well as student training.The goal of this collaborative project is to engineer in-vitro vascularized pancreatic islet organoids from human pluripotent stem cells (hPSCs). Self-organization of hPSCs will be engineered into heterogeneous three-dimensional (3D) constructs with a physiological islet vascular network and endocrine function. The team has developed a novel hydrogel system that closely mimics the 3D islet physiology through self-organization of hPSC derived pancreatic progenitor cells. The hydrogel platform enables precise control over the 3D culture configuration as well as allowing multicellular aggregation. This is a substantive departure from status quo, where hPSCs are randomly aggregated in a stirred suspension resulting in uncontrolled aggregates of varying size and phenotype. In-vitro vascularization will be engineered by incorporating isolated adipose-derived microvessel fragments within the engineered 3D cellular construct. These microfragments retain the endothelial, vessel matrix components, and supporting cells necessary for angiogenesis. This pre-embedment and development of a vascular network is a substantive departure from status quo, where in-vivo implantation and host vasculature integration is relied upon for microvascular network formation. The project perhaps represents the first attempt to generate in-vitro vascularized pancreatic islet organoids from hPSCs. The Research Plan is organized around three aims: 1) to determine culture conditions inducing aggregation of hPSC derived cells; 2) to induce islet-specific microvascular network within the islet organoids; and 3) to induce and characterize mature islet functionality (endocrine phenotype and glucose responsive insulin production) in the vascularized organoids in vitro and in vivo in an immunocompromised mouse model. It is hypothesized that adequate reproduction of islet microenvironment within the organoid will induce islet-specific vascular characteristics and phenotype in the adipose-derived microvessels. Such regenerative islet organoids will be directly relevant for pancreatic tissue and organ engineering, and methods developed have the potential to transform the field of tissue engineering in general.