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

PIS：Banerjee，Ipsita/Rege，Kaushal/Hoying，James建议编号：1706674/1706268/1706742胰岛是产生和释放多种激素的微型器官，主要是胰岛素，进入血流。胰岛健康和功能的恶化导致糖尿病，它已经成为一个世界性的医疗保健问题。糖尿病的一种有希望的治疗方法是胰岛移植--从捐赠者那里分离出来的胰岛被移植到患者体内。在缺乏足够的胰岛捐赠者的情况下，目前的重点是在实验室中利用人类多能干细胞(HPSC)生成胰岛。胰岛主要由荷尔蒙产生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荷尔蒙。除了营养物质的输送，胰岛血管对胰岛的发育和功能也起着至关重要的作用。因此，当从hPSC中获得胰岛器官时，设计胰岛内血管系统将会有多种好处，这也是本项目的目标。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，并将从脂肪组织中获得的微血管碎片(MF)包含进来以促进血管形成。再生类有机物最重要的影响将是糖尿病的细胞治疗。一个更可实现的目标是使用功能性胰岛有机化合物作为体外模型来测试治疗糖尿病的药物化合物的有效性和毒性。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该项目的多学科方法来培训研究生和本科生，并扩大外展计划，以增加不同学生群体的机会。该团队将开发一个联合暑期实习计划，每个机构的少数族裔学生将在其他两所大学实习，从而增加合作机会和学生培训。这个合作项目的目标是从人类多能干细胞(HPSC)中设计体外血管化胰岛器官。HPSCs的自组织将被工程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型的水凝胶系统，通过hPSC来源的胰腺前体细胞的自组织来接近模拟3D胰岛的生理。水凝胶平台能够精确控制3D培养配置，并允许多细胞聚集。这是对现状的实质性改变，在现状中，hPSC随机聚集在搅拌的悬浮液中，导致不受控制的大小和表型不同的聚集体。体外血管形成将通过将分离的脂肪来源的微血管片段整合到工程化的3D细胞结构中来实现。这些微片段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。这种血管网络的预植入和发展是对现状的实质性偏离，在现状中，微血管网络的形成依赖于体内植入和宿主血管系统的整合。该项目可能是从hPSC产生体外血管化胰岛类器官的第一次尝试。该研究计划围绕三个目标组织：1)确定诱导hPSC来源细胞聚集的培养条件；2)在胰岛器官体内诱导胰岛特异性微血管网络；以及3)在体外和体内免疫受损的小鼠模型中诱导和鉴定血管化器官中成熟的胰岛功能(内分泌表型和葡萄糖反应性胰岛素的产生)。假设在类器官内充分复制胰岛微环境将在脂肪来源的微血管中诱导胰岛特有的血管特征和表型。这种再生的胰岛类器官将直接与胰腺组织和器官工程相关，开发的方法有可能改变组织工程领域。

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.