Humanized Mouse Avatars for T1D

 描述（由申请人提供）：迄今为止，人类1型糖尿病（T1 D）的研究未能提供对疾病原因的机制性理解，主要是因为患者必须在自身免疫性攻击开始后很长时间内进行分析。我们对介导人类T1 D启动和进展的关键分子和细胞的无知可能是缺乏重要的新治疗干预措施的原因。在这里，我们提出使用iPS衍生的β细胞、胸腺上皮细胞（TEC）和来自T1 D患者的免疫系统重建人T1 D，所述T1 D患者植入基于NOD-scid-IL 2 rgnull（NSG）菌株的新型免疫缺陷小鼠模型中。为了实现我们的目标，我们提出了两个目标。目的1将在优化的免疫缺陷小鼠（OPTI-MICE）中验证人免疫和胰腺β细胞功能。目前可用的OPTI-MICE将使用遗传技术进行改进，以：1）增强人细胞的植入; 2）允许自发性和诱导性高血糖症; 3）支持人HLA等位基因和细胞因子的表达;以及4）敲除损害人细胞植入和功能的小鼠基因。这套改进将通过植入胎儿人类干细胞和组织来验证。目的2将使用来自1型糖尿病iPS细胞的细胞在小鼠中重建人T1 D。这些iPS细胞将用于产生三种关键细胞类型：将产生免疫系统的造血干细胞（HSC），TEC和β细胞，所有这些都是T1 D病理学的组成部分。这些细胞将通过使用定向分化和重编程策略获得。我们已经成功地从人对照和T1 D患者iPS细胞中产生功能性人β细胞，通过最近开发的用于定向分化的多步骤方案，为我们的研究提供了标准化和可重复的β细胞来源。将使用两种技术产生功能性人HSC：1）iPS细胞的定向分化， 2)分化造血细胞重编程 细胞使用确定的因子来衍生诱导的HSC。将使用与用于实现完全分化的人辟田胞的那些类似的定向分化方案来产生功能性人TEC。每种细胞类型都将在体外和体内进行严格的分析，以确保其完全功能。将来自单个供体的分化细胞共移植到OPTI-MICE中，从而在动物模型中重建个体患者的疾病。将仔细监测移植小鼠是否出现自身抗体和自身反应性抗体。 T-细胞，以及用于破坏β-细胞。这种新的人类T1 D模型将允许详细观察，操作和分析T1 D，使我们能够确定哪些细胞和抗原启动T1 D。这种机械的见解将适当地为治疗甚至预防这种疾病的新方法提供信息。为了实现我们的目标，我们组建了一个互动的研究团队，使用Helmsley慈善信托基金的种子资金。我们已经合作并定期开会超过5年，并在完成该项目所需的相关领域拥有专业知识。

 DESCRIPTION (provided by applicant): To date, studies of human type 1 diabetes (T1D) have failed to provide a mechanistic understanding of the causes of the disease, largely because patients must be analyzed long after the autoimmune attack was initiated. Our ignorance of the key molecules and cells mediating the initiation and progression of human T1D may well underlie the paucity of significant new therapeutic interventions. Here we propose to reconstruct human T1D, using iPS-derived β cells, thymic epithelial cells (TEC), and immune systems derived from T1D patients implanted in a novel immunodeficient mouse model based on the NOD-scid-IL2rgnull (NSG) strain. To accomplish our goal, we propose 2 aims. Aim 1 will validate human immune and pancreatic beta cell functions in optimized immunodeficient mice (OPTI-MICE). Currently available OPTI-MICE will be improved using genetic techniques to: 1) enhance engraftment of human cells; 2) allow for spontaneous and inducible hyperglycemia; 3) support expression of human HLA alleles and cytokines; and 4) knockout mouse genes that impair human cell engraftment and function. This suite of improvements will be validated using implantation of fetal human stem cells and tissues. Aim 2 will reconstruct human T1D in mice using cells derived from Type 1 diabetic iPS cells. These iPS cells will be used to produce the three key cell types: hematopoietic stem cells (HSC) that will generate immune systems, TEC, and β-cells, all integral to the pathology of T1D. These cells will be derived through the use of directed differentiation and reprogramming strategies. We have been successful in generating functional human β cells from human control and T1D patient iPS cells by a recently developed multi-step protocol for directed differentiation, providing a standardized and reproducible source of β cells our studies. Functional human HSC will be generated using two technologies: 1) directed differentiation of iPS cells and 2) reprogramming of differentiated hematopoietic cells using defined factors to derive induced-HSCs. Functional human TEC will be generated using directed differentiation protocols similar to those used to achieve fully differentiated human β cells. Each cell type will be subjected to rigorous analysis in vitro and in vivo to ensur full functionality. Differentiated cells derived from a single donor will be co-transplanted into OPTI-MICE, thus reconstituting an individual patient's disease in an animal model. Transplanted mice will be carefully monitored for the emergence of autoantibodies and autoreactive T-cells, and for destruction of β-cells. This new model of human T1D will permit detailed observation, manipulation, and analysis of T1D as it progresses, enabling us to determine which cells and antigens initiate T1D. Such mechanistic insights will properly inform new approaches to curing, or even preventing, this disease. To accomplish our goal, we have assembled an interactive team of researchers formed using seed monies from the Helmsley Charitable Trust. We have now been working together and meeting regularly for over 5 years and have expertise in the relevant areas required to accomplish this project.