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Differentiation and Integration of Trisomy 21 iPSCs in an Animal Model

动物模型中 21 三体 iPSC 的分化和整合

基本信息

批准号：
9538075
负责人：
Wenbin Deng
金额：
$ 32.58万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9538075
关键词：
Affect Alzheimer&apos s Disease Animal Model Animal Testing Animal Testing Alternatives Animals Area Astrocytes Autologous Autopsy Behavior Biochemical Biological Assay Biological Models Biological Neural Networks Biology Biomedical Research Brain Brain Diseases Candidate Disease Gene Cell Culture Techniques Cell Line Cell Polarity Cell Transplantation Cell model Cells Cellular biology Cerebrum Chromosomes, Human, Pair 21 Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Cognition Cognition Disorders Cognitive deficits Collection Communication Complement Complex Congenital Abnormality Data Defect Development Developmental Biology Developmental Gene Disease Disease Progression Disease model Dissection Down Syndrome Drug Screening Early Intervention Electrophysiology (science)Embryo Engineering Environment Feasibility Studies Foundations Fragile X Syndrome Future Gene Dosage Gene Targeting Generations Genes Genetic Genetic Diseases Genetic Engineering Genetic Predisposition to Disease Genetic Screening Genotype Human Human Chromosomes Human Genome Hydrogels IFNAR1 gene IFNAR2 gene Immune In Vitro Individual Inflammation Inflammatory Integrins Intellectual functioning disability Interferons Intervention Investigation Knock-out Knowledge Learning Mediating Memory Microfabrication Modeling Molecular Molecular Biology Morphology Mus Nature Neonatal Neurodegenerative Disorders Neuroglia Neurologic Neurons Neurophysiology - biologic function Organ Organoids Paper Pathogenesis Pathologic Pathology Pathway interactions Patients Pharmacology Phenotype Physiological Physiology Plant Roots Positioning Attribute Process Property Prosencephalon Publishing Quality of life Rag1 Mouse Reagent Regulator Genes Regulatory Element Research Research Infrastructure Resources Role Sampling Scientist Shapes Source Stem cells Symptoms System Technology Testing Tissue Model Tissue Sample Tissue-Specific Gene Expression Tissues Toxic effect Transgenic Animals Translating Translational Research Transplantation Trisomy Validation Work astrocyte progenitor base behavioral outcome body system cell behavior cell type cohort common treatment developmental disease dosage drug discovery drug testing epigenetic profiling experimental study follow-up gene function genetic approach genetic information genetic profiling genome editing human disease human model human tissue improved in vitro Model in vivo in vivo Model induced pluripotent stem cell innovation insight interest materials science mouse model neuroinflammation neurotoxicity new therapeutic target novel novel strategies novel therapeutics overexpression pleasure pre-clinical prevent public health relevance relating to nervous system response severe intellectual disability small molecule libraries stem cell technology three dimensional structure tissue culture tool two-dimensional

项目摘要

PROJECT SUMMARY / ABSTRACT: Differentiation and Integration of Trisomy 21 iPSCs into Cerebral Tissues: Modeling Down Syndrome using Patient-specific iPSC-derived CNS Organoids and Humanized Chimeric Mice. Down syndrome (DS) is caused by trisomy 21, the triplication of human chromosome 21 (HSA21), and is the most common genetic cause of intellectual disability. We have successfully established and characterized multiple lines of iPSCs derived from DS patients. Particularly, we have established more than 50 DS Trisomy 21 iPSC lines, and obtained multiple pairs of corresponding isogenic disomy 21 control lines from these DS iPSCs. In addition, we have implemented CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) technology in making genetic corrections in iPSCs. Modeling of human genetic diseases has previously been largely dependent upon availability of either pathological analysis of postmortem human tissue samples or recapitulation of human disease in transgenic animal models; better research tools for disease modeling are needed. Patient-specific iPSCs are excellent tools and versatile resources for this kind of translational research. As iPSCs are generated on an individual basis, iPSCs may be the optimal cellular material to use for disease modeling, drug discovery, and development of patient-specific therapies. We have already generated a significant amount of preliminary data. We have used a highly efficient CRSPR system to precisely control and normalize genes of interest on HSA21. We have also developed a system of 3- dimentional (3D) CNS organoid (CO) culture from DS iPSCs, which better recapitulates brain development and disease pathogenesis than the conventional 2-dimentional (2D) flat culture, and allows for in-depth characterization by electrophysiological assays. The CNS organoid technology represents an excellent approach for disease modeling; the cerebral organoids generated from patient iPSCs can be used as a model to recapitulate complex neural developmental diseases such as DS. In addition, we have generated a humanized chimeric mouse model, in which DS iPSC derived astrocytes are grafted to the neonatal mouse brains. The detailed genetic etiology for the various symptoms in DS remains elusive. Taking the advantage of these unique tools and resources, we will create novel in vitro and in vivo models of DS with human iPSCs derived from patients to recapitulate the defects in neural differentiation in DS. In support of the feasibility of this proposal, we have obtained the necessary materials and expertise to be used in this study, and have published a rather massive paper on DS iPSCs [Chen C, Jiang P, Xue H, Peterson SE, Tran HT, McCann AE, Parast MM, Li S, Pleasure DE, Laurent LC, Loring JF, Liu Y, Deng W. (2014) Role of astroglia in Down’s syndrome revealed by patient-derived human-induced pluripotent stem cells. Nature Communications. 5:4430 doi: 10.1038/ncomms5430 (2014)]. Our preliminary data show that both trisomy and the isogenic disomy DS astrocytes are able to repopulate the mouse brain, allowing for further interrogation of in vivo behavior of these cells and examination of their effects on neuroinflammation and cognition of the animals. Building upon prior work on multiple genes in pathways of astrocyte-mediated inflammation, we propose to produce both in vitro CNS organoids and in vivo chimeric mouse models to investigate the critical role of these astrocytic inflammatory genes (S100B, IFNAR1, IFNAR2) in development and function of DS patient-derived iPSCs. Taken together, we will use a novel platform of both an in vitro 3D organoid culture system and an in vivo humanized chimeric mouse model using DS patient-derived iPSCs. These models will provide fundamental insights into neural function in the physiological environment of 3D organoids and in early development of human cells in a living animal. The completion of the project will immensely bolster DS pathogenesis studies using patient iPSCs, as well as biochemical and molecular approaches complemented with investigation into neural network functionality. These insights will undoubtedly impact on the treatment of patients with DS.

项目摘要/摘要： 21 三体 iPSC 向脑组织的分化和整合：向下建模使用患者特异性 iPSC 衍生的中枢神经系统类器官和人源化嵌合小鼠进行综合征。向下综合症 (DS) 是由 21 三体症（人类 21 号染色体 (HSA21) 的三倍体）引起的，是最常见的智力障碍的常见遗传原因。我们已经成功建立并表征了多个来自 DS 患者的 iPSC 系。特别是，我们已经建立了超过50个DS Trisomy 21 iPSC 系，并从这些 DS iPSC 中获得了多对相应的同基因二体性 21 对照系。在此外，我们还实施了 CRISPR/Cas9（成簇规则间隔短回文在 iPSC 中进行基因校正的重复/CRISPR 相关）技术。人类遗传建模疾病以前在很大程度上取决于死后病理分析的可用性转基因动物模型中的人体组织样本或人类疾病的再现；更好的研究工具需要进行疾病建模。患者特异性 iPSC 是出色的工具和多功能资源一种转化研究。由于 iPSC 是在个体基础上生成的，因此 iPSC 可能是最佳选择用于疾病建模、药物发现和患者特异性疗法开发的细胞材料。我们已经生成了大量的初步数据。我们使用了高效的 CRSPR 系统精确控制和标准化 HSA21 上的感兴趣基因。我们还开发了一个系统 3- DS iPSC 的三维 (3D) CNS 类器官 (CO) 培养物，可以更好地概括大脑发育和与传统的二维 (2D) 平面培养相比，疾病发病机制更加清晰，并且可以进行深入研究通过电生理学测定进行表征。 CNS 类器官技术代表了卓越的疾病建模方法；从患者 iPSC 生成的大脑类器官可用作模型概括复杂的神经发育疾病，例如 DS。此外，我们还生成了一个人源化嵌合小鼠模型，其中 DS iPSC 衍生的星形胶质细胞被移植到新生小鼠中大脑。 DS 各种症状的详细遗传病因学仍然难以捉摸。利用这些独特的工具和资源，我们将利用人类 iPSC 创建新颖的 DS 体外和体内模型来自患者以概括 DS 神经分化的缺陷。支持可行性根据这项建议，我们已经获得了本研究中使用的必要材料和专业知识，并且已经发表了一篇关于 DS iPSC 的相当大的论文 [Chen C, Jiang P, Xu H, Peterson SE, Tran HT, McCann AE, Parast MM, Li S, Pleasure DE, Laurent LC, Loring JF, Liu Y, Deng W. (2014) 星形胶质细胞在唐氏综合症中的作用由患者来源的人类诱导多能干细胞揭示的综合征。自然通讯。 5:4430 doi：10.1038/ncomms5430（2014）]。我们的初步数据显示三体性和同基因二体性 DS 星形胶质细胞能够重新填充小鼠大脑，从而可以进一步研究这些星形胶质细胞的体内行为细胞并检查它们对动物神经炎症和认知的影响。建立在先前的基础上研究星形胶质细胞介导的炎症途径中的多个基因，我们建议在体外生产这两种基因 CNS类器官和体内嵌合小鼠模型研究这些星形胶质细胞的关键作用炎症基因（S100B、IFNAR1、IFNAR2）在 DS 患者来源的 iPSC 发育和功能中的作用。总之，我们将使用体外 3D 类器官培养系统和体内 3D 类器官培养系统的新颖平台。使用 DS 患者来源的 iPSC 的人源化嵌合小鼠模型。这些模型将提供基本的深入了解 3D 类器官生理环境中的神经功能以及早期发育中的神经功能活体动物体内的人类细胞。该项目的完成将极大地促进 DS 发病机制研究使用患者 iPSC 以及生化和分子方法并辅以调查神经网络功能。这些见解无疑将对 DS 患者的治疗产生影响。