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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.

项目总结/摘要： 21三体iPSCs向脑组织中的分化和整合：建模使用患者特异性iPSC衍生的CNS类器官和人源化嵌合小鼠的研究。下来综合征（DS）是由21三体，即人类21号染色体（HSA 21）的三倍体引起的，智力残疾的常见遗传原因。我们已经成功地建立和表征了多个来源于DS患者的iPSC细胞系。特别是，我们已经建立了50多个DS Trisomy 21 iPSC 系，并从这些DS iPSC获得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（重复的规则间隔短回文基因），重复序列/CRISPR相关）技术在iPSC中进行遗传校正。人类遗传学建模以前，疾病在很大程度上依赖于死后病理分析或人类组织样本或在转基因动物模型中再现人类疾病;更好的研究工具疾病建模的需要。患者特异性iPSC是这方面的优秀工具和多功能资源一种转化研究。由于iPSC是在个体基础上产生的，因此iPSC可能是最佳的细胞材料用于疾病建模、药物发现和患者特异性疗法的开发。我们已经收集了大量的初步数据。我们使用了高效的CRSPR 该系统用于精确控制和标准化HSA 21上的感兴趣基因。我们还开发了一个3- 来自DS iPSC的三维（3D）CNS类器官（CO）培养物，其更好地再现了大脑发育，疾病的发病机制比传统的二维（2D）平面培养，并允许深入通过电生理学测定进行表征。CNS类器官技术代表了一种优秀的疾病建模的方法;从患者iPSC产生的脑类器官可用作模型来概括复杂的神经发育疾病如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移植到新生小鼠大脑DS各种症状的详细遗传病因仍然难以捉摸。利用这些独特的工具和资源，我们将创建新的体外和体内模型的DS与人类iPSCs 从患者中获得的，以概括DS中神经分化的缺陷。为了支持以下可行性：根据这项建议，我们已经获得了这项研究所需的材料和专门知识，发表了关于DS iPSC的相当大量的论文[Chen C，Jiang P，Xue H，Peterson SE，Tran HT，McCann AE，刘永，李文，李文. 2014年：星胶质细胞在唐氏症中的作用患者来源的人诱导多能干细胞揭示的综合征。Nature Communications.五点四四三○ doi：10.1038/ncomms5430（2014）]。我们的初步数据表明，三体和同基因二体性DS 星形胶质细胞能够重新填充小鼠大脑，允许进一步询问这些星形胶质细胞的体内行为。细胞并检查它们对动物的神经炎症和认知的影响。基于先前的在星形胶质细胞介导的炎症通路中的多个基因的工作，我们建议在体外产生 CNS类器官和体内嵌合小鼠模型来研究这些星形胶质细胞的关键作用。炎症基因（S100 B，IFNAR 1，IFNAR 2）在DS患者来源的iPSC的发育和功能中的作用。总之，我们将使用一种新的平台，即体外3D类器官培养系统和体内图4示出了使用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在活体动物体内植入人体细胞该项目的完成将极大地支持DS发病机制的研究使用患者的iPSC，以及生物化学和分子方法，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。