Temporal Single Cell RNAseq to Identify Genes and Pathways Affected by 15q11.2 Duplication in Autism iPSC-Derived Differentiating Cortical Neurons

时间单细胞 RNAseq 识别自闭症 iPSC 衍生的分化皮质神经元中受 15q11.2 重复影响的基因和通路

• 批准号: 9200627
• 负责人: Wei Zhang
• 金额: $ 22.45万
• 依托单位: JUVOBIO PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-16 至 2018-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9200627
• 关键词: Academic support; Address; Affect; Autistic Disorder; Bioinformatics; Biological Markers; Brain; Cell Count; Cell Line; Cell Proliferation; Cells; Child; Clinical; Companions; Computer Simulation; Copy Number Polymorphism; DNA Sequence Alteration; Data; Data Quality; Data Set; Databases; Deposition; Development; Developmental Disabilities; Diagnosis; Dimensions; Disease; Disease Progression; Disease model; Drug Targeting; Environmental Risk Factor; Epilepsy; Exposure to; Frequencies; Gene Expression; Genes; Genetic Predisposition to Disease; Glutamates; Health Priorities; Heterogeneity; In Vitro; Lateral; Lead; Maps; Marketing; Mendelian disorder; Molecular; Molecular Profiling; Morphology; Mutation; Nature; Nervous System Physiology; Neuronal Differentiation; Neurons; Pathogenesis; Pathology; Pathway interactions; Patients; Pharmacologic Substance; Phase; Phenotype; Play; Population; Price; Process; Protocols documentation; Proxy; Publishing; Reagent; Research; Research Personnel; Research Support; Resources; Risk Factors; Role; Sampling; Schizophrenia; Scientist; Sorting - Cell Movement; Synapses; Synaptic plasticity; System; Therapeutic; Time; Tissue Sample; United States; Up-Regulation; autism spectrum disorder; cell type; database structure; diagnostic biomarker; disease mechanisms study; disease phenotype; disorder control; drug discovery; genetic risk factor; induced pluripotent stem cell; innovation; insight; interest; microdeletion; molecular dynamics; nerve stem cell; nervous system disorder; neuron development; neuronal circuitry; novel therapeutics; peer; public health priorities; reconstitution; relating to nervous system; research study; tool; transcriptome; transcriptome sequencing

PROJECT SUMMARY We aim to establish a single-cell RNAseq database of differentiating cortical neuronal progenitors (NPCs) and neurons derived from patient-specific induced pluripotent stem cells (iPSCs) for neuro- developmental diseases (NDDs). The database will also include data on differentiation, morphology, formation and functionality of synapses, annotations of analytical findings, and will be supported by the most advanced single-cell RNAseq bioinformatics tools. We will provide all this, together with corresponding NPCs and neurons for research reagents, as tool to support academic research and drug discovery for NDDs in the field. One in 68 children born in 2002 are diagnosed with Autism Spectrum Disorder (ASD), a public health priority in the US. Genetic predispositions in ASD are thought to contribute to the primary pathology by altering neuronal development, evidenced partly by altered gene expressions. Patient-specific induced pluripotent stem cells (iPSCs) have been shown to recapitulate specific disease phenotypes through the neurogenic process and can serve as effective disease models. Hence, single-cell RNAseq along the accessible and controlled process of differentiating ASD-specific iPSCs into neural progenitors and subtypes of neurons can provide insights into the temporal and multi-lineage dimensions of ASD pathogenesis and biomarkers for diagnostics, progression, and therapeutics discovery. Copy-number variants (CNVS) at 15q11.2 is a prominent risk factor for neurological disorders including ASD, epilepsy, and schizophrenia. 15q11-q13 duplication/triplication represent the most common CNVs in patients with ASD (up to 3%). Duplications and microdeletions can both lead to the same disorders, suggesting the importance of this region in normal neurological functions and the necessity to study the impact of both duplications and deletions for a full understanding of the mechanisms. Phase I utilizes four 15q11.2 duplication iPSC lines from ASD patients and two control iPSC lines to: Aim 1: Characterize neural differentiation of ASD and control iPSC-derived neurons. Differentiate iPSCs into cortical NPCs, glutamatergic, and GABAergic subtypes. Identify deficits in ASD lines through morphological studies and structural analyses of synapses. Aim 2: Generate single-cell RNAseq datasets of differentiating NPCs and neuronal subtypes at nine time points during differentiation. Aim 3: Perform bioinformatics analyses. Reconstitute the molecular dynamics underlying neuronal differentiation. Validate experimental conditions including sampling frequency and number of cells. Identify, validate molecular signatures underlying 15q11.2 duplication’s impact on neuronal differentiation and functions. Phase II: utilize the experimental conditions established here to generate single-cell RNAseq datasets from multiple ASD iPSC lines that harbor different genetic mutations to be included in the database; build database structure and user interface, and seek to identify aberrant differentiation and functional development caused by ASD mutations as well as genes and pathways that are commonly and differentially affected across multiple ASD mutations. We will devote resources to annotate the database with our own findings and those that are published by peers to enhance its utility to subscribers.

项目概要 我们的目标是建立一个分化皮质神经元祖细胞的单细胞RNAseq数据库 (NPC) 和源自患者特异性诱导多能干细胞 (iPSC) 的神经元，用于神经- 该数据库还将包括分化、形态、形成的数据。 突触的功能和分析结果的注释，并将得到最先进的支持 我们将提供所有这些，以及相应的 NPC 和单细胞 RNAseq 生物信息学工具。 用于研究试剂的神经元，作为支持该领域 NDD 学术研究和药物发现的工具。 2002 年出生的儿童中，每 68 名就有 1 名被诊断患有自闭症谱系障碍 (ASD)，这是一种公共卫生疾病 在美国，自闭症谱系障碍的遗传倾向被认为是通过改变主要病理学来促成的。 神经元发育，部分由患者特异性诱导多能性的改变证明。 干细胞（iPSC）已被证明可以通过神经源性重现特定的疾病表型 因此，单细胞 RNAseq 具有可及性和可作为有效的疾病模型的作用。 将 ASD 特异性 iPSC 分化为神经祖细胞和神经元亚型的受控过程可以 提供对 ASD 发病机制和生物标志物的时间和多谱系维度的见解 诊断、进展和治疗发现。 15q11.2 的拷贝数变异 (CNVS) 是神经系统疾病的一个重要危险因素，包括 ASD、癫痫和精神分裂症是最常见的 CNV。 患有 ASD 的患者（高达 3%）。重复和微缺失都可能导致相同的疾病， 表明该区域在正常神经功能中的重要性以及研究其影响的必要性 重复和删除以充分理解机制。 第一阶段利用来自 ASD 患者的四个 15q11.2 重复 iPSC 系和两个对照 iPSC 系来： 目标 1：表征 ASD 的神经分化并控制 iPSC 衍生的神经元。 分为皮质 NPC、谷氨酸能和 GABA 能亚型，通过识别 ASD 系的缺陷。 突触的形态学研究和结构分析。 目标 2：生成区分 NPC 和神经亚型 9 种的单细胞 RNAseq 数据集 分化过程中的时间点。 目标 3：进行生物信息学分析。重建神经网络的分子动力学。 验证实验条件，包括采样频率和细胞数量。 验证 15q11.2 重复对神经元分化和功能影响的分子特征。 第二阶段：利用此处建立的实验条件生成单细胞 RNAseq 数据集 多个包含不同基因突变的 ASD iPSC 系纳入数据库构建数据库； 结构和用户界面，并寻求识别由以下原因引起的异常分化和功能开发 自闭症谱系障碍 (ASD) 突变以及在多种疾病中普遍受到不同程度影响的基因和通路 我们将投入资源用我们自己的发现和那些发现的结果来注释数据库。 由同行发布，以增强其对订阅者的实用性。