Creation and Evaluation of iPSCs from Children with ASD with Megalencephaly

自闭症谱系障碍 (ASD) 巨脑畸形儿童 iPSC 的创建和评估

• 批准号: 10238008
• 负责人: JOACHIM F HALLMAYER
• 金额: $ 37.68万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-07 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238008
• 关键词: ASD patient; Address; Animals; Antibodies; Apoptosis; Behavioral; Body Size; Brain; Cell Cycle; Cell Cycle Regulation; Cell Line; Cell Proliferation; Cell Survival; Cells; Characteristics; Child; Clinical; Coculture Techniques; Cognitive; Communities; Complex; Cyclins; Data; Defect; Derivation procedure; Development; Disease; Evaluation; Fibroblasts; Flow Cytometry; Gene Expression; Gene Expression Profiling; Generations; Genes; Goals; Heterogeneity; Homeostasis; Human; Image; Immune; In Vitro; Individual; Induced pluripotent stem cell derived neurons; Institutes; Language; Lead; Measures; Medical; Megalencephaly; Microglia; Modeling; Molecular; Neuroglia; Neurons; Oligodendroglia; Onset of illness; PTEN gene; PTPRC gene; Paper; Pathway interactions; Patients; Phagocytes; Phagocytosis; Pharmacological Treatment; Phase; Phenotype; Play; Population; Preventive measure; Proteins; Research; Resources; Role; Severities; Signal Transduction; Skin; Stains; Synapses; Testing; Western Blotting; autism spectrum disorder; autistic children; base; behavioral impairment; behavioral phenotyping; boys; brain behavior; brain overgrowth; brain size; cell injury; cell type; cohort; differentiation protocol; genetic makeup; gray matter; in vitro Model; in vivo; individualized medicine; induced pluripotent stem cell; induced pluripotent stem cell technology; insight; nerve stem cell; neurodevelopment; neuroimaging; oligodendrocyte progenitor; patient population; phenome; pluripotency; precursor cell; predictive marker; prevent; recruit; social communication; stem cells; therapeutic target; transcriptome sequencing; trend; white matter

PROJECT SUMMARY – PROJECT 3 Autism spectrum disorder (ASD) is a complex condition characterized by important changes to the brain and behavior. 15% of boys with ASD have disproportionate megalencephaly (ASD-DM), or enlarged brain relative to body size. An increase in brain size often precedes the first clinical signs of the disorder, suggesting that understanding the mechanisms leading to brain overgrowth could provide a window of opportunity to intervene and possibly prevent disease onset. Here, the research team will use human induced pluripotent stem cell (hiPSC) technology to model ASD-DM and investigate the underlying cellular and molecular mechanisms involved. They will obtain skin fibroblasts from 40 individuals in Project 2 and derive human iPSCs from: A) 10 ASD subjects with megalencephaly, ASD-DM; B) 10 ASD subjects with normal sized brains, ASD-N C) 10 Typically developing (TD) subjects with megalencephaly, TD-DM, and D) 10 TD subjects with normal sized brains, TD-N. Following iPSC generation, they will differentiate each of the iPSC lines into neural progenitor cells (NPCs), oligodendrocyte progenitor cells (OPCs), and microglia (the primary immune cells in the brain that maintain homeostasis). The overarching goals of their project are two-fold: 1) to investigate whether ASD-DM is due to an increase in cell proliferation, increase in cell survival, improper elimination of damaged cells, and/or a combination of all; and 2) to identify therapeutic targets by understanding the underlying cellular and signaling mechanisms involved. In Specific Aim 1, they will identify the cellular mechanisms underlying ASD-DM by investigating changes in the cell cycle, cell proliferation, and apoptosis of iPSC-derived NPCs, OPCs, and microglial cells. In Specific Aim 2, they will investigate the functional activity of microglia in ASD-DM by directly differentiating each of the iPSC lines into microglia and assessing their phagocytic capacity by co-culturing them with mixed neuroglial cultures derived from the same lines. This will test their hypothesis that microglia are compromised in ASD-DM, failing to eliminate damaged cells and synapses and contributing to brain overgrowth. In Specific Aim 3, they will identify the underlying regulatory signaling mechanisms that lead to the changes at the cellular level. They will differentiate the iPSCs into NPCs, OPCs, and microglia, sort them by flow cytometry using antibodies specific for each cell type, and perform RNA-sequencing to identify gene networks and signaling mechanisms that are significantly regulated in each condition. Using these mechanistic insights, they will identify therapeutic targets to directly test in the in vitro models. Their overall goal across the projects is to collect imaging, behavioral, and mechanistic data on the same cohort of subjects. In Specific Aim 4, they will correlate the cellular and mechanistic data obtained in Project 3 with the imaging and behavioral data from Project 2 to identify broader trends and characteristics specific to ASD-DM. This comprehensive body of data will be a valuable resource for the broader research and medical communities in identifying predictive biomarkers of ASD and/or ASD-DM and potentially more tailored therapies.

项目摘要 - 项目3 自闭症谱系障碍（ASD）是一种复杂的疾病，其特征是大脑的重要变化 行为。 15％的ASD男孩患有不成比例的大脑（ASD-DM），或相对于大脑增大 身体大小。大脑大小的增加通常是该疾病的第一个临床征兆，这表明 了解导致大脑过度生长的机制可以提供干预的机会之窗 并可能预防疾病发作。在这里，研究小组将使用人类诱导的多能干细胞 （HIPSC）对ASD-DM进行建模并研究潜在的细胞和分子机制的技术 涉及。他们将从项目2中的40个个体中获得皮肤成纤维细胞，并从：a）10中获得人IPSC。 ASD主体，具有巨型脑，ASD-DM； b）10个具有正常大脑的ASD受试者，ASD-N c）10 通常发育（TD）具有大小正常的受试者，TD-DM和D）10 TD受试者 大脑，TD-N。 IPSC生成之后，它们将将每条IPSC线区分为神经祖细胞 细胞（NPC），少突胶质细胞祖细胞（OPC）和小胶质细胞（大脑中的主要免疫细胞 保持体内平衡）。他们项目的总体目标是两个方面：1）调查ASD-DM是否 是由于细胞增殖的增加，细胞存活增加，不当消除受损细胞和/或 所有人的结合； 2）通过理解潜在的细胞和信号来识别治疗靶标 涉及的机制。在特定的目标1中，他们将通过ASD-DM的细胞机制来确定ASD-DM的细胞机制 调查IPSC衍生的NPC，OPC和OPC的细胞周期，细胞增殖和凋亡的变化 小胶质细胞。在特定目标2中，他们将直接研究ASD-DM中小胶质细胞的功能活性 将每条IPSC系列区分为小胶质细胞，并通过共同培养评估其吞噬能力 与源自相同线的混合神经培养物。这将检验他们的假设，即小胶质细胞是 在ASD-DM中受到损害，无法消除损伤细胞和突触，并导致大脑过度生长。 在特定目标3中，他们将确定导致变化的基本调节信号传导机制 细胞水平。他们将将IPSC区分为NPC，OPC和小胶质细胞，通过流式细胞仪对其进行排序 使用针对每种细胞类型的特异性抗体，并执行RNA测序以识别基因网络和信号传导 在每种条件下都受到显着调节的机制。使用这些机械见解，他们将确定 在体外模型中直接测试的治疗靶标。他们在整个项目中的总体目标是收集成像， 在同一受试者队列上的行为和机械数据。在特定的目标4中，它们将与细胞相关 以及项目3中获得的成像和行为数据中获得的机械数据，以识别更广泛的 ASD-DM特有的趋势和特征。这种全面的数据将是一个宝贵的资源 在识别ASD和/或ASD-DM的预测生物标志物和/或ASD-DM和 可能更量身定制的疗法。