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，其特征是大脑和 行为。 15% 患有自闭症谱系障碍 (ASD) 的男孩患有不成比例的巨脑畸形 (ASD-DM)，即相对于正常人而言大脑增大 车身尺寸。大脑尺寸的增加通常先于该疾病的第一个临床症状出现，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 并可能预防疾病的发生。在这里，研究团队将使用人类诱导多能干细胞 (hiPSC) 技术来模拟 ASD-DM 并研究潜在的细胞和分子机制 涉及。他们将从项目 2 中的 40 个人身上获得皮肤成纤维细胞，并从以下来源获得人类 iPSC：A) 10 患有巨脑畸形的 ASD 受试者，ASD-DM； B) 10 名具有正常大脑大小的 ASD 受试者，ASD-N C) 10 患有巨脑畸形、TD-DM 的典型发育 (TD) 受试者，以及 D) 10 名正常体型的 TD 受试者 大脑，TD-N。 iPSC 生成后，他们会将每个 iPSC 系分化为神经祖细胞 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， 维持体内平衡）。他们项目的总体目标有两个：1）调查 ASD-DM 是否 是由于细胞增殖的增加、细胞存活的增加、受损细胞的不当消除和/或 所有的组合； 2) 通过了解潜在的细胞和信号传导来确定治疗靶点 涉及的机制。在具体目标 1 中，他们将通过以下方式识别 ASD-DM 背后的细胞机制： 研究 iPSC 衍生的 NPC、OPC 和细胞周期、细胞增殖和凋亡的变化 小胶质细胞。在具体目标 2 中，他们将通过直接研究 ASD-DM 中小胶质细胞的功能活性 将每个 iPSC 系分化为小胶质细胞，并通过共培养评估它们的吞噬能力 与来自同一系的混合神经胶质细胞培养物。这将检验他们的假设，即小胶质细胞是 ASD-DM 中的神经元受到损害，无法消除受损的细胞和突触，并导致大脑过度生长。 在具体目标 3 中，他们将确定导致变化的潜在监管信号机制 细胞水平。他们将 iPSC 分化为 NPC、OPC 和小胶质细胞，并通过流式细胞术对它们进行分类 使用针对每种细胞类型的特异性抗体，并进行 RNA 测序来识别基因网络和信号传导 在每种情况下都受到显着调节的机制。利用这些机械见解，他们将识别 直接在体外模型中测试的治疗靶点。他们整个项目的总体目标是收集图像， 同一组受试者的行为和机械数据。在具体目标 4 中，他们将把细胞 项目 3 中获得的机械数据与项目 2 中的成像和行为数据相结合，以识别更广泛的 ASD-DM 的特定趋势和特征。这些全面的数据将成为宝贵的资源 更广泛的研究和医学界识别 ASD 和/或 ASD-DM 的预测生物标志物， 可能更有针对性的治疗方法。