Creation and Evaluation of iPSCs from Children with ASD with Megalencephaly

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%的患有自闭症的男孩有不成比例的巨脑(ASD-DM)，或相对于 体型。大脑体积的增大通常出现在这种疾病的第一个临床症状之前，这表明 了解导致大脑过度生长的机制可以为干预提供机会之窗 并有可能预防疾病的发作。在这里，研究小组将使用人类诱导的多能干细胞 (HiPSC)技术建立ASD-DM模型并研究其潜在的细胞和分子机制 牵涉其中。他们将在项目2中从40个人身上获得皮肤成纤维细胞，并从以下来源获得人类IPSCs： ASD患者合并巨脑畸形，ASD-DM；B)10例ASD患者，脑大小正常，ASD-N C)10 典型发育中(TD)巨脑畸形、TD-DM和D)10例正常大小的TD患者 大脑，TD-N。在IPSC产生后，他们将把每一株IPSC分化为神经前体细胞 细胞(NPC)、少突胶质前体细胞(OPC)和小胶质细胞(大脑中的初级免疫细胞 保持动态平衡)。他们项目的总体目标有两个：1)调查ASD-DM 是由于细胞增殖增加，细胞存活率增加，不适当地清除受损细胞，和/或 ALL的组合；以及2)通过了解潜在的细胞和信号来确定治疗靶点 涉及的机制。在具体目标1中，他们将通过以下方式确定ASD-DM的细胞机制 研究IPSC来源的NC、OPC和OPC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，他们将通过直接研究ASD-DM中小胶质细胞的功能活性 将每一株IPSC分化为小胶质细胞并通过共培养检测其吞噬能力 用来自相同品系的混合神经胶质细胞培养。这将检验他们的假设，即小胶质细胞是 ASD-DM患者受损，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，他们将确定导致以下变化的基本监管信号机制 在细胞水平上。他们将把IPSCs区分为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抗体，并执行RNA测序以识别基因网络和信号 在每种情况下都受到显著调控的机制。使用这些机械的洞察力，他们将发现 治疗靶点直接在体外模型中测试。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学和机械学数据。在特定的目标4中，他们会将细胞 以及在项目3中获得的机械数据和项目2中的成像和行为数据，以确定更广泛的 ASD-DM特有的趋势和特征。这一全面的数据将是 更广泛的研究和医学界在确定ASD和/或ASD-DM的预测生物标志物和 可能会有更多量身定做的疗法。

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.