Creation and Evaluation of iPSCs from Children with ASD with Megalencephaly

项目概要-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巨脑畸形（ASD-DM），或相对于 体型大脑体积的增加通常先于疾病的第一个临床症状，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 并可能预防疾病的发生。在这里，研究小组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 涉案他们将从项目2中的40个个体获得皮肤成纤维细胞，并从以下来源获得人iPSC：A）10 患有巨脑畸形的ASD受试者，ASD-DM; B）10名具有正常大小大脑的ASD受试者，ASD-N C）10名 D）10名具有正常大小的TD受试者， 大脑TD-N在iPSC产生之后，他们将使每一个iPSC系分化成神经祖细胞。 细胞（NPC）、少突胶质祖细胞（OPC）和小胶质细胞（大脑中的主要免疫细胞， 保持体内平衡）。他们的项目的首要目标是双重的：1）调查ASD-DM是否 是由于细胞增殖增加、细胞存活增加、受损细胞的不适当消除，和/或 所有的组合;和2）通过理解潜在的细胞和信号传导来识别治疗靶点 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衍生的NPC、OPCs和NPCs的细胞周期、细胞增殖和凋亡的变化， 小胶质细胞在具体目标2中，他们将通过直接观察小胶质细胞在ASD-DM中的功能活性， 将每个iPSC系分化成小胶质细胞，并通过共培养它们来评估它们的吞噬能力 来自相同细胞系的混合神经胶质细胞培养物。这将验证他们的假设，即小胶质细胞是 在ASD-DM中受损，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潜在调控信号机制： 细胞水平。他们将iPSC分化为NPC，OPCs和小胶质细胞，通过流式细胞术对其进行分类， 使用针对每种细胞类型的特异性抗体，并进行RNA测序以识别基因网络和信号传导 在每种情况下都受到显著调节的机制。利用这些机械的洞察力，他们将识别 在体外模型中直接测试的治疗靶点。他们在这些项目中的总体目标是收集图像， 行为和机械数据。在具体目标4中，他们将把细胞 项目3中获得的机制数据与项目2中获得的成像和行为数据相结合，以确定更广泛的 ASD-DM的特定趋势和特征。这一全面的数据将是一个宝贵的资源， 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， 可能更有针对性的疗法。

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.