Modeling ASD-linked genetic mutations in 3D human brain organoids

项目概要： 现代基因组测序技术使该领域能够识别重要的遗传基因， 与神经发育和神经精神障碍如精神分裂症相关的多态性 (SCZ)和自闭症谱系障碍（ASD）。然而，我们仍然对细胞的理解有限， 与这些病理中的基因突变和变异相关的基因表达缺陷。寻找答案 这些疾病的复杂性使得这些关键问题变得困难（这些疾病影响多种细胞类型， 不同的大脑区域），缺乏单一的，理想的实验模型，这些具体的“人类”病理， 以及需要在许多遗传背景中研究表型异常。啮齿动物模型有 由于其发展、架构和功能的固有差异， 大脑相比，人类;越来越清楚的是，在啮齿动物的工作必须与使用相结合， 灵长类动物模型，包括人脑模型。 使用内源性人脑组织的研究因组织的实际和伦理问题而复杂化 可用性、扩展性和操纵性。然而，最近的进展使细胞的发展成为可能。 通过生成3D大脑类器官来建立人类大脑发育模型，我们提出可以 补充动物模型系统，以模拟人脑发育和病理学的基本方面。 虽然本质上是还原论，但3D人脑类器官适合基因工程和高- 通量分析，使其成为研究基因突变谱的有利平台。 这些模型可以提供一个有价值的平台，将疾病相关基因的突变与特定的 人类神经元和电路的异常，以及帮助确定干预的分子靶点。 CHD 8基因是散发性ASD中最常见的突变基因之一，产生ASD亚型 常伴有大头畸形。尽管已经证明CHD 8调节许多其他的细胞因子， ASD风险基因，有限的信息是在不同细胞类型的细胞和分子缺陷 在CHD 8突变体人体组织中。我们最近建立了一个优化的培养系统， 培养健康的人脑类器官长达13个月，产生异常成熟的类器官， 不同的细胞类型，分子上类似于它们的内源性对应物，以及发育的成熟神经元， 树突棘并参与自发活性网络（Quadrato等，Nature，in press）.我们将 使用该方案来表征个体人脑细胞类型中ASD风险基因的表达谱 在类器官内使用高通量单细胞测序。此外，我们还创造了人类大脑 来自多能干细胞的类器官被工程化以携带CHD 8中的杂合无效突变，我们 显示了在患者中观察到的一些表型变化。我们将使用这个模型来研究 在单细胞水平上由CHD 8突变引起的分子和细胞缺陷。

Project summary: Modern genomic sequencing technologies have allowed the field to identify important genetic polymorphisms associated with neurodevelopmental and neuropsychiatric disorders such as schizophrenia (SCZ) and autism spectrum disorder (ASD). However, we still have a limited understanding of the cellular and gene-expression defects associated with genetic mutation and variation in these pathologies. Finding answers to these key questions is made difficult by the complexity of these diseases (which affect multiple cell types in distinct brain regions), the lack of single, ideal experimental models for these specifically “human” pathologies, and the need to investigate phenotypic abnormalities across many genetic backgrounds. Rodent models have important limitations due to the inherent differences in the development, architecture and function of their brains compared to humans; it is increasingly clear that work in rodents must be integrated with the use of primate models, including models of the human brain. Studies using endogenous human brain tissue are complicated by practical and ethical concerns of tissue availability, expansion and manipulation. However, recent progress has enabled the development of cellular models of the human developing brain via the generation of 3D brain organoids, which we propose can complement animal model systems to model basic aspects of human brain development and pathology. Although reductionist in nature, 3D human brain organoids are amenable to genetic engineering and high- throughput analysis, making them advantageous platforms for investigating a spectrum of genetic mutations. These models can provide a valuable platform to link mutations in disease-associated genes with specific abnormalities in human neurons and circuits, as well as to help identify molecular targets for intervention. The CHD8 gene is one of the most commonly mutated genes in sporadic ASD, producing an ASD subtype frequently associated with macrocephaly. Although it has been demonstrated that CHD8 regulates many other ASD risk genes, limited information is available on the cellular and molecular defects across different cell types in CHD8 mutant human tissue. We have recently established an optimized culture system that is able to develop healthy human brain organoids for up to 13 months, producing unusually mature organoids containing diverse cell types that molecularly resemble their endogenous counterparts, and mature neurons that develop dendritic spines and participate in spontaneously active networks (Quadrato et al., Nature, in press). We will use this protocol to characterize the expression profile of ASD risk genes in individual human brain cell types within organoids using high-throughput single-cell sequencing. In addition, we have created human brain organoids from pluripotent stem cells engineered to carry a heterozygous null mutation in CHD8, which we show recapitulate some of the phenotypic changes seen in patients. We will use this model to investigate the molecular and cellular defects resulting from CHD8 mutation at the single-cell level.