Construction of the integrated human cortical organoids to investigate neurodevelopmental disorders

项目摘要 诊断主要基于症状描述，很难区分其潜在的发病机制 神经精神障碍，包括精神分裂症(SCZ)和自闭症谱系障碍(ASD)。近期 先进的人类遗传学研究已经确定了多种神经精神疾病的遗传基础 疾病，表明大多数SCZ和ASD是遗传异质性的，由罕见的De引起 新星突变。特别是，SET1/COMPASS复合蛋白或相关蛋白的罕见突变 在SCZ或ASD患者中明显发现。我们的中心假设是研究SCZ的功能- 或ASD相关蛋白在皮质和皮质下发育中将揭示分子机制如何 具有相似催化活性的蛋白质的突变会导致临床上不同的疾病。可获得性的匮乏 人脑组织一直在挑战通过使用人脑组织直接解决这些问题。 因此，为了达到这个目标，我们将使用人脑有机化合物，即结构和功能上的 复制发育中的人脑区域。人类多能干细胞(HPSCs)的使用已经 彻底改变了人类大脑的研究。HPSC可无限制自我更新，并可分化为 细胞类型，包括脑细胞。我们已经报道了由以下物质生成三维(3-D)结构 HPSCs概括了发育中的人类皮质(HCO，皮质类器官)、内侧神经节隆起 或间脑丘脑(hThO，丘脑有机物质)。将hCO与hMGEO融合，或 HTho再现了发育不同的两个区域的相互作用，如MGE的切向迁移 细胞至皮质，或相互的皮质丘脑或丘脑皮质连接。使用先进的干细胞 工具，我们将追求实现目标的目标。1)我们将使用CRISPR/Cas9基因编辑工具介绍 将SCZ和ASD基因突变导入hPSC细胞，并研究其细胞和分子功能。 使用hThOS和HCOs的皮质和丘脑发育。2)我们将开发方法来复制 丘脑内多个核团，进一步提高hThos的区域性规范。3)我们将调查 SCZ和ASD基因在皮质和皮质下连接中的作用。总体而言，我们先进的人脑 基于有机物的方法与基因组学和神经生物学工具相结合将定义分子 机制明显受SCZ和ASD相关基因调控，并提供前所未有的独特 构建功能性皮质丘脑连接的平台。

Project Summary Diagnosed mainly based on symptomatic description, it is difficult to distinguish the underlying mechanisms of neuropsychiatric disorders, including schizophrenia (SCZ) and autism spectrum disorders (ASD). Recent advanced human genetic studies have identified the genetic underpinning of the a variety of neuropsychiatric disorders, showing that majority of SCZ and ASD are genetically heterogeneous and caused by with rare de novo mutations. Particularly, a rare mutations at SET1/COMPASS complex proteins or related proteins were distinctly discovered at SCZ or ASD patients. Our central hypothesis is that investigating the function of SCZ- or ASD-associated proteins in cortical and subcortical development will reveal the molecular mechanism how mutations of proteins with similar catalytic activity result in clinically distinct disorders. The paucity of accessible human brain tissues has been challenging to directly addressing these questions by using human brain tissue. Thus, in order to achieve the goal, we will use human brain organoids that structually and functionally reproduce the developing human brain regions. The use of human pluripotent stem cells (hPSCs) has revolutionized the human brain studies. hPSCs undergo unlimited self-renewal and can differentiate into any cell types, including brain cells. We have reported the generation of 3-dimensional (3-D) structures from hPSCs that recapitulate the developing human cortex (hCO, cortical organoids), medial ganglionic eminence (hMGEO, MGE organoid), or diencephalic thalamus (hThO, thalamic organoids). Fusing hCO with hMGEO, or hThO reproduced the interaction of developmentally distinct two regions, such as tangential migration of MGE cells to cortex, or reciprocal corticothalamic or thalamocortical connections. Using the advanced stem cell tools, we will purse the aims to achieve the goal. 1) We will use CRISPR/CAS9 gene editing tools to introduce mutations of SCZ and ASD genes into hPSC lines, and investigate the cellular and molecular function in cortical and thalamic development using hThOs and hCOs. 2) We will develop methods to reproduce the multiple nuclei in thalamus to further improve the regional specification of hThOs. 3) We will investigate the function of SCZ and ASD genes in cortical and subcortical connectivity. Overall, our advanced human brain organoid-based approaches combined with genomics and neurobiological tools will define the molecular mechanism distinctly regulated by SCZ and ASD-associated genes and provide unprecedented unique platforms to construct the functional corticothalamic connection.