Genetically diverse mouse cerebellar organoids for in vitro neurotoxicology

用于体外神经毒理学的遗传多样性小鼠小脑类器官

• 批准号: 10079772
• 负责人: Ted Choi
• 金额: $ 25.21万
• 依托单位: PREDICTIVE BIOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-20 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10079772
• 关键词: 3-Dimensional; Alcohol consumption; Ataxia; Ataxia Telangiectasia; Atlases; Biological Testing; Cell Line; Cells; Cerebellar Ataxia; Cerebellum; Chemicals; Cytoplasmic Granules; ES Cell Line; Environment; Ethanol; Gene Expression; Genetic; Human; Image; Immunologics; In Vitro; Industrialization; Inherited; Mental Depression; Metals; Methods; Modeling; Movement Disorders; Mus; Muscular Atrophy; Neurons; Neurotoxins; Nuclear Pore Complex; Organoids; Pharmaceutical Preparations; Phase; Population; Protocols documentation; Publishing; Spinocerebellar Ataxias; Stains; Testing; alcohol exposure; autism spectrum disorder; cell type; experimental study; genetic signature; neurotoxic; neurotoxicology; response; self assembly; single-cell RNA sequencing; stem cells; toxicant; transcriptome; transcriptomics

Abstract. Cerebellar ataxias, or movement disorders arising from loss of muscle control, have many causes. Inherited conditions include spinocerebellar ataxia, ataxia-telangiectasia and Friedrich’s ataxia. They are also caused by chemicals in the environment including drugs, industrial compounds and metals. The most common cause of ataxia is alcohol consumption. There is also abundant evidence of cerebellar involvement in non-motor disorders such as autism and depression. The overall aim of this project is to develop a population model of cerebellar organoids for testing of potential neurotoxicants. In Phase I we will merge and adapt published protocols for mouse cerebellar neurospheres, NPC differentiation to granule neurons, and assembly of self organizing human cerebellar organoids to establish a robust and scalable method for producing mouse cerebellar organoids. We will characterize the organoids by immunological staining as well as by scRNAseq at different stages of organoid differentiation, and we will test biological replicates to estimate batch effects that can confound assessment of genetic contribution to response. We will assign the single cell clusters to cerebellar cell types and demonstrate authentic cell identity using reference transcriptome atlases. Finally, we will test the organoids with ethanol as a model cerebellar toxicant. From these Phase I experiments we will determine the parameters of a larger screen in Phase II comprised of 24 to 48 cell lines and 12 toxicants.

抽象。 小脑共济失调或由肌肉控制丧失引起的运动障碍， 可使.遗传性疾病包括脊髓小脑共济失调，共济失调毛细血管扩张症和弗里德里希的 共济失调它们也是由环境中的化学物质引起的，包括药物，工业， 化合物和金属。共济失调最常见的原因是饮酒。有 也有大量证据表明小脑参与非运动障碍，如自闭症， 萧条本项目的总体目标是建立一个小脑的群体模型， 用于测试潜在神经毒物的类器官。在第一阶段，我们将合并和改编出版 用于小鼠小脑神经球、NPC向颗粒神经元分化的方案，以及 自组织人类小脑类器官的组装，以建立一个强大的和可扩展的 制备小鼠小脑类器官的方法。我们将通过以下方式来表征类器官： 免疫染色以及scRNAseq在类器官分化的不同阶段， 我们将测试生物重复，以估计可能混淆评估的批次效应， 基因对反应的影响我们将把这些单细胞群归类为小脑细胞类型 并使用参考转录组图谱证明真实的细胞身份。最后我们将 用乙醇作为小脑毒物模型测试类器官。从这些第一阶段的实验中 我们将在第二阶段确定由24至48个细胞系组成的更大筛选的参数 12有毒物质