Genetically diverse mouse cerebellar organoids for in vitro neurotoxicology

抽象的。 小脑性共济失调，或由于失去肌肉控制而引起的运动障碍，有许多 原因。遗传性疾病包括脊髓小脑性共济失调、共济失调-毛细血管扩张症和弗里德里希病 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。的确有 还有大量证据表明小脑参与了非运动性疾病，如自闭症和 抑郁症。这个项目的总体目标是建立一个小脑的种群模型。 用于测试潜在神经毒物的有机化合物。在第一阶段，我们将合并和改编出版 小鼠小脑神经球、NPC分化为颗粒神经元的方案，以及 自组织人小脑有机体的组装建立一个健壮和可伸缩的 一种小鼠小脑器官的制备方法。我们将通过以下方式对有机化合物进行表征 免疫染色以及在器官分化的不同阶段进行scRNAseq检测， 我们将测试生物复制以估计批量影响，这可能会混淆对 基因对反应的贡献。我们将单细胞团划分为小脑细胞类型 并使用参考转录组图谱证明真实的细胞身份。最后，我们会 以乙醇为模型小脑毒物对有机物进行检测。从这些第一阶段的实验中 我们将在第二阶段确定由24到48个细胞系组成的更大屏幕的参数 和12种毒物。

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.