Genetically diverse mouse cerebellar organoids for in vitro neurotoxicology

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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.