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

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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.