Experimental models of the human brain are needed for basic understanding of its development and disease 1. Human brain organoids hold unprecedented promise for this purpose; however, they are plagued by high organoid-to-organoid variability 2,3. This has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility comparable to the endogenous tissue. Here, we show that an organoid model of the dorsal forebrain can achieve reproducible generation of a rich diversity of cell types appropriate for the human cerebral cortex. Using single-cell RNA sequencing of 166,242 cells isolated from 21 individual organoids, we find that 95% of the organoids generate a virtually indistinguishable compendium of cell types, through the same developmental trajectories, and with organoid-to-organoid variability comparable to that of individual endogenous brains. Furthermore, organoids derived from different stem cell lines show consistent reproducibility in the cell types produced. The data demonstrate that reproducible development of complex central nervous system cellular diversity does not require the context of the embryo, and that establishment of terminal Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Genetic risk for autism spectrum disorders (ASD) is associated with hundreds of genes spanning a wide range of biological functions 1-6 . The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals 6,7 . Here, we leveraged organoid models of the human cerebral cortex to identify cell type-specific developmental abnormalities resulting from haploinsufficiency in three ASD risk genes, SUV420H1 (KMT5B), ARID1B, and CHD8, in multiple cell lines from different donors, using single-cell RNA-seq (scRNA-seq) of over 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations demonstrates asynchronous development of two main cortical neuronal lineages, GABAergic neurons and deep-layer excitatory projection neurons, but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This work uncovers cell typespecific neurodevelopmental abnormalities shared across ASD risk genes that are finely modulated Paulsen et al.

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