Summary Organoid techniques provide unique platforms to model brain development and neurological disorders. While several methods for recapitulating corticogenesis have been described, a system modeling human medial ganglionic eminence (MGE) development, a critical ventral brain domain producing cortical interneurons and related lineages, has been lacking until recently. Here, we describe the generation of MGE and cortex-specific organoids from human pluripotent stem cells that recapitulate the development of MGE and cortex domains respectively. Population and single-cell RNA-seq profiling combined with bulk ATAC-seq analyses revealed transcriptional and chromatin accessibility dynamics and lineage relationships during MGE and cortical organoid development. Furthermore, MGE and cortical organoids generated physiologically functional neurons and neuronal networks. Finally, fusing region-specific organoids followed by live-imaging enabled analysis of human interneuron migration and integration. Together, our study provides a platform for generating domain-specific brain organoids, for modeling human interneuron migration, and offers deeper insight into molecular dynamics during human brain development.

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Human brain organoid techniques have rapidly advanced to facilitate investigating human brain development and diseases. These efforts have largely focused on generating telencephalon due to its direct relevance in a variety of forebrain disorders. Despite its importance as a relay hub between cortex and peripheral tissues, the investigation of three-dimensional (3D) organoid models for the human thalamus has not been explored. Here, we describe a method to differentiate human embryonic stem cells (hESCs) to thalamic organoids (hThOs) that specifically recapitulate the development of thalamus. Single-cell RNA sequencing revealed a formation of distinct thalamic lineages, which diverge from telencephalic fate. Importantly, we developed a 3D system to create the reciprocal projections between thalamus and cortex by fusing the two distinct region-specific organoids representing the developing thalamus or cortex.Our study provides a platform for understanding human thalamic development and modeling circuit organizations and related disorders in the brain.(C) qPCR analysis for expressions of regional markers in developing hThOs, hMGEOs, and hCOs. Each data point represents expressions in pooled batch of 3-4 organoids, and 3 batches were collected for analysis. Mean ± SD is shown. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Immunostaining for MAP2 and thalamic marker TCF7L2 in day 41 hThO, hCO, and hMGEO. The scale bar represents 250 mm. (E) Immunostaining for thalamic and cortical progenitor marker PAX6, and cortical marker TBR1 in day 41 hThO, hCO, and hMGEO. The scale bar represents 250 mm. See also Figure S1.

Human brain organoid systems offer unprecedented opportunities to investigate both neurodevelopmental and neurological disease. Single-cell-based transcriptomics or epigenomics have dissected the cellular and molecular heterogeneity in the brain organoids, revealing a complex organization. Similar but distinct protocols from different labs have been applied to generate brain organoids, providing a large resource to perform a comparative analysis of brain developmental processes. Here, we take a systematic approach to compare the single-cell transcriptomes of various human cortical brain organoids together with fetal brain to define the identity of specific cell types and differentiation routes in each method. Importantly, we identify unique developmental programs in each protocol compared to fetal brain, which will be a critical benchmark for the utility of human brain organoids in the future.

Here, we engineered human embryonic stem cells (hESCs) to ectopically express human ETS variant 2 (ETV2) to create in vitro vasculature in cortical organoids (hCOs), namely vhCOs (vascularized hCOs). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. This protocol describes hESC maintenance, the generation of inducible ETV2 expressing hESCs, and the generation of cortical organoid with functional vascular-like network.