Morphogen-Directed, High-Throughput Development of hiPSC-Derived Telencephalic Organoids for Comparative In Vitro...

Pluripotent stem cell (PSC)-derived telencephalic organoids have long been established as an in vitro cell model to study the molecular and cellular features of the developing cerebral cortex and associated brain structures. Numerous directed organoid differentiation protocols have been developed that recapitulate cell subtype specification, cytoarchitectural organization, and cellular function with a high degree of fidelity. Protocol development has focused on the scalability of this in vitro system, and most organoid culture platforms involve a population-based approach in large vessel sizes. This makes comparative study with multiple cell lines both costly and time-consuming, and lowers the throughput for phenotypic screening. We have established a high-throughput approach for in vitro generation of hiPSC-derived telencephalic organoids that can be used to develop comparative phenotypic assays involving high-content imaging platforms. This article first highlights the use of a scaled-down, miniaturized cell culture volume that is amenable to high-throughput production of multiple 384-well plates. Then, using controlled morphogen cues, 3D hiPSC aggregates are directed into an anterior telencephalic identity and further specified into dorsal pallium or ventral subpallium cell fates, which can additionally be combined into assembloids for modeling excitatory and inhibitory networks or left to develop separately as single organoid subtypes. Finally, we introduce a Support Protocol detailing an in vitro 3D cell painting assay for organoid morpho-phenotypic characterization. © 2026 Wiley Periodicals LLC. Basic Protocol 1: High-throughput 3D aggregation of hiPSCs in morphogen-rich media Basic Protocol 2: Recombinant inhibitory protein-directed induction of telencephalic organoids Basic Protocol 3: Morphogen-guided induction of dorsal telencephalic/pallial organoids Basic Protocol 4: Morphogen-guided induction of ventral telencephalic/subpallial organoids Support Protocol: 3D live cell painting for organoid morphometric phenotyping.