Distinct spatial patterning and transcriptomic landscapes of human neural organoids by localized delivery of morphogens.

Positional patterning during human brain development is orchestrated through highly coordinated interplays of locally produced inductive signals. Although animal models have elucidated general signaling pathways during early neurodevelopment, individual morphogens' effects underlying the proper human brain regionalization remain unclear. Current technologies are limited in generating stable, well-confined gradients in neural organoids for robust regionalization. Here, we report a Matrigel-free passive diffusion-based morphogen gradient generator (PdMG) that reliably established a steep exogenous spatial morphogen gradient in human neural organoids. We further established dorsal-ventral forebrain, rostral-caudal fore-midbrain-like, and rostral-caudal fore-hindbrain-like patterning by applying Sonic hedgehog/Wingless/int1 (WNT) inhibitor, WNT, and retinoic acid gradients, respectively. Spatial transcriptomics analysis revealed robust regionalization in early-stage patterned organoids, as well as active neurogenesis and γ-aminobutyric acid (GABAergic) interneuron migrations in late-stage patterned organoids. Together, this study provides a framework for modeling the spatial-temporal morphogen dynamics that regulate key cell fate specifications and axis formations using patterned neural organoid models.