Generation of human nucleus basalis organoids with functional nbM-cortical cholinergic projections in transplanted assembloids

The nucleus basalis of Meynert (nbM), the major cholinergic output of the basal forebrain, regulates cortical modulation, learning, and memory. Dysfunction of the nbM-cortical cholinergic pathway is implicated in neurodegenerative and neurodevelopmental disorders, including Alzheimer’s disease (AD) and Down syndrome (DS). Here, we generated human nbM organoids (hnbMOs) from human pluripotent stem cells (hPSCs) containing functional cholinergic projection neurons. Then we reconstructed long-distance cholinergic projections from nbM to the cortex by co-culturing hnbMOs with human fetal brains and transplanting hnbMOs into immunodeficient mice. We further established nbM-cortical assembloids by fusing hnbMOs with human cortical organoids (hCOs). We also established a human-specific cholinergic projection system in transplanted assembloids. Using viral tracing and functional assays, we validated that cholinergic neurons send projections into hCOs and form synaptic connections. Moreover, we captured projection deficits in DS-derived assembloids, demonstrating the utility of this model for studying nbM-related neural circuits and neurological disorders. • Human nbM organoids generated from hPSCs recapitulate nucleus-specific identity • hnbMOs establish long projection and functional connectivity with hCOs in vitro • Assembloid transplantation formed the functional nbM-cortical cholinergic pathway • nbM-cortical assembloids model projection deficits in Down syndrome The nucleus basalis of Meynert (nbM) regulates cortical function and cognition. Liu and colleagues report a method for creating human nbM organoids (hnbMOs) containing cholinergic projection neurons that form functional connections with the cortex. This work provides a model to investigate nbM circuits and cholinergic dysfunction in neurological disorders.