Construction of Human nucleus basalis Organoids and Cholinergic Projections in nbM-cortical Assembloids

The nucleus basalis, also known as the nucleus basalis of Meynert (nbM), which is considered to be one of the major cholinergic output of basal forebrain, have been found to dynamically modulate activity in the cortex. Dysfunction of nucleus basalis-cortical cholinergic circuit led to cognitive impairment, such as Alzheimer's disease (AD) and Down syndrome (DS). Human nucleus basalis cholinergic neurons derived from human pluripotent stem cells provide powerful tools to study cholinergic neurons-associated diseases and cell therapy. Previous studies reported the generation of 2D human basal forebrain cholinergic neurons which failed to recapitulate the spatial organization, cellular diversity, and crosstalk between different regions. Therefore, a better model to recapitulate human nucleus basalis and cholinergic projections in nbM-cortical is desired. Here we developed a approach for differentiating human pluripotent stem cells into nucleus basalis of Meynert organoids (hnbMOs). We reconstructed hnbM-cortex cholinergic projection by transplanting hnbMOs into immunodeficiency mice to construct chimeric brains and coculturing with human fetal brain. Then we fused hnbMOs with cerebral cortex organoids (hCOs) to form hnbMO-hCO assembloids. We validate the structural and functional connectivity of basal forebrain cholinergic neurons to the cortex in assembloids. An assembloid-chimeric brain was constructed innovatively by transplanting corresponding organoids in the cortex and nbM region to establish a complete human cholinergic projection system. Futhermore, we identified the defects in projection of cholinergic neurons at the morphological and transcriptomic level in Down syndrome patient iPSC-derived assembloids as well as Down syndrome fetal brain tissue. Our work establishes new approach for the study of neurological disorders associated with nbM and nbM-cortical cholinergic neuron circuit.