A system for high-throughput axonal imaging of induced pluripotent stem cell-derived human i³Neurons

Neurons have long, thin axons and branched dendritic processes which rely on an extensive microtubule network that functions as a cellular scaffold and substrate for cargo transport. Microtubule defects are a defining pathological feature of neurological disorders. The highly arborized, long, polarized neuronal processes pose challenges for imaging-based assays. Available methods use either dispersed cultures, which are inefficient for compartment-specific analyses, or microfluidic chambers, which allow clear separation of somatodendritic and axonal compartments but are expensive and difficult to maintain. Here, we introduce an "i³Neurosphere" culture model of induced pluripotent stem cell (iPSC)-derived human cortical i³Neurons that enables high-throughput imaging of hundreds of axons without specialized equipment. We characterize neurite outgrowth, polarization, microtubule dynamics, and motility of diverse cargo, providing a reference for future work on microtubule processes in this system. The high-throughput compartment-specific imaging we present, combined with facile genetic engineering in i³Neurons provides a powerful tool to study human neurons.