Advancing human iPSC-derived motor neuron models using glutamatergic modulators for synaptic function studies

Abstract Understanding human synapse structure and function at nanoscale resolution requires experimental systems that support both high-quality imaging and robust synaptogenesis. While brain tissue and primary neuronal cultures have been widely used, human induced pluripotent stem cells (iPSCs) offer key advantages: they enable patient-specific modeling and provide a renewable source of human neurons without relying on animal models. However, reproducible synapse formation in iPSC-derived neurons, particularly motor neurons (MNs), remains challenging due to variable differentiation efficiencies and low synaptic density. Here, we optimized MN differentiation to enhance synaptogenesis and enable high-resolution structural analysis using cryo-electron tomography (cryo-ET). Within 28 days, human iPSC-derived MNs developed into morphologically and functionally mature neuronal networks, characterized by phase-bright somata, elongated axons, and dense axodendritic branching. To further promote synaptic connectivity, we applied the glutamatergic modulators CX516 and CDPPB during differentiation. Their effects were validated by immunolabeling of synaptic markers, electrophysiology, calcium imaging, live-cell synaptic vesicle recycling assays, and ultrastructural imaging, including cryo-ET. This combined approach of optimized differentiation and targeted neuromodulation yielded reproducible MN networks with enhanced synaptic density and function, providing a robust in vitro platform for investigating human MN physiology, synaptic mechanisms, and disease-relevant synaptopathies.