Rapid and Simplified Induction of Spinal Motor Neurons From Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) provide a critical source for the study of motor neuron diseases (MNDs), which has been hindered by the lack of appropriate disease models for many years. Although many spinal MN differentiation protocols have been established by mimicking in vivo neurogenesis using extrinsic signaling molecules, substantial variations in the duration and efficiency persist due to inconsistencies in concentrations, timing, and delivery methods of these molecules. Here, we present an efficient monolayer culture differentiation strategy that enables the generation of enriched CHAT⁺ spinal MNs (sMNs) in 18 days and functional sMNs exhibiting extensive network activities, as confirmed by multielectrode array (MEA), within 28 days. Therefore, this optimized MN differentiation protocol facilitates the production of mature sMNs for MND research, high-throughput drug screening, and potential cell replacement therapies. Key features • This protocol provides a rapid and simple monolayer culture strategy for differentiating hiPSCs into sMNs in 18 days. • Early administration of the Notch inhibitor Compound E accelerates the generation of hiPSC-sMNs 10 days in advance compared to a previous protocol. • This protocol uses neural stem cells (NSCs) and MNPs as an intermediate to generate functionally mature sMNs.