Derivation of functional neurons from induced pluripotent stem cells using a simple neuromesodermal progenitor generation and rapid spinal cord neuron differentiation process.

To generate spinal cord neurons from pluripotent stem cells via neuromesodermal progenitors (NMPs) is not only an important step for regenerative purposes but also required for human developmental research. This study describes a protocol to obtain spinal cord neurons in culture using induced pluripotent stem-cell-derived NMPs. The protocol starts with a 3D culture of NMPs and continues with the transfer of 3D NMPs to monolayer culture in which retinoic acid and sonic hedgehog pathways were triggered sequentially. The established protocol enabled generation of spinal cord neurons with active calcium signaling, electrophysiological activity, axon elongation capacity, and synaptic vesicle trafficking. The expression profile of marker proteins, including β-Tubulin, NeuroD1, Pax6, NeuN, Mnx-1, Isl1, Isl2, Map2, NF, Sox2 was detected to explore the production of developmental regulatory transcription factors and terminal differentiation markers in a time-dependent manner. Cells during differentiation process acquired a fully neural phenotype, which was confirmed by RNA sequencing at the molecular level. The protein expression profile showed neural differentiation induction pathways based on LS-MS/MS analysis. Since NMPs differentiate into spinal cord neuron cells at the developmental stage, the results of this study highlight the further potential of NMP-derived spinal cord neurons in disease modeling and treatment in the clinics.