ABSTRACT: Mesenchymal stem cell (MSC)-derived neurons offer a promising therapeutic approach for the treatment of spinal cord injury (SCI). In this study, we developed a one-step differentiation system utilizing a cocktail of CYSP (CHIR99021, Y27632, SB431542, and Purmorphamine) to convert rat MSCs into neuron-like cells. After 7 days of differentiation, over 80% of the cells were positive for neuronal markers Tubb3 and Map2. Notably, the cells expressed Synapsin-1, a protein associated with functional neuronal synapses, after 14 days of differentiation. In addition, the differentiated cells exhibited characteristics of motor neurons, as evidenced by the presence of Islet1 and CHAT, markers of motor neuron identity. Transcriptomic analysis revealed significant upregulation of genes related to neuronal development, synapse formation, and synapse transmission at day 7 of differentiation, while genes associated with cell proliferation and the cell cycle were downregulated. Notably, the induction process led to the significant inhibition of the Hippo signaling pathway, while activation of the HIF1α, JAK/STAT, and PI3K/AKT signaling pathways was observed. To further enhance therapeutic potential, we developed a biomimetic silk scaffold (BSS) neurocomplex featuring a 3D nanofiber structure, designed to match the compressive modulus of the human spinal cord, by seeding induced neuron-like cells (iNs) onto the scaffold. We demonstrated that this neurocomplex effectively integrates into the host neural circuit, promoting neural regeneration and significantly enhancing functional recovery in rats with SCI. Collectively, our findings suggest that MSC-derived neurons, in combination with the BSS to form the neurocomplex, offer a promising strategy for SCI treatment and provide valuable insights for future regenerative therapies.
