Development of a 3D Nanofiber Neurocomplex from MSCs-Derived Neuron-like cells for Spinal Cord Injury Regeneration and Functional Recovery

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 HIF1a, 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. Overall design: To investigate the mechanisms underlying CYSP-induced neuronal differentiation of MSCs, we performed RNA sequencing to analyze changes in gene expression at the transcriptional level over a 7-day period. The volcano plot illustrates the number of differentially expressed genes (DEGs) during transdifferentiation, with 1,947 upregulated and 1,919 downregulated genes identified. KEGG enrichment analysis revealed that these DEGs were significantly associated with signaling pathways closely linked to neurogenesis and neuronal differentiation, including MAPK, Hippo, and PI3K/AKT. Further analysis of upregulated and downregulated genes using heatmaps and Gene Ontology (GO) analysis highlighted that DEGs related to nervous system development, neuronal differentiation, synapse formation, and synapse transmission were significantly upregulated, while genes associated with cell proliferation and the cell cycle were notably downregulated. In addition to specifically targeting individual signaling pathways, small molecules also modulate the crosstalk between multiple pathways. Different combinations of these molecules can lead to variations in neuronal subtypes and conversion efficiency. Gene Set Enrichment Analysis (GSEA) revealed that the HIF1a, JAK/STAT, and PI3K/AKT signaling pathways were significantly activated following transdifferentiation, while the Hippo signaling pathway was notably inhibited.