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Spinal bifida aperta (SBA) is a congenital malformation with a high incidence. Bone marrow mesenchymal stem cell (BMSC) transplantation has the potential to repair the structure of damaged tissues and restore their functions. This is an optional treatment that can be used as a supplement to surgery in the treatment of SBA. However, the application of BMSCs is limited, as the neuronal differentiation rate of BMSCs is not satisfactory when used in treating severe SBA. Thus, we aimed to assess the effect of neural stem cell (NSC)-derived exosomes on BMSC neuronal differentiation and observe the therapeutic effect in an ex vivo rat SBA embryo model. We found that NSC-derived exosomes increased the neuronal differentiation rate of BMSCs in vitro and in the SBA embryo model ex vivo. Proteomic analysis showed that NSC-derived exosomes were enriched in Netrin1, which positively regulated neuronal differentiation. Netrin1 increased the neuronal differentiation rate of BMSCs and NSCs and upregulated the expression of the neuronal markers, microtubule-associated protein (Map2), neurofilament, and β3-tubulin. Bioinformatic analysis revealed that Netrin1 treatment increased the expression of the transcription factors Hand2 and Phox2b, related to neuronal differentiation. Furthermore, the Netrin1-induced NSC neuronal differentiation was significantly blocked by Phox2b knockdown. We suggest that NSC-derived exosomal Netrin1 induces neuronal differentiation via the Hand2/Phox2b axis by upregulating the expression of Hand2 and Phox2b. Therefore, NSC-derived exosomes are a critical inducer of BMSC neuronal differentiation and represent a potential treatment agent that can benefit BMSC treatment in SBA.

Neural Stem Cell-Derived Exosomal Netrin1 Contributes to Neuron Differentiation of Mesenchymal Stem Cells in Therapy of Spinal Bifida Aperta