Neuron damage repair with bone-marrow-neural-crest-cell-sourced secretome and small extracellular vesicles with superiority of delivery miRNA-21-5p facilitate gene-therapy for nerve defect

Cytotherapy can enhance peripheral nerve repair and regeneration via paracrine bioactive factors and intercellular communication, rather than just direct cellular interaction or cell substituting. Nevertheless, the contribution complexity of various types of soluble cytokines and extracellular vesicle (EV) cargoes in secretome remain elusive. As that, uncovering the substantial predominance of certain type cell-based therapy is imperative and also indispensable. Previously, we have revealed the repair effect of rat bone marrow derived neural crest cells (BM-NCCs), served as Schwann cell precursors, on long-distance nerve defect. Accordingly, the current work focused on exploring the superiority of BM-NCCs-sourced secretome and small EVs on repairing peripheral nerve injury (PNI). Hypoxic preconditioning and three-dimension spheres culture of NCCs was adopted to optimize the cellular paracrine state, that was verified by cell viability, proliferation, hypoxia inducible factor-1 alpha expression, and increased paracrine function of hypoxia-preconditioned NCCs (H-NCCs), as well as secretome efficacy on oxygen-glucose deprivation (OGD) injured sensory neurons. Then H-NCCs secretome derived small EVs (H-NCC-EVs) were applied to treat OGD- or axotomy- injured sensory neurons. And RNA sequencing of H-NCC-EVs showed the miRNAs profile, demonstrating that the upregulated miRNA-21-5p ranked first and accounted for nearly half of the total miRNAs read in H-NCC-EVs. Subsequently, miRNA-21-5p agomir was further utilized to repair axotomy in vitro and loaded into conduit to restore nerve defect in vivo, and the recovery efficacy on neuro-muscular damage was confirmed functionally and morphologically. Intriguingly in this work, the individual miRNAs profile signature of BM-NCC-EVs presented the overwhelming miRNA-21-5p superiority pattern that has not been reported in other type of EVs. H-NCC-EVs were revealed as a type of native nanoparticles highly enriching miRNA-21-5p. Together, this study nominated miRNA-21-5p as one main contributor among various bioactive factors delivered by H-NCC-secretome for promoting neuroregeneration, that might facilitate to interpret the action paradigm and underlying principles, as well as promote miR-21-5p application for tissue engineering regeneration medicine.