The functional specificity of supernatant from cranial and trunk neural crest cells derived from embryonic stem cells in repairing rat sciatic nerve injury

Background　Peripheral nerve injury is a highly prevalent disabling disease. The immune rejection and tumorigenic risks of cell therapy limit its clinical translation, while the functional specificity effect of the embryonic regionalization characteristics of neural crest cell supernatant on nerve repair remains unclear. Objective　To investigate the reparative effect and functional specificity of the supernatant of cranial neural crest and trunk neural crest cells induced by human embryonic stem cells on sciatic nerve injury in rats. Methods　Cranial neural crest and trunk neural crest cells were induced by adjusting the concentration and action time of WNT (wingless-related integration site) pathway activators. After purification via magnetic bead sorting, quantitative Real-Time PCR was adopted to detect the expression levels of neural crest-specific markers (such as SOX10 and P75NTR) and regional-specific markers of the HOX family, and immunofluorescence staining was used to verify the identity of neural crest cells. A rat model of sciatic nerve transection injury was established, and the experimental animals were randomly divided into the control group (CTRL group), cranial neural crest supernatant group (CNC-S group) and trunk neural crest supernatant group (TNC-S group), with 6 rats in each group. Polycaprolactone conduits loaded with basal medium, cranial neural crest supernatant and trunk neural crest supernatant were used for bridging repair respectively. After surgery, immunofluorescence staining was performed to detect indicators related to angiogenesis (α -SMA, CD31) and axon extension (S100- β, NF200); transmission electron microscopy was used to detect myelin formation-related indicators including myelin thickness and G-ratio. The repair differences among groups were analyzed combined with histopathology. Results　The successfully induced cranial neural crest cells highly expressed HOXA1 and HOXB1, while trunk neural crest cells highly expressed HOXA7 and HOXB7. Both cell types stably expressed neural crest molecules such as SOX10 and P75NTR (P＜0.01). Both the CNC-S group and TNC-S group significantly repaired sciatic nerve defect injury in rats. Compared with the CTRL group, the proportion of α-SMA⁺CD31⁺ positive areas for early angiogenesis in the CNC-S group was significantly increased (P＜0.01); the axon regrowth length (P＜0.01) and myelin thickness (P＜0.01) in the TNC-S group were significantly higher than those in the CNC-S group and CTRL group, and its G-ratio was closer to the optimal value of 0.6, with statistically significant differences compared with the other two groups. Conclusions　This study demonstrates that cranial neural crest cell supernatant (CNC-S) and trunk neural crest cell supernatant (TNC-S) exhibit regionalized functional specificity in repairing sciatic nerve defect: CNC-S predominantly promotes early angiogenesis, whereas TNC-S drives axon extension and myelin maturation. These findings elucidate the mechanism underlying the functional division of regionalized neural crest supernatants, provide a novel strategy for precision and sequential cell-free repair of peripheral nerve injury, and lay an experimental foundation for the development of typed and targeted biological agents for nerve repair.