| BackgroundRecently,due to the occurrence of earthquake,car accident and other disasters,the injury of sciatic nerve is increasing day by day.Treating the injury of peripheral nerve,such as sciatic nerve,is a great clinical demanding.Peripheral nerve regeneration has been the focus of neurobiology research.Autogenous nerve transplantation is still the "gold standard" for repairing peripheral nerve defects,but donor sources are limited,the residual donor area sensory dysfunction and donor trauma would be exacerbated.The development of tissue engineering in recent years has brought new hope for long segment defect injury.Neural stem cells(NSCs)can differentiate into various types of nerve cells.Transplanting it to the injured site could promote axon regeneration and myelin formation.Previously,our team focused on the culture of human embryonic stem cells(hESCs),and found that embryonic stem cells differentiated spontaneously into neural stem cells by chance.We expected that hESCs could be induced to differentiate into high purity neural stem cells under certain conditions(human embryonic stem cell derived-neural stem cells,hESC-NSCs).Because of the limited sources of neural stem cells,NSCs derived from hESCs was particularly important.Whether NSCs could be obtained with high purity and high yield makes it possible to be applied to clinical cell therapy.We conjectured that if hESC-NSCs could promote axonal regenerationfrom as the NSCs from the fetal brain,and further studies showed that hESC-NSCs could promote axonal regeneration through indirect action.Now more and more studies had shown that NSCs played a role in repairing nerve injury not through direct cell integration but through secreting factors,and NSCs was a nucleated cell,which was at risk of tumorigenesis.Microvesicles(MVs)are active and tangible components secreted by cells into conditioned medium,which were the material basis for material transmission and information exchange between cells and their surrounding cells or environment.Therefore,we speculated that microvesicles released from hESC-NSCs(hESC-NSC-MVs)might promote axonal regeneration as well as NSCs,and further study them.ObjectivesWe aimed to investigate the neural stem cells differentiation induced from human embryonic stem cells and the effect of microvesicles secreted by neural stem cells on the repair of 5mm defect of sciatic nerve in rats.Methods(1)Neural stem cells were induced from feeder-free hESCs and identified by immunofluorescence and PCR.(2)PCR and flow cytometry were used to demonstrate that neural stem cells could undergo adherent-sphere transition to improve stemness.(3)Serum-free medium or conditioned medium of hESC-NSCs was collected and hESC-NSC-MVs were isolated through ultracentrifugation.(4)The hESC-NSC-MVs were co-cultured with dorsal root ganglia to determine the length and number of axons.(5)To establish a model of sciatic nerve defect in SD rats,and evaluate the effect of hESC-NSC-MVs on sciatic nerve injury in rats by means of sciatic nerve functional index(SFI)and HE and Masson staining.Results(1)hESCs could differentiate into neural stem cells,and the highest expression of neural stem cell markers Nestin,Pax6 and other was found near P8 hESC-NSCs.(2)The expression of neural stem cells markers Nestin,Pax6 and stemness of neural stem cells increased after adherent-sphere transformation.(3)The length and area of regenerated axons in hESC-NSC-MVs co-cultured with dorsal root ganglion group were better than those in PBS group.(4)A rat model of sciatic nerve defect injury was successfully established.The morphology recovery and the number of regenerated axons in hESC-NSC-MVs groupwere significantly higher than those in hESC-NSCs group 12 weeks after nerve injury.Conclusions(1)hESC could differentiate into NSCs and hESC-NSCs expressed neural stem cell markers.(2)hESC-NSCs could increase the expression of neural stem cells markers and stemness through adherent-sphere transformation.(3)hESC-NSC-MVs could repair sciatic defect nerve injury in rat. |
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