The Contributions And Mechanisms Of Human Somatic Stem Cell In Repair After Spinal Cord Injury

Objective: Spinal cord injury(SCI)is usually caused by an external force,leading to severe dysfunction of the limbs below the injured segment.Human somatic stem cells(h SSCs)have been shown to contribute to the repair of spinal cord injury after transplantation,although there are still certain issues to be addressed.For example,the most appropriate time window of transplantation,the best administrative method,the safe and optimal dosage of human neural stem cells(h NSCs),and whether the combination of allogeneic transplantation with an immunosuppressant can achieve a better therapeutic effect are still unclear.On the other hand,the inflammatory response after microglia activation plays a vital role in the prognosis of SCI,and human umbilical cord mesenchymal stem cell(h UCMSC)transplantation has been proven to improve the repair of SCI by reducing the inflammatory response;our previous research showed that h UCMSCs from 32 donors have different inhibitory abilities on BV2 cell proliferation due to heterogeneity;therefore,it is necessary to reveal whether there are differences in the treatment of spinal cord injuries,and further explore thier mechanism.Methods: A spinal cord clamp injured mice model was established,and hNSCs were administrated by three different transplantation routes: intravenously,intrathecally,and in situ routes at 3 weeks after SCI,h NSCs were injected intravenously with low and high doses at 1 week after SCI,in addition,three different dosages(low,medium,and high)of h NSCs were injected intrathecally at 1 week after SCI;a safer and more effective transplantation method was determined by detecting the survival of transplanted mice combined with the behavioral function recovery detected by BMS and electrophysiology.To test the necessity of combination treatment with an immunosuppression drug,Rapamycin was injected peritoneally before and after the transplantation of h NSCs,the recovery of motor function was monitored by BMS score,Western blot(WB)and real-time fluorescence quantitative PCR(q PCR)were used to detect and analyze the changes of SCI related proteins and m RNA,Hematoxylin and eosin(HE)staining and immunofluorescence staining were used to observe the histological changes in the spinal cord.On the other hand,the three h UCMSC lines with strong,medium and weak ability that inhibit the proliferation of BV2 cells were regarded as high,medium and low selective h UCMSCs,which were labeled as MSC_A,MSC_B and MSC_C.HUCMSCs were transplanted into spinal cord clamp injured mice intravenously at 1d,3d,and 2w after SCI,where hind limb motor function was observed by BMS score and the changes of SCI related proteins and m RNA were analyzed by WB and q PCR.HE staining and immunofluorescence staining were used to observe the histological changes of spinal cord,the underlying mechanisms were analyzed by RNA sequencing and correlation analysis,which were finally verified by q PCR.Results: The autonomous recovery of motor function in mice was limited after SCI according to a standardized behavior test(BMS),and both intrathecal and intravenous injections obtained the best outcomes in behavior results,however,intravenous transplantation recorded an impressively high death rate,which might be caused by lung embolism of aggregated human neural stem cells.Comprehensive consideration,in the first week after SCI,the administration of h NSCs at a medium dose was safer and more effective;h NSCs transplantion not only prevented neuronal loss and glial scar formation,but also shortened the latency of motor potential and improved BMS score.While the intervention of rapamycin had little effect on the BMS score;in the latter stage,it led to the infection of SCI mice,the combined treatment with h NSCs did not achieve a better therapeutic effect on SCI.On the other hand,the therapeutic effect of h UCMSC transplantation on the third day was better than that on the first day and the second week after SCI.The therapeutic effects of the three h UCMSC lines were positively correlated with their inhibitory abilities of BV2 cell proliferation rates in vitro.The MSC_A line had greater effect on reducing the protein and m RNA expression of glial fibrillary acidic(GFAP)and ionized calcium binding adaptor molecule 1(IBA1)and increasing the expression of neuronal nuclei(NEUN),and had a better therapeutic effect on improving the hind limb motor function.After transcriptome sequencing analysis,differentially expressed genes including Zbtb16,Per3,and Hif3 a were probably the key genes involved in the protective mechanism by MSC_A after SCI,q RT-PCR results confirmed that SCI-induced reductions in Zbtb16,Per3,and Hif3 a expression could be reversed by MSC_A application.Conclusion: A medium dose of hNSCs by intrathecal transplantation was shown to be safer and more effective in the first week after SCI.However,the combination treatment with rapamycin did not achieve better therapeutic effect on SCI.The effect of h UCMSC transplantation on acute SCI depends on their inhibitory abilities to inflammation reaction,where high selective h UCMSCs with stronger ability to inhibit the proliferation of BV2 cells had better therapeutic effect which may be related to the regulation of Zbtb16,Per3,and Hif3 a genes.Our study may help to shape future use of h NSCs and h UCMSCs combined with improving the effectiveness of clinical transformation.