Spinal Cord Injury Treated By Neural Stem Cells-derived Umbilical Cord Stromal Cells Combined With BDNF

Background:Spinal cord injury (SCI) is one of the common and serious traumas. The incidence of spinal cord injury increases significantly with the rapid development of economic construction and motor vehicle traffic transportation. Spinal cord injury leads to lifelong paralysis easily, which brings heavy economic burden to the whole family and society. In recent years, studies showed that neural stem cells(NSCs) transplantation could promote recovery of spinal function in experimental animals with SCI,and the transplanted neural stem cells can survive, proliferate and migrate in the damaged area. Brain-derived nerve growth factor (BDNF) is one of the important members of neurontrophic factors (NTFs), it plays an important role in the nervous system development, functional maintainance, forming of neurons and the proliferation, differentiation of neural stem cells in the damaged brain after brain injury. In this study, we used Neural Stem Cells-derived Umbilical Cord Stromal Cells Combined with BDNF to treat Spinal Cord Injury in order to confirm if it is a effective method for SCI.Objective:In our study,An animal model of transected spinal cord injury (SCI) was used to expore the curative effects of cografted human umbilical mesenchymal stem cells-derived neurospheres (HUMSC-NSs) Combined with BDNF and evaluate its feasibility and effectiveness of the restoration for SCI.Methods:(1) We cultivated cord mesenchymal cells (HUMSCs) in vitro to observe their growth, then used our licensed technology to differentiate HUMSCs to HUMSC-NSCs,at last identificated HUMSC-NSCs by Immunofluorescence method. (2) Immunofluorescence method and western blot were used to identify the differentiated neurons, Astrocytes and Oligodendrocytes for two groups(HUMSC-NSs+BDNF and HUMSC-NSs)after HUMSC-NSCs were transformed to them. (3) Sprague-Dawley rats were divided into four experimental groups:(a) control group, culture medium-treated group,(b)HUMSC-NSs-treated group, (c) HUMSC-NSs and BDNF treated group, and (d) Sham group. The spinal cord(T9-T10) was completely transected using a surgical blade. The dura was just opened for Sham group without transecting the spinal cord. (4) A behavioral test (BBB)was performed to measure functional recovery of the hind limb postoperatively. (5) Frozen spinal cord sections were stained by immunofluorescence technique to observe the survival and differentiation of HUMSC-NSs in HUMSC-NSs and BDNF combination therapy group, HUMSC-NSs treatment group and control group (medium) after transplantation. (6) Frozen spinal cord sections of HE dyeing and NF200 immune response (IR) were used to observe the gross anatomical change, regeneration of axons and formation of cavitation in damaged spinal cord,compared with sham group. (7) Frozen sections of FG marked motor area (SMC), red neuclei(RN) and spinal cord rostral to injuried site were observed to understand the repair of spinal cord axons by countering the number of labeled cells in HUMSC-NSs+BDNF treatment group, HUMSC treatment group,control group and sham groups after transplantation in order to evaluate the restoration of damaged axons.Results:In vitro,HUMSC-NSs terminally differentiated into higher percentages of cells expressing neuronal markers:P-tubulin III and MAP2ab by the supplement with BDNF,compared with the control. Following grafted into injured spinal cord, very few grafted cells survived in the HUMSC-NSs, BDNF-treated (<3%) and HUMSC-NSs-treated (<1%) groups in vivo. The survived cells were differentiated into various cells, which were confirmed by double staining of BrdU and neural or glia markers. In comparison, more grafted cells in the HUMSCNSs+BDNF group transformed into mature neural-like cells, while more grafted cells in the HUMSC-NSs group transformed into oligodendrocyte-like cells. HUMSCNSs+ BDNF-treated group had more greatly improved BBB scores, compared with HUMSC-NSs-treated and medium-treated groups. Additionally, axonal regeneration showed significant improvement in rats receiving HUMSCNSs+BDNF, compared with HUMSC-NSs-treated and medium-treated groups, as demonstrated by the NF-200-positive staining and Fluorogold (FG) retrograde tracing study (F=31909.63, P<0.001). Lastly, a significant reduction in the percentage cavitation was seen in the HUMSCNSs+BDNF-treated groups compared with medium control group and HUMSC-NSs.Conclusion:BDNF can promote the neural differentiation of HUMSC-NSs in vitro and in vivo. However, cellular replacement is not the important mechanism to explain the improvement in functional outcome. The neural recovery might more rely on the axonal regeneration and neurotrophic factors. Our study confirms that Neural Stem Cells-derived Umbilical Cord Stromal Cells Combined with BDN is a potential therapy for SCI.