Bone Mesenchymal Stem Cell-conditioned Medium Induces The Upregulation Of Smad6,Which Inhibits The BMP-4/Smad1/5/8 Signaling Pathway

Objectives: According to the current epidemiological survey shows that the incidence of spinal cord injury has gradually increased,while the treatment of spinal cord injury strategy is very limited,often could not get satisfactory results.In recent years,more and more scholars have begun to study stem cells as a material for the treatment of spinal cord injury.Before animal models and clinical trials,it is necessary to explore the differentiation process of neural stem cells under various conditions in vitro.It is possible to simulate changes in the microenvironment after spinal cord injury in order to improve the efficiency of stem cell research.In this study,we aimed to investigate the effect of bone mesenchymal stem cells(BMSCs)conditioned medium on BMP4-induced activation of the Smad signaling pathway during the process of neural stem cells differentiation.Methods: In the course of the experiment,neural stem cells were extracted from the brain tissue of 24 h SD rats,and bone mesenchymal stem cells were extracted from SD rats.The cells were cultured respectively and identified by immunofluorescence staining and flow cytometry.BMSC-CM was obtained according to the standard method,and then neural stem cells differentiated under different conditions.We designed four experimental groups: control group,BMP-4 treatment group,BMSC-CM group,and BMSC-CM(BMSC-conditioned medium)combined with BMP-4 group.The expression of microtube-associated protein-2(MAP-2)and glial fibrillary acidic protein(GFAP)was detected by immunocytochemical staining and western blot.The expression of BMP receptors(BMPR? and BMPR?a)was determined by western blot,while SMAD1/5/8 and SMAD6 m RNA expression was examined by Q-RT-PCR(quantitative real-time polymerase chain reaction).Results: The cells were identified by immunofluorescence staining and flow cytometry were neural stem cells and bone marrow mesenchymal stem cells respectively.We found that the expression of microtube-associated protein-2(MAP-2)decreased in BMP-4 group compared to control group through Western Blot,and the results of neurons via immunocytochemical staining was consistent.In BMSC-CM group,the expression of GFAP was increased and the expression of MAP-2 was decreased compared to control group.This phenomenon was weaker in Co-culture group.We also demonstrated that BMSC-CM could inhibit the activation of BMP signaling by downregulating the expression of BMPR? and BMPR?a proteins.Moreover,BMSC-CM could upregulate the expression of Smad6 m RNA and inhibit the activation of Smad1/5/8 m RNA and REST m RNA.Conclusion: These observations suggest that BMSC-CM could neutralize the effect of BMP-4 on the differentiation of NSCs by upregulating the expression of Smad6 and decrease the expression of GFAP by inhibiting the BMP-4-SMAD1/5/8 signaling pathway,conversely increasing the generation of neurons.It could be seen that bone marrow mesenchymal stem cell culture medium(BMSC-CM)can change the process of differentiation of neural stem cells and improve the differentiation efficiency of neural stem cells.Therefore,the use of bone marrow mesenchymal stem cell conditioned medium(BMSC-CM)and neural stem cell sequential transplantation method may be able to greatly improve the efficiency of treatment for the follow-up animal model test and clinical trial provides a new idea.