| The limited regeneration ability of the central nervous system leads the central nervous system diseases,including spinal cord injury,to be a clinically difficult to overcome.However,with the progress and development of science and technology,cell culture,gene technology and biomaterials in the field of neuroscience,have provided new solutions for spinal cord injury,among which cell transplantation makes a figure.New opportunities are always accompanied by new problems and challenges,the source,survival and function of transplanted cells will be taken into account.Mesenchymal stem cells(MSCs),a widely-studied non-neuronal cell in spinal cord injury,are characterised by high proliferative and differentiation abilities,low immunogenicity,adequate source,autologous transplantation.However,studies have shown that only a small percentage of the transplanted MSCs can reach the lesions,leading to a very low rate of cell replacement.Thus,enhancing the migratory capacity of MSCs is critical to maximize the effectiveness of MSCs-based therapy.The migration ability and the chemotaxis of MSCs are differently dependent on the cell sources,cell cycle stages and differentiation states.In our study,MSCs were induced into neurons with five stages,and 24 h-preinduced MSCs have the strongest migration capacity.Studies have shown that the injured area can release a large number of chemokines,spinal cord injury is also true.In line with it,the expression of SDF-la(cell-derived factor-la)and HGF(hepatocyte growth factor)was continuously increased in the window period(7 days to 14 days)that suitable for cell transplantation for the treatment of spinal cord injury.At the same time,we transplanted Lenti-GFP labeled MSCs after 7 days of transection modeling by intrathecal injection of T9-T10 in the rat spinal cord.It was found that the cells were clustered around the injured area,which further validated the theory of the existence of a large number of chemokines in the injured region.Therefore,we studied the molecular mechanisms of SDF-1? and HGF induced chemotaxis migration of MSCs under varying neural differentiation states.Many growth factors and cytokines have been found to act as strong chemoattractants for MSCs,among which SDF-1? and HGF have received much attention.SDF-la through its cognate receptor CXC chemokine receptor 4(CXCR4),plays a pivotal role in migration,engraftment and survival of MSCs,and stimulates the homing of transplanted MSCs to injuried tissue.Firstly,we took advantage of the direct-viewing Dunn chemotaxis chamber to investigate the migration rate and migration efficiency of MSCs after SDF-la stimulation.MSCs of 24-h preinduction exhibited the strongest chemotactic response as compared with cells in other differentiation states.Cell migration is a complex process that results from the ordered changes in the cytoskeletons and the regulated formation,turnover and distribution of focal adhesion(FA).Studies have shown that tyrosine phosphorylation of focal adhesion proteins,including FAK and paxillin,is increased in migrating cells and that FA signalling plays an important role in SDF-1?-induced cell migration.Since MSCs of varying differentiation states have different migratory response to SDF-1?,we test whether the assembly,turnover of FA and organisation of F-actin vary in SDF-la-stimulated MSCs and the relationship with the differentiation of these cells.We found that the assembly of FA correlated closely with the differentiation of MSCs upon SDF-1? treatment,and that cells of 24-h preinduction have a higher level of FA assembly and ratio of small FA that usually characterise the fast moving cells,which is consistent with the above-rmentioned observations that MSCs of 24-h preinduction exhibit the most effective migration toward SDF-la.Furthermore,the differentiation of MSCs greatly affects the phosphorylation of FAK and paxillin in response to SDF-1?,which might contribute to the assembly of FA and the chemotactic response of MSCs.In addition,PI3K/Akt signalling is involved in the assembly and turnover of FA in all MSCs tested,the effects of signallings via MAPKs,including ERK1/2,p38MAPK and SAPK/JNK are strictly dependent on the differentiation states of MSCs.HGF also promotes the migration,proliferation and proliferation of a variety of cells,including MSCs.We studied the effect of HGF on the migration of MSCs in various differentiation states.More and more evidence suggests that activation of?-catenin can promote the migration of MSCs.The activation of the p-catenin signaling pathway means that ?-catenin protein accumulates in the cytoplasm after being free from the degraded complex,and accumulated p-catenin further transports into the nucleus.In the nucleus it interacts with the T-cell factor/lymphoid enhancer factor(TCF/LEF)family of transcription factors,leading to gene transcription to regulate cell behaviours.We found that the translocation of ?-catenin into the nucleus,the expression of activated ?-catenin,the activation of Top/Fop luciferase and the expression of classical Wnt/?-catenin signal target gene c-Myc were increased in the HGF-treated MSCs.All these results proved that HGF could activate ?-catenin signaling pathway in MSCs.Furthermore,we found that both the activation of ?-catenin signal and the HGF-induced chemotaxis were different in cells of different differentiation states,and the cells of 24-h preinduction showed the strongest.Nedd9(neural precursor cell expressed,developmentally down-regulated 9)is a downstream target gene of ?-catenin signaling pathway,and is involved in the stabilization of cell polarization and polarization direction,FA turnover and cell migration.In our study,activation of the?-catenin signaling pathway in MSCs promoted the expression of Nedd9,while inhibition reduced Nedd9 expression.To further elucidate the molecular mechanism,we used the lentivirus to specifically knockdown ?-catenin and its downstream target gene Nedd9 in MSCs,and found that interfering with ?-catenin and Nedd9 inhibited HGF-induced chemotaxis,indicating that HGF-induced chemotactic migration of MSCs is achieved by up-regulating the expression of ?-catenin signaling pathway target gene Nedd9.One of the ways in which Nedd9 regulates MSCs migration is by regulating the assembly of FA.We can conclude that cells of 24-h preinduction are the best target for transplantation into spinal cord injury among all the differentiation states because of the strongest chemotactic migration capacity towards SDF-la and HGF,which were up-regulated in the damaged region.In the study of the effect of Nedd9 on the chemotaxis of MSCs to HGF,we were surprised to find that the morphology of MSCs with high expression of Nedd9 changed.These cells like a neuron-like cell with long protrusions.This phenomenon leads us to explore whether neuronal differentiation can be acquired by altering the expression of Nedd9.On the one hand,astrocytes and neurons belong to the same neural lineage.On.the other hand,a large number of active glial cells are generated to form glial scars after spinal cord injury.These glial cells are very dense,secreting inhibitory molecules into the extracellular matrix,leading to the impaired neuronal axons being difficult to regenerate through the lesion.Therefore,we through the transformation of astrocytes making it waste into treasure.Since Nedd9 is a highly expressed gene in neural stem cells,we hypothesize that Nedd9 can induce the transdifferentiation of astrocytes into neurons through neural stem cells.We confirmed our hypothesis by lentiviral-mediated induction of Nedd9 expression in a tetracycline inducible Tet-on system.We firstly used Dox to induce astrocytes to express Nedd9 to dedifferentiate into neural stem cells,and further reduced the expression of Nedd9 in neural stem cells to differentiate into neurons in neuron-induced medium.During this process,the expression of Isll,Mytll,Olig2 and Proxl increased.In addition,we found that high expression of Nedd9 promotes the proliferation and survival of astrocytes.It would be benefit for obtaining a large number of transplanted cells and their survival.Finally,we used the transection injury model of rat spinal cord at T9?T10 to test the above in vitro experiments of cell behavior.BBB(Basso,Beattie and Bresnahan)and immunohistochemical experiments showed that compared with the transplanted undifferentiated MSCs,MSCs of 24-h preinduction migrated to the injured area with larger number,reduced the area of cavity with a greater degree and promoted the increase of BBB score in the hind limb of rats.Astrocyte-derived neural stem cells were transplantated into the injuried spinal cord and differentiated into spinal cord specific neurons,and the transplantation increased BBB scores.In this study,we present a new idea for promoting the migration of MSCs and inducing astrocyte transdifferentiation to provide a new idea for cell transplantation to repair spinal cord injury and provide a theoretical basis for the future clinical application of spinal cord injury. |
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