Isolation Of The Mesenchymal Stem Cells Derived Exosomes And Preliminary Study On Functional Recovery After Spinal Cord Injury

Exosomes are cell-derived membrane vesicles virtually secreted by all cells that are loaded with biologically active molecules such as protein,lipid,RNA^[1-3].Due to their low immunogenicity,long half-life in circulation and ability to cross the brain-blood barrier?BBB?,spread to body fluids and reach distant tissues.This prominent spreading ability has suggested that exosomes can be exploited into several different clinical applications ranging from biomarkers to therapeutic carriers^[4-7].Compared to other cells,MSCs can produce large amounts of exosomes^[8].MSCs secreted bioactive factors with strong immunomodulatory activity,inhibits fibrosis and apoptosis,enhances angiogenesis and promotes neuron survival and differentiation^[9].Therefore,it is used extensively for nerve repair.Currently research shows that the therapeutic effect of MSCs may be mainly through secrated exosomes^[10-13].Therefore,MSCs derived exosomes are expected to become a novel therapeutic tool.The placental tissue has a largely number of mesenchymal stem cells,which has largly clinical application value,due to it is easys to culture and has the ability of a large amount proliferation and the ability to produce abundant exosomes.In this article,we first isolated MSCs from term fetal placental tissue,and were able to stably establish long-term passages.Flow cytometric analysis results showed that MSCs express mesenchymal cell surface markers CD44,CD73,CD90,and CD105,instead of Hematopoietic cell surface markers CD11b,CD19,CD44,HLA-DR,three lineage differentiation showed that they were able to differentiate into adipocytes,osteocytes,and chondrocytes,demonstrating that they have three-line differentiation potential.Purification of exosomes with good biological activity is crucial,and the purification of exosomes is influenced by the medium and purification methods.Due to the large number of exosomes contained in the serum medium,the source of exosomes could not be identified during isolated.To overcome this problem,we established a serum-free culture system to culture MSCs,which can maintain the self-renewal and producation substantial exosomes.For the purification of exosomes,we used polyethylene glycol precipitation and ultracentrifugation to purify exosomes.We found that exosomes obtained by ultracentrifugation had a double-layer structure of phospholipids and diameters in the range of 30²00 nm,50 m L of culture medium of MSCs can obtain 30⁵0ug exosomes.MSCs culture in serum-free medium,we can obtain many morphologically intact exosomes through ultracentrifugation.To confirm that the function exosomes,we used spinal cord transect of rat model to detect the rEPair function of exosome to treat injured nerve.Spinal cord injury?SCI?lead to severe nerve injury in central nervous system,result in severe inflammatory reactions and scar formation^[14,15],blocks the regeneration of axons,and leads to motor and cognitive dysfunction^[16].Previous studies have demonstrated that MSCs have the function of rEPairing spinal cord injury.We speculated that MSCs-exosomes may promote functional recovery after SCI.To test this speculation,we first established spinal cord transection model in rat,and then intravenously injected MSCs-exosomes,to test the therapeutic effect of MSCs-exosomes using behavioral assessment and immunofluorescence staining in spinal cord injury.Results we found that positive cells of express pluripotent stem cell marker SOX2,PAX6,Nestin,newborn neuron marker DCX and MAP2,nevel neuron marker TUJ1and NeuN in exosomes the treatment group.BBB score and urinary plaque test results showed that the hindlimb motor function was improved.These results suggest that MSCs-exosomes can activate endogenous neural stem cells and promote differentiation into neurons,which play a role in functional recovery after SCI.Numerous studies have rEPorted that neurotrophin-3?NT3?can activate regeneration of endogenous neural stem cells and spinal neurogenesis,and promote axonal growth^[17].We hypothesized that exosomes load NT3 released into the SCI site may further promote the rEPair of spinal cord injury in the future.To explore this possibility,in this article we established the NT3 overexpressing MSCs cell lines.Over-expression of NT3 lentivirus was packaged using calcium transfer and over-expressed NT3 in MSCs.This will allow us to continue to study the prEParation of exosomes load NT3 in the treatment of spinal cord injury and provide new ideas for the treatment of spinal cord injury.