Exosomes Secrected By Human Urine Derived Stem Cells Promote Functional Improvement After Cerebral Ischemia

ObjectiveThe high mortality and disability of stroke,give a heavy financial burden to society and individuals.At present,the intravascular thrombolytic therapy by tissue-type plasminogen activator（r-tPA）is the main accepted strategy,however,the patients who are not in the time window,still face a high risk of disability due to the limited treatment.The stem cells were found owning a role promoting the functional improvement,in the experiments of cerebral ischemia model and the clinical trials of stroke.Stem cells which have the ability of self-renewal and pluripotent differentiation,mainly include embryonic stem cells（ESCs）and adult stem cells.However,the the application of ESCs are limited for the issuess of ethics and tumorigenicity.And adult stem cells,especially the mesenchymal stem cells cells（MSCs）,show a promising application potential,because they can be obtained from patients themselves.But MSCs are mostly harvested from adipose tissue and bone marrow tissue,the limited resources and traumatic behavior are the main problems of these MSCs.In recent years,our team had established a method for extracting MSCs from urine,by which we can extract large amounts of urine-derived stem cells（USCs）from urine.In addtion,the ability of USCs of adipogenesis,cartilage,osteogenic and neurons differentiation were confirmed;and the ability of repairing skin wound and bone injury by promoting angiogenesis were also confirmed.Meanwhile,USCs are abundant and noninvasive with more advantages compared to the other tissue-derived mesenchymal stem cells.However,the way of direct transplantation of stem cells is still at risk of embolization and MSCs exist the high risk of genetic variation in the long-term vitro culture.In recent years,the paracrine mechanism of MSCs were found,and exosomes had being paid more and more attention to as a paracrine mechanism of stem cells by researchers.Exosomes is a nanoscale biological vesicle with a particle size in the range of30-100 nm,which bring biological effects to effector cells by transporting some bioactive substances,such as miRNAs,mRNAs and proteins.In some experiments study,such as myocardial ischemia,skin damage and stroke,Exosomes derived from MSCs play a similar repair role with MSCs.However,the experimental study of USCs-Exo has not been reported in the field of cerebral ischemia.In view of the advantages of USCs,we established the rat model of cerebral ischemia to study the effect of USCs-Exo on stroke,providing a new method for clinical treatment.Methods1.Isolation and identification of USCs and USCs-ExoThe USCs were extracted from the urine of healthy adults according to the method established by our teams.The morphology of USCs was observed under microscope;USCs were identified by flow cytometry.The expression of Alix,tsg101 and CD63 were detected by Western blotting.The USCs-Exo was extracted from the conditioned medium of USCs by ultracentrifugation and ultrafiltration.The size of exosomes was detected by q-NANO.The morphology and size of USCs-Exo were observed by transmission electron microscopy.Western blotting was used to detect USCs-Exo-specific protein markers Alix,tsg101 and CD63.2.Establishment of Transient Middle Cerebral Artery OcclusionThe MCAO model on the right side was established by the classical suture method.This study was divided into 3 groups:USCs-Exo treatment group,MCAO group and sham operation group,and each group contains 10 rats;Twenty four hours after surgery,1ml 3×10¹¹ number of USCs-Exo were intravenously injected in USCs-Exo treatment group,the other groups were injected with the same amount of PBS solution.3.Effects of USCs-Exo on the neurological function of the stroke ratsThe mNSS score system and adhesive removal test were used to observe the neurological function of the rats before surgery and the 1st,3 th,7 th and 14 th days after operation.4.Effects of USCs-Exo on angiogenesis and nerve regenerationOn the 7th and 14th day after the operation,the proliferation of the brain was observed by Ki67 immunofluorescence staining;the angiogenesis was observed by immunofluorescence staining with CD31 and CD34.;the nerve regeneration was observed by DCX,Nestin and Sox2 immunostaining.5.Effects of USCs-Exo on neuronal apoptosisOn the 7th and 14th day after surgery,the neuronal apoptosis was observed by TUNEL staining.Results1.USCs were successfully extracted from the urineThe fibroblast-like cell clones appeared,and after the third generation,the cells showed homogeneous fibroblast-like growth.Flow cytometry showed that USCs were identical to CD29,CD73 and CD44,and HLA-DR,CD45 and CD34 were not expressed.The results showed that USCs were consistent with the characteristics of mesenchymal stem cells.2.USCs-Exo was successfully enriched from USCs mediumThe USCs-Exo was enriched in the serum-free conditionedmedium of USCs,and the USCs-Exo diameter was observed at 30-100 nm by q-NANO and TEM.Western blotting showed the expression of secreted proteins Alix,Tsg101 and CD63.3.USCs-Exo significantly improve neurological function after cerebral ischemiaOn the 14th day after operation,the neurological function of USCs-Exo group was better than that of control group（P<0.05）.4.USCs-Exo obviously promote angiogenesis and nerve regeneration in ischemic brain tissueThe immunofluorescence staining showed that the number of Ki67,CD31,CD34,Dcx,Nestin and Sox2 positive cells in USCs-Exo group increased（P<0.05）on the 7th and14th day after operation.5.USCs-Exo can reduce the apoptosis of ischemic brain tissueCompared with the control group,TUNEL staining positive cells obviously decreased on the 7th and 14th day after operation（P<0.05）.Conclution1.USCs and USCs-Exo can significantly promote the recovery of neurological function after stroke in rats,providing a new strategy for clinical treatment.2.USCs-Exo repair the ischemic brain by enhancing angiogenesis and nerve regeneration and by inhibition of apoptosis.