Heterogeneity Of Immunomodulatory Function Of Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells

Mesenchymal stem cells(Mesenchymal stem cells,MSC)have a regulatory function of adaptive and acquired immunity.MSC have rapidly used in the treatment of some immune-related diseases in clinical practice since it was successfully applying for graft-versus-host disease(Graft-Versus-Host Disease,GVHD).The study on the mechanism of MSC showed that the function of MSC was influenced by the cell source,the culture condition in vitro and the microenvironment post-input.A large number of clinical trials have demonstrated that MSC has a significant difference heterogeneity in the clinical treatment of GVHD.It is really difficult for MSC survive after intravenous administration.The functions of MSC mainly depend on paracrine and the exosome.It has been demonstrated the exosome interaction channels involve in the process of MSC immune regulation.However,the exosome particularlyhasa smallvolume 30nm~100nmdiameter,which is difficult to be efficient prepared by the existing methods.At present,it has not been studied whether the difference of therapeutic effect of MSC is related to the difference of the immune regulation function of exosome.his study explored the problem employing the following experiments.The experimental MSC were extracted from umbilical cord of healthy pregnant women at Air Force GeneralHospital from 2015-01 to 2015-05,with the amplification of P3-P5.MSCwere divided into 5 groups.Polyethylene glycol 6000(Polyethylene glycol 6000,PEG6000)was used to extracted exosomes from supernatant without serum in 5 groups of MSCs,respectively.Morphological and flow cytometry were used to determine the harvests from supernatants and compare the protein levels of the 5 groups.In additionfive preparedMSC lines were randomly selected from the standard cells from independentdonors.Then extractthe exosomes from thepreparedMSCs using PEG6000 withappropriate concentration.The mononuclear cells from heparinizedumbilical cord blood(Umbilical cord blood mononuclear cells,UBMCs)from healthy donor were extracted and divided into 5 groups(negative control group,experimental group 1,experimental group 2,experimental group 3,positive control group).The cells in negative onlywith serum-containedRPMI1640 medium.T The cells inpositive control group and the experimental groups were stimulated with concanavalin A(Concanavalin A ConA)andrecombinant interleukin 2(rhIL-2).In addition,the cells inexperimental groups 1,2,3 co-cultured withthe hUC-MSCs-exwith the concentration gradientof 2?g/ml,5?g/ml,10?g/ml.The effects of 5 different hUC-MSCs-ex on the proliferation and secretion of UBMCs were studied.The results showed that the hUC-MSCs-ex extracted by the PEG6000 was acirclefigureunder the transmission electron microscope,with a diameter of about 30~100nm,expressing CD9,CD63,CD81 and CD44.It is suggested that the extracted substances were exosomes secreted by MSC.The protein levelofhUC-MSCs-exwas 837±107?g/1×107cells with the PEG6000 final concentration was 8%.In the inhibition of cell proliferation,there was no interaction between the source and concentration factors(F=1.033,P>0.05).Different sources,different concentrations of hUC-MSCs-ex on the proliferation inhibition of UBMCs were statistically different(F=77.362,P<0.01;F=13.862,P<0.01).The inhibition ability of C#hUC-MSCs-ex was higher than that of other experimental groups.In inhibited UBMCs secretion of IFN-?,TNF-?,there were significant differences in ability of different sources,different concentrations of hUC-MSCs-ex(F=63.390,P<0.01;F=14.605,P P<0.01).The ability of hUC-MSCs-ex to inhibit the secretion of UBMCs was statistically different,and the inhibition ability of A#hUC-MSCs-ex was higher than that of other experimental groups.In the inhibition of cell killing ability,the effect of killing ability of different sources of exosomes on UBMCs with concentration is 2? g/ml and5?g/ml was differences(F=12.576 P P<0.05;F=8.702,P<0.05),which inhibits the ability of D#hUC-MSCs-ex were higher than that of other groups.In summary,our study found that PEG6000 with a 8% final concentrationcouldbe used to quickly and efficiently prepare the exosome.The immunoregulatory functions of hUC-MSCs-ex from different donors had heterogeneities.