CD200Expression On The Surface Of Amniotic Mesenchymal Stem Cells And Its Regulation Effects On Microglia Activation

BackgroundWith the gradual maturation of stem cell transplantation, Application of cell therapy inneurological diseases has caused widespread concern. Mesenchymal stem cells (MSCs) arethe more applied type of adult stem cells with multiple differentiation capacity. Recentstudies have shown that amniotic mesenchymal stem cells (AMSCs) extracted from humanamnion at present is an ideal source of seed cells for cell therapy, because comparing withembryonic stem cells and bone marrow stem cells it has advantages of wide sources,readily obtained, easy amplification, multiple differentiation potential and lowimmunogenicity, meanwhile avoiding issues like embryonic stem cells lack, ethics andlegal restrictions. More and more animal experiments show that transplanted amnioticmesenchymal stem cells can improve the symptoms of neurological deficit, but the exactmechanism of action needs to be further explored.Microglia (MI) is now recognized as effector cells in the central nervous system react tobrain injury, inflammatory and various degenerative diseases, with its main feature ofbeing easily activated by any type of brain damage or diseases, and plays dual role ofdamage and repair in kinds of nervous system lesions. Activated microglia have manyfunctions in the innate immune response of the central nervous system, includingphagocytosis, immune inflammation, cytotoxicity, and play a regulatory role throughantigen presented T lymphocytes. MI will release various cytokines after being activated,which can cause inflammation and apoptosis in various different degrees, and constitute"pathological cascade" changes. Thus, we speculate that it will be very favorable incontrolling the entire process of pathology or injury if activation of MI can be regulated during early stage of CNS inflammation or damage.CD200as a leukocyte differentiation antigen is one type of transmembrane glycoprotein oncell surface, and together with its receptor CD200R belong to the immunoglobulinsuperfamily. CD200has its expression in a wide scope, mainly in nerve cells, thymus cells,B cells, T cells and vascular endothelial cells. Studies have shown that CD200can beexpressed on the surface of amniotic mesenchymal stem cell. CD200R expressed mainly inthe mononuclear phagocyte system (including microglia) and a small amount of T cells.CD200and CD200R induce immune tolerance and transfer inhibiting signal by cell contact.It is found that, CD200can down-regulate the activation status of CNS microglia, reduceproduction of microglia induced cytokine, we envisage that amniotic mesenchymal stemcells may also adjust the activation of CNS MI in this way, thereby control the occurrenceof its triggered series of inflammatory cascade, and accordingly play a role in cell therapy.ObjectiveThis experiment studies contact ways among cells related with CD200and CD200R, andexplore CD200effects during amniotic mesenchymal stem cells’ immune regulation inmicroglial, in order to more fully understand the action mechanism of amnioticmesenchymal stem cells.Part I In vitro culture and identification of amniotic mesenchymal stem cells andmicroglialMethods1. In vitro culture and identification of AMSCs, to observe growth situation ofprimary culture and subculture cell and make the growth curve; In vitro inductionand differentiation experiments ofAMSCs.2. In vitro culture, purification and identification of MI3. Activation of MIResults1. AMSCs obtained by successfully separated and cultured from the amniotic membrane with MSCs morphology and cell surface markers, consistent with growth andproliferation features of MSCs. Flow cytometer testing shows that the cells expressCD73, CDl05, rather than CD45and HLA-DR. AMSCs shows no expression of NSEand weak expression of GFAP before induction, but strong expression of neuronal cellspecific markers NSE, GFAP after induction.2. Successfully separated and purified MI is in slender or oval shape, with small andbranched protuberance out of cell body. Positive cells in CD68immunofluorescencestaining are MI, with the purity of more than95%.3. MI got activated after LPS stimulation, with the cells in amoeba-like appearance,positive in MAC-1immunofluorescence. MI in resting status can secrete smallamounts of TNF-α and IL-6by itself, while content in supernatant get obviouslyincreased after activation, and the difference before and after activation is statisticallysignificant (P <0.05).Part II Researches on amniotic mesenchymal cells’in vitro regulation microglialactivity and its possible mechanismMethods1. To test CD200expression on AMSCs surface by immunofluorescence staining; totest CD200expression inAMSCs from P2to P6by ELISA.2. To test CD200expression on MI surface by immunofluorescence staining; to testCD200expression in MI from P1to P4by ELISA.3. AMSCs and MI in vitro co-culture and anti-CD200antibody blocking experiment.Establish AMSCs and MI co-culture system with experiment groups as follows:MI group, MI+LPS group, MI+LPS+AMSCs group, MI+LPS+AMSCs+anti-CD200antibody group, MI+LPS+AMSCs transwell group, and observeshapes of each group after48h co-culture, test expression of inflammatorycytokines TNF-α and IL-6in cell supernatants of each group by ELISA, testCD200R expression in cells of each group by ELISA.Results1. AMSCs expresses CD200, and CD200expression showed an increasing trendfrom P2to P6, expression increasing significantly up to P6, difference between P2 and P6is statistically significant (P <0.0001).2. MI expresses CD200R, and CD200R were expressed in surface of each generationMI, compared with P1, OD value of the rest generations were all increased, but nosignificant differences between generations (P>0.05).3. With the effects of LPS, secretion of LPS TNF-α and IL-6in supernatantsincreased significantly compared with MI group, while in the LPS+MI+AMSCsgroup, the secretion of the two inflammatory factors showed a downward trendwhen there AMSCs exists, two cytokines secretion compared with LPS+MI+AMSCs set to rise after LPS+MI+AMSCs+anti-CD200antibody group beingcultured for48h, but still lower than LPS+MI group; secretion of two cytokinesin Transwell group was significantly increased compared with LPS+MI+AMSCsgroup, there was no significant difference compared with LPS+MI+AMSCs+anti-CD200antibody group.4. In Immunofluorescence method, it was found CD200R positive cells exist in eachgroup, the fluorescence intensity was slightly decreased in MI+LPS group thanthat in MI group, CD200R got significantly enhanced in MI+LPS+AMSCsgroup than MI+LPS group, and there was no obviously difference in anti-CD200antibody group, Transwell group compared with MI+LPS group.5. CD200R expression in MI+LPS group decreases than the MI group, CD200Rexpression showed a significant upward trend when AMSCs exists, the expressionof CD200R got decreased in LPS+MI+AMSCs+anti-CD200antibody groupafter being cultured for48h, compared with LPS+MI+AMSCs group, but nosignificant difference with LPS+MI group. CD200R expression in Transwellgroup was significantly decreased compared with LPS+MI+AMSCs group, butno significant difference compared with LPS+MI+AMSCs+anti-CD200antibody group.Statistical MethodsAll statistical analysis was performed by using SPSS16.0statistical software. Data areshown with mean±standard deviation (x±s), T-test was adopted for comparisonbetween two groups, while One-Way ANOVA analysis was adopted among groups, and P<0.05indicates a statistically significant difference. Conclusion1. AMSCs surface expresses CD200, while MI expresses CD200R.2. It may increase CD200R expression on activated MI surface to co-culture AMSCs andMI, play an immune inhibition role on activated MI through direct contact betweenCD200and CD200R, regulate MI activation, reduce the secretion of inflammatorycytokines, and anti-CD200antibodies are capable of reverse this inhibition.