The Immunosuppressive Effect Of Supernatant From Human Umbilical Cord-derived Mesenchymal Stem Cells

Objective: To investigate the immunosuppressive effect and mechanism of supernatantfrom human umbilical cord-derived mesenchymal stem cells（hUC-MSCs）on humanperipheral blood mononuclear cells (hPBMCs)，then explore the impact of this kind ofinhibitory effect on cytotoxic activity of activated PBMC when cultured with lungadenocarcinoma cancer cells.Method:1. The study on isolation、culture and biological characteristics of human umbilicalcord-derived mesenchymal stem cellshUC-MSCs were isolated from human umbilical cord by tissue adherent method andexpanding cultured in vitro.The morphologic appearance of hUC-MSCs were observed byfluorescent microscope. Surface antigens were detected by flow cytometry. The applicationof different factors induced hUC-MSCs differentiating into osteoblasts、adipocyte、chondrocyte differentiation and identification.2. The impact of hUC-MSCs on the activation、the survival of human hPBMCs andthe proportions of each human lymphoid subgroupPBMCs were isolated from healthy donors by density gradient centrifugation, thencultured in MSC-CM as treatment group after being activated by OKT3. Each lymphoidsubgroup proportion and the cell cycle of PBMCs were analyzed by flow cytometry toobserve the difference between treatment and control group. The level of apoptosis wasassessed by flow cytometry with Annexin-V/PI as fluorescent marker. The effect of MSC-CM on activated PBMCs for the production of IFN-γ and IL-10were tested byELISA.3. PGE2participate in regulating the proportion of Treg by hUC-MSCs and influence theactivity of PBMCs proliferation and apoptosisCollecting hUC-MSCs supernatant after culturing2~3days with NS-398which isspecific antagonist of PGE2. PBMCs were cultured in this kind of hUC-MSC supernatant asexperimental group, cultured in hUC-MSCs supernatant which was collected from normalhUC-MSCs culture system as control group1, cultured in DMEM/F12medium as controlgroup2. FCM compared the cell cycle of PBMCs and Treg proportion betweenexperimental group and control groups; the level of apoptosis was assessed by FCM withAnnexin-V/PI as fluorescent marker; the effect of MSC-CM on activated PBMCs for theproduction of IFN-γ and IL-10were tested by ELISA.4. The impact of hUC-MSCs micro-environment on cytotoxic activity of activatedCIK cells for killing lung adenocarcinoma cancer cellsPBMCs were inducted into cytokine induced killer (CIK) cells after isolated from healthydonors by density gradient centrifugation; FCM detected the immune phenotype of CIKcells and compared the difference of before and after induction; The cell cycle of A549cells after cultured in MSC-CM was observed by FCM; MTT method portray the growthcurve of A549cells; The impact of cytotoxic activity of activated CIK cells on lungadenocarcinoma cancer cells was detected by MTT method.Result: Successfully isolated human umbilical cord-derived mesenchymal stem cells.Thesecells were long fusiform shape and grew against the wall of flask, high expression ofstromal cells markers CD73、CD105、CD44and CD29, adhesion molecules CD90, butnegative for CD14、HLA–DR which belongs to MHC molecules classⅡand hematopoieticstem cell surface marker CD34、CD45. Gomori calcium-cobalt method, oil red O stainingand Alcian blue staining confirmed that hUC-MSCs can be induced to differentiate intoosteoblasts、adipocytes and cartilage cells. Compared with the control group, MSC-CMdown-regulated the ratio of CD4+T cell to CD8+T cell, and increased the proportion ofCD4+CD25+CD127low Treg cell, thus other subgroup had no significant difference. MSC-CM inhibited the production of IFN-γ by PBMCs, but promoted the secretion ofIL-10., and protected PBMCs from apoptosis when activated with OKT3. InhibitorsNS-398test confirmed that immunosuppressive effect of hUC-MSCs supernatant wasmediated by immune effect factor PGE2; Cytotoxicity experiment revealed that hUC-MSCssupernatant can suppress cytotoxic activity of CIK, and the inhibiting effect may beneutralized by serum.Conclusion: hUC-MSC may play a role of immunosuppression by promoting theproliferation and activation of Treg cell which is mediated by supernatant secrete factorsPGE2. This kind of inhibitory activity neither relied direct or indirect contact with thelymphocytes, nor influenced by inducing immune cells apoptosis. And the inhibitoryactivity may reduce killing function of CIK cell on lung adenocarcinoma A549cells.