The Study Of Biological Propetry And Immunosuppression Mechanism On Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) are capable of self-renewal and multi-lineagedifferentiation. They can derive from many tissues. In1970, Friedenstein found theMSC in bone marrow at first. Then MSC was found in many tissues includingadipose tissue, Wharton’s jelly of the umbilical cord, placenta tissue and so on. MSCnot only has strong proliferation and multi-differentiation capacity, but also hasimmunosuppression effects. MSC can also inhibit allograft immune rejection andprolong graft survival time. These characteristics make MSC have importantapplication value in the treatment diseases of immune system and tissue or organtransplantation. At present, the molecular mechanism of T lymphocyte suppressionby WJ-MSC is not very clear. This will not provide the vaule of clinical applicationand security of clinical therapy. Based on above reasons, the study selected the bonemarrow derived mesenchymal stem cell (BM-MSC), adipose tissue derivedmesenchymal stem cell (AT-MSC), Wharton’s jelly of the umbilical cord derivedmesenchymal stem cell (WJ-MSC) and placenta derived mesenchymal stem cellPL-MSC) and compared their biological characteristics. Then we selected a betterMSC to repair the damage tissue. And on this basis we further explored themechanism that MSC inhibited the proliferation of T lymphocyte. This will providethe theoretical and experimental basis for the treatment of medical applications.1．Isolation, culture and identification of BM-MSC, AT-MSC, WJ-MSC andPL-MSC.Objective: To establish a simple and effective way of acquisition and cultureMSC from human bone marrow, adipose tissue, Wharton’s jelly and placenta tissue. And to observe the morphology, detect the surface antigen markers, adipogenic andosteogenic capability of MSC, and provide sufficient stem cell source for subsequentexperiments. Method: AT-MSC, WJ-MSC and PL-MSC were isolated bycollagenase digestion, while BM-MSC was isolated by blood marrow adherentculture. The cell morphology was observed by inverted microscope. The surfaceantigens of MSC isolated from four human tissues were tested by flow cytometry.The pluripotency of MSC from four tissues was detected including the abilities ofadipogenic and osteogenic differentiation. Results: AT-MSC, WJ-MSC andPL-MSC were isolated successfully by collagenase digestion, and also enoughamount of BM-MSC was isolated successfully by bone marrow adherent culture.The MSC from four tissues were spindle-shaped adherent cells. The flow cytometryresults showed that the surface markers of MSC were positive for CD44, CD73,CD90, CD105, and negative for CD14, CD34, and CD45. The MSC from fourtissues have the abilities of adipogenesis and osteogenesis by induction. The aboveresults conformed the define and standard of MSC by International Society for CellTherapy. This indicated that the cells isolated from four tissues were consistent withthe characteristics of MSC. Conclusion: BM-MSC, AT-MSC, WJ-MSC andPL-MSC were isolated successfully from different tissues. The isolated cellsconformed the standard of MSC, including cell morphology, the expression level ofsurface markers and the abilities of multi-pluripotent differentiation. The isolatedcells were qualified for subsequent experiments because of their relatively stablecharacteristics.2．Comparative study of differentiation, proliferation of MSC from four tissuesObjective: To compare the proliferation and the differentiative capacity of MSCfrom four tissues. Method: The proliferative ability of MSC from four tissues wasdetected by living cell number counting method. The cell cycle of MSC isolatedfrom four human tissues was tested by flow cytometry. The MSC differentiation wasinduced by STEMPRO Osteogenesis Differentiation Kit and STEMPRO Adiposeness Differentiation Kit. The adipogenic capacity of MSC was detected by adipogenic cell counting method. The osteogenic capacity of MSC was detected bybone nodules counting. Results: The WJ-MSC displayed the highest proliferationability followed by AT-MSC, PL-MSC and BM-MSC. The AT-MSC displayed thehighest adipogenic capacity followed by WJ-MSC, BM-MSC and PL-MSC. TheWJ-MSC displayed the highest osteogenic capacity followed by PL-MSC, AT-MSCand BM-MSC. Conclusion: The WJ-MSC displayed the higher proliferation ability,higher osteogenic capacity than all other three MSC. However AT-MSC displayedthe highest adipogenic capacity.3．T lymphocyte suppression of MSC and the molecular mechanisms involved3.1Comparative study of T lymphocyte suppression of MSC from four tissuesObjective: To compare T lymphocyte suppression of MSC from four tissues.Method: MSC and T lymphocyte were co-cultured at the ratio of1:1,1:5,1:10,1:20,and1:50respectively. Then the proliferation of T lymphocyte was detected by Brdu.Results: All MSC from four tissue sources significantly suppress T lymphocyteproliferation in a cell number-dependent manner. The inhibitory effects of theWJ-MSC, AT-MSC and PL-MSC on T lymphocyte were more prominent thanBM-MSC (P<0.05). And the WJ-MSC displayed the most prominent inhibitoryeffects on T lymphocyte. Conclusion: All MSC from four tissue sources significantlysuppress T lymphocyte proliferation. And the WJ-MSC displayed more prominentinhibitory effects on T lymphocyte than all other three MSC.3.2The expression and activity of indoleamine2,3-dioxygenase (IDO) inWJ-MSC co-cultured with T lymphocyteObjective: To detect the expression and activity of IDO in WJ-MSC co-culturedwith T lymphocyte, and to study the relationship between IDO and proliferation of Tlymphocyte. Method: We analyzed IDO expression in WJ-MSC co-cultured with Tlymphocyte by RT-PCR and Western blot. We used High-Performance LiquidChromatography-tandem Mass Spectrometry/Mass Spectrometry (HPLC-MS/MS) todetect the activity of IDO in the supernatant of co-cultured system. And we measuredthe quantity of IFN-γ in the co-cultured supernatant by ELISA. Results: The results showed that WJ-MSC did not secrete IFN-γ when growing alone. T lymphocytesecreted large amounts of IFN-γ and the secretion can be inhibited by WJ-MSC whencoculturing together. IFN-γ secreted by T lymphocyte could induce the expression ofIDO in WJ-MSC. The results of RT-PCR, Western blot and HPLC-MS/MS impliedthat WJ-MSC could express activated IDO. And the activity of IDO in co-culturedsystem was higher than control group (P<0.001). After addition1-methyl tryptophan(1-MT), more than80%of the proliferation of T lymphocyte recovered in theco-cultured system. Conclusion: IFN-γ was secreted by T lymphocyte and WJ-MSCwas induced by IFN-γ to express the IDO. The proliferation of T lymphocyte wasinhibited mostly by the activated IDO. And it suggested that IDO was one of themechanisms of immunosuppression by WJ-MSC.3.3WJ-MSC blocked cell cycle progression of T lymphocyteObjective: To study the molecular mechanism of T lymphocyte suppression byWJ-MSC. Method: Flow cytometry was used to detect the cell cycle of T lymphocyteco-cultured with WJ-MSC. The IDO blocker1-MT was added into a group as control.The cell cycle of T lymphocyte was tested by cell cycle detection kit. The cell cyclesrelated genes were detected by qRT-PCR. Results: WJ-MSC increased the G0/G1phase T lymphocyte by arresting T lymphocyte from G1phase to S phase. The SPFand PI value of co-cultured T lymphocyte were significantly less than control Tlymphocyte (P<0.001). We also found that the proliferation related gene CDK4wassignificantly inhibited by MSC (P<0.001). After addition of1-MT, the cell cycle of Tcells were almost reversed to normal as control. At the same time, the mRNAexpression of the cell cycle genes was almost reversed to control too. Conclusion: Ourfindings provide an insight into the mechanism(s) employed by WJ-MSC to stronglysuppress T lymphocyte activation. By expression the activated IDO, WJ-MSCtargeted the cell cycle pathways in T lymphocyte by down-regulation CDK4mRNAlevel.3.4WJ-MSC promoted apoptosis of T lymphocyteObjective: To analyze the effects of WJ-MSC on the apoptosis of T lymphocyte and the expression of related genes. Method: Flow cytometry was used to detect theapoptosis rate of T lymphocyte co-cultured with WJ-MSC. The IDO blocker1-MTwas added into a group as control.The apoptosis of T lymphocyte was tested byAnnexin V-FITC/PI kit. The apoptosis related genes were detected by qRT-PCR.Results: The apoptosis ratio of T lymphocyte induced by MSC was significantlyhigher than control. The apoptosis rate of T lymphocyte reached to (22.8±1.61)%.The appoptosis resistant gene Bcl2was down regulated and appoptosis promotinggene Caspase3was up regulated in T cells by MSC (P<0.001). After addition of1-MT, the apoptosis rate of T cells was almost reversed to normal as control. At thesame time, the mRNA expression of the apoptosis genes was almost reversed tocontrol too. Conclusion: By expression the activated IDO, WJ-MSC could promoteapoptosis of T lymphocyte, and induce apoptosis in T lymphocyte throughBcl2/Caspase pathway.4．Screening of8reference genes used in quantitative RT-PCR on human Tlymphocyte co-cultured with WJ-MSCObjective: To identify the stability of reference genes of T lymphocyteco-cultured with WJ-MSC and co-cultured with WJ-MSC added1-MT. Method: Wescreened8reference genes:18S, ACTB, B2M, GAPDH, HPRT1, RPL13A, PPIA andTBP used in qRT-PCR. These genes were analyzed by the software geNorm,NormFinder and BestKeeper. Results: The overall analysis suggested that B2M wasthe most stably expressed reference genes in these cells. We also demonstrate that thetwo reference genes, ACTB and GAPDH, are frequently used are not alwayssuccessful in many case. Conclusion: It suggested that B2M was the most stablyexpressed reference genes in these T lymphocyte.