| Background:Mesenchymal stem cells can be isolated from many tissues. MSC can modulate the immune response, including modulates T cells, B cells, natural killer cells and dendritic cells. Therefore, MSC might be used for treatment of immune related refractory diseases. The isolation of MSC was based on adhere properties of MSC, and the cultured MSC were a mixture of all kinds of cells. Cells in the same culture condition might be different for ability of proliferation, differentiation and hematopoiesis support. Use specific cell surface or intracellular marker, different subpopulation of MSC can be identified. However, there is little research for subpopulation of MSC from placenta. Also researches for subpopulation of MSC were mostly on proliferation and differentiation ability of MSC, and researches for immune modulation subpopulation of MSC were limited.Objective:Isolate CD106+and CD106" cells from CV-MSC. Compare the shape, colony formation, differentiation,immune modulation ability of CD106+and CD106-CV-MSC.Methods:(1) Phenotypes of MSC isolate from bone marrow, adipose, umbilical cord and chorionic villi were analyzed by flow cytometry. These four types of MSC were co-culture with PHA stimulated human peripheral blood mononuclear cells, and IFN-y concentrations in culture medium were analyzed by ELISA. Real-time PCR were used to analyze gene expression in four types of MSC. Concentrations of PGE2in culture medium from four types of MSC were tested by ELISA.(2) CD106+and CD106-cells were isolated from CV-MSC. Cell morphologies were observed by an inverted microscope. Differentiated CD106+and CD106-CV-MSC under osteogenic and adipogenic condition, and alizarin red S or oil red O solution was used to compare their differentiation ability. Seeded CD106+and CD106-CV-MSC to T75culture flask in a low concentration;14days later, the cells were stained with crystal violet and the colonies generated in the flask were counted.(3) CD106+or CD106-CV-MSC were co-cultured with PHA stimulated PBMC, and concentrations of IFN-γ and TNF-a in culture medium were analyzed by ELISA. CD4+T cells from human cord blood mononuclear cells were isolated by magnetic microbeads. Co-cultured CD106+or CD106CV-MSC with CD4+T cells (stimulated by PHA and IL-2), and concentrations of IFN-y and TNF-a in culture medium were analyzed by ELISA. In some cases, transwell was used to avoid cell-cell contact. The mRNA expression of IFN-y, TNF-α, T-bet, IL-21and IL-22was tested by real-time PCR. Co-cultured CD106+or CD106-CV-MSC with CD4+T cells (stimulated by IL-2) and the proportion of CD4+CD25+Foxp3+cells was tested by flow cytometry.(4) Conditioned medium of CD106+and CD106"CV-MSC was collected. CD34+cells were isolate from human cord blood mononuclear cells by magnetic microbeads. CD34+cells were planted in methylcellulose culture medium, and conditioned medium of MSC was added. Colonies were counted under a microscope after2and3weeks, and the size of the colonies were observed. Cells were isolated and the phenotypes of the cells were analyzed by flow cytometry after3weeks.(5) Using affymetrix microarray, different gene expressions betweeen CD106+and CD106-CV-MSC were compared. Data was analyzed using MAS3.0molecule annotation system. Different gene expressions of CD106+and CD106"CV-MSC were tested by real-time PCR. Concentrations of PGE2in culture medium from CD106+and CD106-MSC were tested by ELISA.(6)CV-MSC were passaged to passage9. Cell morphology of passages3and passage9were observed by an inverted microscope. Phenotypes of CV-MSC of different passages were analyzed by flow cytometry. Real-time PCR were used to analyze gene expression in different passages. CV-MSC of different passages were co-cultured with PHA stimulated PBMC, and IFN-y concentrations in culture medium were analyzed by ELISA.(7)Pro-inflammatory cytokines IFN-y, TNF-a and IL-1β were used to stimulate CD106-CV-MSC or UC-MSC. Gene expressions were analyzed by real-time PCR, and the phenotypes of MSC were analyzed by cytometry.Results:(1) Compare to bone marrow, adipose and umbilical cord derived MSC, CV-MSC possesses highest proportion of CD106+cells and strongest gene expression of COX-2, IL-1α, IL-1β, IL-6and IL-8. CV-MSC secreted PGE2strongly and inhibited IFN-y production of PHA stimulate MSC effectively.(2) High purity of CD106+and CD106-cells can be isolated from CV-MSC with MSC shape. Both of CD106+and CD106-MSC were capable of osteogenic and adipogenic differentiation while CD106"CV-MSC possessed increased colony formation capacity.(3) CD106+CV-MSC inhibit IFN-y and TNF-a production of PHA stimulated PBMC more effectively than CD106-CV-MSC. Also, for inhibition the IFN-y and TNF-a production of CD4+T cells, CD106+CV-MSC were more effective. Cell-cell contact was not decisive for immune modulation ability of CD106+CV-MSC. CD106+CV-MSC also inhibited mRNA expression of IFN-y, TNF-a, T-bet, IL-21and IL-22more strongly, and showed a slightly superior for up-regulate CD4+CD25+Foxp3+Treg cells.(4)Culture medium of CD106+CV-MSC was more effective for supporting cord blood CD34+cells differentiation than culture medium of CD106-CV-MSC.(5)Microarray analyze revealed that CD106+CV-MSC expressed more immune related genes such as COX-2, IL-la, IL-1β, IL-6and IL-8compare to CD106-CV-MSC, and changes of these genes were confirmed by realtime PCR.(6)CV-MSC down regulated CD106and many CD106+CV-MSC related genes as passages. Also, there were changes in cell shape and immune modulation ability after several passages.(7) TNF-a and IL-1β can up regulate CD106, COX-2, IL-1α, IL-1β, IL-6and IL-8in CD106-CV-MSC and UC-MSC, while IFN-y apparently up regulated CD106and IL-6expression, but failed in other genes.Conclusion:CD106could be used as a biomarker for a subpopulation of CV-MSC with strong immunosuppressive activity, and there is a positive correlation between CD106expression and immunosuppressive effect of CV-MSC. TNF-a and IL-1β could induce CD106+CV-MSC related genes. |
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