The Study For Biological Characteristics Of Human Umbilical Cord-derived Mesenchymal Stem Cells With High Expression CD200

Objective:To investigate the biological characteristics of human umbilical cord derived mesenchymal stem cells（h UC-MSCs）with high expression CD200 and its role in the treatment of acute graft versus host disease（a Gv HD）.Contents:1.Culture identification of h UC-MSC,flow cytometry sorting CD200^highh UC-MSC and CD200^lowh UC-MSC cell subpopulations.2.In vitro experiments to explore the biological characteristics and immunomodulatory function of CD200^highh UC-MSC;3.A mouse a Gv HD model was established to explore the efficacy of CD200^highh UC-MSC in the treatment of a Gv HD.Methods:1.h UC-MSC was isolated and cultured in vitro,and the cell morphology was observed by Giemsa staining,the cell phenotype was analyzed by flow cytometry,the osteogenic and adipogenic differentiation ability was detected by alkaline phosphatase（ALP）staining and oil red O staining,respectively,and the expression of key osteogenic and adipogenic transcription factors was detected by real-time fluorescence quantitative PCR（q PCR）.2.Flow cytometry sorted CD200^highh UC-MSC and CD200^lowh UC-MSC two populations of cells and detected the sorting efficiency by flow cytometry,the expression of CD200 at the protein level was analyzed by Western blotting,and the cell morphology and phenotypes of CD200^highh UC-MSC and CD200^lowh UC-MSC by Giemsa staining and flow cytometry.3.CD200^highh UC-MSC and CD200^lowh UC-MSC were induced differentiation in vitro,alkaline phosphatase staining and oil red O staining were performed to detect the osteogenic and adipogenic differentiation ability of cells in vitro,respectively.q PCR was used to detect the expression of key transcription factors for osteogenesis and adipogenesis.CCK-8 experiment,flow cytometry,CFU-F experiment and cell scratch experiment were performed to detect the cell proliferation,cell cycle,cell cloning ability and migration ability of CD200^highh UC-MSC,CD200^lowh UC-MSC and h UC-MSC.4.CD200^highh UC-MSC,h UC-MSC and CD200^lowh UC-MSC were co-cultured with mouse T lymphocytes,and the effect of each group of cells on T cell proliferation ability was detected by flow cytometry CFSE staining.q PCR was used to detect the expression of T cell inflammatory factors at the m RNA level.5.A mouse model of acute graft-versus-host disease（a Gv HD）was constructed and treated with CD200^highh UC-MSC,h UC-MSC,and CD200^lowh UC-MSC via tail vein injections.The survival status of each group was assessed by recording their body weight,clinical Cook score,and survival rate.HE staining was performed to detect pathological changes in the lung,liver,perianal skin and small intestine of each group.The proportion of splenic T lymphocytes polarized to Th1 cells in each group of mice was measured by flow cytometry,and the expression of inflammatory factors at the m RNA level in splenic T lymphocytes was analyzed by q PCR.Results:1.The cultured cells were identified,and the results showed that the cells were all fibroblast-like,vortex-shaped,and adherent growth under inverted microscopy.The cell phenotype was analyzed by flow cytometry,which showed high expression of CD73,CD90,CD105 and low or no expression of CD14,CD34,CD45.After in vitro adipogenic and osteogenic differentiation,compared with the self-differentiated group,the osteogenic induction group showed positive ALP staining and significantly higher m RNA expression levels of ALP and OPN,whereas the adipogenic induction group showed positive oil red O staining and significantly higher m RNA expression levels of ADI and PPAR-γ.The above results indicated that the human umbilical cord-derived MSC were successfully isolated and cultured,and matched with the international standards for MSC identification criteria.2.CD200^highh UC-MSC and CD200^lowh UC-MSC were successfully sorted by flow cytometry,and the expression rate of CD200 in CD200^highh UC-MSC was（97.3±0.01）%by flow cytometry.Western blotting results showed that the expression of CD200 in CD200^highh UC-MSC was significantly higher than that in the h UC-MSC group vs.CD200^lowh UC-MSC group.The biological properties of CD200^highh UC-MSC and CD200^lowh UC-MSC displayed that both cells were morphologically and phenotypically unaltered and both could be induced to differentiate into osteoblasts and adipocytes.3.CCK-8 and cell cycle assays were used to analyzed CD200^highh UC-MSC,h UC-MSC,and CD200^lowh UC-MSC proliferation capabilities,and results showed that the cell proliferation ability of CD200^highh UC-MSC was strong;the results of CFU-F assay and cell scratching assay showed that the clonogenic ability and migration ability of CD200^highh UC-MSC were significantly enhanced than that of CD200^lowh UC-MSC.4.In vitro T lymphocyte proliferation assays showed that CD200^highh UC-MSC significantly inhibited the proliferation of CD3⁺T cells,while the expression of the proinflammatory factor IL-17A was also significantly decreased.5.A mouse a Gv HD model was successfully established,and results showed that the mice in the CD200^highh UC-MSC treated group was with faster weight recovery,higher survival rate and lower Cook score values.The HE staining results showed that the recovery of pathological damage in lung,liver,perianal skin,and small intestine was significantly enhanced in CD200^highh UC-MSC group mice compared with CD200^lowh UC-MSC group and h UC-MSC group.The proportion of Th1 in mouse spleen lymphocytes was analyzed by flow cytometry and results showed that CD200^highh UC-MSC can effectively inhibit the polarization of mouse spleen T lymphocytes to Th1 cells.The m RNA expression levels of inflammatory factors in the spleen T lymphocytes of mice in each group were detected by q PCR,and the results showed that the expression levels of IFN-γin the spleen T lymphocytes of mice in the CD200^highh UC-MSC group were significantly lower compared with the CD200^lowh UC-MSC group.The above results suggest that CD200^highh UC-MSC can significantly improve the efficacy of treatment of a Gv HD.Conclusion:CD200^highh UC-MSC showed significantly enhanced cell proliferation,clone formation and migration ability,and significantly inhibited T cell proliferation,thus effectively enhancing the efficacy of h UC-MSC in the treatment of aGvHD.