Effect Of Human Bone Marrow Mesenchymal Stem Cell On T Cell Activation And Proliferation In Patients With Aplastic Anemia

Objective To explore effect of human bone marrow mesenchymal stem cell (MSC) on T cell activation and proliferation in patients with aplastic anemia (AA) in vitro. Methods In order to isolate human MSC, bone marrow aspirates of 10–20 ml were taken from the iliac crest of normal donors, The harvest of bone marrow from healthy volunteers for isolation and expansion of MSC to study its effect in vitro . Briefly, mononuclear cells were isolated from Percoll-separated bone marrow, resuspended in human MSC medium consisting of Dulbecco's Modified Eagle's Medium-Low Glucose , supplemented with 10^% foetal bovine serum and plated at 2×10⁵ cells/cm2 in Falcon flasks. The serum lot was selected for optimal MSC growth with the retention of osteogenic, chondrogenic and adipogenic differentiation. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 for three to five passages. Using flow cytometry, cultured MSC were uniformly positive for Sca-1, CD105, CD44, CD29, and negative for haematopoietic markers HLA-DR , CD34 and CD45. Purified peripheral blood lymphocytes (PBL) were prepared from buffy coats or haeparinized blood with the help of centrifugation. Mononuclear cells were isolated from Ficoll-Isopaque, resuspended in human T cell medium consisting of RPMI1640, supplemented with 20% foetal bovine serum and plated at 5×10⁷ cells/cm² in Falcon flasks.Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂ for one hour. T cells were harvested by using nylon column. MSCs of various concentrations were added to PHA induction T cell proliferation cultures with AA patients. The proliferation of T cell was measured by MTT method. The CD25 (IL-2R) and CD38 expression rates of CD3⁺ T cells was analyzed by flow cytometry .Results MSCs were planted in 96-well plates(1×10⁴/well for group A)and cocultured with T cell isolated from peripheral blood of AA patients. Peripheral blood T cell noncocultured with MSC acted as the control group. MSCs inhibit significantly PHA induced T cell proliferation and the CD25 and CD38 expression of CD3⁺ T cells in patients with AA (P<0.01).But the OD values and the CD25 and CD38 expression had no significantly difference compared with control group (1×10³/well for group B).Conclusion Human bone marrow MSCs inhibit T cell activation and proliferation in patients with AA in vitro and the inhibitory effects are dependent on the amount of MSCs.