Preliminary study on biological properties of adult human bone marrow-derived mesenchymal stem cells

Abstract

Objective: To establish a method of culture and expansion of adult human bone marrow-derived MSCs in vitro and to explore their biological properties. Methods: Mononuclear cells were obtained from 5 mL adult human bone marrow by density gradient centrifugation with Percoll solution. Adult human MSCs were cultured in Dulbecco’s Modified Eagle’s Medium with low glucose (LG-DMEM) containing 10% fetal calf serum at a density of 2×105 cell/cm2. The morphocytology was observed under phase-contrast microscope. The cell growth was measured by MTT method. The flow cytometer was performed to examine the expression of cell surface molecules and cell cycle. The ultrastructure of MSCs was observed under transmission electron microscope. The immunomodulatory functions of MSCs were measured by MTT method. The effects of MSCs on the growth of K562 cells and the dynamic change of HA, IV-C, LN concentration in the culture supernatant of MSCs was also observed. Results: The MSCs harvested in this study were homogenous population and exhibited a spindle-shaped fibroblastic morphology. The cell growth curve showed that MSCs had a strong ability of proliferation. The cells were positive for CD44, while negative for hematopoietic cell surface marker such as CD3, CD4, CD7, CD13, CD14, CD15, CD19, CD22, CD33, CD34, CD45 and HLA-DR, which was closely related to graft versus host disease. Above 90% cells of MSCs were found at G0/G1 phase. The ultrastructure of MSCs indicated that there were plenty of cytoplasmic organelles. Allogeneic peripheral blood lymphocytes proliferation was suppressed by MSCs and the inhibition ratio was 60.68% (P<0.01). The suppressive effect was also existed in the culture supernatant of MSCs and the inhibition ratio was 9.00% (P<0.05). When lymphocytes were stimulated by PHA, the suppression effects of the culture supernatant were even stronger and the inhibition ratio was 20.91% (P<0.01). Compared with the cell growth curve of the K562 cells alone, the K562 cells cocultured with MSCs grew slowly and the exponential phase of growth wasn’t significant. Seeing from the concentration curve, as time passed, the concentration of HA increased quickly, while those of IV-C and LN didn’t change much. Conclusion: The method for culture and expansion of adult human bone marrow-derived MSCs in vitro has been successfully established in this study. MSCs were a homogenous population that had unique growth phenotype and multilineage potential. Preliminary study proved that it had the abilities of immunomodulatory function, antitumor, hematopoietic supporting and could act as seed cell of tissue engineering.