Slowly-adhered primary bone marrow mesenchymal stem cells may exhibit more potent immuno-regulatory activity

Abstract

This study was to investigate the relationship between the adherent ability of freshly isolated MSCs with their inhibitory effect on lymphocyte activation. Human bone marrow mononucleated cells were maintained in culture for 48 hours, the attached and the non-attached cells were then cultured separately and the adherent cells were collected and passaged. Cellular surface markers were analyzed with flow cytometry. Alkaline phosphatase activity and the intracellular lipid droplets were measured by histological staining, the in vitro osteogenesis and adipogenesis were identified. One-way mixed lymphocyte reaction was used to evaluate the suppressive activity of the adherent cells on lymphocyte proliferation, the prostaglandin E2 level in supernatant of cultured cells was detected by ELISA. Some cells attached to the plastic after the bone marrow mononucleated cells were allowed to adhere for 48 hours. The slowly-attached cells were fibroblast-like in morphology, homogenously positive for CD44 and CD73 and negative for CD31 and CD45. They could be coaxed into osteoblasts and adipoblasts under the standard inductive conditions. These cells were able to inhibit lymphocyte proliferation in mixed lymphocyte reaction and their effect was more potent than those from the adherent cells appeared within 48 hours. The concentration of prostaglandin E-2 in the supernatants of the slowly-adhered cells was significantly higher than that in the MSCs cultured with the traditional method (90.8 ± 10.37 ng/ml vs 70.2 ± 8.98 ng/ml) (P < 0.01). The MSCs exist in the marrow mononucleated cells after adherent culture for 48 hours, and the MSCs may exhibit more potently inhibitory activity on lymphocyte activation.