Multilineage differentiation of human bone marrow mesenchymal stem cells in vitro and in vivo

Abstract

The aim of the present study was to investigate the ability of human bone marrow-derived mesenchymal stem cells (BMSCs) to undergo multilineage differentiation. Human BMSCs were isolated from the ilia of donors by density gradient centrifugation, then purified by adherent separation and cultured in vitro. P3 or P4 BMSC populations were collected and induced for multilineage differentiation into osteoblasts, adipocytes and neuroblasts using an inductive medium in vitro. The BMSCs were cultured in either an osteoblast or chondroblast induction medium, seeded onto porous coral scaffolds and implanted into mice in vivo. The mice were sacrificed by anesthesia overdose at 6 or 9 weeks post-surgery. The scaffolds were then removed for analysis. Lipid vacuoles were observed subsequent to being cultured in an adipogenic medium. These accumulated lipid vacuoles were detected using Sudan Black B and Oil Red O (positive) staining. Deposited calcium was detected using von Kossa and Alizarin Red S (positive) staining subsequent to being cultured in an osteogenic medium. The BMSCs retracted to form neuron-like cells with axon- and dendrite-like processes following induction by β-mercaptoethanol. The cells were positively stained by toluidine blue and glial fibrillary acidic protein (GFAP) immunohistochemistry. Newly formed bone tissues were observed and islands of cartilage tissue were also formed at 9 weeks post-implantation in vivo. The present study demonstrated that human BMSCs were homogeneous and differentiated with high fidelity to osteogenic, adipogenic, neurogenic or chondrogenic lineages. These cells also form bone and cartilage tissues when implanted in vivo and may therefore be used as seed cells in bone tissue engineering.