Mesenchymal stem cells (MSC) that can differentiate to various connective tissue cells may be useful for autologous cell transplantation to defects of bone, cartilage, and tendon, if MSC can be expanded in vitro. However, a short life span of MSC and a reduction in their differentiation potential in culture have limited their clinical application. The purpose of this study is to identify a growth factor(s) involved in self-renewal of MSC and the maintenance of their multilineage differentiation potential. Fibroblast growth factor-2 (FGF-2) markedly increased the growth rate and the life span of rabbit, canine, and human bone marrow MSC in monolayer cultures. This effect of FGF-2 was more prominent in low-density cultures than in high-density cultures. In addition, all MSC expanded in vitro with FGF-2, but not without FGF-2, differentiated to chondrocytes in pellet cultures. The FGF+ MSC also retained the osteogenic and adipogenic potential throughout many mitotic divisions. These findings suggest that FGFs play a crucial role in self-renewal of MSC.

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We have expressed and biologically characterized recombinant human growth/differentiation factor 5 (huGDF5). This protein is composed of a mature homodimer consisting of 15 kD subunits. Using recombinant expressed protein, we have demonstrated that huGDF5 in vitro stimulated mesenchyme aggregation and chondrogenesis in rat limb bud cells. In vivo, partially purified huGDF5 induced cartilage and bone formation in muscular tissues of rodents. However, in contrast to the effects of other BMPs, as for example BMP-2, the osteoblastic MC3T3-E1 cells did not respond to huGDF5 as measured by alkaline phosphatase activity. These results suggest that the action of GDF5 may be relatively specific for chondrogenesis during the entire process of the endochondral bone formation. GDF5 may control the morphogenesis of cartilaginous tissue, including joints, in the skeletal development of limbs.

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