Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environmen t, following treatment with soluble human bone morphogenetic protein-2 (BMP -2). To enhance this process, the human BMP-2 cDNA was cloned into a retrov iral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3H10T1/2 cells with this retrovira l construct resulted in an infection efficiency of 90-95% and was highly ef fective in converting cells in micromass culture to a chondrocyte phenotype , as assessed by positive Alcian blue staining for extracellular matrix pro teoglycans, increased sulfate incorporation, increased expression of the ca rtilage marker genes collagen type Il and aggrecan, and decreased expressio n of collagen type I. Interestingly, BMP-2 expression in the micromass cult ures also induced the expression of the cell cycle inhibitory protein/diffe rentiation factor p21/WAF1, suggesting its functional involvement in chondr ogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected c ells did not chondrify when co-cultured as a nonoverlapping micromass adjac ent to BMP-2 expressing cells. These data indicate that retrovirally expres sed BMP-2 is highly effective at inducing a chondrocyte phenotype in a mult ipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.