FORMATION OF CARTILAGE-LIKE SPHEROIDS BY MICROMASS CULTURES OF MURINEC3H10T1 2 CELLS UPON TREATMENT WITH TRANSFORMING GROWTH-FACTOR-BETA-1/

Formation of cartilage during both embryonic development and repair pr ocesses involves the differentiation of multipotential mesenchymal cel ls. The mouse cell line, C3H10T1/2, has been shown to be multipotentia l and capable of differentiating into various phenotypes normally deri ved from embryonic mesoderm, including myocytes, adipocytes and chondr ocytes. In this study, we have analyzed the induction of chondrogenesi s in C3H10T1/2 cells by transforming growth factor-beta (TGF-beta 1, h uman recombinant form). Treatment of high-density micromass cultures o f C3H10T1/2 cells with TGF-beta 1 resulted in the formation of a three dimensional spheroid structure, which exhibited cartilage-like histol ogy. Extracellular matrix components characteristic of cartilage, type II collagen and cartilage link protein, were demonstrated by immunohi stochemistry. TGF-beta 1 treatment increased collagen synthesis, and i mmunoblot analysis showed the presence of type II collagen in TGF-beta 1-treated micromass cultures, but not in TGF-beta 1-treated monolayer cultures nor in untreated cultures. An increase in radioactive sulfat e uptake relative to DNA synthesis was also seen in TGF-beta 1-treated micromass cultures forming spheroids, indicating the increased synthe sis of sulfated proteoglycans. These observations indicated that the s pheroids formed are of a cartilaginous nature, and that multipotential C3H10T1/2 cells, which do not spontaneously enter the chondrogenic pa thway, can be induced to undergo cellular differentiation towards chon drogenesis in vitro through culture in a favorable environment.