COMBINATION OF OSTEOINDUCTIVE BONE PROTEINS DIFFERENTIATES MESENCHYMAL C3H 10T1/2 CELLS SPECIFICALLY TO THE CARTILAGE LINEAGE/

During embryonic development, cartilage formation involves the condens ation of mesenchymal stem cells and a series of maturation steps that ultimately results in the mineralized hypertrophic chondrocyte. The em bryonic, murine mesenchymal stem cell line, C3H/10T1/2, is pluripotent exposure to azacytidine or to bone morphogenetic protei n-2 or -4 res ults in low rates of differentiation to three mesengenic lineages. In contrast to previous studies, we report conditions for 10T1/2 differen tiation specifically to the cartilage lineage and at high yields. Thes e conditions include high cell density micromass cultures, a purified mixture oi osteoinductive proteins (BP; Intermedics Orthopedics, Denve r, CO), a serum substitute, 50 mu g/ml ascorbic acid, and 10 mM beta-g lycerophosphate. The cartilagenous fate was confirmed by 1) histologic al detection of sulfated proteoglycans, 2) electron microscopic detect ion of proteoglycan and rounded cells separated by extracellular matri x containing short, disorganized collagen fibrils, 3) morphological de tection of chondrocytes surrounded by a territorial matrix and encompa ssed within a distinct perichondrium, and 4) immunocytochemical detect ion of type II collagen and link protein. After 4 weeks in culture, ma ture although unmineralized cartilage was observed, as indicated by hy pertrophic morphology, immunocytochemical detection of osteocalcin, an d histological detection of lacunae. These conditions promote overt ch ondrogenesis for most of the treated cells and preclude lineage determ ination to the fat, muscle, and bone lineages, as assayed by electron microscopy and histomorphology. The faithful recapitulation of cartila ge differentiation that we have established in vitro provides a versat ile alternative to the use of chondrocyte and limb bud explant culture s. We propose this as a model system to study the factors that regulat e commitment to the chondrogenic lineage, exclusion to related mesenge nic pathways, and maturation during chondrogenesis. (C) 1997 Wiley-Lis s, Inc.