Abstract. Mouse endochondral chondrocytes were immortalized with a temperature-sensitive simian virus 40 large tumor antigen. Several clonal isolates as well as pools of immortalized cells were characterized. In monolayer cultures at the temperature permissive for the activity of the large tumor antigen (32°C), the cells grew continuously with a doubling time of ,~2 d, whereas they stopped growing at nonpermissive temperatures (37°C-39°C). The cells from all pools and from most clones expressed the genes for several markers of hypertrophic chondrocytes, such as type X collagen, matrix Gla protein, and osteopontin, but had lost expression of type lI collagen mRNA and failed to be stained by alcian blue which detects cartilagespecific proteoglycans. The cells also contained mRNAs for type I collagen and bone Gla protein, consistent with acquisition of osteoblastic-like properties. Higher levels of mRNAs for type X collagen, bone Gla protein, and osteopontin were found at nonpermissive temperatures, suggesting that the expression of these genes was upregulated upon growth arrest, as is the case in vivo during chondrocyte hypertrophy. Cells also retained their ability to respond to retinoic acid, as indicated by retinoic acid dose-dependent and timedependent increases in type X collagen mRNA levels. These cell lines, the first to express characteristic features of hypertrophic chondrocytes, should be very useful to study the regulation of the type X collagen gene and other genes activated during the last stages of chondrocyte differentiation. C HONDROCYTES are highly specialized cells that are derived from mesenchymal cells during embryonic development to form the various cartilages of vertebrates. Chondrocytes are characterized by the production of cartHage-specific extracellular macromolecules including the collagen types H, IX, and XI, and the proteoglycan aggrecan (16). In permanent cartilages, the phenotype of chondrocytes is stably maintained, whereas in endochondral cartilages, chondrocytes undergo a further differentiation process (16,34). During this process, chondrocytes mature into hypertrophic ceils around which the cartilage matrix becomes calcified, and subsequently undergo apoptosis, making way for invading osteoblasts. Hypertrophic chondrocytes still contain type II collagen and aggrecan RNAs, although at reduced levels (21, 38), they are characterized by the synthesis of type X collagen, which is uniquely expressed by these cells (10,21,39), and by the expression of markers typically associated with osteoblasts. Both hypertrophic chondrocytes and osteoblasts have the ability to induce mineralization of the extracellular matrix and to produce alka-
