KINETICS OF IN-VITRO MINERALIZATION BY AN OSTEOGENIC CLONAL CELL-LINE (C1) DERIVED FROM MOUSE TERATOCARCINOMA

We have previously reported the isolation of an osteogenic clonal cell line (C1) derived from mouse teratocarcinoma and immortalized by the SV 40 oncogenes. In this report we describe the kinetics of osteogenic differentiation of aggregated C1 cells by following the matrix deposition and mineralization and the expression of alkaline phosphatase. We show that after addition of beta-glycerophosphate and ascorbic acid, more than 95% of C1 aggregates synthesize a bone matrix which is deposited as early as 2 days and increases progressively with time in culture. Matrix calcification is evidenced by von Kossa staining and tetracycline incorporation into the mineral whereas no calcification appears in control cultures. Calcium is detectable in mineralizing aggregates at 2 days and calcium content increases linearly with time in culture, being 125-fold higher in mineralizing nodules than in control aggregates at 30 days. Aggregated C1 cells are characterized by a high activity of the bone type isoenzyme of alkaline phosphatase, a marker of osteoblast phenotype. Upon addition of inducers, alkaline phosphatase activity decreases by fivefold after the onset of mineralization and remains stable thereafter. The down-regulation of alkaline phosphatase activity is confirmed at the cellular level by histochemical staining. The mRNA levels for alkaline phosphatase decline during osteogenesis, following a pattern similar to the decrease in protein activity. Analysis of DNA synthesis by (H-3)-thymidine incorporation and quantification of labelled nuclei on autoradiographs shows that C1 cells proliferation is not down-regulated during the time course of differentiation and that proliferating C1 cells still express alkaline phosphatase activity during osteogenic differentiation. The clonal Cl cells represent therefore, an original model of osteoblast differentiation where the high frequency and synchrony of nodule mineralization make it possible to follow the expression of bone-related genes along the kinetics of osteogenesis in vitro.