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 deposi tion 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 depos ited as early as 2 days and increases progressively with time in cultu re. Matrix calcification is evidenced by von Kossa staining and tetrac ycline 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 aggreg ates at 30 days. Aggregated C1 cells are characterized by a high activ ity of the bone type isoenzyme of alkaline phosphatase, a marker of os teoblast phenotype. Upon addition of inducers, alkaline phosphatase ac tivity decreases by fivefold after the onset of mineralization and rem ains stable thereafter. The down-regulation of alkaline phosphatase ac tivity is confirmed at the cellular level by histochemical staining. T he mRNA levels for alkaline phosphatase decline during osteogenesis, f ollowing a pattern similar to the decrease in protein activity. Analys is of DNA synthesis by (H-3)-thymidine incorporation and quantificatio n of labelled nuclei on autoradiographs shows that C1 cells proliferat ion is not down-regulated during the time course of differentiation an d that proliferating C1 cells still express alkaline phosphatase activ ity during osteogenic differentiation. The clonal Cl cells represent t herefore, an original model of osteoblast differentiation where the hi gh frequency and synchrony of nodule mineralization make it possible t o follow the expression of bone-related genes along the kinetics of os teogenesis in vitro.