Clones of nontransformed hormone-responsive bone cells have been isolated in vitro from mixed cell populations of fetal rat calvaria. In several independent isolations, microscopically visible colonies appeared at plating efficiencies of 5-10% of the starting cell numbers. Of these clones, approximately 10% grew to mass populations which could be assayed for a number of growth and biochemical properties. Although some similarities existed among the clones, they could be distinguished from each other and from the mixed cell populations. Population-doubling times (tDs) and saturation densities varied over a wide range: e.g., tDs of 24-72 h and saturation densities of 0.4-5 x 10(5) cells/cm2. Morphologies varied from roughly polygonal multilayering cells to typically spindle-shaped monolayering cells. Hormone responsiveness, as measured by stimulation of cAMP by hormones, indicated that some clones were responsive to both parathyroid hormone (PTH) and prostaglandin E2 (PGE2), while others responded to PTH only. Analysis of extracellular matrix components revealed that all clones produced type I and type III collagens, though in different proportions. Similarly, although all clones synthesized four glycosaminoglycans (hyaluronic acid, heparan sulfate, chondroitin sulfate, and dermatan sulfate), the quantities of each were distinctive from clone to clone. Further investigation of such clones is continuing to define more precisely the heterogeneity of clonal bone cell populations in vitro. They represent an important step in the study of the endocrinology and differentiation of bone.

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