ESTABLISHMENT OF A GROWTH-HORMONE RESPONSIVE CHONDROGENIC CELL-LINE FROM FETAL-RAT TIBIA

Reproducible effects of growth hormone (GH) on primary isolated cells in monolayer are highly dependent on the culture conditions and/or the fraction of GH responsive cells. To study the effect of GH at the cellular level, a homogenous cell line with both GH responsiveness and chondrogenic properties was established. Primary isolated cells from 18-day-old fetal rat tibia were subcultured using a strict protocol for passages (every third day and a seeding density of 15,000/cm2). Of six established cell lines, one fetal tibia cell line No.5 (FTC 5) expressed adipogenic and chondrogenic properties at a low frequency. Cells from FTC 5 were subcultured in soft agar suspension with the addition of bovine GH (100 ng/ml). After 14 days in culture eight monoclonal cell lines were established from individual large colonies. Two subclones, FTC 5:3 and FTC 5:6, expressed a chondrogenic phenotype as demonstrated by chondrocyte foci, alcian blue staining and production of type II collagen. Further characterization of FTC 5:3 revealed specific binding of bovine GH with an affinity of 1.7 x 10(9) M-1, and approximately 7300 receptors/cell. Northern blot analysis of FTC 5:3 with a P-32-labeled RNA probe complementary to an extracellular part of the rat GH receptor, revealed two major labeled bands (4.0 and 1.2 kilobases). Both GH and insulin-like growth factor-I (IGF-I) stimulated H-3-thymidine uptake in FTC 5:3 (194 +/- 28% and 405 +/- 127% over control, respectively), while proteoglycan synthesis, as measured by [S-35]sulphate uptake, was stimulated by IGF-I only (101 +/- 18% over control). In conclusion, a chondrogenic cell line was established that expressed GH receptors and exhibited GH responsiveness.