ESTABLISHMENT AND CHARACTERIZATION OF CONDITIONALLY IMMORTALIZED STROMAL CELL-LINES FROM A TEMPERATURE-SENSITIVE T-AG TRANSGENIC MOUSE

We established bone marrow stromal cell lines from a transgenic mouse that harbors a temperature-sensitive mutant of the simian virus 40-der ived large T-antigen under the control of an major histocompatibility complex (MHC) I promotor, These cell lines were screened for their abi lity to induce the formation of osteoclasts in a spleen cell/stromal c ell coculture system, By means of this screen, five clones, referred t o as marine bone marrow stromal clone 1 (mBMS-B1) mBMS-B2, mBMS-B14, m BMS-B18, and mBMS-B21, were selected for detailed characterization, Ce ll growth depends on culture conditions, i.e., cells grow at 33 degree s C in the presence of murine interferon-gamma, whereas cell prolifera tion ceases at 39 degrees C. The phenotype of the cells is also correl ated with the culture conditions because the osteoclast inductive capa city is only seen at 39 degrees C, indicating that the cells undergo d ifferentiation when the transforming agent is inactivated, These condi tionally immortalized stromal cells can be induced to express a variet y of markers that are typical for mature osteoblasts, e.g., alkaline p hosphatase activity and expression of functional parathyroid hormone r eceptor after stimulation with soluble osteogenic protein 1 (sOP-1). m RNA analysis revealed the expression and regulation of osteopontin, os teonectin, and collagen alpha 1(I) as well as the inducibility of oste ocalcin upon treatment with sOP-1. The cells have the potential to for m mineralized nodules in supplemented medium, We observed expression o f vascular cell adhesion molecule-1, which is stimulated upon treatmen t of the cells with 1 alpha,25-dihydrocholecalciferol after 4 days, in dicating the presence of the receptor for this steroid, These cell lin es represent a model to study mechanisms and factors involved in osteo blast differentiation.