DEVELOPMENT AND CHARACTERIZATION OF CONDITIONALLY IMMORTALIZED OSTEOBLAST PRECURSOR CELL-LINES FROM HUMAN BONE-MARROW STROMA

Although the differentiation of mature osteoblasts has been well studi ed, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for is olating small numbers of authentic osteoblast precursor cells from hum an bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996). He re we describe the conditional immortalization of these cells by retro viral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature-sensitive mutant form of the simian virus la rge T-antigen. At the permissive temperature of 34 degrees C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5 degrees C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reve rse transcriptase PCR after 4 days of culture at 39.5 degrees C, the s ix cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Hex) and 1 alpha,25-dihydroxyvitamin D-3 ( 1,25(OH,)D,) for osteoblast (alkaline phosphatase [ALP], type I collag en [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH-R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10 (-8) M Dex, gene expression for ALP, PTH-R, and LPL increased, but tha t for OC decreased. Stimulation with 10(-8) M 1,25(OH,)D, increased ge ne expression for ALP, OC, and Col I. Changes in protein production fo r ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D-3 were similar to changes in mRNA levels. When cultured at 39.5 degrees C,vith ascorbate and beta-glycerolphosphate for 21 days, mineralizatio n of matrix occurred, whereas culture with Dex plus 1,25(OH2)D-3, or r abbit serum led to enhanced formation of cytoplasmic lipid droplets wi thin 6 days. Thus, these cell lines are capable of bipotential differe ntiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte diffe rentiation in humans.