Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2.
Rat stromal bone-marrow cells cultured in the presence of dexamethasone, ascorbic acid, beta-glycerophosphate, and fibroblast growth factor-2 (FGF-2) express the osteogenic phenotype (Pitaru et al., J. Bone Miner. Res. 8:919-929, 1993). The purpose of this study was to establish a long-term homogeneous culture expressing the osteogenic phenotype. The cultures were routinely passaged every 5 days in the absence or presence of either or both dexamethasone and FGF-2, and the cumulative doubling number and the expression of the osteogenic phenotype were determined. Cultures treated with dexamethasone (10(-7) M) ceased proliferation and only upon addition of FGF-2 (3 ng/ml) was a spontaneous immortalization achieved, as expressed by sustained proliferation for about 1 year, with a doubling time of 22 h and more than 300 doublings in 72 passages. Both FGF-2 and dexamethasone are required and act synergistically to maintain cell propagation, alkaline phosphatase expression, and osteocalcin secretion; however, protein content was FGF-2 dependent and the mineralization was dexamethasone dependent. Repetitive single-cell cloning tested the homogeneity and stability of the cells expressing the osteogenic phenotype in these long-term cultures. It was shown that 25% to 50% of subclones derived from clones with an osteogenic phenotype do not further express the osteogenic phenotype. In conclusion, we have established a spontaneously immortalized dexamethasone- and FGF-2-dependent rat stromal bone-marrow-derived long-term culture expressing the osteogenic phenotype. The cultures tend to lose the osteogenic phenotype, and dexamethasone supports the long-term preservation of the osteogenic phenotype.
Kotev-Emeth
Shlomo
S
Maurice and Gabriela Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Pitaru
Sandu
S
Pri-Chen
Sara
S
Savion
Naphtali
N
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000880:Anthraquinones; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology*; D004338:Drug Combinations; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005938:Glucocorticoids / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D000880:Anthraquinones; D004338:Drug Combinations; D005938:Glucocorticoids; D016222:Fibroblast Growth Factor 2; D015675:Osteocalcin; C004468:Alizarin Red S; D003907:Dexamethasone; D000469:Alkaline Phosphatase