Progesterone stimulates osteoprogenitor proliferation and differentiation in osteoblast-like cell populations derived from adult female rat vertebrae

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A significant contribution to the bone loss associated with aging is likely to be a decline in bone formation. We have characterized and compared the number, capacity for proliferation and differentiation and the self-renewal ability of osteoprogenitors of aged (24-week-old) and young (6-week-old) female Wistar rats using limiting dilution analyses and continuous subculture experiments. Cells were obtained from outgrowths of explants of lumbar vertebrae (L1-L6) and grown in α-minimal essential medium (α-MEM), 10% FBS and 50mg/ml ascorbic acid with or without dexamethasone (Dex) or progesterone (Prog). Growth curves for cell populations of both age groups were similar with population doubling times of 28.3 and 27.5 h for the aged and young animals, respectively. Osteoprogenitors from both age groups formed bone nodules when cultured in the presence of either Dex or Prog. Limiting dilution analysis in the presence of 10 nM Dex showed no difference between the aged and young rats in the n … More umber of colony forming units-fibroblast (CFU-F), alkaline phosphatase-positive colony forming units-fibroblast (AP+CFU-F) or colony forming units-osteoblast (CFU-O). Limiting dilution analysis in the presence of 3 mM Prog showed no differences in the numbers of CFU-F, AP+CFU-F or CFU-O between the aged and young groups. Frequencies and numbers of both progenitor types were determined for up to six subcultures using continuous subculturing, limiting dilution analysis, and colony assays. In Dex-containing medium, subculturing resulted in a significant increase in the total number of Dex-responsive osteoprogenitors in second subculture cells over first subculture cells without a significant increase in the frequency of these progenitors. From the third subculture onward, the frequency of osteoprogenitors decreased in a linear manner and none were observed after six subcultures. Similar results were obtained in Prog-containing medium. Results indicate that the cell population doubling times, growth characteristics, and the number of CFU-F and osteoprogenitors in vertebral bone cell populations from aged rats and young rats are similar. This suggests that the bone loss associated with aging is not caused by a decrease in osteoprogenitor cell number. However, cell populations from the aged rats showed a reduced capacity for self-renewal in vitro, which would ultimately translate into a reduced number of osteoblasts and might be partly responsible for a decrease in bone formation in aged animals.Previous experiments have demonstrated that bone cell populations derived from explants of lumbar vertebral bone of adult female rats contain osteoprogenitors that require Dex or Prog to proliferate and differentiate into fully differentiated bone-forming osteoblasts. We have shown that the Prog-dependent population can not be detected in male rats after sexual maturation but is present in prepubertal rats of both sexes, and can be induced in adult male-derived populations by culturing the explants in media containing 17 β-estradiol (10^<-9>-10^<-8>M). This suggested that the Frog- and Dex- dependent osteoprogenitors in adult female-derived populations were likely to be distinct populations and that the survival of the Prog-dependent osteoprogenitors and/or their ability to proliferate is dependent on the presence of estrogen. The results imply that Prog plays an important role in maintaining bone mass through regulating the class of osteoprogenitors responsive to Prog. Less