Proliferation, differentiation and self-renewal of osteoprogenitors in vertebral cell populations from aged and young female rats

Abstract

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 (17–26-month-old) and young (1.5-month-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 50 μg/ml ascorbic acid with or without dexamethasone (Dex; 0.3–300 nM) or progesterone (Prog; 0.01–10 μM). Growth curves for cell populations of both age groups were similar with population doubling times of 27.1 and 26.7 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 number of colony forming units-fibroblast (CFU-F), alkaline phosphatase-positive colony forming units-fibroblast (AP+ CFU-F) or colony forming units-osteoblast (CFU-O). No differences were also found for any progenitor within the aged group. Limiting dilution analysis in the presence of 3 μM Prog showed no differences in the numbers of CFU-F, AP+ CFU-F or CFU-O between the aged and young groups or within the aged group. Continuous subculture of cells in the presence of 10 nM Dex revealed that the number of nodules per 10 4 plated cells increased in second subculture over first subculture cells in the young group but decreased in the aged group. Also, in third to fifth subculture cells, the number of nodules was lower in the aged group than in the young group. A similar pattern was observed in the presence of 3 μM Prog. 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.