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

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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 104 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.

Introduction

Loss of bone is a common occurrence during aging of both humans and rats. Usually a distinction is made between postmenopausal bone loss occurring in women primarily in the years surrounding and following menopause and age-related bone loss occurring in both males and females at the same rate. Current views are that both age-related bone loss in males and females and the rapid postmenopausal bone loss are related to decreases in the effective concentration of circulating estrogen (for review, see Riggs et al., 2002). A significant contributor to both types of bone loss is thought to be a decline in bone formation resulting from decreased osteoblastic activity or number. Since osteoblasts arise from osteoprogenitor cells, and ultimately from multipotential mesenchymal stem and progenitor cells (for review, see Aubin et al., 1993), a decrease in the number of active osteoblasts could result from a decrease in the number of proliferating and differentiating osteoprogenitors. Although osteoprogenitor cells are present within cortical and cancellous bone as well as in the bone marrow stromal cell compartment, most studies addressing the effect of aging on osteoprogenitor proliferation and differentiation in either the human, rat or mouse have focused on those found in the bone marrow. However, cell populations derived from human cancellous bone are similarly capable of differentiating into osteoblasts, chondrocytes and adipocytes, suggesting that progenitors or stem cells in cancellous bone-derived cells are similar to those present in bone marrow (Sottile et al., 2002). It may be, however, that differences exist between osteoprogenitor cells present in bone tissue and in bone marrow since the former normally progress to differentiate and form bone while the latter appear not to unless appropriately stimulated, although this may be related more to the environment or the neighboring cells than to differences between the osteoprogenitors.

In human iliac crest bone marrow cell populations, the number of alkaline phosphatase-positive colony forming units-fibroblast (AP+ CFU-F) has been found to decrease sharply between 10 and 20 years of age and then only very gradually after age 20 (Nishida et al., 1999). Similarly, in cell populations derived from human vertebral bone marrow, D'Ippolito et al. (1999) found a significant decrease with age in the number of AP+ CFU-F within a 3–36-year-old group but no significant change with age in a 41–70-year-old group, also suggesting that the major decrease in progenitor cells with osteogenic potential occurs following skeletal maturation and not during senescence. This is consistent with the marked decrease in the number of osteoblastic precursor cells in cell populations obtained from human cancellous bone explants in the second and third decades of life to a number that is maintained into old age (Shigeno and Ashton, 1995). Others have also reported decreases with age in the number of osteogenic colonies or AP+ CFU-F in human bone marrow (Majors et al., 1997) as well as in bone marrow from the rat (Tsugi et al., 1990, Dodson et al., 1996, Egrise et al., 1992) and mouse (Bergman et al., 1996) and in mitogenic response and cell growth in populations derived from human cancellous bone (Pfeilschifter et al., 1993, Shigeno and Ashton, 1995). With regard to differences between marrow-derived and bone derived populations, Egrise et al., 1992, Egrise et al., 1996 found an age-related decrease in CFU-F in bone marrow cultures but an increased rate of proliferation in cancellous bone cell cultures from tibiae and femora of 4- and 21-month-old rats.

In contrast to these findings, Oreffo et al., 1998a, Oreffo et al., 1998b found no difference in the number of CFU-F or AP+ CFU-F present at different ages in human bone marrow cell populations but noted a significant decrease in CFU-F size with increasing age. Stenderup et al. (2001) also found no difference in the number of CFU-F or AP+ CFU-F in human iliac crest bone marrow populations obtained from patients of different ages. In addition, in mouse femoral bone marrow, no difference was found in the number of CFU-F between 2- and 16-month-old mice (Brockbank et al., 1983) and in rat femoral bone marrow, Quarto et al. (1995) found that while the number of CFU-F in 24-month-old rats was decreased compared with 6- and 12-month-old rats, the decrease could not account for the considerable loss of bone tissue with advancing age.

Further complicating the issue of changes with age in the osteoprogenitor cell population is the possibility of sex-dependent differences. In human iliac crest bone marrow cells, a significant decrease in AP+ CFU-F number with age was found in women but not in men (Muschler et al., 2001) and in human bone-derived cells, osteoblasts from men did not show the same age-dependent differences as observed in women (Katzberg et al., 1999). Age-related osteopenia may also be different in different parts of the skeleton, as illustrated by the observation that decreases with age in osteocalcin secretion and osteocalcin mRNA content were much greater in cortical than in cancellous bone (Martinez et al., 2001).

Dexamethasone (Dex) and progesterone (Prog) both stimulate bone nodule formation from osteoprogenitors present in vertebral cell cultures of adult female rats (Ishida et al., 1996, Ishida et al., 1997, Ishida and Heersche, 1997). Also, by using replica plating techniques, the osteoprogenitors responding to Prog were found to be different from those responding to Dex (Ishida and Heersche, 1999). The objective of the present study was to compare the number, and the proliferative, differentiative and self-renewal characteristics of osteoprogenitors colony forming units-osteoblast (CFU-O) and CFU-F in vertebral cell populations (primarily cancellous bone) of aged (17–26 months) and young (1.5 months) female Wistar rats to determine if changes such as a reduction in the number and characteristics of the osteoprogenitor and progenitor cell pool could account for the bone loss associated with aging. In view of the possibility that differences between males and females could result in changes occurring in the Prog-responsive osteoprogenitors, we specifically investigated changes in the number of Prog- and Dex-responsive osteoprogenitors.

Section snippets

Cell isolation and culture procedures

The cell isolation and culture procedures were as described previously (Ishida et al., 1996). Vertebral bodies were dissected aseptically from the lumbar vertebrae (L1–L6) of female Wistar rats aged 1.5 months and 17–26 months. After removal of the adherent soft connective tissues, vertebral bodies were cut into ∼1–2 mm3 fragments. These fragments (explants) were placed in plasma clots (10 μl of 15% citrated bovine plasma (Life Technologies, Grand Island, NY) and 85% α-minimal essential medium

Results

Growth curves for the populations of RV cells obtained from aged (17–26 months) and young (1.5 months) animals are shown in Fig. 1. Mean maximal population doubling times for six populations obtained from aged rats was 27.1±3.0 h (varying from 21.2 to 31.1 h). For two populations of 1.5-month-animals, the mean maximal population doubling time was 26.7 h. No differences were found within the 17–26-month-old group (r2=0.012; P=0.836) or between the aged and young animals.

Dex stimulated bone

Discussion

The aim of this study was to compare the number, and the proliferative, differentiative and self-renewal characteristics of Dex- and Prog-responsive osteoprogenitors in vertebral cell populations of aged and young female Wistar rats in order to determine whether changes with age in any of these parameters were likely to contribute to bone loss associated with aging. Female rats between 17- and 26-month-old were selected for the aged group since rats have been reported to become menopausal

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