Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells^,

Abstract

Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18–29 years, n = 6) and old (aged 68–81 years, n = 5) donors. MSC were serially passaged until reaching maximal life span. Cell growth, markers of cellular senescence, and osteogenic and adipogenic potential were determined in early-passage and late-passage cells established from young and old donors. MSC from old donors exhibited a decreased maximal life span compared with cells from young donors (24 ± 11 population doublings [PD] vs 41 ± 10 PD, P < 0.05) and mean PD rate was lower in old donor cells (0.05 ± 0.02 PD/day) compared with young donor cells (0.09 ± 0.02 PD/day) (P < 0.05). No differences were detected in number of senescence-associated β-galactosidase positive (SA β-gal⁺) cells and mean telomere length in early-passage cells obtained from young and old donors. However, MSC from old donors exhibited accelerated senescence evidenced by increased number of SA β-gal⁺ cells per PD as compared with young (4% per PD vs 0.4% per PD, respectively). MSC from young and old donors were able to form similar amounts of mineralized matrix in vitro and of normal lamellar bone in vivo. In adipogenic medium similar numbers of adipocytes formed in cultures of young and old donors. In conclusion, aging is associated with decreased proliferative capacity of osteoprogenitor cells, suggesting that decreased osteoblastic cell number, and not function, leads to age-related decrease in bone formation.

Introduction

Several clinical and histomorphometric studies have demonstrated that aging is associated with decreased bone mass and that decreased bone formation is an important pathogenetic factor [1], [2], [3]. However, the cellular and molecular mechanisms underlying decreased bone formation are not known in detail. Osteoblasts (the bone-forming cells) are recruited continuously from stem and osteoprogenitor cells in the bone marrow during the bone formation phase of skeletal remodeling [3]. While the identity of these stem cells has not been determined, it is generally accepted that they are present in bone marrow stromal cells (MSC) based on their ability for extensive cellular proliferation and multipotentiality [4], [5], [6].

Depletion of stem cells has been suggested to contribute to a large number of degenerative diseases of brain, liver, skin, and bone marrow [7]. Previous studies examining age-related effects on the size of the osteoprogenitor cell population in bone marrow have yielded variable results. Some studies in rodents and humans reported an age-related decrease in the number of osteoprogenitor cells [8], [9], [10], [11], [12] while other studies found no effects or an age-related increase [13], [14], [15], [16]. We have previously examined the number of osteoprogenitor cells generated in Stro-1 purified MSC cultures from a large number of normal healthy young and old donors as well as patients with osteoporosis [17]. We found maintenance of the number and the proliferative capacity of osteoprogenitor cells with aging and in patients with osteoporosis. However, this study examined the proliferative capacity of the cells in short-term cultures (2 weeks).

Long-term in vitro culture of normal diploid cells (the so-called Hayflick model of cellular senescence) has been employed extensively in the field of biogerontology to identify the cellular mechanisms of age-related impairment of functions [18], [19], [20]. In this model, cultured cells exhibit arrest of proliferation after a variable number of population doublings (PD) and characteristic biochemical and molecular changes that are dependent on the age of the donor [21], [22], [23] and on disease condition [24]. We have also, previously, employed this model to study the senescence phenotype of human osteoblasts derived from trabecular bone explants [25], [26], [27]. In the current study, we characterized the long-term in vitro growth and biological characteristics of MSC cultures obtained from both young and old healthy donors.