Dr Hui B. Sun and Dr Evan L. Flatow, Department of Orthopaedics, Box 1188, Mount Sinai, School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. Tel.: +1(212) 241 3767; fax: +1(212) 876 3168; e-mail: email@example.com@msnyuhealth.org
Aging is a major risk factor for tendon injury and impaired tendon healing, but the basis for these relationships remains poorly understood. Here we show that rat tendon-derived stem/progenitor cells (TSPCs) differ in both self-renewal and differentiation capability with age. The frequency of TSPCs in tendon tissues of aged animals is markedly reduced based on colony formation assays. Proliferation rate is decreased, cell cycle progression is delayed and cell fate patterns are also altered in aged TSPCs. In particular, expression of tendon lineage marker genes is reduced while adipocytic differentiation increased. Cited2, a multi-stimuli responsive transactivator involved in cell growth and senescence, is also downregulated in aged TSPCs while CD44, a matrix assembling and organizing protein implicated in tendon healing, is upregulated, suggesting that these genes participate in the control of TSPC function.
Age is a major risk factor for tendon disorders. Age-related changes in structural and mechanical properties may predispose tendons to injury; moreover, tendon healing is often impaired in the elderly (Vogel, 1978; Birch et al., 1999; Magnusson et al., 2003; Couppe et al., 2009). Tenoblasts and tenocytes, the major tendon cellular elements that produce and organize tendon extracellular matrix (ECM) (Kannus, 2000), also undergo age-dependent changes in number and activity (Ippolito et al., 1980; Nakagawa et al., 1994), but the mechanisms behind these changes remain unclear. Recently, tendons of several species (Bi et al., 2007; Rui et al., 2009; Zhang & Wang, 2010) were shown to contain a minor population of cells with stem cell properties, termed tendon-derived stem/progenitor cells (TSPCs). Because adult or tissue-resident stem/progenitor cells are considered essential for tissue maintenance and repair, we investigated whether the self-renewal capacity and differentiation potential of rat TSPCs are influenced by age.
Results and discussion
Tendon-derived stem/progenitor cells were prepared from rat tendon tissues based on their preferential attachment and potent clonal expansion over the majority of tendon resident cells (Bi et al., 2007). We found that nearly 100% of both young and aged TSPCs stained positively for three stem cell markers: nucleostemin, Oct-4, and SSEA-4 (Fig. S1), indicating that this population retains features reflecting their stemness regardless of age. Rat TSPCs also expressed surface antigens CD44 (Fig. 1A, left panels) and CD90.1 (Fig. 1A, middle panels), but not the endothelial cell marker CD106 (VCAM-1) (Fig. 1A, right panels), as shown for murine and human TSPCs (Bi et al., 2007) and recently reported for rat TSPCs (Rui et al., 2009). Aged TSPCs expressed lower levels of CD90.1 than young cells, but higher levels of CD44 as determined by both the percentage of positive cells (Fig. 1A, left panels) and mean fluorescence intensity (Fig. 1B). The differences in CD44 expression were also seen at mRNA levels by RT–PCR (Fig. 1C). CD44, implicated in healing of many tissues, is downregulated during scarless fetal tendon healing (Favata et al., 2006); moreover, mice genetically deficient in CD44 show improved patellar tendon healing (Ansorge et al., 2009). These findings suggest that the increased CD44 expression in aged TSPCs may contribute to reduced TSPC repair capacity with age.
Tendon-derived stem/progenitor cells accounted for a dramatically smaller fraction of total tendon cells in old rats (0.171 ± 0.06%) than young rats (6.26 ± 0.55%, P < 0.001) (Fig. 1D) based on the number of colonies formed by whole tendon-derived cells (Bi et al., 2007; Delorme & Charbord, 2007). This corresponded to a 70% reduction in the total number of cells recovered from old versus young tendons. However, colony formation assays using P0 TSPCs from aged and young rats showed only small differences (data not shown). These results indicate that diminished colony formation by aged TSPCs primarily is due to low TSPC numbers likely reflecting a depleted TSPC pool in aged tendon tissues.
Proliferation of aged and young TSPCs also differed. Cell numbers after 8 days of culture were lower in aged TSPCs (1.08 ± 0.09 × 106 vs. 1.91 ± 0.16 × 106, P < 0.05, Fig. 1E, upper panel), with a corresponding increase in estimated mean population doubling time (DT) (18.8 ± 0.22 vs. 17.4 ± 0.20 h, P < 0.05, Fig. 1E, lower panel). Similar differences were observed in CFSE-dilution assays (Lyons, 2000) assessed by flow cytometry (data not shown). Analysis of cell cycle phase distribution using propidium iodide further showed that aged TSPCs contained a higher fraction in G2/M (Fig. 1F), suggesting that aged TSPCs were preferentially subject to late cell cycle arrest. This could result from accumulated genetic and/or epigenetic damage, as has been reported in other stem/progenitor cell populations (Rossi et al., 2007). Additionally, differences in apoptotic rates between young and old TSPCs could also contribute to the observed disparities in population size. However, to our knowledge, neither issue has yet been examined in TSPCs. Collectively, these data indicate that TSPCs undergo age-related declines in self-renewal capacity (Nishimura et al., 2005; Levi & Morrison, 2008) that could account for reduced stem cell numbers with aging.
The ability of TSPCs to differentiate into tenocytes was also diminished by age. Expression of two tendon lineage-specific genes (Perez et al., 2003; Shukunami et al., 2006; Murchison et al., 2007), Scleraxis (Scx) and Tenomodulin (Tnmd), was lower in aged TSPCs than in young cells (Fig. 2A). Aged TSPCs also showed diminished induction of these markers by TGF-β3 (Kovacevic & Rodeo, 2008). Whether these differences also extend to other tenocyte lineage-inductive stimuli like mechanical loading (Juncosa-Melvin et al., 2006; Kuo & Tuan, 2008) and whether they reflect differentiation capacity in vivo remains to be determined. Interestingly, however, aged TSPCs formed adipocytes more readily than younger cells (Fig. 2B) and expressed higher levels of adipogenic markers PPARγ2 (PPARGC1A), C/EBPa (Cebpa/CEBPA) and leptin following induction (Fig. 2C). Young and old TSPCs showed no apparent difference in the ability to form osteoblasts or chondrocytes (Fig. S2). These data may help explain the higher levels of adipose tissue normally associated with older tendons (Kannus & Jozsa, 1991), a pattern similar to that observed in bone marrow, where adiposity was found to correlate inversely with the functionality of hematopoietic stem/progenitor cells (Naveiras et al., 2009).
Finally, we explored age-dependent changes in a potential regulator of TSPC function. Cited2 is a transcription factor implicated in the control of growth and senescence in several cell types (Sun et al., 1998; Kranc et al., 2003; Yokota et al., 2003; Sun, 2009). Recent studies further reveal that Cited2 is required to maintain adult hematopoietic stem cells (Chen et al., 2007; Kranc et al., 2009). We found that Cited2 expression in aged TSPCs was reduced at both the mRNA (Fig. 2D) and protein levels (Fig. 2E). These data are consistent with positive roles for Cited2 in TSPC self-renewal. Furthermore, the coordinated expression of Cited2 and Scx suggests that it may also regulate TSPC differentiation.
This study has demonstrated remarkable changes in number and function of tendon stem/progenitor cells with advancing age. It remains to be determined whether these age-related changes are also influenced by factors such as activity, which could affect tendon loading history. Second, while the functional characterizations of rat TSPCs in vitro may reflect properties of these cells, including the adaptation to an in vitro environment, further studies are also needed to determine whether TSPCs exhibit age-dependent differences in the ability to repopulate functional tenocyte pools in vivo.
The authors thank Drs Zhengzhe Li, Li Sun and Damien M. Laudier for scientific and technical support. Supported by NIH grants AR050968, AR 047628 (Sun H.B.), AR52743 (Flatow E.L.).