Figure 7. Age-dependent capacity of Fp and Fr to contract a collagen gel. Dermal equivalents were generated with Fp and Fr samples from 3 young (19 yr, 21 yr, and 26 yr) and 3 old donors (57 yr, 62 yr, and 74 yr). Diameter of lattices (cm) was measured at days 1, 2, 3, and 4. (A) Evolution of the diameter of lattices generated with Fp from young and old donors (means6SEM, n = 3, independent samples). (B) Evolution of the diameter of lattices generated with Fr from young and old donors (means6SEM, n = 3). (*p,0.05; n.s. = not significant). doi:10.1371/journal.pone.0004066.g007 promote epidermal morphogenesis and full differentiation, as compared to the other fibroblast populations tested (Fig. 8I to 8L). Age-related impact of Fp and Fr on the morphology of reconstructed skin after grafting into nude mice To complete the observations performed in vitro, similar reconstructed skin samples were grafted onto nude mice. Grafting confirmed that young Fp were more potent to promote the development of a correctly stratified and differentiated epidermis (Fig. 9A) than Fr of a similar age (Fig. 9B). In addition, we detected that under these in vivo conditions only, young Fp promoted the formation of rete-ridge structures in the grafted reconstructed skin samples. Rete-ridge structures were not observed in grafted skin samples containing old Fp (Fig. 9C) or old Fr (Fig. 9D). It had to be mentioned that dermal fibroblasts of recipient mice did not migrate into the dermis of human reconstructed skin after grafting during the time of experiments, as demonstrated by species specific staining of vimentin (not shown). Discussion Well known characteristics of skin aging in humans are a reduction of thickness [7] and alterations in its biomechanical properties [41]. The dermis is for long thought to play a key role in this multi-causal process, since molecular studies have confirmed profound changes in the synthesis and degradation of the dermal matrix molecules with increasing age [7,8]. Because fibroblasts which reside in the dermis can both produce and degrade extracellular matrix components, exploration of the possible evolution of their properties throughout aging is particularly important to progress in the understanding of this complex Fp and Fr in Skin Aging process. This study was conducted to explore the possibility that fibroblast sub-populations which reside within the superficial and the deep regions of the dermis, respectively papillary and reticular fibroblasts (Fp and Fr), may play different roles in skin aging. We found that Fp exhibit a higher growth rate in culture than Fr, which is in agreement with previous reports [42]. However, we found that aging was associated with a reduction of this difference. Since the growth characteristics of Fr with increasing age appeared unchanged, both in term of colony growth at low culture density and in term of growth rate in mass cultures, it is the marked decrease of Fp growth potential that contributes to this leveling of the growth rates. This was taken as a first indication that Fp might be more affect by aging than Fr. Analysis of protein secretion by the two fibroblast populations led to a similar conclusion. Most of the secreted proteins were equivalent in young and aged Fr with the exception of MCP-1, which was found down-regulated in Fr from aged donors. On the contrary, protein secretion by Fp was widely affected by aging. Whereas the growth potential of Fp decreased with age, secretion levels of the growth factor KGF, the metalloproteinases MMP-1, MMP-2, MMP-3, as well as the metalloproteinase inhibitors TIMP-1 and TIMP-2, was all increased in old compared to young Fp. Thus, the levels of different protein secretion, which were globally lower in young Fp compared to the young Fr appeared similar in Fp and Fr samples from older donors. A general increase in MMP production by dermal fibroblasts with age is described in previous reports [43–45]. However, none of these studies identified different secretion profiles for the specific fibroblast sub-populations, as shown here. Our comparative studies of Fp and Fr performed at the global level of cell populations have revealed some differences in biological properties of the two fibroblast populations. However, analyses of cell morphology by flow cytometry and evaluation of colony-forming efficiency in culture have revealed that fibroblast samples from the two tissue localization present a broad heterogeneity. We routinely observed a higher proportion of fibroblasts with small size cells and low granularity in Fp samples compared to Fr samples. Dermal fibroblasts are for long known to produce clones with heterogeneous characteristics in low density cultures [23,46]. Analysis of the clonogenic potential of Fp and Fr indicated that formation of large clones was more frequently observed in Fp compared to Fr cells. Interestingly, the high clonogenic potential of Fp faded with aged, indicating that probably only minority of the young Fp cell population accounts for the observed higher growth potential of young Fp. Loss of this particular cell subpopulation in the papillary dermal compartment during aging could be an important factor in the process of skin aging. A decrease of dermal fibroblast growth potential with age has been observed long ago [47], but the papillary localization of the fibroblasts responsible for this phenomenon was not estab- lished in this report. In this context, it is interesting to note that, in long-term cultures of fibroblasts used as an in vitro model of cell aging, a decrease of the growth potential [18,19], a reduced number of small cells [48], and an increased protein production [21], have been reported, which is in agreement with our results. Fp and Fr are known to differ in their sensitivity to growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-b1) [37]. The observed decreased growth capacity of Fp with increasing age could result from a reduction of responsiveness to mitogenic factors, as previously suggested [24]. In a next step, comparative transcriptional profiling of Fp and Fr using full genome DNA arrays will be particularly informative to identify specific molecular characteris- tics of these two fibroblast populations. PLoS ONE | www.plosone.org 9 December 2008 | Volume 3 | Issue 12 | e4066