were clearly observed in sections of young skin, whereas old skin is characterized by a marked flattening of the dermo epidermal junction. A structural disorganization of the collagen fibers was observed in the aged dermis. Secretion of cytokines and MMPs/TIMPs by Fp and Fr as a function of donor’s age Age-related secretion of cytokines and MMPs/TIMPs by Fp and Fr was analyzed in a cohort of skin samples from 16 independent donors, ranging from 19 to 74 years old, by linear regression. Age-related evolution of cytokine secretion by Fp and Fr was characterized by distinct profiles. Secretion of KGF increased with age in Fp (Fig. 3A), and remained constant in Fr (Fig. 3B). Consequently, the ratio of KGF secretion by Fr and Fp (Fr/Fp) in paired samples from the same skin biopsy decreased with donor’s age (Fig. 3C). The evolution of VEGF secretion resembled that of KGF, however, its up-regulation in Fp from old donors was not statistically significant. MCP-1 secretion was did not vary in Fp, but was decreased with age in Fr. Secretion of IL-6 was not affected by aging in both fibroblast populations. For all analyzed MMPs and TIMPs a progressive increase of secretion level was observed in Fp, whereas no significant alterations with age were detected for Fr. All values are detailed in Table 1. Evolution with age of Fp and Fr morphology Populations of Fp and Fr from young (19 yr, 21 yr, and 26 yr) and older donors (57 yr, 62 yr, and 74 yr) were examined by flow cytometry, according to the morphological parameters of size (forward scatter) [FSC] and granularity (side scatter) [SSC]. Dot plots corresponding to Fp and Fr from a typical young donor (19 yr) (Fig. 4A and 4B, respectively), and from a typical older donor (74 yr) (Fig. 4C and 4D, respectively), indicated that all cell samples contained cells with heterogeneous morphologies. However, in the young age group, median FSC (Fig. 4E) and SSC (Fig. 4F) values Figure 2. Histological characteristics of young and old human skin. Photographs shown correspond to histological sections of mammary skin biopsies obtained from a 19 year old (A) and a 74 year old donor (B). Sections were stained with hematoxylin, eosin, and saffron (HES). Scale bars = 50 mm. Dp = papillary dermis. Dr = reticular dermis. doi:10.1371/journal.pone.0004066.g002 Fp and Fr in Skin Aging were significantly lower in Fp than in Fr (p,0.05), indicating that Fp samples contained a higher proportion of small sized cells with low granularity than Fr samples. Morphological differences between Fp and Fr appeared less marked in the older group. Indeed, median SSC values were still significantly lower in old Fp than in old Fr (p,0.05), but this difference was less important. Median FSC values of Fp and Fr were still not significant in the old group. Age-related growth characteristics of fibroblasts isolated from papillary (Fp) and reticular (Fr) dermis Papillary and reticular fibroblasts (Fp and Fr) isolated from skin samples from 3 young (19 yr, 19 yr, and 26 yr) and 3 old individuals (57 yr, 58 yr, and 67 yr), were studied for their respective growth rate in short-term culture. Mean cumulative expansion curves indicated that, in the young age group, Fp exhibited a higher growth rate than Fr (Fig. 5A), in particular when comparing the respective rates at day 7, day 14, and day 21 (p,0.05). In the old age group, this difference was less marked, but still significant at day 14 and day 21 (p,0.05) (Fig. 5B). It is interesting to note that Fr growth rates were not significantly affected by age with a cellular growth index at day 21, for young Fr and old Fr of 12.260.5 and 9.0762.03, respectively. On contrary, a marked decrease of cellular growth index with age was observed in Fp at day 21, with 21.661.6 for young and 14.361.3 for aged cells (p,0.05). Evolution with age of the clonogenic capacity of Fp and Fr Investigation of the clonogenic capacity of Fp and Fr with aging on cell samples prepared from skin biopsies of 4 young (19 yr, 21 yr, 23 yr, and 26 yr) and 4 old (57 yr, 60 yr, 63 yr, and 74 yr) donors indicated that the colony-forming efficiency (CFE) of Fp was significantly higher than that of Fr, whatever the age of donors (Fig. 6A). This difference was particularly marked in samples from young donors: for an equivalent of 100 plated cells, 59.7567.6 and 32.9767.6 colonies were obtained from young Fp and Fr, respectively (p,0.05). The CFE of Fp remained significantly more elevated than that of Fr in old donors, but this difference was reduced: respectively 45.465.6 and 36.6765.2 colonies were obtained / 100 plated Fp and Fr (p,0.05). Colonies produced by the different Fp and Fr samples were then classified according their size (i.e. area occupied by fibroblasts in individual colonies). As shown in Fig. 6B, small colonies (area ,0.5 mm2) were characterized by a low fibroblast density, whereas cell density was markedly higher in large 2 size colonies (area .1.5 mm ). Cell density was intermediate in 2 medium size colonies. Correspondence between colony area (mm ) and fibroblast number indicated that small size, medium size, and large size colonies contained respectively 2.66610260.616102, 1.86610360.456103, and 4.65610360.276103 fibroblasts (Fig. 6C). Analysis of colony size also indicated marked functional differences between Fp and Fr in the young age group (Fig. 6D). In particular, the higher clonogenic growth capacity of young Fp, as compared to that of Fr of similar age, was clearly illustrated by the increased number of large size colonies generated by Fp: respectively 1166 and 362 large colonies were obtained / 100 plated young Fp and Fr (p,0.05). This difference in the growth capacity of Fp and Fr was lost with increasing age. Figure 1. Experimental definition of Fp and Fr cells. (A) and (B), principle of papillary (Fp) and reticular (Fr) fibroblast isolation. (C) Histological preparation of a mammary skin sample after dermatome cutting at a depth of 0.3 mm to obtain the superficial dermis with the epidermis. (D) Full thickness histology of the same skin sample. (E) Histological preparation of the skin sample after dermatome cutting at a depth of 0.7 mm to reach the deep dermis. Scale bars = 50 mm. (F) and (G) Photographs of typical cultures of Fp and Fr in light microscopy. Scale bars = 10 mm. (H) and (I) Fluorescence photographs of Fp and Fr after actin and vinculin immuno-staining. Scale bars = 2.5 mm. doi:10.1371/journal.pone.0004066.g001 PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e4066