Fp and Fr in Skin Aging Figure 6. Evolution with age of Fp and Fr clonogenic capacity. The growth capacity of Fp and Fr samples from 4 young (19 yr, 21 yr, 23 yr, and 26 yr) and 4 old donors (57 yr, 60 yr, 63 yr, and 74 yr) was analyzed in term of colony-forming efficiency (CFE) and size of the colonies generated by these different fibroblast samples. (A) Compar- ison of the CFE of Fp and Fr from young and old donors. Data are expressed as number of colonies obtained from 100 plated fibroblasts (means6SEM, n = 4, independent samples). Colonies were classified 2 according to their individual area (mm ). (B) Photographs of colonies of typical ‘small’, ‘intermediate’, and ‘large’ size. Scale bars = 50 mm. (C) 2 Correspondence between colony area (mm ) and fibroblast number (means6SEM, n = 10 colonies per size category). (D) Comparison of the sizes of colonies generated by Fp and Fr from young and old donors. The histogram shown corresponds to the number of colonies obtained from 100 plated fibroblasts (means6SEM, n = 4, independent samples), (*p,0.05; n.s. = not significant). doi:10.1371/journal.pone.0004066.g006 (p,0.05). Differences were less marked when epidermis was generated in the presence of Fp and Fr from older donors. Detection of filaggrin, a marker of differentiated keratinocytes, confirmed the highest capacity of Fp from young donors to Figure 5. Age-related growth characteristics of Fp and Fr. Fp and Fr samples from 3 young (19 yr, 19 yr, and 26 yr) and 3 old donors (57 yr, 58 yr, and 67 yr) were cultured for 21 days. Cellular growth rate was estimated every 7 days after culture initiation. Data are expressed as cumulative expansion curves (means6SEM, n = 3 independent samples). (A) Comparison of growth rates of Fp and Fr from young donors. (B) Comparison of growth rates of Fp and Fr from old donors (*p,0.05; n.s. = not significant). doi:10.1371/journal.pone.0004066.g005 contraction of Fr. Differences were particularly important at day 1: diameter of lattices containing Fp and Fr were 2.506 cm60.068 and 1.928 cm60.078, respectively. Differences in the lattice retraction rate between Fp and Fr became less important with increasing age. At day 1, diameter of lattices containing Fp and Fr were respectively 2.264 cm60.035 and 1.976 cm60.078 (p,0.05). Differences were still significant at days 2 and 3, but not at day 4. In general, the lattice contraction capacity of Fr did not change with age (Fig. 7B), whereas in Fp the contraction rate clearly increased with age (p,0.05) (Fig. 7A). Age-related impact of Fp and Fr on the epidermal compartment in reconstructed skin The effect of Fp and Fr from young and old donors on skin reconstruction was evaluated in a reconstructed skin model comprising a dermal and epidermal equivalent. Keratinocytes from the same batch were seeded onto dermal equivalents containing either Fp or Fr from young or old donors. Histological sections revealed that young Fp were more potent to promote epidermal morphogenesis than Fr of similar age. Epidermis reconstructed on lattices containing young Fp had more cell layers and was fully differentiated (Fig. 8A), whereas lattices with young Fr generated a thin and incompletely differentiated epidermis (see granular and horny layers) (Fig. 8B). The differentiation of epidermis generated in the presence of old Fp (Fig. 8C) and Fr (Fig. 8D) was equivalently poor, indicating alterations of Fp with age. In particular, epidermis obtained in the presence of young Fp was characterized by higher thicknesses of total nucleated keratinocyte (Fig. 8E), cornified (Fig. 8F), granular (Fig. 8G), and spinous (Fig. 8H) layers, as compared with epidermis generated in the presence of Fr of similar young ages PLoS ONE | www.plosone.org 8 December 2008 | Volume 3 | Issue 12 | e4066