In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Recently, genetically engineered telomerase-deficient mice were generated which exhibited characteristics of premature skin aging (Flores clonogenicity and are unresponsive to mitogen stimulation < 0.09; Fig. 3C). Tail whole-mount labelling for Ki67 revealed that there were no differences in K15 positive, bulge-associated stem cell proliferation between young and old samples (yellow arrowheads, Fig. 3GCI). Nevertheless, flow cytometry revealed an increase in Vemurafenib G1 phase cells concomitant with reductions in S and G2/M-phase cells in aged telogen-phase skin [total or 6(+) keratinocytes; Fig. 3DCF]. This is consistent with a modest decrease in proliferation with age. Fig. 3 Interfollicular epidermal proliferation declines with aging. (A, B) Representative young (A) and old (B) epidermal whole mounts stained for keratin 14 (green) plus Ki67 (red). (C) Quantification of Ki67(+) nuclei per interfollicular epidermis unit (defined ... Altered peripheral immunity in aged skin In addition to altered morphology, skin aging is frequently associated with reduced peripheral immunity and increased infection (McCullough & Kelly, 2006). To study whether aging influences murine skin immunity, epidermal Vemurafenib whole mounts were stained for the pan-haematopoietic cell antigen CD45. There appeared to be fewer CD45 reactive cells within the IFE of old mice but this was not statistically significant (Fig. 4ACC). In young but not old mice, most of these CD45 positive cells exhibited a dendritic morphology. CD45 positive cells present in old skin were localized to the follicularCinterfollicular junction (infundibulum; Fig. 4A,B). Fig. 4 Aging results in altered skin leukocyte abundance. (A, B) Representative whole mounts from young (A) and old (B) tail epidermis stained for the pan-haematopoietic marker CD45 (red) and the keratinocyte-specific marker keratin 14 (green). (C) Quantification ... To further characterize the Vemurafenib haematopoietic cells present in young and aged skin, we performed immunostaining to identify Langerhans cells (DEC205 reactive), Vemurafenib T cells (CD3 reactive), and atypical dendritic epidermal T cells (DETCs, TCR reactive). There were no significant differences in Langerhans cell abundance or localization between young and aged skin (Fig. 4DCF). In contrast, there was a dramatic and statistically significant loss of both epidermal T cells (CD3) and DETCs (CD3+ TCR) associated with skin aging (Fig. 4GCI). This near-complete loss of DETCs despite retention of rare CD3(+), T cells suggests that aged murine skin may have impaired innate immunity, and therefore, may be more susceptible to infection following injury. Skin aging is associated with reduced Igfbp3 expression Based on our observation that skin aging did not correlate with a reduction in the number of epidermal stem cells, we hypothesized that aging might instead be regulated by environmental components. The Igf signalling pathway has previously been identified as a key mediator of both epidermal proliferation (Edmondson throughout their lifetime, and maintained on a 12-h light/dark cycle. The mice were sacrificed by CO2 asphyxiation, and samples were immediately fixed, frozen, or processed for further analysis. No experimental procedures were performed on any mice prior to sacrifice. Immunohistochemistry and imaging TMPRSS2 Tissue was processed for tail epidermal whole-mount immunohistochemistry as previously described (Braun = 3C6 replicates per age and genotype (Igfbp3WT or KO). Error bars in Fig. 5 represent standard deviation as determined by GEO profile MAS5 signal intensity. Over 100 HF clusters were counted per individual mouse (= 4) for HF cluster analysis. The Student’s < 0.05. Acknowledgments We gratefully Vemurafenib acknowledge the assistance of Daniel Pearce, Dominique Bonnet, Kirsty Allen, and Kristin Braun. This research was supported by an NIH postdoctoral fellowship (Adam Giangreco) and Cancer Research UK..