Epidermal stem cells are retained in vivo throughout skin aging - PubMed (original) (raw)

Epidermal stem cells are retained in vivo throughout skin aging

Adam Giangreco et al. Aging Cell. 2008 Mar.

Abstract

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. It is currently unknown whether epidermal stem cells influence or are affected by skin aging. We therefore compared young and aged skin stem cell abundance, organization, and proliferation. We discovered that despite age-associated differences in epidermal proliferation, dermal thickness, follicle patterning, and immune cell abundance, epidermal stem cells were maintained at normal levels throughout life. These findings, coupled with observed dermal gene expression changes, suggest that epidermal stem cells themselves are intrinsically aging resistant and that local environmental or systemic factors modulate skin aging.

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Figures

Fig. 1

Fig. 1

Age-associated changes in murine skin. (A, B) Haematoxylin-and-eosin-stained sections of young (A) and old (B) telogen murine dorsal skin showing epidermis, dermis, hypodermis, and underlying muscle. Abnormal follicular architecture, dermal thinning, and hypodermal thickening are present in aged skin. (C, D) Keratin 14-stained skin whole-mount images from young (C) and old (D) mice [bracket indicates hair follicle (HF) bulge]. (E) Average dermis (measured from epidermis to hypodermis) and hypodermis (measured from dermis to underlying muscle) thickness in young (green) and old (red) mice. (F) Average number of HFs per cluster in young (green) and old (red) tail epidermis. (G) Quantitative polymerase chain reaction analysis of p16/Ink4a/Arf gene expression in skin of mice of increasing age. Scale bars = 100 µm (A, B, D, E). (n = 4 mice/age; *P < 0.05).

Fig. 2

Fig. 2

Epidermal stem cells are maintained during skin aging. (A, B) Representative epidermal whole mounts from young and old mice stained for keratin 14 (green) plus the stem cell marker keratin 15 (red) to identify hair follicle bulge stem cells. (C–E) Representative flow cytometry plots from 2-month-old (C), 6-month-old (D), and 22-month-old (E) murine epidermal skin preparations stained for CD34-PE and α6 integrin–FITC. Three distinct epidermal populations are shown: α6(+) basal cells (blue gate), α6(+)/CD34(+) stem cells (orange gate), and α6(dim)/CD34(+) cells (grey gate). (F) Quantification of the per cent of viable cells represented within the α6(+) gated population from epidermal preparations of n = 3 mice/age at each of 2 months, 6 months, 12 months, 18 months, or 22 months. (G) Quantification of the per cent of viable cells represented as α6(+)/CD34(+) stem cell (orange bars) or α6(dim)/CD34(+) stem cell (grey bars) populations. No significant differences in stem cell abundance were observed between 6 months and 22 months age. (H) Quantitative polymerase chain reaction analysis of whole-skin RNA for α6 (black bars) and keratin 15 (green bars) levels in 2-month-old, 6-month-old, 18-month-old, and 22-month-old mice. Scale bars = 100 µm (A, B). Asterisk (G, H) indicates significant difference versus 2-month sample; P < 0.05.

Fig. 3

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 in Silva-Vargas et al., 2005) in young and old mice. (D–F) Flow cytometric analysis of total (left side graphs, C–E) or α6(+) keratinocyte (right side graphs, C–E) cell cycle status in young (green) versus old (red) skin preparations. (G, H) Keratin 15 (green) plus Ki67 (red) immunostaining in young and old mice to determine bulge stem-cell-specific proliferation. (I) Quantification of Ki67(+) cells per bulge in young and old mice. Scale bars (A, B) = 100 µm. (n = 4 mice/age; *P < 0.05).

Fig. 4

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 of CD45(+) cells present per square millimetre of tissue. (D, E) Young (D) and old (E) epidermal whole mounts stained to reveal haematopoietic Langerhans cells (DEC205, red). (F) Quantification of DEC205(+) cells per square millimetre of epidermis. (G, H) Whole-mount immunostaining for T cell populations using antibodies to the pan-T cell marker CD3 (red) and γδ-T cell receptor (γδ-TCR; green) to identify dendritic epidermal T cells (DETCs, orange dual stain). (I) Quantification of CD3 (red) and γδTCR/CD3 dual (green) positive cells per square millimetre of epidermis. All images and quantification represent at least n = 3 individuals/age. Scale bars = 200 µm (A, B); 100 µm (C–F).

Fig. 5

Fig. 5

Skin aging results in changes in Igfbp3 transcript abundance. (A) Bioinformatic analysis of relative Igf/Igfbp signalling pathway gene abundance in matrix (green), outer root sheath (ORS, black), dermal fibroblast (DF, red), dermal papilla (DP, yellow), or melanocyte (blue) cell populations relative to ORS/basal keratinocytes. Most Igf pathway members were enriched within dermal compartments (DF and DP). (B) Quantitative polymerase chain reaction of whole-skin cDNA to determine Igf/Igfbp expression in 2-month-old, 6-month-old, 18-month-old, and 22-month-old mice. (n = 3 mice/age; *P < 0.05).

Fig. 6

Fig. 6

Igfbp3 does not directly influence epidermal immune cell abundance but results in increased anagen follicles. (A, B) Representative whole mounts from age- and sex-matched wild-type (WT) (A) and Igfbp3KO (B) tail epidermis immunolabelled for keratin 14 (green) and CD45 (red). (C) Quantification of CD45 cell abundance in WT and knock-out (KO) epidermis per square millimetre. (D, E) WT (D) and KO (E) whole mounts stained for γδTCR (green) and CD3 (red) identify CD3(+) T cells and dendritic epidermal T cells (orange). (F) Quantification of CD3(+) and γδTCR(+) cell abundance in WT and KO skin. (G, H) WT (G) and KO (H) tail whole mounts stained for keratin 15 (green) and Ki67 (red) to identify bulge stem cells and mitotic keratinocytes, respectively. (I) Quantification of percentage of total growing (anagen) follicles present in WT and KO tail epidermal whole mounts (e.g. see arrowheads, H). Scale bars = 100 µm (A, B, D, E, G, H). (n = 4 mice/genotype; *P < 0.05; I).

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