Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling

Abstract

In mammalian skin, multiple types of resident cells are required to create a functional tissue and support tissue homeostasis and regeneration. The cells that compose the epithelial stem cell niche for skin homeostasis and regeneration are not well defined. Here, we identify adipose precursor cells within the skin and demonstrate that their dynamic regeneration parallels the activation of skin stem cells. Functional analysis of adipocyte lineage cells in mice with defects in adipogenesis and in transplantation experiments revealed that intradermal adipocyte lineage cells are necessary and sufficient to drive follicular stem cell activation. Furthermore, we implicate PDGF expression by immature adipocyte cells in the regulation of follicular stem cell activity. These data highlight adipogenic cells as skin niche cells that positively regulate skin stem cell activity, and suggest that adipocyte lineage cells may alter epithelial stem cell function clinically.

Figure 1. Intradermal adipocytes regenerate via a proliferative precursor cell during the hair cycle

A. Histogram plot of the size distribution of Lipidtox⁺/caveolin⁺ cells during the hair cycle. n=80–100 cells from 3–4 mice for each hair cycle stage. Asterisks indicate significance compared to other hair cycle stages. B. Caveolin immunostaining (green) and Lipidtox staining (red) marks intradermal adipocytes during the telogen (P49) and anagen (P56) of the second hair cycle. A, anagen; T, telogen. Dashed lines outline epidermis and hair follicles. Box and whisker plots of the distance of caveolin⁺ cells from hair follicle bulge at P56. n= 100 follicles from 2 individual mice for each box. C. Schematic of 3-day BrdU labeling experiments during catagen (P18–P21) and anagen (P21–P24). Representative images for perilipin (green), nuclei (blue) and BrdU (red) immunostaining of skin sections. Dashed lines outline epidermis and hair follicles. Arrows indicate perilipin⁺, BrdU⁺ cells. Quantification of the number of BrdU⁺, perlipin⁺ adipocytes in 20X microscopy fields. n= 3–5 mice; >15 follicles per timepoint. Quantification the % of BrdU⁺ nuclei of mature adipocytes as analyzed by FACS. n= 3–5 mice for each bar. All data are ± SEM, *p<0.05. Bars=100µm. See also Figure S1.

Figure 2. Resident skin adipocyte precursor cells display dynamic activity associated with the hair cycle

A. Representative FACS plots of Sca1⁺, CD24^+/− adipogenic cells within the CD31/CD45 negative (Lin-), CD34⁺, and CD29⁺ gated cell populations in subcutaneous adipose tissue or P21 skin. B. Representative FACS plots of adipocyte precursor cells from skin in catagen (P18) or early anagen (P22). C. Graphs quantify the % of adipogenic cells and the % of BrdU⁺ adipogenic cells within the Lin⁻, CD29⁺, and CD34⁺ cell population at P18 (catagen), P22 (initial anagen) or P25 (mid-anagen). D. Real-Time PCR analysis of PPARγ mRNA expression in P21 adipogenic cells and mature intradermal adipocytes compared to total isolated stromal vascular fraction (SVF) in the skin. n=3 independently isolated cell populations. All data are ± SEM, *p<0.05. See also Figure S2.

Figure 3. Defects in the generation of immature adipocyte lineage cells blocks follicle stem cell activation

A. FACS analysis of Sca1⁺ adipogenic cells derived from WT, Ebf1^−/− and Azip skin reveals an absence of adipocyte precursor cells in Ebf1^−/− mice. n=3 mice. B. Analysis of BrdU incorporation during a 3-day pulse in caveolin⁺ cells in the skin of WT, Ebf1^−/− and Azip mice after P21. Arrows indicate BrdU⁺ (red), caveolin⁺ (green) cells within the intradermal region of the skin. C. Analysis of PPARγ expression (green, arrows) in the dermis of WT, Ebf1^−/− and Azip mice. Dotted lines outline hair follicles. Dots indicate non-specific immunostaining. n=3 fields from 3 mice for each genotype. D. Analysis of anagen (Ana), catagen (Cata), and telogen hair stages in WT, Ebf1^−/− and Azip mice based on morphology at indicated ages. n=5–7 mice; >37 follicles for each bar. All data are ± SEM, *p<0.05, *** p<0.0001. Bars=100µm. See also Figures S3 and S4.

Figure 4. PPARγ antagonists abrogate intradermal adipogenesis and hair follicle stem cell activation

A. FACS analysis of Sca1⁺ adipogenic cells derived from skin shows increased adipocyte precursor cells in BADGE and GW9662 (GW)-treated mice. n=3 mice. B. PPARγ immunostaining in vehicle and mice treated with BADGE either P18–P24 or P21–27. Arrows indicate PPARγ⁺ cells. SG, sebaceous gland. n=3 mice; 3 fields of view. C. Analysis of anagen induction in mice injected with vehicle, BADGE, or GW for 6 days at indicated ages. All data are ± SEM, *p<0.05, **p<0.005, ***p<0.0005. Bars=100µm. See also Figure S5.

Figure 5. Adipogenic cells are sufficient to induce hair follicle regeneration

Analysis of anagen induction in mice injected with 50,000 stromal vascular fraction cells (SVF) or Sca1⁺ adipocyte precursor cells from indicated genotypes. A. Luciferase imaging of adipocyte differentiation 6 weeks after injection of adipocyte precursor cells derived from subcutaneous adipose tissue of FVB leptin-luciferase mice into shaved, 7-week old FVB WT recipient mice. B. After two weeks, WT adipocyte precursor cells induce hair growth when injected intradermally into shaved 8 week old backskin. Graph indicates quantification of anagen induction based on morphology of 35 follicles from 2 independent experiments. Histology of hematoxylin and eosin staining follicles from SVF or adipocyte precursor injected skin regions is shown. C. Intradermal injection of WT adipocyte precursor cells induces anagen as indicated by proliferation (Ki67⁺ cells, arrow) within hair germs of P21 Ebf1^−/− mice after 3–5 days (D) or full anagen after 2 weeks (wks). In situ hybridization on skin sections of injected tissue reveals Y chromosome localization (arrows) in female Ebf1^−/− skin after intradermal injection with FACS isolated adipocyte progenitors derived from male WT skin. A, anagen follicle; T, telogen follicle; AP, adipocyte precursor. Dots indicate autofluorescence. n>35 follicles from 2 independent experiments. D. Adipocyte precursor cells derived from Azip skin induce anagen in WT P49 skin 3D after intradermal injection as indicated by Ki67⁺ hair germ cells (arrows). Flag epitope immunostaining of transplanted skin reveals the localization of Flag⁺ transplanted cells in skin injected with adipocyte precursors. Arrows indicate anagen induction as indicated by enlarged, hair germs and Ki67⁺ staining. AP, adipocyte precursor. n>27 follicles from 2 independent experiments. All data are ± SEM, *p<0.05. Bars=100µm.

Figure 6. PDGF signaling in the skin requires intradermal adipocyte precursor cells

A. Immunostaining for phospho-SMAD(1, 5, 8), β-catenin, and phosphorylated p42/44 (MAP kinase) in skin sections of P7 WT and Ebf1^−/− mice. Arrows indicate positive cells. B. Phospho-MAP kinase staining in vehicle and BADGE-treated mice at P24. Arrows indicate positive cells. Bu, bulge; HG, hair germ; DP, dermal papillae. C. Real-Time PCR analysis of mRNA expression in adipocyte precursor cells and mature intradermal fat compared to total isolated skin cells. n=3 independently isolated cell populations. D. PDGF receptor expression in telogen stage follicles (P19) localizes to the dermal papilla (DP) beneath the α6 integrin⁺ border with the hair follicle cells. E. Analysis of PDGF receptor activation in DP from WT (telogen (P49) and anagen (P56)), BADGE-treated (P24) and Ebf1^−/− (P21) mice. Dashed lines mark the hair follicle, while solid lines mark the dermal papilla (DP). n=50–75 follicles in ≥2 mice for each sample. F. Hematoxylin and eosin stained follicles from Ebf1^−/− mice injected with control or PDGF coated beads 5 days post-injection. Quantification of the dose response of anagen induction with beads coated with BSA or indicated concentrations of PDGFA. n=15–24 follicles from two independent experiments. All data are ± SEM, *p<0.05. ***p<0.001. Bars=100µm.

Figure 7. Model for the role of adipocytes in the skin in WT and mouse models with defects in adipogenesis

During the hair follicle cycle, intradermal adipose tissue increases size in part due to the activation of adipocyte precursor cells (green and blue stars) that generate new mature adipocytes (orange circles) de novo. Proliferation of adipocyte lineage cells is stimulated during follicle regression (catagen) to increase adipocyte precursor cell number during telogen and anagen initiation. Azip mice lack mature adipocytes but have adipocyte precursor cells and regenerate follicles similar to WT mice. In Ebf1^−/− mice, adipocyte precursor cells are absent within the skin after catagen, leading to a reduction in intradermal adipose tissue and defects in stem cell activation. Similarly, treatment of mice with PPARγantagonists during catagen blocks subdermal adipose tissue growth and follicle regeneration. However, the most immature adipocyte precursor cells are maintained in mice treated with PPARγ antagonists, suggesting that intradermal adipocyte precursor cells that express PPARγ(green star) are essential for sterm cell activation in the hair follicle.