Obesity accelerates hair thinning by stem cell-centric converging mechanisms.

Methods

Mice
Animal care was in accordance with the guidance of the Tokyo Medical and Dental University for animal and recombinant DNA experiments. Mice were housed individually in cages at a maximum density of 5 mice per cage and kept on a 12-h light-dark cycle. The ambient temperature was maintained at 25° C with 40-80 % humidity. C57BL/6N and C57BL/6J mice were purchased from the Sankyo Labo Service. The following mouse cell lines were used: K15CrePR31, Rosa-H2B-EGFP (Riken CDB), Rosa-lsl-rtTA (Jackson lab, #005670), tetO-Gli2ΔC24
IL-1Ra KO28
K19CreER (Riken BRC), TNFα KO32, Nrf2 KO33, COL17A1 Tg34 and tetO-Gli2ΔN30. More than 3 mice were used for each experiment. Hair depilation was conducted by plucking or by a hair removal cream (Epilat, Kracie) as described in each Figure Legend. When using a hair removal cream, the dorsal skin was shaved and then treated with a hair removal cream for 3 min, after which the cream was washed off with warm water. K15CrePR induction was performed by topical application of 20 mg/ml Ru486 for 5 days on the dorsal skin of 7-week-old mice. GFP-positive cells were counted in telogen stage at the times indicated in the Figures. K19CreER induction was performed by an intraperitoneal injection of 100 μl 20 mg/ml tamoxifen for 5 days. The 60 kcal% HFD (D12492) and the doxycycline diet (D10001+0.0625% Doxycycline) were purchased from Research Diets. For treatment with α-lipoic acid, a HFD containing 2% w/w α-lipoic acid was used for four months during the HFD treatment35. For treatment with SAG, 100 μl 100 μM SAG (Calbiochem, 364590-63-6) in DMSO was applied using a Pipetman on the dorsal surface after hair depilation for 5 days.

Histology and immunofluorescence
Immunofluorescence was performed as described previously2. The following antibodies or chemical compounds were used: COL17A1 (Abcam, ab186415, 1:400), Keratin 14 (Biolegend, 906004, 1:500), ITGA6 (BD, 555734, 1:200), Keratin 15 (Biolegend, 833904, 1:300), NFκB (Santa Cruz, sc-101749, 1:50), CD11b (BD, 557686, 1:100), F4/80 (Bio-Rad, MCA497A488, 1:100), KI67 (Invitrogen, 14-5698-82, 1:300), CD3 (Biolegend, 100235, 1:100), MHC2 (Biolegend, 107605, 1:100), CD31(BD Biosciences, 557355, 1:200), anti-DNA/RNA Damage (Abcam, ab62623, 1:2000), Tom20 (Thermo Fisher, 11802-1-AP, 1:30), Keratin 1 (Abcam, ab185628, 1:300), survivin (Cell signaling, 2808, 1:100), casp3 (Cell signaling, 9661, 1:200), Tuj1 (Covance, PRB-435P, 1:500), CD34-FITC (Invitrogen, 11-0341-85, 1:100), ITGA6-PE (BD, 555736, 1:100), Sca1-APC (Miltenyi Biotec, 130-102-343, 1:100), CD45-APC-cy7 (BioLegend, 103116, 1:100). FV10-ASW4.2 was used for capturing confocal images. CellSens Standard v1.13 (Olympus) was used for collecting HE data.

Whole mount immunofluorescence
Whole mount staining of dorsal skin was performed as described previously with slight modifications36. Briefly, mouse skin was fixed with 4% paraformaldehyde (PFA) for 1 h on ice following by washing with 0.2% Tween 20/PBS, and subsequent blocking by PBSTM buffer (0.25% Fish skin gelatin, 1% skim milk, 0.5% Triton X-100 in PBS) for 1 h at room temperature (RT). Primary and secondary antibody reactions were performed in PBSTM buffer at 4°C overnight. After washing, skin samples were rendered transparent with BABB (benzyl alcohol mixed with benzyl benzoate at 1:2) as follows: Samples were dehydrated with 50% (vol/vol) methanol/PBS, rocked at RT for 10 min, then transferred to 100% methanol for 10 min at RT. After replacement of the methanol with 50% (vol/vol) BABB/methanol, samples were incubated for 5 min followed by the replacement of BABB/methanol with 100% BABB. Samples were then further incubated for 5 min. To obtain confocal images, samples were transferred to a slide glass with silicone grease to help hold the coverslips. To observe GFP by whole mount staining, the CUBIC transparent method was performed instead of BABB as follows: Briefly, after fixing with 4% PFA, samples were incubated with the 1st CUBIC buffer (25% urea, 25% N,N,N’,N’-tetrakis(2-hydroxypropyl) ethylenediamine, 15 wt% Triton X-100) overnight at 4°C. After washing, the primary and secondary antibody reactions were performed at 4°C overnight or for 4 h, respectively. The samples were then incubated with the 2nd CUBIC buffer (50% sucrose, 25% urea, 10% aminoalcohol, 0.1% (v/v) Triton X-100) overnight at 4°C.

Streptozotocin and glucose assessment
C57BL/6N 7-week-old male mice, each weighing 20–25 g, were purchased from Sankyo Labo Service. Obliteration of pancreatic β-cells was achieved with intraperitoneal injections of 50 mg/kg streptozotocin (STZ, Sigma-Aldrich) in 50 mmol/L sodium citrate buffer (pH 5) for 5 consecutive days. Seven days after the initial injection, mice with fasting blood glucose levels of 120 mg/dL after 18 h were deemed diabetic, and measurement of blood glucose levels were performed with terminal tail vein blood using a Breeze 2 (Bayer). A total of 9 diabetic mice were used in this experiment.

Single cell isolation from mouse dorsal skin
Single cells from mouse dorsal skin were obtained as described previously37. Briefly, dorsal skin was placed dermis side down in 0.25% trypsin (Gibco) or 0.2% Dispase II (Roche) for 12-14 h at 4°C. The epidermis and dermis were then obtained by scraping the skin gently. The epidermis was minced with a razor blade and then filtered with strainers (40 μm) (BD Falcon). The dermis was minced using scissors and the pieces were incubated in 0.2% collagenase (Sigma) for 1 h at 37°C with gentle agitation. Those suspensions were filtered with 70 μm strainers and epidermal and dermal cells were obtained after two washes with PBS.

FACS
Epidermal and dermal cell suspensions were incubated with the appropriate antibodies for 1 h at 4°C. Staining with 7-AAD (BD Bioscience) was used to exclude dead cells. Cell isolations were performed using a FACS AriaII or AriaIIIu equipped with Diva software version 6.1.3 (BD Bioscience). For RNA extraction of HFSCs, populations were sorted directly into Buffer RLT (Qiagen).

Microarray analysis
HFSCs (CD34highITGA6highSCA1−) were harvested from ND- and from HFD- fed mice after 3 months of treatment (each mouse is then 5 months old) using FACS. Repeated hair depilation was not performed for microarray analysis. Telogen HFSCs were isolated from telogen region and anagen HFSCs were obtained 3 days after hair depilation using hair plucking at the telogen region. FACS-sorted HFSCs were stored into Buffer RLT (Qiagen). 10,000 cells per sample were obtained from one mouse for telogen HFSCs and from four (ND) or eight (HFD) mice for anagen HFSCs. Microarray analysis was performed by the Chemicals Evaluation and Research Institute (CERI). All array data were deposited in the GEO public database (accession no. GSE131958).

RNA seq analysis
Anagen HFSCs were obtained 3 days after hair plucking at telogen region from ND- and from HFD-fed mice after 4 months treatment. 5,000 to 10,000 cells per sample were obtained from one mouse. Library preparation and sequencing analysis on an Illumina NovaSeq 6000 was performed by GENEWIZ Inc. The expression of each gene derived from the read alignments was normalized to fragments per kilobase of exon model per million (FPKM) for further analysis. RNA-seq data were deposited in the GEO public database (accession no. GSE169173).

Assay for Transposase Accessible Chromatin with high-throughput (ATAC)-sequencing
FACS-sorted HFSCs (20,000 cells) or epidermal keratinocytes (ITGA6high SCA1+) were lysed in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) on ice for 10 min. After centrifugation, nuclei in the pellets were resuspended in 50 μl transposase reaction mix (25 μl Tagment DNA buffer (Illumina), 2.5 μl Tagment DNA enzyme (Illumina) and 22.5 μl water), incubated at 37°C for 35 min and then purified with a MinElute PCR Purification Kit (Qiagen). After optimization of PCR cycle numbers using SYBR GreenI Nucleic Acid gel Stain (Takara Bio), transposed fragments were amplified using NEBNext High Fidelity 2x PCR Master mix and index primers, and were purified with a MinElute PCR Purification Kit (Qiagen). Library DNAs were size-selected (240-360 bp) with BluePippin (Sage Science). Sequencing was performed using HiSeq1500 (Illumina) with a single-read sequencing length of 60 bp. Bowtie2 (version 2.2.6; with default parameters) was used to map reads to the reference genome (UCSC/mm10) with annotation data from iGenomes (Illumina). Reads mapped to mitochondria were removed. To ensure even processing, reads were randomly downsampled from each sample to adjust to the smallest read number of samples. MACS (version 2.1.1; with default parameters) was used to call peaks in the downsampled reads. The catalogue of all peaks called in all samples was produced by merging all called peaks that overlapped by at least one bp using bedtools. The MACS bdgcmp function was used to compute the fold enrichment over the background for all populations, and the bedtools map function was used to identify the maximum fold enrichment observed at each peak in the catalogue in each population. Maximum fold enrichment at each site in the catalogue was quantile normalized between samples using the PreprocessCore package in R (3.3.2). We used the Homer package with command annotatePeaks.pl using default parameters to annotate regions with promoter and distal labels and the nearest gene, and with command findMotifsGenome.pl using default parameters to identify enriched motifs, and the catalogue of all called peaks as a background.

DAVID functional enrichment analysis
To perform gene pathway analysis, we used the web-based Database for Annotation, Visualization and Integrated Discovery (DAVID). For analysis of anagen HFSCs, upregulated genes in the HFSCs of HFD-fed mice with a fold change ≥2 (Extended Data Fig. 8f) and downregulated genes with a fold change <0.5 (Fig. 2a) were used for BioCarta pathway enrichment analysis. For telogen HFSCs, upregulated genes in HFD-fed mice with a fold change >2.0 and p<0.1 were used for BioCarta pathway enrichment analysis (Fig. 3a). All pathways with p<0.05 are shown.

Gene set enrichment analysis (GSEA)
GSEA was provided by the Broad Institute of MIT and Harvard University. GSEA was used to investigate the potential mechanisms upregulated in telogen HFSCs or downregulated in anagen HFSCs in HFD-fed mice using c2 (c2.cp.biocarta.v5.0.symbols or c2.cp.biocarta.v7.2.symbols, respectively) as the gene set correlation. All pathways with p<0.1 for upregulated in telogen HFSCs or those with p<0.05 for downregulated in anagen HFSCs were shown.

Real time qPCR
Total RNAs were purified from FACS-sorted cells by directly sorting into Buffer RLT (Qiagen) using a RNeasy micro Kit (Qiagen). cDNAs were then synthesized in 20 μl reaction mixtures using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to standard procedures. Quantitative PCR was performed using a SYBR Green qPCR Kit (Agilent Technology). Relative levels of expression were determined by normalization to β-actin using the ddCt method. The reactions were run in a Mx3000P Real-Time QPCR System (Agilent Technology). The primer sequences used were as follows: β-actin: 5′- GGCACCACACCTTCTACAATG-3′, 5′-GTGGTGGTGAAGCTGTAG-3′, mKeratin1: 5′-AGGATCTTGCCAGATTGCTG-3′, 5′-CTACTGCTTCCGCTCATGCT-3′, mGli1: 5’-GGTGCTGCCTATAGCCAGTGTCCTC-3’, 5’-GTGCCAATCCGGTGGAGTCAGACCC-3’, mGli2: 5’-TACCTCAACCCTGTGGATGC-3’, 5’-CTACCAGCGAGTTGGGAGAG-3’, mPtch1: 5’-ATCTCGAGACCAACGTGGAG-3’, 5’-TAGCGCCTTCTTCTTTTGGA-3’, mIl-1b1: 5’-GCAACTGTTCCTGAACTCA-3’, 5’-CTCGGAGCCTGTAGTGCAG-3’. mIl-1b2: 5’-CAACCAACAAGTGATATTCTCCATG-3‘, 5’-GATCCACACTCTCCAGCTGCA-3’. Uncropped gel images are shown in Supplementary Fig. 1.

IL-1β administration
Dorsal hairs of 7-week-old or 21-month-old male mice were depilated to induce anagen. Two days after depilation, an atelocollagen sponge (KOKEN, CSH-10) impregnated for 2 h at RT with 50 μl PBS or 1 μg recombinant IL-1β (Sigma, SRP-3083) was implanted subcutaneously in the dorsal skin of each mouse. The next day, HFSCs (CD34high, ITGA6high, SCA1−) were collected by FACS following qPCR analysis.

DCFDA and MitoSOX assays
The oxidation-sensitive fluorescent probes 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Invitrogen, D399) and MitoSOX Red (Invitrogen, M36008) were used to analyze the total intracellular content of ROS and mitochondrial superoxide production, respectively. Dorsal skin cells were harvested as described previously and incubated with 200 μM DCFDA or 20 μM MitoSOX Red with antibodies (CD34, ITGA6, SCA1) at 4°C for 60 min. Fluorescence was measured in the HFSC populations (singlet live cells, CD34high, ITGA6high, SCA1−).

Immunostaining of 8-oxoguanine
8-Oxoguanine (8G) staining was performed as described previously38,39. Briefly, antigen retrieval was performed for 20 min at 95°C with citrate buffer (pH 6.0) on 4 μm paraffin sections. Sections were permeabilized for 20 min with 0.05% Triton X-100 in PBS, and were then blocked with 10% goat serum in PBS for 1 h at RT. The 1st antibody (anti-DNA/RNA Damage, ab62623, 1:2000) was incubated overnight at 4°C and the 2nd antibody (anti-mouse IgG, A21202 1:300) for 2 h at RT. Both reactions were performed in DAKO mounting medium (S3023).

Flux analyzer
The oxygen consumption rate (OCR) was measured using a Seahorse XF96 extracellular flux analyzer according to the manufacturer’s instructions. Briefly, 1 x 105 cells from the total epidermis (many of which are basal keratinocytes) from 8 week old mice were transferred to a culture plate pre-coated with Cell-Tak containing SF-03 medium and centrifuged at 400 x g for 5 min. A 4% lipid mixture or 200 μM palmitate were added to the medium before measurement. The OCR was measured with sequential addition of 1 μM oligomycin, 2 μM FCCP, 0.5 μM rotenone/antimycin and 50 mM 2-deoxy-D-glucose.

Statistics
Statistical analyses were performed with GraphPad Prism 6 or 8 and Excel 2013. Two-tailed Student’s t-test, two-tailed Mann–Whitney U-test, one-way ANOVA with two-tailed Dunnett’s test or one-way ANOVA with two-tailed Tukey’s test was used as described in the legend. P-values were adjusted for all multiple comparisons. DAVID pathway analysis used a modified Fisher’s exact test to measure the significance of the gene-enrichment in annotation pathways. Image J version 1.51 was used for image analysis.

Extended Data

Supplementary Material
Supplementary Figs. 1-4