Stem cell (SC) exhaustion is a hallmark of aging. However, the process of SC depletion during aging has not been observed in live animals, and the underlying mechanism contributing to tissue deterioration remains obscure. We find that, in aged mice, epithelial cells escape from the hair follicle (HF) SC compartment to the dermis, contributing to HF miniaturization. Single-cell RNA-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) reveal reduced expression of cell adhesion and extracellular matrix genes in aged HF-SCs, many of which are regulated by Foxc1 and Nfatc1. Deletion of Foxc1 and Nfatc1 recapitulates HF miniaturization and causes hair loss. Live imaging captures individual epithelial cells migrating away from the SC compartment and HF disintegration. This study illuminates a hitherto unknown activity of epithelial cells escaping from their niche as a mechanism underlying SC reduction and tissue degeneration. Identification of homeless epithelial cells in aged tissues provides a new perspective for understanding aging-associated diseases.
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All sequencing data were deposited to NCBI–GEO SuperSeries under accession number GSE133648.
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We thank T. Cech (University of Colorado Boulder, HHMI), K. Green and R. Lavker (Northwestern University) for comments; J. Siegenthaler (University of Colorado School of Medicine) for Foxc1fl/fl and Foxc1-LacZ mice; E. Fuchs (Rockefeller University, HHMI) for Krt14-H2BGFP and Krt14-Cre mice; D. Sheppard (University of California San Francisco) for Itgb6 -KO mice; H. Fujiwara (RIKEN, Japan) for anti-NPNT and anti-EGFL6 antibodies; M. Allen (University of Colorado Boulder) for discussing bioinformatic analysis; J. Orth (University of Colorado Boulder), J. Lopez and R. Goldmeyer (Olympus) for two-photon imaging; and all members of the Yi laboratory for suggestions. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award numbers AR059697, AR066703 and AR071435 (to R.Y.) and by the National Institutes of Health under award number GM125871 (to R.D.). C.Z. was supported by an NCI Predoctoral to Postdoctoral Fellow Transition Award (F99CA253738). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The authors declare no competing interests.
Peer review information Nature Aging thanks the anonymous reviewers for their contribution to the peer review of this work.
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a, Two-photon longitudinal tracking of hair follicles in young mice during the anagen to telogen hair cycle. Red numbers designate the same hair follicle in each image. Red dotted lines annotate the bulge region. Scale bar, 50μm. b, Two-photon intravital imaging of hair follicles from young (left panel) and old (middle and right panels) mice. White arrowheads point to miniaturized hair follicles and cells located outside of the HF-SC compartments. Red dotted lines outline miniaturized hair follicles. Scale bar, 50μm. c, Boxplot of the percentage of miniaturized hair follicles, quantified from 3-D scan of live animals. (mean ± s.d.= 0 ± 0 (young), 14 ± 17 (old); n=205 HFs from 5 young mice; n=327 HFs from 3 old mice). d, Representative images of hair follicles with KRT5 and activated caspase 3 (acCas3) signals in young (6~8mo) and old (20mo) mice. (n=50 hair follicles from young mice; n=62 hair follicles from old mice, 3 pairs of mice). Scale bar, 20μm. e-f, Boxplot of number of acCas3+ HFSCs(e) and HG(f) per hair follicle (HFSCs: mean ± s.d.= 0.058 ± 0.31 (young), 0.048 ± 0.22 (old); HG: mean ± s.d.= 0.077 ± 0.33 (young), 0.065 ± 0.31 (old); n=50 hair follicles from young mice; n=62 hair follicles from old mice, 3 pairs of mice). g, 3-D view of hair follicles in 24mo old mice. White arrowheads point to numerous escaped epithelial cells scattering in the dermis. Scale bar, 50μm. h, 3-D view of β4 integrin immunofluorescence signals in 24mo old mice. White arrowheads point to HF-SCs with protruding integrin signals in the new bulge side. Scale bar, 20μm. Data in c, e, f are two-sided t-test.
Extended Data Fig. 2 Quality control and clustering of single-cell RNA-seq data from young and old mice.
a, Quality control and filtering of single cells from old and young samples. Cells were filtered with detected genes numbers (200<nFeature_RNA<5000), transcripts numbers (nCount_RNA) and mitochondrial percentage (percent.mt < 10). b, Track plot of marker genes for each cluster. c, Table shows cluster names, cell numbers and percentage of cells for each cluster after filtering of old and young scRNAseq data.
a-b, UMAP re-clustering and projection of IFE cells, color coded by sample identity (a) and cluster identity (b). c-d, Feature plots of marker genes for basal progenitor cells (Krt14, Krt5) and suprabasal cells (Krt1, Krt10). e-f, Highly enriched GO terms for upregulated genes in old HFSCs (e) and downregulated genes in old IFE (f). g, Violin plots of selected gens in young and old HF-SC and IFE cell clusters. h, Feature plot of monocle3 pseudotime score of hair follicle cells from old and young mice. i, Violin plot of HFSCs pseudotime score in young and old samples. j, Highly enriched GO terms for HFSCs in the ground state with lower pseudotime score (<5.5).
a, Transcriptional activity of Foxc1 locus (Foxc1-LacZ knockin) is detected in anagen bulge but not detected in telogen bulge. Scale bar, 20μm. b, Transcriptional activity of Foxc1 locus (Foxc1-LacZ knockin) is not detected in telogen bulge. Scale bar, 20μm. c, Robust transcriptional activity of Foxc1 locus (Foxc1-LacZ knockin) is detected in both bulge and IRS regions of anagen hair follicles. Scale bar, 20μm.
a, Krt5 and Ki67 staining of hair follicles at early anagen (anagen III, P25) in control and dKO mice, arrowheads indicate Ki67+ HF-SCs, Right panel, quantification of Ki67+ HF-SCs per hair follicle (n=30 hair follicles from 3 pairs of mice). Scale bar, 20μm. b, Ki67 staining of hair follicles at late anagen (anagen V-VI) in control, Foxc1 cKO, Nfatc1 cKO, and dKO mice, arrowheads indicate Ki67+ HF-SCs. Scale bar, 20μm. c, Images of hair coat in the same control and dKO mice on P41 (left panel) and P64 (right panel), the right half of back skin was shaved on P41 and imaged again on P64. d, Images of hair coat of control and dKO mice at ~16mo old. e, Two-photon images of hair follicles in control and dKO mice at 12mo old, red arrowheads point to escaped cells outside of hair follicles. Scale bar, 70μm. f, Boxplot of the percentage of miniaturized hair follicles in young, old and dKO mice. (mean ± s.d.= 0 ± 0 (young), 14 ± 17 (old), 77 ± 25 (dKO); 5 young mice; 3 old mice; 5 dKO mice). g, Representative Two-Photon images for the quantification of HFSCs, red asterisks mark HFSCs(g). h, Boxplot of the number of HFSCs per HF in different samples. (mean ± s.d.= 19.33 ± 2.44 (young), 15.33 ± 4.04 (old), 10.95 ± 3.97 (dKO);91HFs, 5 young mice; 122 HFs, 3 old mice; 58 HFs, 5 dKO mice). i, Longitudinal tracking of dKO hair follicles over 26 days. Red numbers indicate the identical hair follicles in each image. Scale bar, 70μm. j, Boxplot of the percentage of HFs undergo regeneration, degeneration and quiescence in dKO samples (mean ± s.d.= 70 ± 24 (regeneration), 25.23 ± 25 (degeneration), 4.12 ± 7.49 (quiescence); 78 HFs from more than 3 mice were tracked for at least 16 days). Data in a, f, h are two-sided t-test.
Extended Data Fig. 6 Quality control and clustering of single-cell RNA-seq data from control and dKO.
a, Quality control and filtering of single cells from both control and dKO samples at P38. Cells were filtered with detected genes numbers (200<nFeature_RNA<5000), transcripts numbers (nCount_RNA) and mitochondrial percentage (percent.mt < 10). b, Track plot of marker genes for each cluster. c, Table shows cluster names, cell numbers and percentage of cells for each cluster after filtering of both control and dKO single-cell RNA-seq data.
Extended Data Fig. 7 Isolation and transcriptomic analysis of Foxc1 and Nfatc1 single KO and induced dKO HF-SCs.
a, Immunofluorescence staining of Foxc1 and Nfatc1 in Krt15-CrePR induced dKO hair follicles. tdT is tdTomato signals from ROSA26-LSL-tdT allele, indicating Cre+ dKO HF-SCs. Red arrowheads indicate HFSCs without Foxc1 and Nfatc1 signals, white arrowheads indicate inner bulge region, which is negative for tdT. Scale bar, 20μm. b, Highly enriched GO terms of upregulated genes and selected differentially expressed in induced dKO HF-SCs. c, Gating strategy for flow cytometry analysis. d-f, Flow cytometry analysis and quantification of HF-SCs during the first anagen (P28-P31) in control, Krt14-Cre-mediated dKO hair follicles (d), Foxc1 cKO and Nfatc1 cKO hair follicles (e). The rectangle regions are CD34-APChi and Cd49f-PE hi HF-SC populations. Representative plots for 3~5 sets of experiments are shown. f, Flow cytometry analysis of Krt15-CrePR-mediated dKO hair follicles with ROSA26-LSL-tdT allele to mark Cre+ dKO cells. The rectangle regions are CD34-APChi and tdTomatohi populations. Representative plots for 5 sets of experiments are shown.
Extended Data Fig. 8 Single-cell ATAC analysis of Foxc1 and Nfatc1 controlled open chromatin in HF-SCs.
a, tSNE plots of control and dKO total epithelial cells (Krt14-H2bGFP+). The HF-SC populations in each sample are highlighted in blue color and circled. The selected populations show the strongest open chromatin signatures of Cd34, the marker for HF-SC, and the weakest signatures of Gata6, a differentiation marker. b, K-means clustering of control and dKO open chromatin regions from aggregated scATAC-seq data from the HF-SC populations. Cluster 8 is reduced in dKO and cluster 10 is enhanced in dKO. c, Top 3 most highly enriched transcription factor motifs in cluster 8. d-f, Aggregated scATAC-seq tracks of Actg1 (d), Npnt (e) and Col6a1/2 (f) loci annotated with FOXC1 and NFATC1 motifs. Location of FOXC1 (green marks) and NFATC1 (red marks) motifs are indicated. Arrows point to HF-SC-specific open chromatin regions that are lost in dKO and the dashed rectangles mark the TSS of Actg1, Npnt and Col6a1/2, respectively.
Extended Data Fig. 9 Enhancer-promoter interactions are inferred by using aggregated Cicero scores computed from scATAC-seq.
a-f, Enhancer-promoter interactions of Actg1 (a), Bmp2 (b), Krt14 (c), Col6a1 (d), Fgf18 (e) and Itgb6 (f) are illustrated in ctrl IFE, dKO IFE, ctrl HF-SC and dKO HF-SC. The aggregated Cicero score is calculated by the summation of Cicero scores of all enhancer-promoter interactions to the TSS region of each gene. The vertical lines mark the TSS and the dashed line indicates the cutoff of Cicero score (0.2) used for calculation.
a, Hair coat is normal in both control and Itgb6 KO animals at ~9-mo old. b, HF-SC compartment is normal in both control and Itgb6 KO animals at ~9-mo old. Scale bar, 20μm.
Legends and captions for Supplementary Tables and Videos
Supplementary Tables 1–9.
An animated illustration identifies an escaped epithelial cell (KRT5+) in the dermis. The movie is related to Fig. 1h.
An animated illustration shows a number of abnormally located, escaped cells outside of the bulge region of a regressing HF. The movie is related to Fig. 4j.
Cellular activities of telogen HFs in control mice. A 5-h time-lapse movie of telogen HFs from control mice (P45). The time interval of each time point was 6 min. No discernible cellular activities including migration and cell division were observed. The entire sequences were looped twice during movie production. The movie is related to Fig. 7a.
A 5.8-h time-lapse movie of anagen HFs from control mice (P75). The time interval of each time point was 6 min. Limited cellular activities such as migration were observed mostly in HF-SCs (red arrowhead) and IRSs (yellow arrowhead) of growing HFs. The movie is related to Fig. 7b.
Cellular activities of anagen HFs in dKO mice. A 2.5-h time-lapse movie of anagen HFs from dKO mice. The time interval of each time point was 6 min. Robust cell migration activities were observed. In the upper HF, ORS progenitor cells migrate downward along the surface of the HF; IRS differentiated cells (elongated nuclei) migrate upward in the middle of the HF. In the lower HF, ORS progenitor cells migrate horizontally along the surface of the HF. The entire sequences were looped twice during movie production.
Escape of HF-SCs in dKO mice. A 4-h time-lapse movie of anagen HFs from dKO mice. The time interval of each time point was 6 min. HF-SCs escaped from a defective niche as described in the following sequence: (1) in the first 30 min, an HF-SC (blue arrow) detached from the bulge region; (2) in the first 2 h, a pair of HF-SCs (red and green arrows) escaped together from the bulge region; they went through a small orifice in the BM, judging by the altered morphology of the nuclei during escape; (3) in the final 30 min, an escaped HF-SC (red arrow) migrated ~16 μm further away from the HF; (4) more HF-SCs followed the escaping cells and migrated toward the breach point. The move is related to Fig. 7c.
Cellular activities in a disintegrating HF. A 2.5-h time-lapse movie of a miniaturized HF from a dKO mouse. The time interval of each time point was 6 min. Two HF cells (green arrowheads) disintegrated and were released into the dermis. An HF cell (red circle) was dividing in the same miniaturized HF. An escaped epithelial cell (yellow arrowhead) migrated away from the HF. The miniaturized HF was next to an SG and already moved upward. The movie is related to Fig. 7e.
Cell migration in a miniaturized HF. A 2.5-h time-lapse movie of a miniaturized HF from a dKO mouse. The time interval of each time point was 6 min. Less than 20 cells remained in the HF located in the upper portion of the movie. One cell (red arrowhead) migrated downward, away from the HF. The entire sequences were looped twice during movie production.
Escaped HF cells scattered in the dermis. A 3.2-h time-lapse movie of disintegrating HFs from dKO (~15-mo-old) mice. The time interval of each time point was 6 min. In the HF located in the lower right of the movie, epithelial cells were released from the HF to the dermis. In the upper part of the movie, a disintegrating HF had loosely packed HF cells, with several of them scattering in the nearby dermis. The entire sequences were looped twice during movie production. The movie is related to Fig. 7f.
Statistical source data for Fig. 1c–e.
Statistical source data for Fig. 3b,c.
Statistical source data for Fig. 4h,i.
Statistical source data for Extended Data Fig. 1c,e,f.
Statistical source data for Extended Data Fig. 5f,h,j.
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Zhang, C., Wang, D., Wang, J. et al. Escape of hair follicle stem cells causes stem cell exhaustion during aging. Nat Aging 1, 889–903 (2021). https://doi.org/10.1038/s43587-021-00103-w