Single cell analysis of transcriptome and open chromatin reveals the dynamics of hair follicle stem cell aging

Abstract

Aging is defined as the functional decline of tissues and organisms, leading to many human conditions, such as cancer, neurodegenerative diseases, and hair loss. Although stem cell exhaustion is widely recognized as a hallmark of aging, our understanding of cell state changes-specifically, the dynamics of the transcriptome and open chromatin landscape, and their relationship with aging-remains incomplete. Here we present a longitudinal, single-cell atlas of the transcriptome and open chromatin landscape for epithelia cells of the skin across various hair cycle stages and ages in mice. Our findings reveal fluctuating hair follicle stem cell (HF-SC) states, some of which are associated with the progression of the hair cycle during aging. Conversely, inner bulge niche cells display a more linear progression, seemingly less affected by the hair cycle. Further analysis of the open chromatin landscape, determined by single-cell Assay for Transposase-Accessible Chromatin (ATAC) sequencing, demonstrates that reduced open chromatin regions in HF-SCs are associated with differentiation, whereas gained open chromatin regions in HF-SCs are linked to the transcriptional control of quiescence. These findings enhance our understanding of the transcriptional dynamics in HF-SC aging and lay the molecular groundwork for investigating and potentially reversing the aging process in future experimental studies.

Keywords: cell adhesion; hair follicle stem cell; quiescence; scATACseq; scRNAseq; single cell genomics; stem cell exhaustion.

FIGURE 1

Single-Cell transcriptome of aging hair follicles. (A) Integration of scRNAseq samples from P38, P53, 6 months, 12 months, and 24 months. (B) UMAP visualization of all epidermal cell lineages. Color-coded by cell types. IFE, interfollicular epidermal basal cells; Supra, suprabasal cells; UpHF1/2, differentiated hair follicle cells in the upper portion; SG, sebaceous gland; UpHFSC, Lgr6+ HF-SCs; HF-SC, hair follicle stem cells; Niche, inner layer niche cells; HG, hair germ; MigNiche, migratory niche cells; DP, dermal papillae; Prolif, proliferating cells; Fibroblast, Fibroblast cells; Langer, Langerhans cells; Tcells1/2, T cells; APM, arrector pili muscle; Melano, melanocytes; Endo, endothelial cells. (C,D) Marker gene expression of different epithelial cell populations, interfollicular epidermal cells, upper hair follicles (C) and hair follicle stem cell compartment (D). (E,F) Feature plot and violin plot of young (E) and old (F) feature genes sets. The young and old feature genes were genes enriched in young and old HF-SCs. The module scores were the average expression of aggregated feature genes on single cell level compared with control gene sets. The p-value of feature genes between UpHF1 and HF-SC population were calculated using wilcoxon rank sum test. ****: p-value <0.0001.

FIGURE 2

Transcriptomic analysis of HF-SCs aging. (A) UMAP visualization of HF-SCs, color coded by samples. (B) UMAP visualization of HF-SCs color-coded by pseudo-time. (C,D) Highly enriched gene ontology (GO) terms of 6 months (C) and 24 months (D) branch cells. (E,F) Selected gene expression plot along the pseudotime trajectory of 6 months (E) and 24 months (F) branch cells.

FIGURE 3

Aging trajectory of the Niche cells. (A) UMAP visualization of Niche cells during aging, color-coded by samples. (B) Monocle3 pseudo-time plots of aging Niche cells color-coded by pseudo-time values. (C,D) Aggregated expression of genes modules in different co-expression modules along the aging Niche cells. (E,F) Enriched GO terms of gene modules corresponding to (C) and (D).

FIGURE 4

Integration of scRNAseq and scATACseq data. (A,B) UMAP visualization of integrated scRNAseq and scATACseq data, color-coded by cell populations. (C) Dotplot of marker genes expression in scRNAseq (left) and inferred gene activities in scATACseq (right). Gene expression levels were indicated by color intensity. Dot size represents the percentage of cells with the inferred activities. (D) Gene expression, inferred gene activities, motif activities and motif plots of lineage specific transcription factors.

FIGURE 5

Open chromatin dynamics during HF-SC aging. (A) Feature plot and violin plot of open chromatin regions enriched in young HF-SCs. (B) Feature plot and violin plot of open chromatin regions enriched in old HF-SCs. (C,D) Violin plot of young and old open chromatin features in HF-SCs of different samples. (E) Example tileplot of chromatin regions gradually open during aging. (F,G) Enriched motifs in young and old HF-SC open chromatin regions.