Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming

Abstract

Fibroblasts are an essential cell population for human skin architecture and function. While fibroblast heterogeneity is well established, this phenomenon has not been analyzed systematically yet. We have used single-cell RNA sequencing to analyze the transcriptomes of more than 5,000 fibroblasts from a sun-protected area in healthy human donors. Our results define four main subpopulations that can be spatially localized and show differential secretory, mesenchymal and pro-inflammatory functional annotations. Importantly, we found that this fibroblast 'priming' becomes reduced with age. We also show that aging causes a substantial reduction in the predicted interactions between dermal fibroblasts and other skin cells, including undifferentiated keratinocytes at the dermal-epidermal junction. Our work thus provides evidence for a functional specialization of human dermal fibroblasts and identifies the partial loss of cellular identity as an important age-related change in the human dermis. These findings have important implications for understanding human skin aging and its associated phenotypes.

Fig. 1. Single-cell RNA sequencing analysis of sun-protected whole human skin identifies seventeen distinct cell populations.

a Uniform manifold approximation and projection (UMAP) plot depicting single-cell transcriptomes from whole human skin (n = 5). Each dot represents a single cell (n = 15,457). Coloring is according to the unsupervised clustering performed by Seurat. b Heatmap showing the five most differentially expressed genes of each cell cluster, as provided by Seurat. Each column represents a single cell, each row represents an individual gene. Two marker genes per cluster are shown on the right. Yellow indicates maximum gene expression and purple indicates no expression in scaled log-normalized UMI counts. c Average expression of 3–5 well-established cell type markers was projected on the UMAP plot to identify all cell populations (see Methods for details). Red indicates maximum gene expression, while blue indicates low or no expression of a particular set of genes in log-normalized UMI counts. DC dendritic cells, EC endothelial cells.

Fig. 2. Dermal fibroblast subpopulations display specific spatial and functional transcriptomic signatures.

a Left: UMAP plot displaying dermal fibroblasts from young donors (n = 2). Each dot represents a single cell (n = 1792). Coloring is according to the original unsupervised clustering performed by Seurat. Right: Bar plots indicating the percentage of fibroblasts corresponding to each subpopulation and donor. b Top 8 enriched Gene Ontology (GO) terms in each fibroblast subpopulation, sorted by p-value. c Heatmap showing the expression of all collagen genes in the distinct fibroblast subpopulations. Each column represents a single cell and each row an individual collagen gene. Yellow indicates maximum gene expression while purple indicates no expression in scaled log-normalized UMI counts. d Average expression of the genes constituting the papillary and reticular gene signatures for predicting dermal localization of the fibroblasts from the four clusters. In all UMAP gene expression projections, red indicates maximum expression and blue indicates low or no expression of each particular set of genes in log-normalized UMI counts. In the violin plots, X-axes depict cell cluster number and Y-axes represent average expression of each set of genes in log-normalized UMI counts. Statistical significance of the expression changes in the gene signatures between cell clusters is indicated below through the p-values of the corresponding Wilcoxon rank sum tests.

Fig. 3. RNA FISH detection of fibroblast subpopulations in young skin.

a Representative confocal images showing mRNA expression of CTHRC1 (green) and APCDD1 (red), selected markers for the secretory-reticular and secretory-papillary fibroblast subpopulations, respectively. Details from the papillary and reticular regions of the images above are shown in the lower panels (left and center, respectively), and percentage of positive cells for each gene and per dermal region are shown in the lower right panel. b Representative confocal images showing mRNA expression of CCL19 (green) and APOE (red), selected markers for the pro-inflammatory fibroblast subpopulation. A detail of a vessel of the images above is shown in the lower panel. c Representative confocal images showing mRNA expression of ASPN (green), selected marker for the mesenchymal fibroblast subpopulation. A detail of the hair follicle bulb of the images above is shown in the lower panel. Dashed lines in a and b denote the papillary dermis regions while in c denote the dermal papilla. Nuclei were counterstained with DAPI. Each assay was performed in three independent young FFPE skin sections (28–37 y/o). Images are shown at ×40 original magnification. Scale bar: 50 μm for main images and 10 μm for detail images. Pap papillary dermis, Ret reticular dermis, Deep ret deep reticular dermis, HF hair follicle, DP dermal papilla. Statistical analyses were performed using a two-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); error bars represent the standard deviation.

Fig. 4. Aging leads to loss of dermal fibroblast priming.

a Left: UMAP plot displaying dermal fibroblasts from old donors (n = 3). Each dot represents a single cell (n = 4156). Coloring is according to the original unsupervised clustering performed by Seurat. Right: Bar plots indicate the percentage of fibroblasts corresponding to each subpopulation and donor. b Percentage of fibroblasts of each subpopulation that were in the G1, S or G2/M phase of the cell cycle in young and old skin samples, respectively. c Top 8 enriched Gene Ontology (GO) terms in each old fibroblast subpopulation sorted by p-value. Coloring is according to the unsupervised clustering performed by Seurat. d UMAP and violin plots displaying the average expression of all collagen genes in the fibroblasts of all subpopulations, for young and old skin. e UMAP and violin plots displaying the expression of the papillary and reticular gene signatures in the fibroblasts of all subpopulations, for young and old skin. In all UMAP gene expression projections, red indicates maximum expression and blue indicates low or no expression of each particular set of genes in log-normalized UMI counts. In the violin plots, X-axes depict fibroblast subpopulations and Y-axes represent average expression of each set of genes in log-normalized UMI counts. For comparing the ratio of G1 cells between young and old subpopulations, a two-sided two-proportion z-test was used (b). Statistical analyses in d and e were performed using the Wilcoxon Rank Sum test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Y young, O old, S.R Secretory-reticular, INF Pro-inflammatory, S.P Secretory-papillary, MES Mesenchymal.

Fig. 5. Other age-related changes in dermal fibroblast subpopulations.

a Expression of genes encoding skin aging-associated secreted proteins (SAASP) (rows) that are differentially (fold-change > 1.25) expressed between young and old fibroblasts in at least one subpopulation (columns). The heatmap shows the mean relative expression by cluster. b Bar plots showing the number of ligand-receptor interactions predicted for the four observed fibroblast subpopulations with the rest of the cell types identified in human skin, in the two young (≤27 y/o) (up) and the two oldest (≥69 y/o) (down) samples. The medium-aged sampled (53 y/o) showed an intermediate phenotype in this analysis and was therefore omitted. Coloring and numbering are according to the original unsupervised clustering performed by Seurat. c Summary of the top four exclusive interactions lost between each fibroblast subpopulation and undifferentiated keratinocytes, sorted by p-value. The table shows interactions in both directions for each pair. Y young, O old, S.R Secretory-reticular, INF Pro-inflammatory, S.P Secretory-papillary, MES Mesenchymal.