SFRP2/DPP4 and FMO1/LSP1 Define Major Fibroblast Populations in Human Skin

Abstract

Fibroblasts produce matrix, regulate inflammation, mediate reparative processes, and serve as pluripotent mesenchymal cells. Analyzing digested normal human skin by single-cell RNA sequencing, we explored different fibroblast populations. T-distributed stochastic neighbor embedding and clustering of single-cell RNA sequencing data from six biopsy samples showed two major fibroblast populations, defined by distinct genes, including SFRP2 and FMO1, expressed exclusively by these two major fibroblast populations. Further subpopulations were defined within each of the SFRP2 and FMO1 populations and five minor fibroblast populations, each expressing discrete genes: CRABP1, COL11A1, FMO2, PRG4, or C2ORF40. Immunofluorescent staining confirmed that SFRP2 and FMO1 define cell types of dramatically different morphology. SFRP2⁺ fibroblasts were small, elongated, and distributed between collagen bundles. FMO1⁺ fibroblasts were larger and distributed in both interstitial and perivascular locations. Differential gene expression by SFRP2⁺, FMO1⁺, and COL11A1⁺ fibroblasts suggests roles in matrix deposition, inflammatory cell retention, and connective tissue cell differentiation, respectively.

Figure 1.. T-SNE shows groupings and SLM clustering of normal skin cell populations.

Panel a: Each point represents a cell. Two-dimensional t-SNE shows dimensional reduction of cell transcriptomes. Cells are colored by K-nearest neighbors graph based on Euclidean distance in PCA space using a smart local moving algorithm (SLM) to iteratively group cells. Panel b: Cells are grouped by t-SNE as in panel A, but are colored according to the subject identity. Panel c shows transcriptomes of 8,522 cells from six normal skin biopsies clustered using Seurat (SLM clustering). Each column represents a cell. The five genes most differentially expressed between each cluster are shown, and two of these five genes are enlarged to help identify each cluster. Cluster numbers, indicated at the bottom are as shown in Figure 1a, t-SNE.

Figure 2.. Feature plots of genes defining different cell types in normal skin.

The intensity of purple color indicates the normalized level of gene expression. DES: smooth muscle/arrector pili; RGS5: pericytes; VWF: endothelial cells; KRT1 and KRT14: keratinocyte populations; PMEL: melanocytes; SCGB1B2P: gland cells; LOR: cornified keratinocytes; COL1A1: fibroblasts; CD3D: T cells; AIF1: macrophages/dendritic cells; IGJ: B cells

Figure 3:. Hierarchical clustering of fibroblast transcriptomes from normal skin.

Fibroblasts in SLM cluster #0, 3 and 4 were hierarchically clustered. Genes identified to define cell types selected by t-SNE and SLM clustering, as well as visual inspection were used to identify clusters showing gene expression discrete to various cell types. Names of genes associated with each cluster are enlarged to the right of the clustering. The SLM clusters that define the different clusters and cell types are shown to the far right.

Figure 4.. Gene expression by SFRP2+ fibroblasts and CD55/PCOLCE2 and WIF1/NKD2 fibroblast subpopulations.

Intensity of purple color indicates the level of gene expression on t-SNE feature plots for SFRP4 and DPP4 (Panel a), CD55 and PCOLCE2 (Panel b) and WIF1 and NKD2 (Panel c). Violin plots adjacent to each t-SNE plot show the expression level of genes in each of the SLM clusters shown in Figure 5

Figure 5.. Gene expression by fibroblast subpopulations.

Intensity of purple color indicates the level of gene expression on t-SNE feature (a, c, e). Violin plots adjacent to each t-SNE plot (b, d, f) show the expression level of genes in each of the SLM clusters shown in Figure 5.

Figure 6.. Immunofluorescent staining of normal skin showing two distinct major populations of dermal fibroblasts.

Single IF staining of SFRP2 and DPP4 detects cytoplasmic staining of morphologically smaller, elongated cells (panel a, red). IF staining of FMO1 and LSP1 detects nuclear (FMO1) and cytoplasmic (LSP1) staining of larger, more round cells (panel b, red). Double IF staining is shown of FMO1 (green) and SFRP2 (red, panel c); FMO1 (green), DPP4 (red) and SFRP2 (red) DPP4 (green, panel d); FMO1 (green) and LSP1 (red, panel e); and FMO1 (green) and SFRP2 (red, panel f) Dual staining with higher magnification insets are included for FMO1 and LSP1 (panel e) and FMO1 and SFRP2 (panel f). For all panels nuclei are counterstained with DAPI (blue). Scale bar=50 μM.