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. 2018 Apr;138(4):811-825.
doi: 10.1016/j.jid.2018.01.016. Epub 2018 Jan 31.

Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations

Christina Philippeos  1 , Stephanie B Telerman  1 , Bénédicte Oulès  1 , Angela O Pisco  1 , Tanya J Shaw  2 , Raul Elgueta  3 , Giovanna Lombardi  3 , Ryan R Driskell  4 , Mark Soldin  5 , Magnus D Lynch  6 , Fiona M Watt  7
Affiliations

Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations

Christina Philippeos et al. J Invest Dermatol. 2018 Apr.

Abstract

Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis.

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Figures

Figure 1
Figure 1
Differential expression of genes associated with Wnt, ECM, and immune signaling in mouse fibroblast subpopulations. (a) Gene Ontology term analysis of differentially expressed pathways in mouse fibroblast subpopulations. (b–d) Heat maps illustrating differential expression (Affymetrix microarray) of genes implicated in (b) Wnt signaling, (c) inflammation, and (d) ECM regulation. (e) qPCR validation of selected differentially expressed genes. (f) Heatmap comparing expression (Affymetrix microarray) of genes implicated in adipogenesis. (g, h) qPCR analysis showing up-regulation of CD36 expression in (g) pre-adipocyte populations and (h) CD39 in papillary fibroblasts. (e, g, h) Gene expression is normalized to GAPDH and expressed as mean ± standard deviation for three biological replicates. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005. EC, extracellular; ECM, extracellular matrix; qPCR, quantitative PCR.
Figure 2
Figure 2
Spatial profiling of gene expression in human dermis. (a) Hierarchical clustering of gene expression patterns from papillary and reticular dermis in three separate individuals (breast skin samples derived from three donors). Expression was quantified by RNA sequencing (blue, similar; red, dissimilar). (b) Genes up-regulated in papillary dermis (>3-fold). (c) Genes up-regulated in reticular dermis (>10-fold). (d) Cell surface markers for which expression was at least 2-fold up-regulated in papillary dermis. Expression level is indicated separately for each of the three biological replicates (green, high; red, low) and compared with expression in six different cultured fibroblast lines and seven other cultured cell lines. (e) Cell surface markers at least 2-fold up-regulated in reticular dermis. (f) Cell surface markers up-regulated in papillary dermis that are conserved between human adult dermis and mouse P2 dermis. P2, postnatal day 2.
Figure 3
Figure 3
Immunofluorescence labeling of human dermis with antibodies to candidate fibroblast subpopulation markers identified by spatial transcriptomics. (a, b) Expression of COL6A5 is restricted to the papillary dermis (female breast skin, donor age 22 years). The basal layer of the epidermis is labeled with anti-K14 (COL6A5, green; K14, red). (c, d) Expression of APCDD1 is enriched in the papillary dermis (APCDD1, green; K14, red; female back skin, donor age 44 years). (e, f) Expression of HSPB3 is enriched in the papillary dermis (HSPB3, green; K14, red; female breast skin, donor age 22 years). (g, h) Expression of WIF1 is enriched in vascular structures that are more prominent in the upper dermis (WIF1, green; K14, red; female abdominal skin, donor age 27 years). (i, j) Expression of CD36 is highly enriched in the lower dermis (female abdominal skin, donor age 44 years). (k, l) CD39 is enriched in the papillary dermis (CD39, green; podoplanin, red; female abdominal skin, donor age 43 years). Scale bars = 200 μm. K14, keratin.
Figure 4
Figure 4
Human dermal fibroblast subpopulations maintain functional differences in vitro. (a, b) Expression of LUM and COL6A5 is enriched in CD90+ population compared with an unfractionated dermal cell suspension. Gene expression normalized to GAPDH and expressed as mean ± standard deviation for three replicates. (c) CD39 expression is detectable in primary CD31–CD45–ECad– cells but is lost after a single passage in culture (d). However, expression of (d, e) CD90 and (e) CD36 is retained. (f) lin–CD90+CD39+ and lin–CD90+CD36+ cells exhibit morphological differences in vitro, there is considerable intersample variation. Breast skin donor age, 19 years; abdominal skin donor age, 43 years; and thigh skin donor age, 46 years. Scale bars = 50 μm). (g) Quantitative PCR showing retention of LUM and loss of COL6A5 in culture. Gene expression normalized to GAPDH and expressed as mean ± standard deviation for three biological replicates. (h–j) Expression of genes associated with (h) Wnt signaling, (i) inflammation and immunity, and (j) ECM remodeling. Gene expression is normalized to 18S and is expressed as mean ± standard deviation for three biological replicates derived from female (age 42 years) abdominal skin, female (age 46 years) thigh skin, and female (age 64 years) abdominal skin. (k–o) Modulation of expression of cell surface markers in response to IFN-γ stimulation in culture (blue, CD39+IFN-γ; yellow, CD36+IFN-γ; red, unstained control). Top row: representative flow plots. Bottom row: quantitation of data from three independent experiments derived from female (age 42 years) abdominal skin, female (age 46 years) thigh skin, and female (age 64 years) abdominal skin. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001. ECM, extracellular matrix; FSC, forward scatter detector; lin–, lineage negative; MFI, mean fluorescence intensity; SSC, side scatter detector.
Figure 5
Figure 5
Comparison of the ability of different fibroblast subpopulations to support epidermal growth on DED. (a) Isolation of fibroblast subpopulations by flow cytometry. (b–f) Hematoxylin and eosin staining (scale bars = 100 μm) and (g–k) immunofluorescence staining (scale bars = 200 μm) of DED organotypic cultures (b, g) without fibroblasts or (c, h) seeded with unfractionated (lin–CD90+) fibroblasts, (d, i) CD90+CD39+ (enriched in papillary) fibroblasts, (e, j) CD90+CD39– (depleted of papillary) fibroblasts, and (f, k) CD90+CD36+ (reticular/pre-adipocyte) fibroblasts. Keratin 14 (green) marks keratinocytes, and vimentin (white) marks mesenchymal cells (fibroblasts). Experiments were repeated for a minimum of two biological replicates (donor cells derived from female [age 19 years] breast skin and female [age 37 years] breast skin; DED derived from male [age 47 years] abdominal skin, and representative images are displayed. In the case of the CD90+CD39+ cells, two of three experiments involved additional selection for CD26– cells to further enrich for the papillary cell population (CD90+CD39+CD26–). (l) Quantification of epidermal thickness, (m) density of fibroblasts within 300 μm of the epidermis, and (n) relative abundance of fibroblasts at different depths from the epidermis. adipo, adipocyte; DED, decellularized human dermis; FB, fibroblasts; FSC, forward scatter detector; K14, keratin 14; lin–, lineage negative; ns, not significant; SSC, side scatter detector.
Figure 6
Figure 6
Single-cell RNA sequencing of human adult dermal fibroblasts. (a) Isolation of lin– cells and lin–CD90+ cells from human dermis by flow cytometry. Single live cells were isolated from a single donor (female age 64 years, abdominal skin) by gating for forward scatter, side scatter, and DAPI-staining. lin– cells were isolated by gating for CD31–CD45–ECad–. (b) PCA analysis of gene expression patterns. (c, d) tSNE analysis of gene expression patterns (red, lin–; blue, lin–CD90+). (d) Automated clustering of tSNE analysis identifies five dermal fibroblast subpopulations. (e) Expression patterns of markers differentially expressed in each of five clusters (red, high expression; yellow, low expression. (f) Violin plots illustrating differential expression of marker genes in each of five dermal fibroblast subpopulations. (g–j) Immunostaining for candidate fibroblast markers in adult human skin: (g) COL6A5, female breast skin, donor age 37 years; (h) CD26, female breast skin, donor age 62 years; (i) MFAP5, female breast skin, donor age 37 years; and (j) RGS5, female back skin, donor age 29 years. Scale bars = 200 μm. (k) Isolation of lin–CD39+CD26– and CD39– dermal fibroblasts by flow cytometry. (l–p) Expression of (l) CD39, (m) COL6A5, (n) WNT5A, (o) RSP01, and (p) LEF1 in lin–CD39+CD26– and CD39– dermal fibroblasts. Gene expression is normalized to GAPDH and TBP and is expressed as mean ± standard deviation for three biological replicates: male (age 53 years) thigh skin, female (age 52 years) breast skin, and female (age 62 years) breast skin. ∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.0005, ∗∗∗∗P < 0.0001. FSC, forward scatter detector; K14, keratin 14; lin–, lineage negative; PCA, principle component analysis; SSC, side scatter detector; tSNE, t-distributed stochastic neighbor embedding.
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