Alteration of skin fibroblast steady state contributes to healing outcomes

Abstract

Fibroblasts display complex functions associated with distinct gene expression profiles that influence matrix production and cell communications and the autonomy of tissue development and repair. Thrombospondin-2 (TSP-2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell (sc) sequencing analysis on WT and TSP2KO skin fibroblasts demonstrate distinct cell heterogeneity. Specifically, we found an enrichment of Sox10+ multipotent progenitor cells, identified as Schwann precursor cells, in TSP2KO fibroblasts, while fibrosis-related subpopulations decreased. Immunostaining of tissue and cells validated the increase of this Sox10+ population in KO fibroblasts. Furthermore, in silico analysis suggested enhanced pro-survival signaling, including WNT, TGF-β, and PDGF-β, alongside a reduced BMP4 response. Additionally, the creation of two TSP2KO NIH3T3 cell lines using the CRISPR/Cas9 technique allowed functional and signaling validation in a less complex system. Moreover, KO 3T3 cells exhibited enhanced migration and proliferation, with elevated levels of pro-regenerative molecules including TGF-β3 and Wnt4, and enrichment of nuclear β-catenin. These functional and molecular alterations likely contribute to improved healing and increased neurogenesis in TSP2-deficient wounds. Overall, our findings describe the heterogeneity of dermal fibroblasts and identify pro-regenerative features of TSP2KO fibroblasts.

Keywords: Extracellular matrix; Fibroblasts; Schwann cells; Thrombospondin-2; Transforming growth factor beta; Wnt/β-catenin; tissue state.

Figure 1.. Single cell transcriptomics reveals distinct WT and TSP2KO dermal fibroblast heterogeneity.

(A) Diagram of dermal fibroblast isolation and sequencing process; (B) Cell cluster UMAP plot; (B) Cell type distribution in each genotype; (D) Dot plot of selected marker genes for each cell cluster; (E) Feature plots of representative markers for Schwann cell development within Sox10+ population; (F) Histogram of Sox10–700 intensity of unstained and stained dermal fibroblasts and quantification of Sox10+ cells in WT and TSP2KO groups (n = 3, Unpaired T test, two-tailed, *, p<0.05).

Figure 2.. Cell-cluster-specific gene differential analysis identifies highly regulated genes by TSP2 depletion in each cluster.

(A) Heatmap of highly differential genes (score ≥ 6, |power| > 1), (B) GO pathway analysis, and (C) violin plots of highly regulated genes between WT and KO within Schwann Precursors, DCN+, and Rspo3+ fibroblasts.

Figure 3.. NICHE analysis indicates enhanced PDGFb and reduced Bmp4 intercellular signaling in TSP2KO fibroblasts.

(A) The distribution of WT and KO signaling edges in a 2D UMAP plot; (B) Sending cell type and receiving cell type of each edge; (C) Signaling Archetypes defined via Louvain clustering; (D) Representative signaling mechanism for each cluster; (E) Volcano plot of highly regulated ligand-receptor mechanisms perturbed by KO; (F) Circuit plot of PDGFb and Bmp4 family signaling in WT and KO cells (edge thickness is proportional to mean connectivity and frequency, respectively).

Figure 4.. Bulk-RNA sequencing confirms perturbation in ECM proteins and suggests a positive regulation of Wnt pathway in KO fibroblasts.

(A) Heatmap of genes that are differential expressed between two genotypes. (B) GO analysis. (C) Network map of ECM proteins identified. (D) GSEA analysis. (E) qPCR of genes that are shown up in multiple unbiased analysis in WT and KO primary fibroblasts. (F) Western blot of TGF-β3 in cell lysates isolated from WT and TSP2KO (TK) mouse dermal fibroblasts. (n = 3–8, unpaired T-test, two-tailed, *, p<0.05, **, p<0.001)

Figure 5.. Depletion of TSP2 in NIH 3T3 cells using CRISPR/Cas9 creates a clean system to study function and pathway.

(A) Schematic diagram of TSP2KO CRISPR NIH3T3 cell lines. Validation of (B) KO-1 and (C) KO-2 cell lines using western blot. (D) Proliferation and (E) scratch assay of KO and Control cells (n = 3–6, Unpaired T-test, two-tailed, ***, p<0.0001).

Figure 6.. Depletion of TSP2 induces β-catenin activation with increased Wnt4/TGF-β3.

(A) qPCR, (B) western blot and densitometrical analysis of TGF-β3. Western blot of (C) Wnt4 and (D) active β-catenin in Control and KO cells. (E) Immunofluorescence staining and quantification of nuclei enrichment ratio of β-catenin in WT, Control, KO-1 and KO-2 cells. (F) Western blot of Wnt4 and TGF-β3 in TSP2 rescued KO cells. (G) Immunofluorescence staining and nuclei intensity and (H) western blot of β-catenin in KO cells transfected with pcDNA3.1 c or pcDNA GFP and TSP2 (n=3–5, *, p<0.05, **, p<0.01).

Figure 7.. TSP2KO skin wounds demonstrate increased neurogenesis.

(A) Immunofluorescence staining of NFH, Sox10, TGFb3 with Vimentin in D7 WT and TSP2KO mouse skin wounds. Quantification of (B) number of nerve bundles (NBs) in the center, (C) diameter of NBs (μm), (D) Sox10+ area/HPF (%), and (E) Tgfb3+ area/HPF (%) across the whole wounds (dashed circles indicate nerve bundles, n = 3–4, Unpaired T-test, two-tailed, *, p<0.05, **, p<0.01).