using-r-seurat-cellchat-to-analyze-single-cell-transcriptomics

Using R, Seurat, and CellChat to Analyze a Single-Cell Transcriptomics Dataset of Mouse Skin Wound Healing

The process of wound healing is regulated by complex interactions between different cell types across space and time. Through the profiling of individual ...

University of Illinois Chicago

Many factors regulate scar formation, which yields a modified extracellular matrix (ECM). Among ECM components, microfibril-associated proteins have been minimally explored in the context of skin wound repair. Microfibril-associated protein 5 (MFAP5), a small 25 kD serine and threonine rich microfibril-associated protein, influences microfibril function and modulates major extracellular signaling pathways. Though known to be associated with fibrosis and angiogenesis in certain pathologies, MFAP5's role in wound healing is unknown. Using a murine model of skin wound repair, we found that MFAP5 is significantly expressed during the proliferative and remodeling phases of healing. Analysis of existing single-cell RNA-sequencing data from mouse skin wounds identified two fibroblast subpopulations as the main expressors of MFAP5 during wound healing. Furthermore, neutralization of MFAP5 in healing mouse wounds decreased collagen deposition and refined angiogenesis without altering wound closure. In vitro, recombinant MFAP5 significantly enhanced dermal fibroblast migration, collagen contractility, and expression of pro-fibrotic genes. Additionally, TGF-ß1 increased MFAP5 expression and production in dermal fibroblasts. Our findings suggest that MFAP5 regulates fibroblast function and influences scar formation in healing wounds. Our work demonstrates a previously undescribed role for MFAP5 and suggests that microfibril-associated proteins may be significant modulators of wound healing outcomes and scarring.

Microfibril-associated protein 5 and the regulation of skin scar formation.

Compared to skin wounds, oral mucosal wounds heal quicker with less inflammation, faster re-epithelialization, and minimal scarring. Site-specific keratinocytes may be one differentiating factor. This study used immortalized skin and oral keratinocytes (HaCaT and TIGK), which maintain fidelity to their primary cell counterpart, to examine functional and transcriptional differences that might contribute to the differential wound healing at the two sites. Oral keratinocytes were found to have an enhanced migratory and proliferative capacity. To examine the transcriptomic differences, we generated an mRNA-sequencing gene expression dataset utilizing HaCaT and TIGK. Differentially expressed genes (DEGs) were identified between HaCaT and TIGK at baseline and throughout in vitro healing. DEGs in HaCaT and TIGK following injury were also identified when compared to each respective cell type's unwounded gene expression levels. Gene set enrichment analyses were performed to understand the biological significance of the DEGs. Processes related to interferon (IFN) signaling were uniquely enriched in TIGK. TIGK also exhibited a faster transcriptional response to injury and differential expression of integrins and matrix metalloproteinases (MMPs). When grown on extracellular matrix (ECM) proteins, TIGK retained its enhanced migratory capacity over HaCaT. Lastly, TIGK displayed a post-injury secretome that promoted keratinocyte migration. Our comparative analyses suggest that specific transcriptomic differences between oral and skin keratinocytes at unwounded baseline and in response to injury may underlie the distinct wound healing phenotypes observed in these two tissues. This work also provides a new resource of HaCaT and TIGK gene expression data that can be used for future analyses.

Article	Year
Microfibril-associated protein 5 and the regulation of skin scar formation. Scientific reports\| PubMed ID: 37253753	2023
Transcriptomic Differences Between Immortalized Oral and Skin Keratinocytes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 40540308	2025

Shalyn Keiser

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