gms | German Medical Science

Introduction: Hypertrophic skin scarring is one of the main long-term complications for burns patients, causing high morbidity, pain, and reduced quality of life. Treatment of scars is often difficult, with limited number of therapeutic options and few new developments in the field. Although understanding the mechanisms of scar tissue formation is crucial for the development of new therapeutic agents, the underlying cellular mechanisms are still poorly understood.

Methods: We used single-cell RNA sequencing (scRNAseq) to compare mature hypertrophic human scar tissue with healthy human skin. After informed consent, tissue from patients who underwent scar resection surgery (n=3) or abdominoplasty (n=3, for healthy skin samples) was enzymatically digested to gain a viable cell suspension of all celltypes present in skin. Cells were then processed to a Gel-bead-in-emulsion (GEM) by a 10X-Chromium instrument, cDNA-library was prepared, and sequenced. After demultiplexing and counting, the resulting cell-gene matrix was used for downstream analysis. Secondary bioinformatics analysis was performed using the „Seurat“-package (Satija Lab, New York Genome Center, USA) in R.

Results: Our scRNAseq yielded data of 10.261 single cells from scar tissue, and 5754 cells from healthy skin, with an average of ~1000 detected genes per cell. In total, 21 clusters were found, and all celltypes typical for skin, including keratinocytes, melanocytes, mast cells, fibroblasts, dendritic cells, macrophages, T-cells, endothelial cells, lymphatic endothelial cells, smooth muscle cells and pericytes were identified in the dataset. Strikingly, our analyses identified one distinct cell cluster of fibroblasts, the main mediators of fibrotic effects, exclusively present in scar tissue. These distinct fibroblasts highly expressed genes relevant for scar tissue formation, such as collagens and TGFbeta 1, but also genes hitherto not described in the context of scarring, e.g. COPZ2, a coatamer subunit, OGN (osteoglycin), MDK (midkine), or C1QTNF3 (C1q/TNF-related protein-3). Furthermore, transcription factor analysis with genes upregulated in this fibroblast subset revealed enrichment for SRF (serum response factor), which was described in the context of fibrosis, and RMBX (RNA binding motif protein X-linked), a potential new transcriptional regulator of genes involved in scarring.

Conclusion: Taken together, our study provides thus far unknown high-resolution insights into the gene regulation of scar tissue on a single cell level, and identifies numerous new potential targets for the development of new therapeutics to prevent or reverse skin scarring.