P28 Keratinocyte heterogeneity and dynamics in acute human skin wound healing

Abstract Introduction and aims Human chronic skin wounds have been extensively characterized, but ethical limitations of collecting and studying wounds from healthy individuals means there is a sparsity of data regarding re-epithelialization in normal wound healing. Here, we characterized an ex vivo model of wound closure in human skin to identify markers related to re-epithelialization in normal wound healing. Methods We conducted single-cell RNA sequencing (scRNAseq) from ex vivo abdominal skin explants 2 days postwounding (N = 4). Immunostaining was also conducted on sections from healing skin explants. Additionally, scratch assays were conducted to evaluate migration in vitro. Knockdown experiments were conducted using nanoparticles to deliver small interfering RNAs (siRNAs) to keratinocytes in vitro and ex vivo. Results We found that partial re-epithelialization and full healing of 2-mm wounds occurred 2–3 days and 4 days postwounding, respectively. Hence, the 2-day timepoint was selected for scRNAseq data generation. Dimension reduction of the 29 000 cells in the dataset revealed 13 total clusters, with 10 different keratinocyte clusters. Analyses of the dataset revealed a keratinocyte subcluster expressing known wound-response markers, such as S100A7, which we found was expressed at the suprabasal leading edge of the regenerating epidermis using immunostaining. The importance of S100A7 is further confirmed with the changes in migration speed with the application of siRNA targeting S100A7. A different KRT5-expressing basal keratinocyte population was also identified, and ontology analysis revealed differentially expressed migration- and adhesion-related genes. Conclusions S100A7 was previously reported to be increased at the wound edge in human keratinocytes, while we found this in our scRNAseq data and with immunostaining in our ex vivo model. This shows that our ex vivo model reflects changes occurring in wounds in vivo, at least within keratinocytes. We are now leveraging this data to identify novel genes involved in acute skin wound healing.