P13 Keratinocyte heterogeneity and dynamics in 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 characterize ex vivo models of wound closure in human skin to better understand the keratinocyte dynamics of normal healing. Methods We conducted single-cell RNA sequencing (scRNA-Seq) from ex vivo skin explants 2 days postwounding (n = 4) to interrogate heterogeneity in keratinocytes relative to the wound edge. Immunostaining was also conducted on sections from skin explants at different timepoints during wound closure. Scratch assays were conducted using isolated human keratinocytes. Specific zones of keratinocytes were separated relative to the original scratch, 6 h postscratch, and used for real-time polymerase chain reaction analyses. Results We found that partial re-epithelialization and full healing of 2-mm wounds occurred 2–3 days and 4 days postwounding, respectively. Analyses of the scRNA-Seq data showed an increase in S100A7 expression in suprabasal keratinocyte subclusters in wounded skin compared with unwounded skin. Using immunostaining we also showed that S100A7 is expressed at the leading edge of the regenerating epidermis. In an in vitro scratch wound model, the keratinocytes nearest to the wound edge also show an upregulation of S100A7, EGFR, EPHA4 and TIMP1, as well as a downregulation in PCNA, implying a decrease in cell proliferation in leading keratinocytes. Conclusions S100A7 was previously reported to be increased at the wound edge in human keratinocytes, highlighting that transcriptional changes in our ex vivo and in vitro human wound healing models reflect those occurring in vivo. The decrease in PCNA in keratinocytes in the leading edge also recapitulates Results previously shown in mouse models of wound healing. These data on normal re-epithelialization will be compared with published data on human chronic wounds and other skin diseases.