P31 Single-cell RNA sequencing reveals transcriptional changes induced by cell passage in keloid and normal skin derived stromal cells

Abstract Introduction and aims Keloids are scars characterized by excessive collagen deposition and expansion beyond the wound margin, causing aesthetic concerns as well as pain and itching. However, the cellular origins of these sensations remain poorly understood. This study aimed to identify the cellular sources of keloid pain and itch, and explore their potential for in vitro investigation. Methods Single-cell RNA sequencing (scRNAseq) datasets of keloid scars were analysed to identify pain/itch mediators. Expression of key mediators was validated using quantitative polymerase chain reaction (qPCR). The impact of cell passage on keloid and normal-adjacent dermal cells was examined by comparing scRNAseq from primary cells at passage 0 (P0) and passage 4 (P4) from a single patient. Results Analysis of three scRNAseq datasets identified mesenchymal fibroblasts and ACKR1+ endothelial cells as more abundant in keloid samples, and able to express prurogenic and algesic mediators such as CXCL12, tenascin-C, periostin, endothelin-1, interleukin (IL)-6 and IL-33. However, qPCR validation on expanded primary dermal fibroblasts (passages 5–8) showed discordant results, potentially due to changes during culture. To address this, scRNAseq analysis at P0 and P4 revealed that stromal cell subsets amalgamated and became transcriptionally distinct by P4. Moreover, the differential expression of prurogenic and algesic mediators identified from the literature was decreased or absent in P4 cell subsets, likely attributable to relative proportions of subsets normalizing; this explains the previous discordance between scRNAseq and qPCR observations. These changes demonstrate that prolonged culture alters the representation of stromal cell populations and can impact their expression of pain/itch mediators. Conclusions This study identified keloid-enriched subsets that express pain and itch-related mediators. However, their expression was lost or reduced through culture in standard conditions. This indicates key biological differences may have been historically missed, and more sophisticated models or fresh tissue analysis may be required to capture certain disease-relevant features.