O21 Single-cell RNA sequencing and proteomic analysis of dermal fibroblasts cultured from female scalp identifies significant alterations in cell populations with age

Abstract We have previously reported that significant in situ detrimental changes occur in the female scalp with age. We also reported that primary cultures of female scalp dermal fibroblasts (DF) significantly increase the secretion of proteins such as cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5), secreted protein acidic and cysteine rich (SPARC) and smooth muscle actin (ACTA2). This highlights changes in DF cellular identity with age, altering the immediate hair follicle environment, presumably driving hair ageing. To gain a better understanding of age-related molecular and functional changes in DFs, we compared both the transcriptional and proteomic profile of primary cultures of scalp DFs (passage < 3) from female donors (n = 8) aged 21–77 years. Transcriptional profiles were analysed via 10× Genomics scRNAseq. Data profiles were generated by evaluation of cellular lysates by liquid chromatography mass spectrometry proteomics and compared by MS1 precursor intensities using PEAKS software. A total of 16 166 DF cells from young (n = 3) and older (n = 3) donors were sequenced. Across the two populations, 11 transcriptionally distinct clusters were detected, highlighting the heterogeneity in DFs. The percentage of cells in these clusters changed with age, and gene set enrichment analysis ascertained a significant increase of cells in cluster 2, which represented a higher number of gene sets related to fibrosis, senescence, collagen synthesis and degradation. Proteomic analysis of DF cellular proteins (four donors in each age group) identified 11 significantly differentially expressed proteins between the two age groups. Two proteins (serine/threonine-protein kinase 38 and rho GTPase activating protein 21) were significantly reduced in the older cohort, while nine proteins (smoothelin, retinol dehydrogenase 5 tetranectin, oestradiol 17-beta-dehydrogenase 11, CD14, carboxypeptidase M, gamma-enolase, plexin-B1 and endoplasmic reticulum membrane protein complex subunit 4) were significantly elevated in the older cohort. Gene Ontology and STRING analysis of the data revealed modifications to key pathways with age (e.g. collagen and elastic fibre formation, autophagy, ubiquitination and metabolic markers). This study highlights the loss of scalp DF cell signature and identity with age. We hypothesize that these changes impact on the ability of the scalp dermal environment to support and maintain healthy hair follicles, contributing to the age-related changes observed in female hair growth.