1424 Single-cell RNA sequencing of different fibroblast populations cultured from female scalp reveals significant alterations to transciptional profiles with age

With increasing age, female scalp skin undergoes considerable structural and molecular changes. Previously we reported the relevance of the age-related changes in the dermal environment on scalp ageing (Williams et al, J Invest Dermatol, 141: 1041-1051; 2021). The aim of this study was to interrogate global age-related changes in cultured female scalp interfollicular dermal fibroblasts (DF), and hair follicle dermal sheath (DS) and dermal papilla (DP) cells via single cell RNA sequencing (scRNAseq). Primary cultures (passage <3) were established from donors (20-77yrs; n=18), and transcriptional profiles analyzed via 10x Genomics scRNAseq. Data was processed with Cell Ranger, and downstream analysis with Seurat package version 3.1.157, R version 3.5.1 and 10X Genomics Loupe Cell Browser v.6.0.0. A total of 51,774 DF, DS and DP cells from young (20-23yrs) and older (57-77yrs) donors were sequenced. Across the 6 populations, 19 transcriptionally distinct clusters were detected, identifying diverse cellular profiles for DF, DS, and DP cells. Gene set enrichment analysis ascertained significant changes in several age-related pathways. The age-related changes in hair follicle DS and DP cells were more striking than those of the DFs, and affected different pathways. In the older cohort there was a higher number of fibrosis related gene-sets in the DFs, while there was a loss of contractile markers in DS cells, and elevated DNA damage and senescence in DP cells. This study has identified female scalp DF, DS and DP cells are distinct and diverse fibroblast populations with little overlap at the transcriptional level, and all show a loss of cell signature and identity with age. We hypothesize that changes to these cells affect the ability of the scalp dermal environment to support and maintain healthy hair follicles, which contributes to the age-related changes observed in the female scalp.