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Although human dermis contains distinct fibroblast subpopulations, the functional heterogeneity of fibroblast lines from different donors is under-appreciated. We identified one commercially sourced fibroblast line (c64a) that failed to express α-smooth muscle actin (α-SMA), a marker linked to fibroblast contractility, even when treated with transforming growth factor-β1 (TGF-β1). Gene expression profiling identified insulin-like growth factor 1 (IGF1) as being expressed more highly, and Asporin (ASPN) and Wnt family member 4 (WNT4) expressed at lower levels, in c64a fibroblasts compared to three fibroblast lines that had been generated in-house, independent of TGF-β1 treatment. TGF-β1 increased expression of C-X-C motif chemokine ligand 1 (CXCL1) in c64a cells to a greater extent than in the other lines. The c64a gene expression profile did not correspond to any dermal fibroblast subpopulation identified by single-cell RNAseq of freshly isolated human skin cells. In skin reconstitution assays, c64a fibroblasts did not support epidermal stratification as effectively as other lines tested. In fibroblast lines generated in-house, shRNA-mediated knockdown of IGF1 increased α-SMA expression without affecting epidermal stratification. Conversely, WNT4 knockdown had no consistent effect on α-SMA expression, but increased the ability of fibroblasts to support epidermal stratification. Thus, by comparing the properties of different lines of cultured dermal fibroblasts, we have identified IGF1 and WNT4 as candidate mediators of two distinct dermal functions: myofibroblast formation and epidermal maintenance.

Human scalp hair follicles (hHF) harbour several epithelial stem (eHFSC) and progenitor cell sub-populations organised into spatially distinct niches. However, the constitutive cell cycle activity of these niches remains to be characterized in situ . Therefore, the current study has studied these characteristics of keratin 15+ (K15), CD200+ or CD34+ cells within anagen VI hHFs by immunohistomorphometry, using Ki-67 and 5-ethynyl-2′-deoxyuridine (EdU). We quantitatively demonstrate in situ the relative cell cycle inactivity of the CD200+/K15+ bulge compared to other non-bulge CD34+ and K15+ progenitor compartments and found that in each recognized eHFSC/progenitor niche, proliferation associates negatively with eHFSC-marker expression. Furthermore, we also show how prostaglandin D2 (PGD2), which is upregulated in balding scalp, differentially impacts on the proliferation of distinct eHFSC populations. Namely, 24 h organ-cultured hHFs treated with PGD2 displayed reduced Ki-67 expression and EdU incorporation in bulge resident K15+ cells, but not in supra/proximal bulb outer root sheath K15+ progenitors. This study emphasises clear differences between the cell cycle behaviour of spatially distinct stem/progenitor cell niches in the hHF, and demonstrates a possible link between PGD2 and perturbed proliferation dynamics in epithelial stem cells.

Background SIRT1 is likely to play a role in the extension in healthspan induced by dietary restriction. Actions of SIRT1 are pleiotropic, and effects on healthspan may include effects on DNA methylation. Polycomb group protein target genes (PCGTs) are suppressed by epigenetic mechanisms in stem cells, partly through the actions of the polycomb repressive complexes (PRCs), and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation. We hypothesised that SIRT1 would affect DNA methylation particularly at PCGTs. To map the sites in the genome where SIRT1 affects DNA methylation, we altered SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA. DNA was enriched for the methylated fraction then sequenced (HuVEC) or hybridised to a human promoter microarray (Caco-2). Results The profile of genes where SIRT1 manipulation affected DNA methylation was enriched for PCGTs in both cell lines, thus supporting our hypothesis. SIRT1 knockdown affected the mRNA for none of seven PRC components nor for DNMT1 or DNMT3b. We thus find no evidence that SIRT1 affects DNA methylation at PCGTs by affecting the expression of these gene transcripts. EZH2, a component of PRC2 that can affect DNA methylation through association with DNA methyltransferases (DNMTs), did not co-immunoprecipitate with SIRT1, and SIRT1 knockdown did not affect the expression of EZH2 protein. Thus, it is unlikely that the effects of SIRT1 on DNA methylation at PCGTs are mediated through direct intermolecular association with EZH2 or through effects in its expression. Conclusions SIRT1 affects DNA methylation across the genome, but particularly at PCGTs. Although the mechanism through which SIRT1 has these effects is yet to be uncovered, this action is likely to contribute to extended healthspan, for example under conditions of dietary restriction.