King's College London

Dermal fibroblasts arise from a multipotent progenitor, forming two clear populations: the papillary (upper) dermis and the reticular/pre-adipocyte (lower) dermis. The contribution of each population to hair follicle formation (papillary) and early wound healing (reticular/preadipocyte) has been well characterised in the developing mouse dermis. Recent work has identified markers of fibroblast heterogeneity in human dermis. Transforming growth factor- β1 (TGF-β1) promotes fibroblast-to-myofibroblast differentiation, characterised by the expression of α-smooth muscle actin (α-SMA). Myofibroblasts are important for successful wound healing but have also been associated with skin abnormalities including hypertrophic/keloid scarring and systemic sclerosis. Normal human dermal fibroblasts (NHDF), treated with TGF-β1, were assayed for differentiation, proliferation and cell shape using the Operetta imaging system. One donor NHDF, derived from female 64-year-old breast skin, expressed decreased levels of α-SMA protein. Added to a skin reconstitution assay, this donor NHDF failed to support epidermal stratification. The gene expression profile of this donor NHDF was determined using the Agilent microarray system. Four gene candidates (Asporin, ASPN; C-X-C motif chemokine ligand 1, CXCL1; Insulin-like growth factor 1, IGF1; and Wnt family member 4, WNT4) were chosen based on expression values and validated by TaqMan qPCR. Successful knockdown of IGF1 and WNT4 was achieved using MISSION shRNAbased lentiviral treatment. Fibroblast IGF1 knockdown (shIGF1) increased α-SMA mRNA and protein expression; no effect was seen with fibroblast WNT4 knockdown (shWNT4). shWNT4 increased epidermal stratification in skin reconstitution assays; no effect was seen with shIGF1. Here I have characterised NHDF phenotype and gene expression with regard to population heterogeneity. This work highlights the role of IGF-1 signalling on α-SMA expression and dermal fibroblast fate. Targeting IGF/Wnt signalling could provide therapeutic benefit for skin disorders involving aberrant wound healing and excessive fibrosis.