109 Fibroblast extracellular vesicles modulate keratinocyte differentiation during skin aging

Abstract

Small extracellular vesicles (EVs), ie, microvesicles and exosomes, play different roles in skin homeostasis and functions. Here, we want to clarify the interactions between fibroblasts and keratinocytes through EVs during skin aging. Small EVs were purified by ultracentrifugation from monolayer culture supernatants of dermal fibroblasts derived from breast skin of young adult donors (18-20 years old). Both particles mean size of 100 nm (NTA analysis) and expression of CD63, CD81 and TSG-101 (western blotting) allowed us to confirm an enrichment of small EVs in the purified samples. Fibroblast small EVs were then used to treat child/adult-derived keratinocytes grown in monolayer culture and 3D reconstructed epidermis (RE). In monolayer culture, keratinocyte proliferation was not modified by fibroblast small EVs as shown by DNA quantification in a 72-hours study. However, EVs affected keratinocyte differentiation according to the age of keratinocyte donors. Indeed, in monolayer culture, an increase of expression of late differentiation markers (IVL, LOR, FLG, TGM1) was observed in keratinocytes from child donors. In contrast, a decrease of expression of both early (KRT1/10) and late differentiation markers (LOR, FLG) was seen in keratinocytes from adult donors. Such changes were also observed in RE treated by fibroblast small EVs and confirmed by KRT10, LOR and IVL expression using immunofluorescence analysis. Moreover, the granular layer thickness was respectively increased and decreased in RE cultured with keratinocytes derived from child and adult donors. Finally, the mRNA expression of transcription factors involved in the control of keratinocyte differentiation (RUNX1, KLF4, GATA3) was also modulated by fibroblast small EVs treatment. Taken together, our work shows that fibroblast small EVs modulate keratinocyte differentiation and suggests that aging can deeply modify keratinocyte responses to fibroblast small EVs.