Fine-Tuning of Organotypic Skin Equivalents for Preclinical Research and Their Utilization to Study Epidermal-Dermal Crosstalk

Abstract

With the studies performed for realization of this thesis, more insights into the complex intercellular crosstalk between epidermal keratinocytes and dermal fibroblasts were gained. These data demonstrated that dermal fibroblasts are critical for adequate epidermal differentiation and maturation and provided new insights into signalling pathways involved in these processes. When fibroblasts were lacking, consistently an impaired differentiation and dysregulated expression of skin barrier and tight junction proteins, increased skin permeability, and a decreased skin lipid/protein ratio have been shown. Most interestingly, impaired Ras/Raf/ERK/MEK signalling was evident when fibroblasts were not present in the skin equivalents, meaning that fibroblasts orchestrate epidermal differentiation processes. Furthermore, this project, utilised and confirmed the methods for EVs isolation and characterization from cell culture medium which is still under development. Here, it has been proved that fibroblasts-derived EVs – exosomes, which were present in fibroblasts condition medium. A more detailed analysis of these EVs should be of importance for further detailed understanding of the role EVs play in intercellular communication between fibroblasts and keratinocytes. The results described in this thesis demonstrated development of completely human-based skin equivalents with dermal equivalent based on primary human fibroblasts-derived ECM with included endothelial cells. These skin equivalents showed features of excessive epidermal differentiation and maturation potentially conditioned by growth factors produced by fibroblasts and endothelial cells from dermal compartment that have direct impact on keratinocytes and/or the keratinocytes themselves as a result of their cellular interactions in the co-culture. New insights about the effect of endothelial cells on epidermal differentiation and their interactions with keratinocytes and fibroblasts have been noticed. Although the authentic fibroblast-derived ECM and endothelial cells have impact on epidermal morphogenesis, they have also shown a key role in the development of complex dermo-epidermal junction in the skin equivalents leading to their resemblance to native human skin. Due to the better understanding of the influence of endothelial cells on keratinocyte differentiation, this study could be helpful for the development of fully vascularised skin equivalents.