Mathematical modeling of the re-epithelialization of a 3 dimensional (3d) human skin equivalent

Re-epithelialization is the dominant mode of healing in partial thickness wounds. In order to develop better strategies to monitor such wound healing, 3D HSE models and mathematical models have long been utilized to improve our understanding of epidermis formation and re-epithelialization. Using both approaches we endeavoured to generate two mathematical models of epidermis formation and one mathematical model of wound closure based on in vitro studies of epithelialization of 3D HSE models and re-epithlialization of wounded HSE models. We used immunohistochemistry to evaluate keratinocyte proliferation, differentiation and migration. The mathematical models investigated first the role of nutrient concentration (simple model of epidermis formation) and the concurrent roles of ATP, nutrient and calcium concentrations (improved model) in epidermis formation and homeostasis, to eventually lead to a two-dimensional model of wound closure. The 3D HSE model presented similar histology and immunohistochemistry as that of native skin after 11 days of culture. A complete differentiated epidermis filled the wound by day 14 after injury. Finally the contribution of basal cell proliferation in the neoepithelium has been demonstrated during wound closure. The improved mathematical model of epidermis formation produced reasonable representations with a complex cell layer dynamic, while the initial mathematical model presented the potential role of nutrient concentration in determining epidermis thickness. The model of wound closure further illustrated the critical role of nutrient concentration in the process of re-epithelialisation. The major drawback of existing wound closure models is that they neglect the differentiation and stratification; the models developed in this project innovatively integrates by both the second dimension and differentiation, and could consequently bring more insights into the regulation of epidermis homeostasis and wound repair

RhoV, a Potential Notch Target Gene in Murine Skin, Does not Play an Essential Role in Epidermis Development and Homeostasis

April Bezdek Pomey

The evolutionarily conserved Notch signaling cascade plays a pivotal role in the regulation of many fundamental processes such as stem cell maintenance, proliferation, and differentiation. Recently, we aimed to identify downstream target genes regulated by Notch1 during epidermal differentiation. For this purpose an Affymetrix mouse gene chip array of 14.000 genes was performed in both Notch1 gain and loss of function experiments using murine primary keratinocytes. Thirteen genes positively or negatively regulated, based on expression levels and relevance of the involved signaling pathways, were further validated by real-time PCR. Among these, we decided to focus on the Rho GTPase RhoV because it showed robust up- and down-regulation under both gain- and loss-of-function conditions. Therefore, we generated different transgenic mice expressing either a dominant active or a dominant negative form of RhoV specifically in the skin. Morphological and histological analyses did not reveal any overt skin phenotype in transgenic mice. In addition, challenging the system by performing wound healing assays demonstrated that the healing process in transgenic mice occurred normally and that the migration and/or differentiation of keratinocytes was unimpaired. Taken together, our data suggest that gain or loss of RhoV function does not perturb skin development and homeostasis, and does not affect the wound healing process. In addition to the investigation of the potential Notch target gene RhoV, we sought to generate both a floxed Jag2 gene targeted mouse line and a Notch2-specifc reporter mouse. The former was intended to enable conditional deletion of the Notch ligand Jagged2 and therefore allow its role to be studied in a loss-of-function context. The Notch2 reporter mouse is a tool enabling the in vivo study of cells that receive a Notch2-mediated signal. The strategy employs elements of a Tet-Off system and allows cells receiving a Notch2 specific signal to be identified in any tissue at any given time point.

EPFL2010

RhoV, a Potential Notch Target Gene in Murine Skin, Does not Play an Essential Role in Epidermis Development and Homeostasis

April Bezdek Pomey

EPFL2010