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Élodie Metral

Response of keratinocyte stem cells and progenitors to UVA radiation: Implication of DNA damage and repair systems and new strategies of photoprotection

Published on 24 April 2017


Thesis presented April 24, 2017

Abstract:
Skin is daily exposed to sun radiation. Among them, UVA reach the basal layer of epidermis composed of keratinocyte stem cells (KSC) and progenitors commonly called transitory amplifying cells (TA). KSC and TA, responsible of the epidermal renewal, are sensitive to genotoxic agents and more particularly to UV. Indeed, KSC, usually quiescent but composing the stem cell pool all lifelong, as well as TA, are specific targets for photocarcinogenesis and photoageing. In this context, the aim of the project was to develop a method able to isolate KSC and TA for characterizing their response to UVA, and in a more industrial objective, to value a photoprotective and/or genoprotective ingredient by investigating their mechanisms of action. A parallel aim was to define culture conditions suitable for maintaining the stemness of KSC in culture, which is quickly lost from their enter into a proliferative state. Thus we showed that adjunction of adipose derived stem cells (ASC) to fibroblasts in dermis of a skin equivalent model (SE) in order to reproduce the physiological environment of KSC, significantly increases the thickness of the epidermis and preserves the keratinocytes from their senescence. ASC act partially via the increase of fibroblasts proliferation in dermis and potentially via a synergic effect of factors secreted by the combination of ASC and fibroblasts. To compare the behavior of KSC to the one of TA, we firstly optimized the rapid adhesion method; then compared it to the cell sorting by flow cytometry following the alpha6high/CD71low phenotype, which appeared more efficient. Thus, KSC (alpha6high/CD71low) and TA (alpha6high/CD71high) were then irradiated to UVA. KSC showed a photoresistance compared to TA with a better cell viability and a clonogenic potential superior as well as a better ability to reconstruct a pluristratified epidermis in vitro. We also investigated mechanisms of resistance. Our results demonstrate that the induction of the three types of DNA damage immediately induced by UVA is similar in both populations, but that the repair of single strand breaks (SSB) and of thymin dimers (CPDs) is faster for KSC. Finally, PE1, ingredient preselected by Gattefossé was characterized for its photoprotective and genoprotective effect. We showed that PE1 is able to i) preserve capacity of keratinocytes to form holoclones after UVA radiation, ii) decrease DNA damage, notably 8oxoGuanin and CPDs, and iii) improve several repair genes expression and activities. To conclude, this thesis project showed for the first time that KSC are more resistant to UVA radiation than their direct progeny, TA notably via improved DNA repair systems. Moreover, it allowed to identify a plant extract (PE1) able to protect genome of proliferative keratinocytes to UVA radiation.

Keywords:
Keratinocyte stem cell, Progenitor, UVA, DNA damage, Photoprotection, Genoprotection