Abstrakt

Background Epidermal progenitor cells (EPCs) have been under extensive investigation due to their increasing potential of application in medicine and biotechnology. Cultured human EPCs are used in the treatment of chronic wounds and have recently became a target for gene therapy and toxicological studies. One of the challenges in EPCs culture is to provide a high number of undifferentiated, progenitor cells displaying high viability and significant biological activity. Objectives The goal of this study was to characterize the in vitro cultured progenitor cells and the assessment whether the cells with the progenitor phenotype are able to enhance wound healing. Additionally, we aimed to estab lish the complete procedure of the culture, analysis and clinical application of epidermal progenitor cells. Methods In this study we present a method of cell isolation and culture followed by a technique of transplantation of the cultured cells onto the wound bed. The applied isolation technique involves two enzymatic steps (dispase, trypsin) and it is characterized with a high yield of cells. The obtained cells were cultured in vitro up to the second passage in serum-free and xeno-free keratinocytes-dedicated medium. Key stem cell markers were determined with means of flow cytometry and quantitative real-time PCR. Results The in vitro expanded cells displayed high proliferative activity without features of either apoptosis or necrosis. The flow cytometry and transcriptomic analyses showed the enhanced expression of stem cell mark ers (i.e. ΔNp63, CD29, CD49f and BNC1, CDKN1A transcripts) in the expanded cells. In the presented com passionate use study, cultured autologous cells from an oncological patient were suspended in fibrin sealant and transplanted directly to a non-healing wound, resulting in wound closure within 2 months. Conclusion The cells cultured in serum-free media display epidermal stem cells features and a potential to stimulate wound healing. This promising procedure of isolation, culture and application warrants further clinical trials in the treatment of chronic wounds.

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