The treatment of chronic or hard-to-heal skin wounds remains a significant challenge in healthcare, with an estimated annual cost of approximately 60 billion euros in Europe for wound management.

Cutaneous wound healing requires coordinated activation of stem cells, epidermal cells, and extracellular signaling networks. While most wounds are resolved efficiently, impaired healing remains a major clinical challenge. Increasing evidence suggests that regenerative capacity is governed not only by cellular presence but by dynamic regulation of gene regulatory programs.

This thesis investigates how epigenetic modulation influences regenerative behavior in adipose-derived stem cells (ASCs) and keratinocytes in a wound-healing context. By targeting histone acetylation using the histone deacetylase inhibitor, SAHA, and analyzing microRNA-mediated regulation. In parallel, proteomic analyses were performed to explore the skin proteome using the Total Protein tissue Extraction reagent (TPER) as an efficient isolation method in clinical settings.

The findings demonstrate that SAHA modulates ASCs behavior by altering chromatin structure, DNA replication, and cell fate determination. The microRNA-based approach showed that key signaling pathways, such as those involving epidermal growth factor (EGF) and interleukin-1 alpha (IL-1α), are implicated in promoting skin cell proliferation and repair. Furthermore, skin proteomic analyses, including TPER reagent for protein isolation, may enhance clinical wound treatment.

Together, these results advance the understanding of the interplay between gene regulation and protein function in skin healing and the development of skin regeneration future therapies.

Behandling av kroniska eller svårläkta sår är fortfarande en betydande utmaning inom hälso- och sjukvården. I Europa uppskattas den årliga kostnaden för sårbehandling till cirka 60 miljarder euro.

Hudens sårläkning är en komplex process som kräver samordnad aktivering av stamceller, epidermala celler och extracellulära signalsystem. Även om de flesta sår läker effektivt utgör försämrad sårläkning fortfarande en stor klinisk utmaning. Forskning visar i allt högre grad att vävnadens regenerativa kapacitet inte enbart beror på vilka celler som finns närvarande, utan även på hur genuttrycket regleras dynamiskt.

Denna avhandling undersöker hur epigenetisk reglering påverkar regenerativ kapacitet hos stamceller från fettvävnad (ASC) och keratinocyter vid sårläkning. Fokus har varit på histonacetylering med hjälp av histondeacetylashämmaren Vorinostat samt analys av mikroRNA-medierad genreglering. Parallellt genomfördes proteomikanalyser för att undersöka proteinprofiler med hjälp av Total Protein Extraction Reagent (TPER) som en potentiellt effektiv metod för proteinisolering i kliniska miljöer.

Resultaten visar att Vorinostat påverkar stamceller från fettvävnad genom att förändra kromatinstrukturen, DNA-replikationen och cellernas differentiering. Studier av mikroRNA visade att viktiga signalvägar, inklusive de som involverar epidermal tillväxtfaktor (EGF) och interleukin-1 alfa (IL-1α), spelar en viktig roll för att främja hudcellers proliferation och reparation. Dessutom visade proteomikanalyser att TPER kan vara en lovande metod inom klinisk sårbehandling.

Sammantaget bidrar resultaten till en ökad förståelse för samspelet mellan genreglering och proteinfunktion vid hudläkning och kan bidra till utvecklingen av framtida behandlingar.

Introduction Regenerative solutions of the skin represent a hope for burn victims with extensive skin loss and chronic wound patients. The development of xeno-free workflow is crucial for clinical application in compliance with the directives of the European Medicines Agency. This study aimed at evaluating the outcome of the xeno-free isolation workflow of keratinocytes from human skin biopsy. Methods Skin biopsies were obtained from volunteers. The epidermis was digested with TrypLE™ Select, which was deactivated by dilution or with trypsin, deactivated by media with fetal bovine serum. Freshly isolated cells were compared for total cell number, viability, activity of caspase 3, gene expression and the presence of the keratinocyte surface markers cytokeratin 14. The cells were cultured in xeno-free conditions for one week and characterized regarding the number and viability as well as the metalloproteinase secretion. Results The number of obtained cells was similar in both workflows. The cell viability was less in the TrypLE group, with slight reduction of the cell surface marker cytokeratin 14. Caspase 3 activity was comparable as well as the gene expression of the apoptotic markers BAX, BCL2 and SLUG, as well as the keratinocyte markers cytokeratin 14, stratifin and filaggrin. Upon culture, the number of keratinocytes, their viability and secretion of matrix metalloproteinases 1 and 10 were equal in both groups. Conclusion This study reports the possibility of isolating functioning and viable keratinocytes through a xeno-free workflow for clinical application.