Mesenchymal stem cell-derived extracellular vesicles in an ex vivo model of cutaneous wound healing

Abstract

Wound healing is a process to ensure the integrity of the skin and efficient barrier repair. Cell therapy and tissue engineering have been indicated as important strategies for treating wounds and generating skin substitutes and adjuvants that can accelerate epidermal repair and regeneration. Also, it has been demonstrated that stem cells can regenerate damaged skin tissue through their paracrine functions. Specifically, stem cell extracellular vesicles (EVs) were shown to stimulate wound healing process through vertical transfer of microRNAs (miRs). Different models have been proposed to study wound healing, however essential requirements are met by the ex vivo human skin model, which also provides important tools for understanding the mechanisms involved in cutaneous disorders and effects of topically applied therapeutics. Since the molecular features of miR indicate their potential application as diagnostic and prognostic markers for wound healing disorders, we are proposing here the use of ex vivo skin model to study RNA differential expression, in the presence or absence of stem cell-derived EV, with perspectives to improve outcomes, maximize treatments and reduce risks associated with skin pathologies and injuries. Human skin will be obtained from patients undergoing plastic surgery. A 3 mm wound will be created in a full thickness 8 mm skin punch all the way through the dermis. MSC will be cultured in conventional culture conditions. EVs (exosomes) will be extracted and purified by standardized procedures and analyzed by Western blot. Exosomes will be applied to each skin sample. Wounds collected at different time points will be histologically analyzed by hematoxylin-eosin and immune staining for keratin-6. RNA sequencing will be carried out using total RNA for the RiboZero Transcriptome Directional RNAseq sample prep and each sample will be sequenced to 40 million raw reads, demultiplexed and converted to FastQ. FastQ files will be analyzed by NIAMS Biodata Mining and Discovery Section. ANOVA will be performed to find differentially expressed genes, heatmap and volcano plots will be created with Partek Genomic Suites 7.18.0723. Pathway analysis will be conducted using Ingenuity Pathway Analysis (IPA). The knowledge generated in this project may provide useful tools for the study of skin biology that are essential for the development of therapeutic strategies for different diseases and conditions.