Abstract
BACKGROUND: Regenerative medicine therapies offer hope for conditions previously limited to supportive care. The COVID-19 pandemic underscored the urgent need for treatments targeting systemic inflammation. Mesenchymal stem cells (MSCs) are widely recognized for their regenerative and immunomodulatory properties. Recent research shows that hypoxic priming enhances MSCs' therapeutic potential, particularly by increasing the release and modifying the cargo of their extracellular vesicles (EVs). These EVs imitate many of the regenerative and immunomodulatory effects of MSCs, making them a promising cell-free therapeutic option. This study aimed to compare the regenerative and immunomodulatory capacities of small EVs (sEV; less than 200 nm) derived from bone marrow (BM) and wharton's jelly (WJ) MSCs, cultured under normoxic and hypoxic conditions, and decipher their underlying functional mechanisms. METHODS: BM and WJ-MSCs were isolated and expanded. Hypoxia exposure was provided at 1% oxygen concentration for 24 h. sEV were isolated using ultracentrifugation, and in vitro functional assessments were performed using skin specific human sourced cell lines for fibroblasts, keratinocytes and macrophages. miRNA modulations were performed via transfection technique and mechanism of action of sEV were determined. Additionally, in vivo analysis was performed in a traumatic wound rat model, and molecular pathway analysis was performed in animal wound tissues. RESULTS: Through comprehensive in vitro and in vivo evaluations, this study aimed to identify the most potent candidate for regenerative medicine while elucidating the underlying molecular mechanisms. By analysing sEV cargo and the associated molecular pathways within a traumatic wound model, the study revealed that WJ-MSCs exhibit superior regenerative potential. Furthermore, it demonstrated that hypoxia priming enhances their therapeutic efficacy through the involvement of miR-125b-5p and miR-145a-5p, which drive immunomodulation and regeneration by targeting IL-6R/NFκB axis and TGF-β2/SMAD4 pathway, respectively. These findings underscore the significance of WJ-sEV as a promising avenue for personalized regenerative therapies, positioning miR-125b-5p and miR-145a-5p as key therapeutic targets for bioengineering of sEVs. CONCLUSION: By exploring these comparative functions, the study provides insights into the optimization of sEV-based therapies as a cutting-edge approach in regenerative medicine, and reflects the suitability of WJ-sEV as an ideal acellular therapeutics' candidate for translational regenerative and immunomodulatory applications.