Wound healing is a multifaceted biological process that requires tightly regulated cellular activities, including proliferation, migration, and extracellular matrix remodeling. Mesenchymal stromal cells (MSCs) derived from Wharton's Jelly (WJMSC) have emerged as powerful agents in regenerative medicine, largely attributed to their secretome, a dynamic mixture of bioactive molecules and extracellular vesicles. Recent advancements suggest that priming MSCs can potentiate their secretome's therapeutic efficacy. However, the ability to selectively enhance wound repair mechanisms via tailored priming remains unexplored. In this study, we introduce recombinant netrin-1, a multifunctional guidance cue known for its dual role in angiogenesis and immune modulation, as a novel priming agent for WJMSCs. We demonstrate that netrin-1 priming significantly enriches the WJMSCs secretome, amplifying its regenerative potential. Conditioned media collected post 48 hours of netrin-1 PRIMING markedly enhanced fibroblast proliferation and migration, boosted angiogenic tube formation in HUVECs, and skewed macrophage polarization toward an anti-inflammatory M2 phenotype, required as key processes that orchestrates effective tissue repair. This study clearly establishes for the first time the role of netrin-1 priming in creating a functionally superior WJMSCs secretome that targets multiple facets of wound healing synergistically. Unlike conventional single-factor treatments, this approach leverages the natural versatility of MSC secretome augmented with a well-characterized bioactive cue, offering a sophisticated and cell-free therapeutic avenue. The netrin-1 WJMSC secretome presents a promising platform with translational potential to address complex wound healing challenges, especially chronic and non-healing wounds with persistent inflammation and impaired angiogenesis. Altogether, the N1-primed WJ-MSC secretome represents a promising, cell-free therapeutic strategy for enhancing wound healing and tissue regeneration. Future studies will focus on the development of targeted delivery systems to optimize their therapeutic efficacy in preclinical wound models, paving the way for advanced regenerative medicine interventions.
