Growth Factor-Primed WJ-MSC Secretome Enhances Fibroblast Expansion <em>In Vitro</em>

Background: In regenerative medicine, there is interest in using acellular therapy based on the secretome of mesenchymal stem cells (MSC) to promote wound healing. Wharton's jelly cells (WJ-MSCs) are a readily available source. Their secretion has been optimized when stimulated with bFGF and EGF to induce proliferation and prevent senescence. Therefore, evaluating the effect on proliferation and wound closure of human fibroblasts in vitro with different concentrations of the secretome of WJ-MSCs stimulated with growth factors is necessary to identify the most efficient work concentration. Methods: The secretome of human WJ-MSC was collected from passage 1 to passage 2 stimulated with bFGF and EGF (W bFGF/EGF) and the unstimulated secretome (WO bFGF/EGF). The immunophenotype of WJ-MSCs after stimulation was evaluated by flow cytometry for the markers: CD105+, CD73+, CD90+, HLA-ABC+, CD44+, HLA-DR-, CD34-, CD11b-, CD19-, and CD45-. The presence of 14 growth factors in the secretome was evaluated using LEGENDplex through flow cytometry. Fibroblasts were cultured, and their culture medium was supplemented with two different concentrations: one of 1.25 mg/ml and another of 6.25 mg/ml of both stimulated and unstimulated secretome. Proliferation, cellular metabolism, and wound closure were evaluated in vitro. Results: The immunophenotype of WJ-MSCs after stimulation remained unchanged, and the production of growth-assessed factors was increased in stimulated WJ-MSCs. The optimal concentration that induced proliferation and wound closure in vitro was 1.25mg/ml of stimulated WJ-MSC secretome. Conclusions: This study demonstrates that stimulation of WJ-MSCs with FGF and EGF enhances the secretion of growth factors, and that a concentration of 1.25 mg/ml of their secretome promotes optimal fibroblast proliferation and wound closure in vitro. These findings support the potential of optimized WJ-MSC secretome as a promising acellular strategy for regenerative medicine.