Optimizing a 5-factor cocktail to prepare reparative macrophages for wound healing

The treatment of nonhealing wounds, such as diabetic ulcers, remains a critical clinical challenge. Recent breakthroughs in cell therapy have shown great promise, with one primary focus on preparing cells with comprehensive reparative functions and foreseeable safety. In our previous study, we recapitulated the proregenerative and immunosuppressive functions of tumor-associated macrophages in non-tumor-derived macrophages, endowing the latter with characteristics for promoting diabetic wound healing-termed tumor-associated macrophage-educated macrophages. To eliminate the use of tumor-derived sources and devise a more controllable method to prepare tumor-associated macrophage-educated macrophage-like cells, in this study, we identify a cocktail comprising 5 recombinant proteins as an essential condition to induce nonpolarized macrophages into therapeutic cells with prohealing functions. The screened 5 factors are osteopontin, macrophage inflammatory protein 2, chemokine (C-C motif) ligand 8, vascular endothelial growth factor B, and macrophage colony-stimulating factor. We demonstrate the rationale for screening these factors and the phenotype of the 5 factor-induced tumor-associated macrophage-educated macrophage-like macrophages prepared from murine bone marrow-derived macrophages, which exhibit angiogenic and immunomodulatory effects in vitro. Then, we induce primary human monocytes from periphery blood into the 5 factor-induced tumor-associated macrophage-educated macrophage-like macrophages, which show prohealing effects in a human primary cell-based ex vivo model (T-Skin (TM)). Our study demonstrates a simple, effective, and controllable approach to induce primary macrophages to possess repairing activities, which may provide insights for developing cell-based therapeutics for nonhealing wounds clinically. A 5-cytokine cocktail induces primary macrophages to exert prohealing activities.