Cell-free secretome of CD56 ^bright CD16 ^bright directly reprogrammed NK cells enhances wound healing via CCL3/4/5-CCR5 signaling

<b>Rationale:</b> Natural killer (NK) cells are emerging as a promising source of immunomodulatory secretomes with regenerative potential. However, heterogeneity in primary NK cell populations limits the reproducibility of NK-derived cell-free therapies. To address this, we developed directly reprogrammed NK (drNK) cells with a stable CD56^brightCD16^bright phenotype and investigated the therapeutic potential of their conditioned medium (drNK-CM) in wound healing, focusing on underlying molecular mechanisms such as chemokine signaling and angiogenesis. <b>Methods:</b> drNK cells were generated by transcription factor-mediated reprogramming (OCT4, SOX2, KLF4, MYC) and characterized via flow cytometry and RNA-seq. The secretome profile of drNK-CM was evaluated using proteomic analysis. Human epidermal keratinocytes (HEKs), dermal fibroblasts (HDFs), and endothelial cells (HUVECs) were treated with drNK-CM to assess proliferation, migration, and extracellular matrix (ECM) remodeling. Chemokine receptor involvement was evaluated using CCR1, CCR3, and CCR5 antagonists. <i>In vivo</i> efficacy was tested in mouse excisional wound models, with histological and immunofluorescence evaluation of angiogenesis, re-epithelialization, and collagen deposition. <b>Results:</b> drNK-CM significantly promoted proliferation and migration of HEKs, HDFs, and HUVECs, accompanied by enhanced expression of Type I/III collagen, VEGF, and MMPs. Transcriptomic profiling revealed that drNKs uniquely upregulated genes associated with ECM remodeling, chemokine signaling (CCL3/4/5), and angiogenesis. Notably, CCR5 inhibition by maraviroc abrogated drNK-CM-induced cell migration and delayed wound closure <i>in vivo</i>, highlighting the central role of the CCL3/4/5-CCR5 axis. Furthermore, drNK-CM activated AKT and ERK pathways and promoted anti-inflammatory macrophage polarization. <i>In vivo</i> application of drNK-CM accelerated wound closure, improved neovascularization, and supported organized tissue regeneration compared to controls. <b>Conclusion:</b> This study demonstrates that drNK-CM enhances wound healing through coordinated actions on epithelial, stromal, and endothelial compartments. The reparative effects are primarily mediated via the CCL3/4/5-CCR5 signaling axis and pro-angiogenic cascades. Given their consistent phenotype and reproducible secretome, drNKs represent a scalable and safe source for cell-free regenerative therapeutics.