HUC-MSC-derived exosomal miR-16-5p attenuates inflammation via dual suppression of M1 macrophage polarization and Th1 differentiation.

Nowadays mesenchymal stem cell-derived exosomes (MSC-Exos) have emerged as a promising cell-free therapeutic alternative to MSC-based therapies, demonstrating efficacy in treating degenerative diseases, inflammatory disorders, and autoimmune diseases. MSC-Exos transport bioactive cargoes such as proteins, lipids, mRNAs, and microRNAs (miRNAs) to the recipient cells, mediating intercellular communication to regulate immunomodulation and tissue repair. However, the exosomal miRNA profile varies dynamically based on the culture conditions and tissue sources. Thus, elucidating the specific exosomal miRNA profile and regulatory targets is critical for the precise clinical applications and development of MSC-Exos-based cell-free therapies. Here we established an optimized serum-free culture system for human umbilical cord-derived MSCs (hUC-MSCs) and determined the critical 48-72-h harvest window for exosome secretion. High-throughput sequencing identified miR-16-5p as the predominant exosomal miRNA, functioning as a core immunosuppressive effector by suppressing LPS/IFN-γ-induced M1 macrophage polarization and Th1 cell differentiation. Mechanistically, miR-16-5p was found to target key nodes in NF-κB and JAK-STAT pathways, validated via dual-luciferase assays. Additionally, miR-125b-5p and miR-34a-5p enhanced this immunosuppressive effect by co-targeting overlapping pathway components in NF-κB and JAK-STAT pathways, suggesting a multilayered regulatory network. Taken together, our findings highlight the potential of miRNA-engineered exosomes as standardized therapies for inflammatory disorders, emphasizing the importance of optimizing culture conditions and profiling miRNA expression over time in advancing clinical translation.