Exosomes, nanosized extracellular vesicles, may be key to translating multipotent stromal cell (MSC) therapy to the bedside. We previously found that nuclear factor erythroid 2-related factor 2 (Nrf2) regulates MSC promotion of tissue repair in diabetes. Here, we explore a novel role of Nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects MSCs have on wound healing in mice with type 2 diabetes. Exosomes were harvested by differential ultracentrifugation of conditioned human bone marrow derived MSC media. For Nrf2-active exosomes, MSCs were incubated with potent Nrf2 activator, CDDO-Im. MSC characterization demonstrated robust tri-lineage differentiation, >95% expression of positive markers (CD44/CD73/CD90/CD105/CD106), and <5% expression of negative markers (CD45/CD31/CD14/CD19/HLA-DR). Immunoblotting of exosome isolates demonstrates enrichment for CD81, CD9 and TSG101. Nanoparticle tracking analysis demonstrates that Nrf2-active MSCs increase exosome secretion by 54%, compared to Nrf2-baseline MSCs (p<0.05). Exosomes were injected into the wound margin of full-thickness wounds on Leprdb/db (db/db) mice. Both Nrf2-baseline and Nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p<0.05). This reduction eliminated the delay in closure time compared to wounds of C57/B6 mice. Nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated C57/B6 wounds. At day 10, exosome treated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of CD31+ vessels compared to vehicle-treated wounds. We conclude that activating Nrf2 in MSCs increases isolation efficiency by amplifying exosome yield and content. MSC exosome based therapies have potential for rapid translation to improve clinical wound healing outcomes.
J. Kuhn: None. A. Hassan: None. S. Sharma: None. J. Kwong: None. M. Rahman: None. S. Adam: None. J. Lee: None. A.P. Villarreal Ponce: None. P.S. Rabbani: None.
Plastic Surgery Foundation; Wound Healing Society; New York University Clinical and Translational Science Institute