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Human Nrf2-Active Multipotent Stromal Cell Exosomes Reverse Pathologic Diabetic Wound Healing
Joseph Kuhn, MD, Absara Hassan, Sonali Sharma, Jennifer Kwong, BA, Montaha Rahman, Salma Adam, MD, Alvaro Villarreal-Ponce, PhD, Rafa Gras Pena, PhD, Daniel J. Ceradini, MD, Piul S. Rabbani, PhD.
NYU Langone Health, New York, NY, USA.

PURPOSE: Multipotent stromal cells (MSCs) have attracted much attention for their capacity to accelerate wound healing. However, there are no approved MSC based therapies. Exosomes, nanosized extracellular vesicles, may be key to translating MSC therapy to the bedside. We previously found that nuclear-factor-erythroid-2-related -factor-2 (Nrf2) regulates MSC multipotency and promotion of diabetic tissue repair. Here, we explore a novel role of Nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects MSCs have on diabetic wound healing.
METHODS: Whole bone marrow was subcultured from long bones of non-diabetic human donors. Adherent cells were characterized by microscopy, tri-lineage differentiation assays, and flow cytometry. Exosomes were harvested by differential ultracentrifugation of conditioned MSC media. For Nrf2-active exosomes, MSCs were incubated with potent Nrf2 activator, CDDO-Im. Exosomes were characterized by immunoblotting, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Full-thickness humanized-stented wounds were created on adult Leprdb/db diabetic mice (db/db). Exosomes were injected intradermally and circumferentially to the wound margin 1-day post-excision and photographed regularly until closure. Tissues were harvested at day 10 post-wounding for histological/biomolecular analysis.
RESULTS: MSCs adopt an adherent fibroblast morphology and demonstrate robust differentiation along osteogenic, chondrogenic, and adipogenic lineages in culture. >95% of MSCs express positive markers (CD44, CD73, CD90, and CD105) and less than 5% express negative markers (CD45, CD31, CD14, CD19, or HLA-DR). Immunoblotting of MSC exosomes shows enrichment for positive exosomal markers CD81, CD9 and TSG101 and no detection of negative markers, Calnexin and GM-130. NTA shows a nanoparticle population with mode diameter of 168.06.5nm. TEM of exosomes reveals flattened cup-like spheres and confirms the size determined by NTA. NTA demonstrates that Nrf2-activated human MSCs increase exosome secretion by 54%, compared to Nrf2-baseline MSCs (p<0.05). When administered to wounds, 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 of diabetic mice (p<0.05 for both treatments when compared to vehicle). Importantly, exosome treatment of diabetic wounds eliminated the delay in healing compared to C57/B6 wounds (16.6 days; p>0.05 compared to exosome-treated db/db wounds). Nrf2-active exosome treatment reduces closure time by 2.6 days compared to untreated C57/B6 wounds, though this benefit is nonsignificant. Histological analysis at day 10 shows that exosome-treated db/db wounds have significant decreases in epithelial gap and expanded granulation compared to vehicle-treated wounds. CD31 Immunoreactivity of 10 day wounds confirms a greater density of blood vessels in the wounds exosome-treated vs vehicle-treated diabetic wounds.
CONCLUSIONS: Enhancing Nrf2 function in MSCs multiplies exosome yield. Exosome therapy harnesses MSC ability to promote angiogenesis, a process critical for ensuring swift diabetic wound closure. MSC exosome-based therapies hold tremendous promise to improve chronic wound outcomes for patients with diabetes, and our results demonstrate the need for further investigation for rapid translation.


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