Epidermal Stem Cell Derived Exosomes Improve Diabetic Wound Healing By Modulating Fibroblast And Macrophage Functions
Georgios Theocharidis, Peng Wang, Antonio Lobao, Biaoliang Wu, Aristidis Veves.
Beth Israel Deaconess Medical Center, Boston, MA, USA.
Despite advancements in regenerative medicine, healing of diabetic foot ulcers (DFU) still remains a challenging clinical problem. Exosomes are nanosized vesicles released by different cells and implicated in numerous physiological processes. Epidermal stem cells (ESC) reside in the basal epidermal layer and have been shown to contribute to cutaneous wound healing. However, whether they secrete exosomes which also influence the wound repair process has yet to be reported.
To investigate the role of ESC derived exosomes in DFU healing, we first isolated ESC from telogen murine skin. Purity of ESC was demonstrated with Itga6/Cd71 double staining. Exosome isolation was achieved through ultracentrifugation of ESC conditioned media and their presence was confirmed via transmission electron microscopy imaging, nanoparticle tracking analysis and western blot detection of teraspanin exosomal markers CD63 and CD81.
RNA-seq analysis revealed the presence of 87 miRNAs in the ESC exosomal cargo. Exosomes were internalized by primary mouse fibroblasts and macrophages in vitro, as evidenced by PKH-67 dye labelling and increased cell proliferation measured with Ki67 staining and alamar blue assay in a dose-dependent manner. In addition, they accelerated wound closure in a diabetic fibroblast scratch assay model and induced alternative or M2 polarization of macrophages with elevated expression of Ym1, Cd206 and Arg1 markers. Strikingly, intradermal injection of ESC exosomes in the wound edges of db/db mice led to 49% improved wound closure (n=10, p<0.01). Histological evaluation revealed increased vascularization, cell proliferation and M2 macrophages presence.
In summary, we report for the first time detailed characterization of ESC exosomes and demonstrate their in vivo efficacy in improving diabetic wound healing. They could therefore be an alternative to cell therapy and be employed as a novel treatment for DFU.
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