Extracellular Vesicles from Adipose-Derived Mesenchymal Stem/Stromal Cells Accelerate Migration and Activate AKT Pathway in Human Keratinocytes and Fibroblasts Independently of miR-205 Activity

Abstract

Mesenchymal stem/stromal cells (MSCs) are promising tools in cell therapy. They secrete extracellular vesicles (EVs) that carry different classes of molecules that can promote skin repair, but the mechanisms are poorly understood. Skin wound healing is a complex process that requires the activity of several signaling pathways and cell types, including keratinocytes and fibroblasts. In this study, we explored whether adipose tissue MSC-derived EVs could accelerate migration and proliferation of keratinocytes and fibroblasts, activate the AKT pathway, and promote wound healing in vivo. Furthermore, we evaluated if EV effects are miR-205 dependent. We found that MSC EVs had an average diameter of 135 nm. Keratinocytes and fibroblasts exposed to EVs exhibited higher levels of proliferation, migration, and AKT activation. Topical administration of EVs accelerated skin wound closure. Knockdown of miR-205 decreased AKT phosphorylation in fibroblasts and keratinocytes, whereas migration was decreased only in keratinocytes. Moreover, knockdown of miR-205 failed to inhibit AKT phosphorylation in fibroblasts and keratinocytes exposed to EVs. About the mechanism of EV effects, we found that incubation with EVs prevented inhibition of AKT activation by miR-205 knockdown, suggesting that EVs activate AKT independently of miR-205. In conclusion, we demonstrated that EVs are a promising tool for wound healing.

Mesenchymal Stem Cell Derived Extracellular Vesicles for Tissue Engineering and Regenerative Medicine Applications

“…Specifically, collagen I and III distributions secreted by fibroblasts were increased in the early stage of wound healing while, in the late stage, collagen synthesis was diminished to reduce scar formation [152]. EVs from the same source were reported to trigger the migration and proliferation of keratinocytes and fibroblasts in vitro and in vivo in excisional wound-splinting rat models, by a mechanism involving the activation of Akt pathway [153]. lncRNA metastasis-associated lung adenocarcinoma transcript 1 found in human ADMSC-EVs contributed significantly to the migration and proliferation of dermal fibroblasts, promoting wound closure in a rat model of ischemic wound healing [154].…”

Secretome of Adipose Tissue-Derived Stem Cells (ASCs) as a Novel Trend in Chronic Non-Healing Wounds: An Overview of Experimental In Vitro and In Vivo Studies and Methodological Variables

“…Accordingly, angiogenesis-stimulating genes were also significantly upregulated with Exos from hypoxic conditions while angiogenesis inhibitory genes were downregulated. The group of Ferreira et al [76] explored the ability of ASC-EVs to accelerate in vitro migration, proliferation, and protein kinase B (Akt) pathway activation in primary human keratinocytes and HDFs. ASCs isolated from human subcutaneous adipose tissue, at passage 4 or 5, were incubated with serum-free DMEM for 48 h at 37 • C, after which CM was submitted to multiple centrifugation steps at 4 • C; that is, 300× g for 7 min, 1000× g for 15 min, and 100,000× g for 70 min.…”

“…Increased cell proliferation and migration rate. Upregulated Akt pathway [76] Human Lipoaspirates (mean age of the patients: 20 ± 1.5 years).…”

“…Of interest, intravenous injection of Exos was significantly more effective in promoting wound repair than local injection. The Wistar rat excisional wound-splinting model was used by the group of Ferreira et al [76] to evaluate the ability of ASC-EVs to promote skin repair. ASCs isolated from human subcutaneous adipose tissue, after expansion in complete medium, were incubated with serum-free DMEM for an additional 48 h, after which the CM was submitted to successive centrifugation steps, as yet described in Section 3.1.…”

Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display

“…Exosomes function as shuttles with intercellular signaling capabilities. These EVs promote cancer metastasis [ 7 , 8 ], play crucial roles in embryonic development [ 9 , 10 ], modulate immune responses [ 11 , 12 ], accelerate soft tissue wound healing [ 13 , 14 ], improve skeletal myopathy in Duchenne muscular dystrophy (DMD) models [ 15 ], and support heart repair after myocardial infarction (MI) [ 16 , 17 ]. Here, we refer to the collective extracellular fraction of vesicles, including exosomes, as EVs, and routinely characterize our EV preparations for vesicle size, concentration, RNA content and surface protein phenotype.…”