MSC-derived exosomes reduce oxidative damage and scarring to promote wound regeneration

Author(s):
Ayesha Aijaz; Dea Metko; Marc Jeschke

Background:

Wound regeneration is a dynamic and complex process that requires a collaborative effort between the extracellular matrix, skin-resident cells and growth factors. Wound healing and closure continue to be the rate-limiting factors for survival from burn wounds. Mesenchymal stem cells (MSC) have emerged as promising therapy for wound healing. Contrary to the initial hypothesized mechanism of action that stem cells exert their regenerative potential by differentiating into cells required to replace damaged cells, more and more studies indicate that the observed therapeutic benefit associated with MSC therapy may be attributed to paracrine signaling mediated by secreted factors and extracellular vesicles (EVs). EVs and exosomes present several advantages over transplantation of cells.

Hypothesis:

Herein, we hypothesize that MSCs harvested from burn tissue are primed and the thermal injury affects the nature and functionality of the secretome conducive to burn wound healing.

Methods:

MSCs derived from burn tissues and umbilical cords were cultured in vitro and supernatant was collected. Exosomes from the supernatant were purified using polyethylene glycol. Exosome markers and exosome protein concentration were quantified. Exosomal bioactivity was assessed via acetylcholinesterase activity, oxidative stress, scratch, and TGF-b1 induced fibroblast to myofibroblast differentiation assays.

Results:

Presence of purified exosomes was confirmed by expression of HSP 90, CD81 and CD63 (Fig 1A). No significant difference was observed in protein concentration (PC) or acetylcholinesterase activity (AA) between exo UC (PC:9.01 ± 2.7 mg/10⁶ cells; AA: 251.8 ± 43.57 mU/mL) and exo BD(PC: 8.44 ± 1.10 mg/10⁶ cells; AA: 240.6 ± 46.77 mU/mL) (Fig 1B, C).  Exo-BD demonstrated significant reduction in oxidative stress levels in dermal fibroblasts compared to controls, serum and exo-UC groups (Fig 1D).  Dermal fibroblasts demonstrated lower expression of p-p38 and NRF2 following treatment with both exoUC and exoBD (Fig 1E). TFurthermore, fibroblast treatment with exoUC and exoBD reduced NRF2 to basal levels indicating reduction in fibroblast senescence following oxidative damage that occurs with excessive expression of NRF2. Both exo-UC and exoBD accelerated in vitro scratch wound closures in dermal fibroblasts (Fig 1F). Dermal fibroblasts were induced towards myofibroblast differentiation by exposure to TGF- β1. Both exoUC and exoBD inhibited fibroblast to myofibroblast transition as demonstrated by lower expression of α-SMA (Fig G).

Conclusions:

Our results suggest exosomes from burn tissue may be an effective cell-free therapeutic strategy for wound healing.