Extracellular vesicles derived from human mesenchymal stem cells promote corneal wound repair by increasing epithelial cell proliferation and reducing neovascularisation in a rat corneal alkali burn model

Abstract

Purpose : To investigate if topically applied mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) promote corneal wound repair in a rat alkali burn model

Methods : EVs were isolated from media conditioned by human bone marrow-derived MSCs (n=4) using a series of centrifugation, filtration and ultracentrifugation steps followed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA; NanoSight) quality control. Pro-healing effects of hMSC-derived EVs (hMSC-EV) were examined in vitro by cell migration and scratch wound healing assays using human corneal epithelial (HCE) and endothelial (HCEC) cell lines. Human cornea wound healing rate was also determined ex vivo using fluorescein penetration test. Rat corneal alkali burn injury was induced using 1M NaOH and injury was treated with or without 30mg of hMSC-EVs. Efficacy of treatment was monitored by biomicroscopy and optical coherence tomography (OCT) followed by extensive tissue analysis at the end of the observation period.

Results : Successful isolation of EVs was confirmed by immune-gold TEM showing expression of CD63 and TSG101 on their surface. NTA showed the average size of EVs ranged between 50-130nm. Significant increases in HCE and HCEC cell migration in the presence of MSC-EVs were observed compared to PBS control and EV-depleted MSC conditioned media. Moreover, a significant increase in scratch wound closure of cells treated with hMSC-EVs was observed. In vivo, analysis of images taken at day 0, 1, 3 and 7 of fluorescein stained corneal injury showed significantly enhanced healing of the corneal surface at day 1 and 3 when treated with hMSC-EVs compared to PBS-control. OCT analysis revealed that hMSC-EVs reduced inflammation and maintained the physiological structure of the eye. Importantly, a reduction in corneal damage and inflammatory infiltrating cells and an increase in proliferating epithelial cells (Ki67-positive) was observed following hMSC-EV treatment. Finally, human corneas treated with hMSC-EVs healed faster than corresponding PBS-control by 12.1%±9 and 6.1%±3.3 at 24 and 48 hours respectively.

Conclusions : Our results show that topical hMSC-EV application is a novel promising cell-free therapy with high potential for translation in ocular surface disease treatment.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.