Abstract
Purpose :
Mesenchymal stem cells from human corneal stroma (CSSC) have been shown to induce regeneration of transparent stromal tissue during wound repair in mice. Exosomes, nano-sized extracellular vesicles, have recently been implicated in the ability of bone marrow mesenchymal stem cells to repair damaged tissue. In this study we examined the hypothesis that exosomes produced by CSSC can prevent corneal scarring in a manner similar to that of the live CSSC cells.
Methods :
Passage 3-4 CSSC and HEK (human embryonic kidney) cells were cultured 48 hr in exosome-free medium, and exosomes isolated from the conditioned medium using polymer based precipitation followed by ultracentrifugation at 100,000 x g. Exosomes were characterized using Nanosight light scattering, transmission electron microscopy (TEM), and flow cytometry. Suppression of scarring was examined in a mouse model of corneal wound healing with exosomes in a fibrin gel applied at the time of wounding. Scar area 14 days after wounding was calculated in a masked fashion from ex-vivo light microscope images. Mouse corneal fibrotic gene expression was examined by qPCR. Neutrophil infiltration was assessed by ELISA for myeloperoxidase at 48 hr after wounding. Statistical significance was determined with t-test and Dunn’s test analysis using p<0.05 as a criterion.
Results :
Isolated exosomes from CSSC were microvesicles of 20-200 nm as visualized by TEM and light scattering. Flow cytometry found both CD63 and CD81 on the exosome surface. Addition of exosomes to corneal wounds produced a significant reduction of stromal scar area compared to control wounds and wounds treated with exosomes produced by HEK cells (p<0.001, n=8). CSSC exosomes completely suppressed neutrophil infiltration into wounds at 24 hr (p<0.001, n=8). At 14 days, expression of fibrotic markers (Tnc, Acta2, Col3a1, Sparc) was significantly reduced in CSSC-exosome treated wounds (p<0.05, n=4), to a level similar to that of unwounded tissue.
Conclusions :
Exosomes derived from CSSC (but not those of HEK cells) embody a high regenerative potential. In vivo, they suppressed inflammation and prevented corneal scarring and fibrosis in a mouse wounding model. These results suggest that exosomes may allow a non-cell based therapy for corneal scars in the future.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.