BMSC-derived exosomes promote retinal ganglion cell survival

Abstract

Purpose : The loss of retinal ganglion cells (RGC) and their axons is a leading cause of blindness and includes traumatic (optic neuropathy) and degenerative (glaucoma) eye diseases. Mesenchymal stem cells (MSC) have demonstrated significant neuroprotective and axogenic effects on RGC in both of the aforementioned models. The present study aimed to isolate exosomes from bone marrow-derived MSC (BMSC) and test them in a rat optic nerve crush (ONC) and glaucoma model.

Methods : Exosomes were isolated from human BMSC and characterized by electron microscopy, flow cytometry and CD63 ExoELISA. Using an in vitro axotomized rat RGC model and in vivo rat models of ONC and glaucoma, we treated/injected 3x10⁹ exosomes into the cell culture well/vitreous. To measure their neuroprotective and axogenic capacity, we used immunochemistry, optical computerized tomography (OCT) and electroretinography (ERG). The composition of miRNA in exosomes from human BMSC and control human fibroblasts was investigated by RNA sequencing and used to identify candidate target mRNA in RGC.

Results : Both BMSC and fibroblasts secrete similar numbers of exosome (1.03x10⁹ and1.17x10⁹/24h/100,000 cells, respectively). Treatment of RGC cultures with exosomes led too significant RGC survival (299 ± 24.1 RGC/well) compared to both fibroblast exosome treated (72.3 ± 6.4RGC/well) and untreated (121.3 ± 6.2) cultures. Following intravitreal transplantation, exosomes successfully integrated into the inner retinal layers, including RGC. After ONC (21d), BMSC exosomes provided significant therapeutic effects as compared with fibroblast exosomes or untreated eyes. For the three measured outputs, the thickness of the retinal neve fibre layer was 33.8 ± 4.8 μm, 21.6 ± 1.5 μm, and 18.0 ± 2.1 μm, respectively; RGC density was 73.3 ± 7.8/mm of retina, 20 ± 2.2/mm of retina, and 23.6 ± 7.7/mm of retina, respectively; and positive scotopic threshold response was 28.6 ± 8.1 μv, 13.2 ± 3.4 μv, and 13.7 ± 1.1 μv, respectively. The significant therapeutic benefits were also seen in the treatment of glaucoma models. The therapeutic benefit of BMSC exosomes was reduced significantly if isolated following knockdown of Argonaute 2, a protein that complexes with miRNA and is integral to their function.

Conclusions : We demonstrate for the first time that BMSC-derived exosomes offer significant therapeutic benefit to the protection of RGC, an effect mediated at least partially by their miRNA.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.