Extracellular vesicle therapy for retinal diseases

Abstract

Extracellular vesicles (EV), which include exosomes and microvesicles, are secreted from virtually every cell. EV contain mRNA, miRNA, lipids and proteins and can deliver this expansive cargo into nearby cells as well as over long distances via the blood stream. Great interest has been given to them for their role in cell to cell communication, disease progression, or as biomarkers, and more recent studies have interrogated their potential as a therapeutic that may replace paracrine-acting cell therapies. The retina is a conveniently accessible component of the central nervous system and the proposed paradigm for the testing of many cell therapies. Recently, several studies have been published demonstrating that the delivery of EV/exosomes into the eye can elicit significant therapeutic effects in several models of retinal disease. We summarize results from currently available studies, demonstrating their efficacy in multiple eye disease models as well as highlighting where future research efforts should be directed.

Keywords: Exosomes; Extracellular vesicles; Glaucoma; Mesenchymal stem cells; Optic nerve crush; Retina.

Copyright © 2020 Elsevier Ltd. All rights reserved.

Figures

Fig. 1.

Retinal ganglion cell (RGC) counts in a rat model of glaucoma after mesenchymal stem cell (MSC) treatment. Glaucoma was modeled through intracameral injections of transforming growth factor-β for 35d. Treatments consisted of intravitreal transplantation of dental pulp stem cells (DPSC), bone marrow MSC (BMSC), adipose-derived stem cells (ADSC) and dead DPSC (sham-treated; A). Retinae were stained with the phenotypic RGC marker BRN3A (red) and the nuclear marker DAPI (blue; scale bar: 50 μm). In (B), GFP+ MSC stained for the MSC marker STRO1 are identified in the vitreous, adhering to the inner limiting membrane. In (C), the mean number of BRN3A+ RGC in a 1 mm region of retina either side of the optic nerve head is shown from each of the above groups. Note the significant neuroprotective effect elicited by the transplanted MSC. Black lines indicate significant difference between groups (p < 0.01). Modified Fig. 4 from Mead et al. (2016), re-used under the Creative Commons Attribution 4.0 International (CCBY4.0) licence.

Fig. 2.

Schematic diagram detailing exosomal treatment of the retina. Exosomes and microvesicles are isolated through ultracentrifugation of culture medium, conditioned by the proposed cell source. Lower speeds of centrifugation can be used in protocols that utilize polyethylene glycol while other techniques such as passing through a sucrose gradient are employed to further specify the vesicle size obtained. To partially-purify the 30–150 nm exosomes from the 100–1000 nm microvesicles, passage through a 0.22 μm filter is utilized. Following purification, exosome identity can be confirmed with Nanoparticle Tracking Analysis and Western blot before injection into the eye (vitreous or subretinal).

Fig. 3.

Electron microscopy images of exosomes before and after filtration through a 0.22 μm filter along with corresponding Nanosight/Nanoparticle Tracking Analysis of quantity and size. Modified Fig. 2 from Mead et al. (2018) and Mead et al., 2017, re-used under the Creative Commons Attribution 4.0 International (CCBY4.0) licence. The figure inset shows a higher quality electron microscopy image of an exosome (EXO), microvesicle (MV), and apoptotic body (APO). Reused from Osteikoetxea et al. (2015) with permission under the Creative Commons Attribution 4.0 International (CCBY4.0) licence.

Fig. 4.

Publications with the keyword “exosome” or “extracellular vesicle” in the abstract/title from Jan 1st, 1980–Jan 1st, 2020. Note the exponential rise in publications referencing exosomes along with the historical popularity of “exosome” over “extracellular vesicle”, with the gap narrowing significantly in 2019.

Fig. 5.

Differential effects of exosomes and microvesicles on retinal ganglion cells (RGC)/neurons. In three separate studies, one in cortical neurons (A) and 2 in RGC (B/C), exosomes demonstrated a neuritogenic/neuroprotective effect with microvesicles exerting the opposite. The first study (A) showed that exosomes were neuritogenic whereas the effect of microvesicles was worse than untreated controls. The second (B) demonstrated the efficacy of extracellular vesicles diminished at higher doses and this was due to the contamination of microvesicles. A third study (C) showed the same but did not confirm the effect was due to contaminating microvesicles. Modified Fig. 3 from Loppez-Verrilli et al. (2016) (A), Fig. 3 from Mead et al. (2017) (B), and Fig. 1 from van der Merwe et al. (2019), re-used under the Creative Commons Attribution 4.0 International (CCBY4.0) licence.

Fig. 6.

miRNA in L cell exosomes. miRNAseq was performed on exosomes derived from L cells with those detected displayed (A) as mean estimated abundance (derived from the reads) ± standard error mean (SEM). Mouse L cell exosome miRNA that are homologues to their human miRNA counterpart were selected and compared to human bone marrow mesenchymal stem cell (BMSC) and dermal fibroblast exosome miRNA. Those miRNA also shown to be abundant in BMSC exosomes and L cell exosomes in comparison to fibroblast exosomes are displayed (B) as mean estimated abundance (derived from the reads) ± SEM. Comparative data for miRNA expression in BMSC exosomes/fibroblast exosomes is from a previous publication (Mead et al., 2018b).

Fig. 7.

Exosome treatment of human retina. Heterogeneous retinal cultures were generated from a human embryonic stem cell line expressing a fluorescent marker under the brn3b (retinal ganglion cell (RGC) specific) promoter. To induce RGC degeneration, colchicine, a microtubule poison, was added to cultures (B) and led to significant RGC loss compared to uninjured controls (A). Ciliary neurotrophic factor (CNTF) led to significant neuroprotection of RGC (positive control, C), as did mesenchymal stem cell (MSC) exosomes (D), and tumor necrosis factor-α (TNF-α) primed MSC exosomes (E; scale bar: 250 μm). The quantified number of BRN3B+ RGC is shown in F. Fig. 2 from Mead et al. (2020) re-used under the Creative Commons Attribution 4.0 International (CCBY4.0) licence.