Bone Marrow-Derived Mesenchymal Stem Cells-Derived Exosomes Promote Survival of Retinal Ganglion Cells Through miRNA-Dependent Mechanisms

Abstract

The loss of retinal ganglion cells (RGC) and their axons is one of the leading causes of blindness and includes traumatic (optic neuropathy) and degenerative (glaucoma) eye diseases. Although no clinical therapies are in use, mesenchymal stem cells (MSC) have demonstrated significant neuroprotective and axogenic effects on RGC in both of the aforementioned models. Recent evidence has shown that MSC secrete exosomes, membrane enclosed vesicles (30-100 nm) containing proteins, mRNA and miRNA which can be delivered to nearby cells. The present study aimed to isolate exosomes from bone marrow-derived MSC (BMSC) and test them in a rat optic nerve crush (ONC) model. Treatment of primary retinal cultures with BMSC-exosomes demonstrated significant neuroprotective and neuritogenic effects. Twenty-one days after ONC and weekly intravitreal exosome injections; optical coherence tomography, electroretinography, and immunohistochemistry was performed. BMSC-derived exosomes promoted statistically significant survival of RGC and regeneration of their axons while partially preventing RGC axonal loss and RGC dysfunction. Exosomes successfully delivered their cargo into inner retinal layers and the effects were reliant on miRNA, demonstrated by the diminished therapeutic effects of exosomes derived from BMSC after knockdown of Argonaute-2, a key miRNA effector molecule. This study supports the use of BMSC-derived exosomes as a cell-free therapy for traumatic and degenerative ocular disease. Stem Cells Translational Medicine 2017;6:1273-1285.

Keywords: Exosomes; Mesenchymal stem cells; Neuroprotection; Optic nerve crush; Retinal ganglion cells.

Figure 1

Experimental design of in vivo study. Timeline detailing the ERG/OCT recordings, exosome treatments and sacrifice of animals with respect to the day of the optic nerve crush surgery. The injury and treatment (asterisks) for each animal group is also given. Abbreviations: BMSC, bone marrow‐derived mesenchymal stem cell; ERG, electroretinography; OCT, optical coherence tomography.

Figure 2

Exosome secretion from fibroblasts and BMSC. (A): The number of exosomes, assayed and quantified by CD63 ExoELISA, secreted by human fibroblasts and human BMSC, given as exosomes per 100,000 cells, per 24 hours. Numbers were corrected to take into account CD63 expression percentage as determined by flow cytometry. No significant difference was found (p < .05). (B): The percentage of exosomes that express various CD surface expression markers assayed and quantified using a MACSPlex Exosome Kit (human) in conjunction with flow cytometry. The surface markers CD2, CD3, CD4, CD14, CD25, CD31, CD40, CD42a, CD45, CD49e, CD56, CD69, CD133/1, CD146, and CD326 were not detected in either sample. Black lines indicate significant difference (p < .05). Abbreviation: BMSC, bone marrow‐derived mesenchymal stem cell.

Figure 3

Effects of exosome treatment on RGC neuroprotection and neuritogenesis. The number of RGC (A), number of RGC bearing neurites (B) and the length of the longest neurite (C) in heterogeneous retinal cultures after treatment with different quantities of EV, before (exosomes + microvesicles) and after (exosomes) filtration. Black lines indicate significant difference between groups whereas asterisks indicate significant difference between filtered and unfiltered exosomes (p < .05). Representative images of heterogeneous retinal cultures either untreated (D) or treated with BMSC (E), 3 × 10⁹ BMSC exosomes with microvesicles (F), 7.5 × 10¹⁰ BMSC exosomes with microvesicles (G) or 7.5 × 10¹⁰ BMSC exosomes (H). All images are representative of the entire culture, nine separate culture wells/treatment, with every 3 wells using a different animal (scale bars: 50 μm). Sections were stained for βIII‐tubulin (green) and DAPI (blue). Abbreviations: BMSC EV, bone marrow‐derived mesenchymal stem cells extracellular vesicles; RGC, retinal ganglion cells.

Figure 4

RNFL thickness of rats after ONC and exosome treatment. (A): Graph depicting the mean RNFL thickness (μm) of rats before and 21 days after ONC. Black lines indicate significant difference between groups whereas asterisks indicate significant difference from the same group pre‐ONC (p < .05). (B): RNFL measurements were done from a section of retina surrounding the optic nerve head (green line). (C): Representative images of retina from Group 1, 2, 3, and 6, as measured by OCT (scale bars: 200 μm). Abbreviations: BMSC, bone marrow‐derived mesenchymal stem cells; ONC, optic nerve crush; RNFL, retinal nerve fiber layer.

Figure 5

Brn3a⁺ and RBPMS⁺ RGC counts in exosome treated retina after ONC. (A): Graph depicting the mean number of Brn3a⁺ and RBPMS⁺ RGC in a 1‐mm region of retina either side of the optic nerve head. Percentage of surviving RGC in comparison to intact controls is given above each group. Asterisks indicate a significant difference from intact, ONC + BMSC Exosomes and ONC + SiScr Exosomes groups (p < .05). (B): Image of retina immunohistochemically stained for RBPMS (red) from animals injected with ExoGreen labeled exosomes (green). (C): Representative images of retina from Group 1 to 6, immunohistochemically stained for Brn3a (green) and RBPMS (red). All images show tissue counterstained with the nuclear marker DAPI (blue; scale bars: 50 μm). Abbreviations: BMSC, bone marrow‐derived mesenchymal stem cells; ONC, optic nerve crush; RGC, retinal ganglion cells.

Figure 6

pSTR responses after ONC and exosome treatment. (A): Mean amplitude of pSTR from intact or ONC animals receiving intravitreal exosome treatments after receiving flash intensity of 1 × 10⁻⁵ mcd/s. Black lines indicate significant difference between groups whereas asterisks indicate significant difference from the same group pre‐ONC (p < .05). (B): Representative traces of observable pSTR from groups 1‐6. Abbreviations: ONC, optic nerve crush; pSTR, positive scotopic threshold response.

Figure 7

GAP‐43⁺ RGC axon counts in the optic nerve after ONC. (A): Graph depicting the mean number of GAP‐43⁺ RGC axons in the optic nerve at 100–1200 µm from the laminin⁺ crush site. Asterisks indicate significant difference between ONC/ONC + fibroblast exosomes and ONC + BMSC exosomes/SiScr exosomes (p < .05). (B): Representative images of optic nerves immunohistochemically stained for GAP‐43 (green) and laminin (red) from groups 3 and 4 (scale bars: 100 μm). Abbreviations: BMSC, bone marrow‐derived mesenchymal stem cells; ONC, optic nerve crush; RGC, retinal ganglion cells.