Ben Mead

Mesenchymal Stem Cells (MSC) possess a diverse secretome with well-established neuroprotective effects. Form among the materiel released by these cells, extracellular vesicles (EVs) have gained particular interest lately, owing to their good safety profile, stability, and relative ease of use as a cell-free therapy. These lipid-enclosed nano-carriers can significantly alter the survival of recipient cells through the delivery of a wide variety of signalling molecules, the exact composition of which is highly dependent on the type, age, and environment of the donor cells. Glaucoma is a chronic progressive optic neuropathy characterised by the loss of Retinal Ganglion Cells whose axons make up the optic nerve. Preservation of these neurons via the administration of the right EVs represents a promising approach for slowing disease progression, thereby preventing vision loss. Here, we evaluate and compare the protective and neuritogenic potential of small extracellular vesicles (sEVs), a subset of EVs with a diameter smaller than 220 nm, from six different cell types using a rodent in vitro model of RGC degeneration. Our findings showed that Adipose Mesenchymal Stem Cells release the most potent sEVs, with Bone Marrow being a close second. EVs released by cells of the Umbilical Cord, Dental Pulp, Dermal Fibroblasts, and an Oral Mucosal Lamina Propria-Progenitor cells did not have an observable benefit. Thus, our study provides greater insight into how the efficacy of different EVs compares to each other.

Retinal ganglion cell (RGC) axons form the optic nerve (ON). Numerous age-related ON diseases, including glaucoma, the second most common cause of worldwide blindness, result from multiple RGC stressors. Nearly all ON astrocytes in the optic nerve head (ONH): the junctional region between the ON and the retina in young-adult rodents expresses the homeodomain only (Hopx) protein. Hopx(+) ONH astrocytes are depleted during aging. ONH primary cultures which include Hopx(+) astrocytes secrete extracellular vesicles (ONH-EVs) which selectively enhance RGC survival and neurite extension in culture, while extracellular vesicles (EVs) secreted from distal ON cultures lacking Hopx(+) astrocytes do not. ONH-EVs also enhance RGC survival in vivo in a rodent model of glaucoma. Combining rat ONH single-cell (scRNA-seq) sequencing with EV proteomic analysis, we identified ONH-Hopx(+) astrocyte secreted factors. We interrogated the online Broad institute scRNA-seq database for rat RGC gene expression in control animals and following rodent ON crush, an RGC stress model, to correlate ONH-astrocyte secreted factors with RGC gene expression changes. Following stress, RGCs upregulate the complementary pathways involving Hopx(+) astrocytic-associated factors, suggesting reciprocal communication. Using a highly selective transgenic Hopx-cre ONH knockdown strategy, we demonstrate that eliminating Hopx(+) astrocytes also results in upregulation of RGC stress responses. Our results implicate age-related loss of young ONH-astrocytes as a crucial factor in the development of age-related optic nerve diseases, and discuss replacing ONH associated factors as a paradigm shift for ON disease treatment.

JOURNAL/nrgr/04.03/01300535-202605000-00041/figure1/v/2026-04-20T030116Z/r/image-tiff Glaucoma is characterized by chronic progressive optic nerve damage and retinal ganglion cell death. Although extensive research has been conducted on neuroprotection for retinal ganglion cells, there is still no treatment for clinical use. Recent evidence shows that extracellular vesicles isolated from a variety of stem cells are efficacious in retinal ganglion cell neuroprotection. In this study, we tested the novel extracellular vesicle source of the retinal progenitor R-28 cell line in vitro and in vivo . We isolated and characterized extracellular vesicles from R-28 cells and tested their therapeutic efficacy in terms of retinal ganglion cell survival in vitro and in an in vivo glaucoma model, measuring retinal ganglion cell survival and preservation of their axons. Additionally, we tested extracellular vesicles for their neuroprotective capacity in retinal ganglion cells differentiated from human embryonic stem cells. Finally, we investigated miRNA changes in retinal ganglion cells with R-28 extracellular vesicle treatment, and predicted possible pathways that may be modulated. R-28 extracellular vesicles improved retinal ganglion cell survival but failed to preserve axons significantly. Moreover, the results also illustrated the neuroprotection of R-28 extracellular vesicles on human retinal ganglion cells. Finally, we also showed changes in hsa-miRNA-4443, hsa-miRNA-216a-5p, hsa-let-7e-5p, hsa-miRNA-374b-5p, hsa-miRNA-331-3p, and hsa-miRNA-421 expressions, which may have neuroprotective potential on retinal ganglion cell degeneration. This study will pave the way for miRNA and extracellular vesicle-based neuroprotective therapies for glaucoma.

MiRNA, short non-coding RNA, are rapidly emerging as important regulators in cell homeostasis, as well as potential players in cellular degeneration. The latter has led to interest in them as both biomarkers and as potential therapeutics. Retinal ganglion cells (RGC), whose axons connect the eye to the brain, are central nervous system cells of great interest, yet their study is largely restricted to animals due to the difficulty in obtaining healthy human RGC. Using a CRISPR/Cas9-based reporter embryonic stem cell line, human RGC were generated and their miRNA profile characterized using NanoString miRNA assays. We identified a variety of retinal specific miRNA upregulated in ESC-derived RGC, with half of the most abundant miRNA also detectable in purified rat RGC. Several miRNA were however identified to be unique to RGC from human. The findings show which miRNA are abundant in RGC and the limited congruence with animal derived RGC. These data could be used to understand miRNA’s role in RGC function, as well as potential biomarkers or therapies in retinal diseases involving RGC degeneration.

With an evolving understanding and new discoveries in extracellular vesicle (EV) biology and their implications in health and disease, the significant diagnostic and therapeutic potential of EVs for vision research has gained recognition. In 2021, the National Eye Institute (NEI) unveiled its Strategic Plan titled ‘Vision for the Future (2021–2025),’ which listed EV research as a priority within the domain of Regenerative Medicine, a pivotal area outlined in the Plan. In alignment with this prioritization, NEI organized a workshop inviting twenty experts from within and beyond the visual system. The workshop aimed to review current knowledge in EV research and explore gaps, needs and opportunities for EV research in the eye, including EV biology and applications of EVs in diagnosis, therapy and prognosis within the visual system. This perspective encapsulates the workshop's deliberations, highlighting the current landscape and potential implications of EV research in advancing eye health and addressing visual diseases.

Glaucoma is an irreversible blinding disease characterized by the loss of retinal ganglion cells. Development of therapeutics relies on suitable models and, given its complex nature, these are typically in vivo models. A widely used model, owing to its relative simplicity, is the microbead model. This model involves the injections of beads into the anterior chamber, which are suitably sized to block the aqueous outflow pathway, leading to an elevation in intraocular pressure and ultimately, retinal ganglion cell death. In this chapter, we describe in detail the materials and methods for modelling glaucoma using microbeads (both magnetic and nonmagnetic). Key words Ocular hypertension Glaucoma Rodent model Microbead Retinal ganglion cells Neuroprotection