Evaluation of Neuroprotective And Immunomodulatory Properties of Mesenchymal Stem Cells in An Ex-vivo Retinal Explant Model

Abstract

Background: Mesenchymal stem cells (MSCs) have raised considerable hope for the treatment of glaucoma. Their neuroprotective and regenerative potentials are particularly interesting for this degenerative neuropathy in which retinal ganglion cell (RGC) death leads to a progressive loss of visual field and eventually vision. Yet, despite promising results in animal models, no definitive treatment has been developed, and safety concerns have been reported in human trials. Microglial immunomodulation represents a promising therapeutic approach in which MSCs might play a crucial role. In the present study, we investigated the neuroprotective and immunomodulatory properties as well as the safety of MSCs in an ex vivo neuroretina explant model.Methods: Labeled rat bone marrow MSCs were placed in co-culture with rat retinal explants after optic nerve axotomy. We analyzed the neuroprotective effect of MSCs on RGC survival by immunofluorescence using RBPMS, Brn3a and NeuN markers. Gliosis and retinal microglial activation were measured using GFAP, CD68 and ITGAM mRNA quantification and GFAP, CD68 and Iba1 immunofluorescence staining. We also analyzed the mRNA expression of both ‘M1’ or classically activated state inflammatory cytokines (TNFα, IL1β and IL6), and ‘M2’ or alternatively activated state microglial markers (Arginase 1, IL10, CD163, and TNFAIP6).Results: The number of RGCs was significantly higher in retinal explants cocultured with MSCs compared to the control group at Day 7 following optic nerve axotomy. Retinal explants co-cultured with MSCs showed decreased mRNA markers of gliosis and microglial activation, and immunostaining revealed that GFAP, Iba1 and CD68 were limited to the internal layers of the retina compared to controls showing expression of activated microglia throughout the retina. In addition, MSCs inhibited the M1 phenotype of the microglia. However, edema of the explants was observed in the MSC co-culture group, with an increase of fibronectin labelling at the surface of the explant corresponding to an epiretinal membrane-like phenotype. Conclusion: Using an ex vivo model, we demonstrated a neuroprotective and immunomodulatory effect of MSCs on RGCs. Unfortunately, the presence of MSCs also led to explant edema and epiretinal membrane formation, as described in human trials. Using the MSC secretome might offer the beneficial effects of MSCs without their potential adverse effects, through paracrine signaling.