Abstract
PurposeTo determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, particularly tumor necrosis factor α (TNFα), increase their exosomes’ neuroprotective efficacy on retinal ganglion cells (RGCs).MethodsMSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell–derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Luminex assays were used to compare primed versus unprimed exosomes.ResultsMSC conditioned medium and exosomes exerted a significant neuroprotective effect on injured rat and hRGC. This effect was significantly increased after MSCs were primed with retinal conditioned medium or TNFα. Blocking of TNFα signaling with etanercept prevented priming-induced RGC neuroprotective efficacy. Priming increased Pigment epithelium-derived factor (PEDF) and VEGF-AA exosomal abundance.ConclusionsMSC exosomes promote RGC survival not just in rodent retinal cultures but also with hRGC. Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of PEDF and VEGF-AA.
Highlights
To determine whether priming of bone marrow mesenchymal stem cells (MSCs) by signals from injured retina, tumor necrosis factor α (TNFα), increase their exosomes’ neuroprotective efficacy on retinal ganglion cells (RGCs)
MSC exosomes promote RGC survival not just in rodent retinal cultures and with human stem cell–derived retinal ganglion cell (hRGC). Their efficacy can be further enhanced through TNFα priming with the mechanism of action potentially mediated, at least in part, through increased levels of Pigment epithelium-derived factor (PEDF) and VEGF-AA
Retinal cells cultured with the positive control Ciliary neurotrophic factor (CNTF) showed 276.3 ± 12.7 surviving RGCs and 113.7 ± 5.7 RGCs with neurites (Figs. 2B, 2E, 2F) while treatment of retinal cells with unprimed MSC conditioned medium promoted the survival of 110.7 ± 10.7 RGCs and 58.3 ± 16.6 RGCs with neurites (Figs. 2C, 2E, 2F)
Summary
MSCs were primed with retinal cell culture conditioned medium, with or without the TNFα blocker etanercept or TNFα prior to isolation of exosomes. MSC conditioned medium or exosomes were added to rat retinal cultures or human stem cell–derived retinal ganglion cell (hRGC) cultures, and RGC neuroprotective effects were quantified. Human MSCs (bone marrow derived) were purchased from Lonza (Walkersville, MD, USA) and represented pooled samples from three donors. CD29+/CD44+/CD73+/ CD90+/CD45− (confirmed by supplier) MSCs were seeded into T25 or T75 flasks, in a total volume of 5 mL or 15 mL Dulbecco’s modified Eagle’s medium (DMEM), respectively, containing 1% penicillin/streptomycin and 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA) and at a quantity of 1 × 106 cells and 2 × 106 cells, respectively. Cultures were maintained at 37°C in 5% CO2, the supplemented medium was changed every three days, and the cells were passaged when 80% confluent using 0.05% trypsin/EDTA. MSCs were used at passages 2 to 5
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