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D. S. Sakaguchi, M. M. Harper, L. Adamson, B. Blits, M. B. Bunge, S. D. Grozdanic; Brain-Derived Neurotrophic Factor (BDNF) Released From Engineered Mesenchymal Stem Cells (MSCs) Attenuates Glutamate- and Hydrogen Peroxide-Mediated Death of Staurosporine Differentiated RGC-5 Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2074.
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The purpose of this study was to determine the efficacy of cell-based delivery of BDNF from genetically modified MSCs for neuroprotection of RGC-5 cells.
RGC-5 cells were differentiated with staurosporine (SS) and exposed to the cellular stressors glutamate and H2O2, and remaining cells quantified as a percentage of control. BDNF was provided to glutamate or H2O2 treated RGC-5s to examine its neuroprotective capacity via co-culture across a porous membrane insert using MSCs engineered with a lentiviral vector (BDNF-MSCs). As a positive control, recombinant human BDNF (rhBDNF) was added to RGC-5 cells treated with cellular stressors.
After SS differentiation RGC-5s developed complex morphologies, and a significant increase in the proportion of RGC-5s immunoreactive for TUJ 1 and Brn3a was observed. Differentiated RGC-5s also had prominent TrkB staining, demonstrating they had the high-affinity BDNF receptor. Treatment of SS differentiated RGC-5s with glutamate and H2O2, produced significant cell death (56.0 Â± 7.02 and 48.90 Â± 4.58% of control cells, respectively) as compared to carrier-solution treated cells. We confirmed that cell death was occurring by using Sytox to label dying cells. Our results demonstrate that BDNF-MSCs were able to preserve more RGC-5 cells after treatment with glutamate (80.0 Â± 5.40% cells remaining) than control GFP expressing MSCs (GFP-MSCs, 57.29 Â± 1.89%, p < 0.01). BDNF-MSCs also preserved more RGC-5s after treatment with H2O2 (65.6 Â± 3.47%) than GFP-MSCs (46.0 Â± 4.20%, p < 0.01). These results were confirmed by using purified rhBDNF.
Staurosporine differentiated RGC-5s have phenotypic and morphological properties consistent with retinal neurons, and they respond to the cellular stressors glutamate and H2O2 in similar fashion. Additionally, we have demonstrated BDNF can attenuate RGC-5 cell death due to these stressors. Our results demonstrate that genetically-modified MSCs are effective vehicles to deliver BDNF to compromised neural-like cells in an in vitro disease model, suggesting this may be a useful in vivo treatment for chronic retinal diseases.
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