title: Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells.
abstract: The purpose of this study was to determine the viability of cell-based delivery of brain-derived neurotrophic factor (BDNF) from genetically modified mesenchymal stem cells (MSCs) for neuroprotection of RGC-5 cells. RGC-5 cells were differentiated with the protein kinase inhibitor staurosporine (SS) and exposed to the cellular stressors glutamate or H2O2. As a neuroprotective strategy, these cells were then co-cultured across a membrane insert with mesenchymal stem cells (MSCs) engineered with a lentiviral vector for production of BDNF (BDNF-MSCs). As a positive control, recombinant human BDNF (rhBDNF) was added to stressed RGC-5 cells. After SS-differentiation RGC-5s developed neuronal-like 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 expression of the high-affinity BDNF receptor. Treatment of SS-differentiated RGC-5s with glutamate or H2O2, produced significant cell death (56.0 +/- 7.02 and 48.90 +/- 4.58% of control cells, respectively) compared to carrier-solution treated cells. BDNF-delivery from MSCs preserved more RGC-5 cells after treatment with glutamate (80.0 +/- 5.40% cells remaining) than control GFP expressing MSCs (GFP-MSCs, 57.29 +/- 1.89%, p < 0.01). BDNF-MSCs also protected more RGC-5s after treatment with H2O2 (65.6 +/- 3.47%) than GFP-MSCs (46.0 +/- 4.20%, p < 0.01). We have shown survival of differentiated RGC-5s is reduced by the cellular stressors glutamate and H2O2. Additionally, our results demonstrate that genetically modified BDNF-producing MSCs can enhance survival of stressed RGC-5 cells and therefore, may be effective vehicles to deliver BDNF to retinal ganglion cells affected by disease.
authors.affiliation: Iowa State University, Ames, IA 50011, USA.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Harper
authors.forename: Laura
authors.initials: L
authors.lastname: Adamson
authors.forename: Bas
authors.initials: B
authors.lastname: Blits
authors.forename: Mary Bartlett
authors.initials: MB
authors.lastname: Bunge
authors.forename: Sinisa D
authors.initials: SD
authors.lastname: Grozdanic
authors.forename: Donald S
authors.initials: DS
authors.lastname: Sakaguchi
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005818:Genetic Engineering; D018698:Glutamic Acid / Q000633:toxicity*; D006861:Hydrogen Peroxide / Q000633:toxicity*; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology; D051381:Rats; D020813:Receptor, trkB / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*; D050819:Transcription Factor Brn-3A / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D004791:Enzyme Inhibitors; D018696:Neuroprotective Agents; D011994:Recombinant Proteins; D050819:Transcription Factor Brn-3A; C488416:Tubb3 protein, rat; D014404:Tubulin; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; D020813:Receptor, trkB; D019311:Staurosporine