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Sonia Labrador, Girish Kumar Srivastava, Maria Teresa Garcia-Gutierrez, Soraya Tabera-Bartolome, Jose-Carlos Pastor, Ivan Fernandez-Bueno; Evaluation of neurotrophic factors secreted by mesenchymal stem cells in co-culture with porcine neuroretinas. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4565.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGCs) could be neuroprotected by a variety of neurotrophic factors potentially secreted by Mesenchymal Stem Cells (MSCs). Our proposal is to evaluate the effect of Brain-Derived Neurotrophic Factor (BDNF), Ciliary Neurotrophic Factor (CNTF) and Glial Derived Neurotrophic Factor (GDNF), secreted by MSCs in a porcine neuroretina (NR) culture.
Six NR explants were obtained from the area centralis of three adult porcine eyes and cultured alone (controls) on culture membranes or supplemented with MSC in the same wells but physically separated by the culture membrane. Human Bone Marrow Mesenchymal Stem Cells (hBMMSC) were commercially provided by Citospin S.L. (Valladolid, Spain). Co-cultures were maintained during 72 hours. Culture supernatants were collected at culture media exchange at 24, 48 and 72 hours. The presence and concentration of BDNF, CNTF and GDNF was determined in the culture supernatants. Neurotrophic factor levels were measured with quantitative enzyme-linked immunosorbent assays (ELISA) specific for Human BDNF (RayBiotech, Norcross, GA), Human CNTF (Cloud-Clone Corp., Houston, TX) and Human GDNF (RayBiotech), respectively. HBMMSC immunophenotyping after co-culture was performed with MSCs characterization kit (Millipore, Billerica, MA). Data were analyzed using SPSS (version 24.0, SPSS, Inc., Chicago) Analysis of means were performed by repeated measures analysis of variance (ANOVA). The statistical significance level was set at 5%.
NR co-cultured with MSCs preserved their structure better than NR cultured alone. hBMMSC maintained their immunophenotype (immunopositive for CD44, CD90, STRO-1 and CD146, and negative for CD14 and CD19) 72 hours after co-culture with porcine NR. BDNF levels in co-culture supernatants were significantly higher than controls at all time points studied (p<0.05). CNTF levels in the co-cultures were significantly higher than controls at 72 hours (p<0.05). GDNF was not detected in any culture supernatants.
MSCS preserved porcine neuroretina from spontaneous degenerative changes. MSCs secreted neurotrophic factors (BDNF and CNTF) relevant to RGC survival in co-culture with porcine NR. The next step of this study will compare the survival of the RGC on the porcine NR to determine the potential neuroprotective effects of these hBMMSC.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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