PMC

Retinal ganglion cell neuroprotection by mesenchymal stem cell conditioned media. DMEM was conditioned by 72–96 h incubation with rat or human mesenchymal stem cells (hMSCs) or human fibroblasts. Control DMEM was unconditioned. Conditioned media was then concentrated 25 to 55-fold by centrifuge filtration. Concentrated rat MSC (A) or human MSC (B) conditioned media was added to retinal explant media and to the retinal ganglion cell (RGC) surface of retinal explants daily for 7 days. Neuronal survival in the RGC layer was quantified. Human MSC conditioned media was fractionated by fast protein liquid chromatography (C). Retinal explants were treated with individual human MSC conditioned media fractions (F1, F2, F3) or with human MSC co-culture as a positive control, and RGC survival was quantified (D). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent standard error of the mean.

Retinal ganglion cell neuroprotection by factors enriched in the MSC secretome. Retinal explants were cultured using standard protocols with or without the addition of various purified proteins or vehicle (control) to the culture media. A cocktail of 13 factors detailed in was added to the culture media (A, B). Factors were sub-divided as indicated in and added to the culture media (C, D). Factors in the PDGF group were further sub-divided and added to the culture media at the same final concentrations as indicated in (E, F). The dose-dependence of PDGF neuroprotection was evaluated by adding each of these factors at the indicated concentration to the culture media (G). Treatment with rat MSC (rMSC) or human MSC (hMSC) co-culture was used as a positive control for comparison. Neuronal survival in the RGC layer was quantified. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent standard error of the mean.

Retinal ganglion cell neuroprotection by human mesenchymal stem cells. Human mesenchymal stem cells (hMSCs), human fibroblasts (hfibroblast), or mouse embryonic fibroblasts (NIH-3T3) were co-cultured on the retinal ganglion cell (RGC) surface of retinal explants for 7 days. Human MSCs were visualized by anti-human nuclear antigen immunofluorescence (A and B, red). Glial cells were visualized with glial fibrillary acid protein (GFAP) immunofluorescence (A and B, green). Müller glial endplate processes at the inner limiting membrane delineated the boundary of the neural retina. Red fluorescence at the outer retina (B) represents autofluorescence. All nuclei were stained with DAPI. Scale bars: A and B = 100 µm. Quantification of neuronal survival in the RGC layer (C–E). **P < 0.01; ***P < 0.001; NS = not significant. Error bars represent standard error of the mean. Control indicates without cell co-culture.

Role of PDGF signalling in retinal ganglion cell neuroprotection by MSCs. Retinal explants were cultured alone (A); with human MSCs (B, E and F); with PDGF-AA + PDGF-AB (50 ng/ml) added to the culture media (C); or with PDGF-AA + PDGF-AB (50 ng/ml) plus anti-PDGF IgG (35 µg/ml) added to the culture media (D). Retinal ganglion cell (RGC) layer neurons were visualized with NeuN (A–E) or β-III-tubulin (F) immunofluorescence (red) and PDGF signalling was detected with phospho-PDGF receptor immunofluorescence (green). Nuclei were visualized with DAPI (blue). Higher magnification image of the RGC layer in B is depicted as individual channels in E’ and E’’ and merged in E. Scale bars: A–E = 200 µm; F = 25 µm. Retinal explants were cultured alone, or with the addition of PDGF-AA + PDGF-AB (50 ng/ml) to the culture media, or with human MSC co-culture for 7 days. Half of the explants from each group were additionally treated with non-specific anti-goat IgG (35 µg/ml, G); anti-PDGF IgG (35 µg/ml, G–I); small molecular inhibitor of PDGF receptor kinase, AG1296 (45 µM, J–K); or small molecule inhibitor of PI3 kinase, LY294002 (75 µM, L–M). *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent standard error of the mean. Control indicates without cell co-culture or addition of PDGF to growth media.

Retinal ganglion cell neuroprotection by rat mesenchymal stem cells. Green fluorescent protein-expressing rat MSCs (rMSC, green) were co-cultured on the RGC surface of retinal explants for 7 days (B, D, G, H). All nuclei in the RGC layer (RGCL) were visualized by DAPI staining (blue). RGC layer neurons were visualized by Islet-1 (A and B) and NeuN (C and D), or β-III-tubulin (E–H) immunofluorescence. RGC axons and dendrites were visualized with β-III-tubulin immunofluorescence (E–H). TUNEL was carried out and quantified as described (J and K). Arrowhead: TUNEL⁺ karyorrhexic RGCL nucleus. Scale bars: A, B, F, H = 100 µm; C, D, E, G = 200 µm; J = 50 µm. White boxes in E and G shown in higher magnification in F and H, respectively. Quantification of neuronal survival in the RGC layer (I). Inner plexiform layer (IPL) thickness was measured in three areas per section, and in at least five sections per explant (L). *P < 0.05; ***P < 0.001. Error bars represent standard error of the mean. Control indicates without cell co-culture.

Optic nerve protection by PDGF treatment in an in vivo glaucoma model. Unilateral ocular hypertension (OHT) was induced by laser trabecular meshwork photocoagulation on Days 0 and 7 of the experiment, immediately preceded by intravitreal injection of PBS (3 µl, n = 15), PDGF-AA (1.5 µg, n = 15), or PDGF-AB (1.5 µg, n = 15). Intraocular pressure (IOP) measurements over time are shown in A (PBS), B (PDGF-AA), and C (PDGF-AB). Peak intraocular pressure (IOP; D), mean intraocular pressure (E), and cumulative integral intraocular pressure exposure (F) were calculated for ocular hypertension and control eyes in each group. Optic nerve survival was quantified as per cent surviving RGC axons in the ocular hypertension optic nerve as compared to the contralateral control eye (G). The rate of RGC axon loss normalized to cumulative excess intraocular pressure exposure is shown in H. Representative photomicrographs of optic nerve cross sections are shown for treatment with PBS (I), PDGF-AA (J), and PDGF-AB (K). *P < 0.05, ***P < 0.001, compared to the PBS control group. Error bars for intraocular pressure data represent standard deviation. Error bars for RGC survival data represent standard error of the mean. Scale bar = 10 µm.