Characterization of the effects of retinal pigment epithelium-conditioned media on porcine and aged human retina.Graefes Arch Clin Exp Ophthalmol. 2013 Jun; 251(6):1515-28.GA
Retinal pigment epithelium (RPE) cells produce neurotrophic factors that rescue photoreceptors from degeneration. Previously, we showed that conditioned medium (CM) from fetal vs adult RPE cells resulted in significantly better porcine retinal preservation, and possessed significantly higher levels of hepatocyte growth factor (HGF) and pigment epithelium-derived factor (PEDF). This study aimed to further describe the effects of human fetal RPE-CM on porcine and aged human retina, and to characterize its effects biochemically.
RPE-CM was harvested from passage-2 fetal RPE, 7 days after passage, 24-hours after exposure to basal medium. After culture in RPE-CM, porcine retinal morphology was assessed with confocal microscopy. The effects of RPE-CM on porcine and aged human retina survival were assessed by cytotoxicity and apoptosis biochemical assays. To characterize RPE-CM biochemically, effects of heating, digesting with proteinase-K, dilution, concentration, and fractionation were tested. Recombinant proteins and neutralizing antibodies were used to identify proteins that might contribute to the salutary effects of RPE-CM on porcine retina.
Culturing porcine retina in RPE-CM significantly preserved outer nuclear layer width and the number of nuclei in cross-section, and significantly decreased photoreceptor axon retraction. RPE-CM decreased porcine retinal death by 17-34 % (p<0.05) compared to basal medium. Human retina from age-related macular degeneration (AMD) and non-AMD donors responded similarly after culture in RPE-CM. Heating, proteinase-K digestion, and dilution significantly diminished RPE-CM-mediated preservation of porcine retina, whereas concentrating RPE-CM significantly enhanced its preservation of porcine retina. Molecular cut filtration identified retina-preserving activity in the 3-100 kDa filtrate. PEDF or HGF at 90 % receptor occupancy significantly improved retinal preservation over 48 h of culture compared to basal medium. Neutralizing PEDF in RPE-CM decreased its ability to reduce retinal apoptosis by 23-27 % (p<0.05).
RPE-CM reduced biochemically and histologically measured degeneration in porcine retinae. This effect was concentration-dependent, and can be attributed to a protein component(s) in a 3-100 kDa molecular cut fraction. Human retina (including non-AMD and AMD Caucasian and non-AMD African-American) responds to culture in RPE-CM similarly to porcine retina. Receptor occupancy calculations and retinal viability data indicate that PEDF may be one of the components that contribute to retina preservation by RPE-CM.