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Characterization of the effects of retinal pigment epithelium-conditioned media on porcine and aged human retina

  • Basic Science
  • Published:
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Abstract

Background

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.

Methods

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.

Results

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).

Conclusion

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.

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Acknowledgements

The authors thank Dr. Weifeng Wang for assistance with in-vitro porcine retina culture.

Support

The Janice Mitchell Vassar and Ashby John Mitchell Fellowship (MAZ), the Joseph J. and Marguerite DiSepio Retina Research Fund (MAZ), an unrestricted grant from Research to Prevent Blindness (MAZ), Research to Prevent Blindness Medical Student Fellowship (AMK), Midwest Eye Banks Student Stipend (AMK), and American Foundation for Aging Research Graduate Student Fellowship (AMK).

Conflict of interest

AM Kolomeyer, IK Sugino, MA Zarbin. U.S. patent application No. 13/420,502 “Compositions and Methods for Cell based Retinal Therapies”

Data

The authors have full control of all primary data, and agree to allow data to be reviewed upon request.

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Authors and Affiliations

  1. The Institute of Ophthalmology and Visual Science, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 90 Bergen St., DOC6155, Newark, NJ, 07101, USA

    A. M. Kolomeyer, I. K. Sugino & M. A. Zarbin

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  1. A. M. Kolomeyer
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  2. I. K. Sugino
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  3. M. A. Zarbin
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Correspondence to M. A. Zarbin.

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Kolomeyer, A.M., Sugino, I.K. & Zarbin, M.A. Characterization of the effects of retinal pigment epithelium-conditioned media on porcine and aged human retina. Graefes Arch Clin Exp Ophthalmol 251, 1515–1528 (2013). https://doi.org/10.1007/s00417-013-2326-3

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  • DOI: https://doi.org/10.1007/s00417-013-2326-3

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