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      Polarized secretion of PEDF from human embryonic stem cell-derived RPE promotes retinal progenitor cell survival.

      Investigative ophthalmology & visual science
      Animals, Blotting, Western, Cattle, Cell Culture Techniques, Cell Differentiation, physiology, Cell Survival, Coculture Techniques, Embryonic Stem Cells, cytology, metabolism, Enzyme-Linked Immunosorbent Assay, Eye Proteins, Gene Expression Profiling, Gestational Age, Humans, In Situ Nick-End Labeling, Karyotyping, Microscopy, Confocal, Nerve Growth Factors, Phagocytosis, Retinal Pigment Epithelium, Reverse Transcriptase Polymerase Chain Reaction, Rod Cell Outer Segment, Serpins

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          Abstract

          Human embryonic stem cell-derived RPE (hES-RPE) transplantation is a promising therapy for atrophic age-related macular degeneration (AMD); however, future therapeutic approaches may consider co-transplantation of hES-RPE with retinal progenitor cells (RPCs) as a replacement source for lost photoreceptors. The purpose of this study was to determine the effect of polarization of hES-RPE monolayers on their ability to promote survival of RPCs. The hES-3 cell line was used for derivation of RPE. Polarization of hES-RPE was achieved by prolonged growth on permeable inserts. RPCs were isolated from 16- to 18-week-gestation human fetal eyes. ELISA was performed to measure pigment epithelium-derived factor (PEDF) levels from conditioned media. Pigmented RPE-like cells appeared as early as 4 weeks in culture and were subcultured at 8 weeks. Differentiated hES-RPE had a normal chromosomal karyotype. Phenotypically polarized hES-RPE cells showed expression of RPE-specific genes. Polarized hES-RPE showed prominent expression of PEDF in apical cytoplasm and a marked increase in secretion of PEDF into the medium compared with nonpolarized culture. RPCs grown in the presence of supernatants from polarized hES-RPE showed enhanced survival, which was ablated by the presence of anti-PEDF antibody. hES-3 cells can be differentiated into functionally polarized hES-RPE cells that exhibit characteristics similar to those of native RPE. On polarization, hES-RPE cells secrete high levels of PEDF that can support RPC survival. These experiments suggest that polarization of hES-RPE would be an important feature for promotion of RPC survival in future cell therapy for atrophic AMD.

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