title: Polarized secretion of PEDF from human embryonic stem cell-derived RPE promotes retinal progenitor cell survival.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California 90089, USA.
authors.forename: Danhong
authors.initials: D
authors.lastname: Zhu
authors.forename: Xuemei
authors.initials: X
authors.lastname: Deng
authors.forename: Christine
authors.initials: C
authors.lastname: Spee
authors.forename: Shozo
authors.initials: S
authors.lastname: Sonoda
authors.forename: Chih-Lin
authors.initials: CL
authors.lastname: Hsieh
authors.forename: Ernesto
authors.initials: E
authors.lastname: Barron
authors.forename: Martin
authors.initials: M
authors.lastname: Pera
authors.forename: David R
authors.initials: DR
authors.lastname: Hinton
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000378:metabolism*; D020869:Gene Expression Profiling; D005865:Gestational Age; D006801:Humans; D020287:In Situ Nick-End Labeling; D007621:Karyotyping; D018613:Microscopy, Confocal; D009414:Nerve Growth Factors / Q000378:metabolism*; D010587:Phagocytosis / Q000502:physiology; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012374:Rod Cell Outer Segment / Q000502:physiology; D015843:Serpins / Q000378:metabolism*; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D005136:Eye Proteins; D009414:Nerve Growth Factors; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor