title: Comparison of FRPE and human embryonic stem cell-derived RPE behavior on aged human Bruch's membrane.
abstract: OBJECTIVE
  To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruch's membrane (BM) from aged and AMD donors.
  
  
  METHODS
  hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21.
  
  
  RESULTS
  hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67-positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2.
  
  
  CONCLUSIONS
  Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM.
authors: The Institute of Ophthalmology and Visual Science, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 90 Bergen Street, Newark, NJ 07101, USA.
authors: Ilene K
authors: IK
authors: Sugino
authors: Qian
authors: Q
authors: Sun
authors: Jianqiu
authors: J
authors: Wang
authors: Celia F
authors: CF
authors: Nunes
authors: Noounanong
authors: N
authors: Cheewatrakoolpong
authors: Aprille
authors: A
authors: Rapista
authors: Adam C
authors: AC
authors: Johnson
authors: Christopher
authors: C
authors: Malcuit
authors: Irina
authors: I
authors: Klimanskaya
authors: Robert
authors: R
authors: Lanza
authors: Marco A
authors: MA
authors: Zarbin
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology*; D016570:Bruch Membrane / Q000166:cytology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D005260:Female; D053686:Fetal Stem Cells / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020287:In Situ Nick-End Labeling; D016023:Integrins / Q000235:genetics; D007633:Keratins / Q000378:metabolism; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D008268:Macular Degeneration / Q000473:pathology; D008297:Male; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D009924:Organ Culture Techniques; D012333:RNA, Messenger / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D016023:Integrins; D019394:Ki-67 Antigen; C497234:MITF protein, human; D051739:Microphthalmia-Associated Transcription Factor; D012333:RNA, Messenger; D007633:Keratins