title: Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue.
abstract: OBJECTIVE
  We tested what native features have been preserved with a new culture protocol for adult human RPE.
  
  
  METHODS
  We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used.
  
  
  RESULTS
  Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 μM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 μL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01).
  
  
  CONCLUSIONS
  The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.
authors.affiliation: Icahn School of Medicine at Mount Sinai New York, New York, United States.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Blenkinsop
authors.affiliation: Neural Stem Cell Institute, Rensselaer, New York, United States.
authors.forename: Janmeet S
authors.initials: JS
authors.lastname: Saini
authors.affiliation: National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Arvydas
authors.initials: A
authors.lastname: Maminishkis
authors.affiliation: National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Kapil
authors.initials: K
authors.lastname: Bharti
authors.affiliation: National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Qin
authors.initials: Q
authors.lastname: Wan
authors.affiliation: National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Tina
authors.initials: T
authors.lastname: Banzon
authors.affiliation: National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Lotfi
authors.affiliation: National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Janine
authors.initials: J
authors.lastname: Davis
authors.affiliation: Yale University, New Haven, Connecticut, United States.
authors.forename: Deepti
authors.initials: D
authors.lastname: Singh
authors.affiliation: Yale University, New Haven, Connecticut, United States.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Rizzolo
authors.affiliation: National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States.
authors.forename: Sheldon
authors.initials: S
authors.lastname: Miller
authors.affiliation: Neural Stem Cell Institute, Rensselaer, New York, United States.
authors.forename: Sally
authors.initials: S
authors.lastname: Temple
authors.affiliation: Neural Stem Cell Institute, Rensselaer, New York, United States.
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Stern
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016764:Cell Polarity; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008875:Middle Aged; D009414:Nerve Growth Factors / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000648:ultrastructure; D013234:Stem Cells / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D005136:Eye Proteins; D009414:Nerve Growth Factors