title: Functional analysis of serially expanded human iPS cell-derived RPE cultures.
abstract: OBJECTIVE
  To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures.
  
  
  METHODS
  Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE).
  
  
  RESULTS
  RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1-P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein-coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels.
  
  
  CONCLUSIONS
  Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.
authors.affiliation: Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin.
authors.forename: Ruchira
authors.initials: R
authors.lastname: Singh
authors.forename: M Joseph
authors.initials: MJ
authors.lastname: Phillips
authors.forename: David
authors.initials: D
authors.lastname: Kuai
authors.forename: Jackelyn
authors.initials: J
authors.lastname: Meyer
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Martin
authors.forename: Molly A
authors.initials: MA
authors.lastname: Smith
authors.forename: Enio T
authors.initials: ET
authors.lastname: Perez
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Wallace
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Capowski
authors.forename: Lynda S
authors.initials: LS
authors.lastname: Wright
authors.forename: David M
authors.initials: DM
authors.lastname: Gamm
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007150:Immunohistochemistry; D010587:Phagocytosis; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000196:embryology* / Q000378:metabolism
chemical_list: D005136:Eye Proteins; D012313:RNA
keywords: induced pluripotent stem cell; passaging; retinal pigment epithelium
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