title: Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool.
abstract: OBJECTIVE
  To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC).
  
  
  METHODS
  Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated.
  
  
  RESULTS
  We obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation.
  
  
  CONCLUSIONS
  We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.
authors.affiliation: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.|Department of Ophthalmology & Visual Science, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Iwasaki
authors.affiliation: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.
authors.forename: Sunao
authors.initials: S
authors.lastname: Sugita
authors.affiliation: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Mandai
authors.affiliation: Ultrastructural Research Team, RIKEN Center for Life Science Technologies, Kobe, Hyogo, Japan.|Department of Cell Biology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Yonemura
authors.affiliation: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.
authors.forename: Akishi
authors.initials: A
authors.lastname: Onishi
authors.affiliation: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Ito
authors.affiliation: Department of Ophthalmology & Visual Science, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Mochizuki
authors.affiliation: Department of Ophthalmology & Visual Science, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Ohno-Matsui
authors.affiliation: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.
authors.forename: Masayo
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D000818:Animals; D002450:Cell Communication; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D017364:Collagenases / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000166:cytology; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D008214:Lymphocytes / Q000166:cytology; D051379:Mice; D010447:Peptide Hydrolases / Q000737:chemistry; D010587:Phagocytosis; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: C575294:accutase; D054460:rho-Associated Kinases; D010447:Peptide Hydrolases; D017364:Collagenases