title: Canonical/β-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells.
abstract: OBJECTIVE
  The purpose of this study was to better understand the role canonical/β-catenin Wnt signaling plays in the differentiation of human embryonic stem cells (hESCs) into retinal pigmented epithelium (RPE), with the goal of improving methods for derivation.
  
  
  METHODS
  Fluorescent reporters were generated to monitor RPE differentiating from hESCs by using a previously described 14-day derivation protocol. Reporters were used to test the effects of the canonical/β-catenin Wnt pathway agonist CHIR99021 on differentiating RPE. Cells derived from differentiation studies were characterized by lineage-specific transcription factor expression, morphology, pigmentation, and function. The RPE derivation efficiency was determined from percentage positive PMEL17 expression.
  
  
  RESULTS
  Fluorescent reporters mimicked expression of endogenous genes during 14-day differentiation to RPE. Analysis of Wnt pathway gene expression showed that the pathway components are expressed in differentiating RPE cells. Addition of CHIR99021 improved RPE derivation based on morphology, expression of RPE-specific lineage markers, and genes involved in melanogenesis. Additionally, expression of the neural retina marker CHX10 was suppressed during differentiation with CHIR99021. Addition of soluble WNT3A, but not WNT5A, had the same result. The CHIR99021-modified protocol yielded cell populations that were 97.77% ± 0.1% positive for the RPE marker PMEL17 at day 14. After cells were expanded to passage 3, they were shown to express RPE markers, carry out phagocytosis of rod outer segments, and secrete pigment epithelium-derived factor apically and vascular endothelial growth factor basally.
  
  
  CONCLUSIONS
  Our findings demonstrated the importance of canonical/β-catenin Wnt signaling in RPE differentiation and showed that manipulating the pathway significantly improves RPE derivation from hESC.
authors.affiliation: Center for Stem Cell Biology and Engineering, University of California, Santa Barbara, California, United States.
authors.forename: Lyndsay L
authors.initials: LL
authors.lastname: Leach
authors.affiliation: Center for Stem Cell Biology and Engineering, University of California, Santa Barbara, California, United States.
authors.forename: David E
authors.initials: DE
authors.lastname: Buchholz
authors.affiliation: Center for Stem Cell Biology and Engineering, University of California, Santa Barbara, California, United States California State University, Channel Islands, California, United States.
authors.forename: Vignesh P
authors.initials: VP
authors.lastname: Nadar
authors.affiliation: Department of Molecular, Cellular & Developmental Biology, University of California, Santa Barbara, California, United States.
authors.forename: Stefan E
authors.initials: SE
authors.lastname: Lowenstein
authors.affiliation: Center for Stem Cell Biology and Engineering, University of California, Santa Barbara, California, United States.
authors.forename: Dennis O
authors.initials: DO
authors.lastname: Clegg
mesh_terms: D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D005136:Eye Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007150:Immunohistochemistry; D008268:Macular Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009414:Nerve Growth Factors / Q000378:metabolism*; D012313:RNA / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000196:embryology / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism*; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000235:genetics* / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D005136:Eye Proteins; D009414:Nerve Growth Factors; D012313:RNA; D015843:Serpins; D051176:beta Catenin; D000099174:Pigment Epithelium-Derived Factor
keywords: Wnt signaling; age-related macular degeneration; fluorescent reporters; human embryonic stem cells; retinal pigmented epithelium