April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Microphthalmia-associated Transcription Factor (MITF) affects optic vesicle cell proliferation and retinal pigment epithelium maturation in human ES cell (hESC) cultures
Author Affiliations & Notes
  • Anna Petelinsek
    University of Wisconsin-Madison, Madison, WI
  • Elizabeth E Capowski
    University of Wisconsin-Madison, Madison, WI
  • Sara Howden
    Morgridge Institute for Research, Madison, WI
  • Lynda S Wright
    University of Wisconsin-Madison, Madison, WI
  • Isabel Pinilla Lozano
    Ophthalmology, University Hospital Lozano Blesa, Zaragoza, Spain
    IIS Aragon, Aragon Health Sciences Institute, Zaragoza, Spain
  • Kyle Wallace
    University of Wisconsin-Madison, Madison, WI
  • Eric Clark
    University of Wisconsin-Madison, Madison, WI
  • Joe Phillips
    University of Wisconsin-Madison, Madison, WI
  • David M Gamm
    Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI
    McPherson Eye Research Institute, Madison, WI
  • Footnotes
    Commercial Relationships Anna Petelinsek, None; Elizabeth Capowski, None; Sara Howden, None; Lynda Wright, None; Isabel Pinilla Lozano, None; Kyle Wallace, None; Eric Clark, None; Joe Phillips, None; David Gamm, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 697. doi:
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      Anna Petelinsek, Elizabeth E Capowski, Sara Howden, Lynda S Wright, Isabel Pinilla Lozano, Kyle Wallace, Eric Clark, Joe Phillips, David M Gamm; Microphthalmia-associated Transcription Factor (MITF) affects optic vesicle cell proliferation and retinal pigment epithelium maturation in human ES cell (hESC) cultures. Invest. Ophthalmol. Vis. Sci. 2014;55(13):697.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Loss of MITF expression results in microphthalmia and RPE defects in rodent models, but its role in human retinogenesis remains unknown. We created a MITF double knockout (dKO) hESC line to examine the consequences of its absence during early retinal development.

Methods: The second common exon of MITF was targeted by two rounds of BAC-mediated homologous recombination to inactivate both alleles in WA09 hESCs. Correct targeting and gene inactivation were confirmed by genomic PCR, RT-PCR, and Western blots. Light microscopy, immunocytochemistry (ICC), RT-PCR, and RT-qPCR were then performed at different stages of retinal cell development to examine the impact of MITF loss on the production, proliferation, and maturation of early neuroretinal cells and RPE.

Results: RT-PCR and ICC analyses confirmed the absence of MITF transcripts and protein in dKO-MITF hESCs. Upon differentiation, ICC revealed that dKO-MITF hESCs sequentially adopted anterior neuroectoderm and early eye field fates in a manner similar to isogenic control WA09 hESCs. However, optic vesicle-like structures (OVs) generated from dKO-MITF hESCs were significantly smaller than control OVs (>60% reduction; p<0.0001). In addition, expression levels of PAX6, RX, and SIX6 were reduced in dKO-MITF vs. control OVs (54, 76, and 79% reductions, respectively), as was the number of proliferating cells (Ki67+ cells: 15.4% vs. 23.4%; p≤ 0.01). Following onset of VSX2 expression, cell proliferation within dKO-MITF OVs matched that of control OVs. Furthermore, dKO-MITF OVs produced CRX/Recoverin+ precursors in a temporal and spatial pattern indistinguishable from control OVs. However, RPE generated from dKO-MITF hESCs failed to fully mature; instead, dKO-MITF RPE remained unpigmented and lacked typical RPE organization even after several months in culture.

Conclusions: Using a genetically engineered hESC line, we showed that MITF is specifically involved in human OV proliferation and RPE maturation. Such information may lead to improvements in retinal cell production in vitro and/or provide insight into retinal development at stages previously inaccessible in humans.

Keywords: 721 stem cells • 739 transcription factors • 701 retinal pigment epithelium  

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