July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Characterization of Stargardt iPSC-derived RPE cells
Author Affiliations & Notes
  • Nermin Kady
    Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States
    Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Anna Matynia
    Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States
    Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Saravanan Karumbayaram
    Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, California, United States
    Eli and Edythe Broad Stem Cell Research Center, University of California, Los Angeles, California, United States
  • Jane Hu
    Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States
    Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Marcia Lloyd
    Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States
    Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Dean Bok
    Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States
    Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Michael B Gorin
    Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States
    Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Roxana A. Radu
    Stein Eye Institute, David Geffen School of Medicine, University of California, Los Angeles, California, United States
    Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Nermin Kady, None; Anna Matynia, None; Saravanan Karumbayaram, None; Jane Hu, None; Marcia Lloyd, None; Dean Bok, None; Michael Gorin, None; Roxana Radu, None
  • Footnotes
    Support  NIH-EY025002, Stein Eye Institute Core Grant for Vision Research EY000331 and an unrestricted grant from Research to Prevent Blindness and Sarkaria philanthropic award (RAR).Sarkaria philanthropic award and a Stein/Oppenheimer Endowment Award to MGB/AM, RPB and the Harold and Pauline Price Foundation (MBG). Esther B. O’Keeffe Foundation, The Jean Perkins Foundation and UCLA E&E BSCRC ( SK)
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3045. doi:
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    • Get Citation

      Nermin Kady, Anna Matynia, Saravanan Karumbayaram, Jane Hu, Marcia Lloyd, Dean Bok, Michael B Gorin, Roxana A. Radu; Characterization of Stargardt iPSC-derived RPE cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3045.

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

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Abstract

Purpose :
Stargardt disease (STGD) is a blinding disease of children and young adults, caused predominantly by mutations in the ABCA4 gene. The underlying STGD pathogenesis resides mainly within the supportive cells, the retinal pigment epithelium (RPE). We generated induced pluripotent stem cell (iPSC) derived RPE cells from three STGD patients with single pathogenic variants in the ABCA4 gene for whom other STGD genes have been excluded. In the current study, morphological and molecular features were evaluated in iPSC-derived RPE cells from one STGD patient with a heterozygous ABCA4 variant (R1129L / C.3386G>T).

Methods :
Fibroblasts from skin biopsies of STGD patients were reprogrammed to generate three independent iPSC lines, using the non-integrating SimpliconTM RNA Reprogramming method. One of these STGD patient-derived lines and a normal control iPSC line (Source: UCLA BSCRC iPSC Core) were differentiated into RPE cells that were analyzed at passage 2, after 2 and 5 months (mo) in culture. Transcription levels of ABCA4 and markers of cell pluripotency, development, melanogenesis, and RPE-specificity were determined by qRT-PCR. Protein levels were evaluated by immunohistochemistry and immunoblotting. Morphological features of the iPS-derived RPE cells were analyzed by light and electron microscopy (EM). Transepithelial resistance (TER) was measured and formation of tight junctions was evidenced by immunofluorescence.

Results :
Normalized RPE-specific marker (RPE65, LRAT, BEST1) mRNA levels were several-fold lower in STGD iPSC-derived RPE cells compared to control in both 2 and 5 mo cultures. ABCA4 mRNA expression was reduced at 2 mo in culture compared with control cells and normalized by 5 mo. By EM analysis, STGD iPSC-derived RPE cells were highly polarized demonstrating basal nuclei, basal infoldings, apical microvilli and intercellular junctions, similar to the controls. By immunohistochemistry, ZO-1 localized on the lateral border of the cells with a heterogeneous distribution in the STGD-derived RPE. TER was significantly reduced in STGD iPSC-derived RPE cells compared to control.

Conclusions :
Differentiation of the STGD patient iPSCs into RPE cells was similar to the control iPSCs. Both STGD and control iPSC-derived RPE cells express robust levels of ABCA4. This will allow further molecular and biological analyses, including vitamin A metabolism and complement reactivity.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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