June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Characterization of Danon disease human induced pluripotent stem cell derived retinal pigment epithelial cells
Author Affiliations & Notes
  • Shyamanga Ram Borooah
    Ophthalmology, University of California at San Diego Department of Ophthalmology at the Shiley Eye Institute, La Jolla, California, United States
  • Paul Bushway
    University of California San Diego, La Jolla, California, United States
  • Joshua Fong
    University of California San Diego, La Jolla, California, United States
  • Yasmin Yacoubian
    University of California San Diego, La Jolla, California, United States
  • Kristyn Huffman
    Ophthalmology, University of California at San Diego Department of Ophthalmology at the Shiley Eye Institute, La Jolla, California, United States
  • Eric Adler
    University of California San Diego, La Jolla, California, United States
  • Footnotes
    Commercial Relationships   Shyamanga Borooah None; Paul Bushway None; Joshua Fong None; Yasmin Yacoubian None; Kristyn Huffman None; Eric Adler None
  • Footnotes
    Support  Knights Templar Eye Foundation and Foundation Fighting Blindness CDA grant CD-GT-0918-0746-SEI
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2446 – F0390. doi:
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      Shyamanga Ram Borooah, Paul Bushway, Joshua Fong, Yasmin Yacoubian, Kristyn Huffman, Eric Adler; Characterization of Danon disease human induced pluripotent stem cell derived retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2446 – F0390.

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

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Abstract

Purpose : Danon disease (DD) is a rare X-linked syndromic disorder resulting from mutations in the gene LAMP2 leading to dysfunctional autophagy. The exact contribution of lysosome-associated membrane protein 2 (LAMP2) deficiency to retinal degeneration in human DD remains unclear. We performed a phenotypic and functional characterization of human-induced pluripotent cell (hiPSC) derived RPE (hiPSC-RPE) cells possessing harboring a frameshift insertion in exon 2 of LAMP2 to better understand the pathogenesis of retinopathy in DD.

Methods : HiPSCs were generated using Sendai virus expression of Yamanaka factors in donor cells from an affected male patient with DD and unaffected control patient. The hiPSCs were differentiated to hiPSC-RPE using our in house protocol. We performed basic hiPSC-RPE characterization using immunostaining, electron microscopy (EM), rod outer segments (ROS) phagocytosis and autophagy analyses by performing LC3-II immunostaining and western blot as well as qPCR for LAMP2 isoforms

Results : Both DD and control hiPSC-RPE demonstrated expression of ZO-1, MITF, Bestrophin-1 and CRALBP RPE markers. Control hiPSC-RPE demonstrated a significantly greater expression of LAMP2A (p<0.001) and 2B (p<0.001) isoforms compared with 2C (Fig 1).
Phagocytosis of ROS in Danon hiPSC-RPE cells at 6 hours (21.4±18.9 ROS) was significantly greater (p<0.05) compared to control hiPSC-RPE cells (45.7±6.6 ROS) Western blot found no significant difference in LC3-II expression between the DD line and control line under normal conditions but did find a significant difference under conditions promoting autophagy including serum starvation (p<0.05) and under conditions blocking fusion of the autophagsomes and lysosome with the addition of hydroxychloroquine (p<0.01)(Fig 2).

Conclusions : Functional hiPSC-RPE were successfully differentiated from both a DD and control hiPSC line. Our findings suggest that there is a cell specific isoform expression signature in RPE. In addition, autophagic flux is impaired in DD hiPSC-RPE under stress, likely resulting in autophagosome accumulation and our finding of increased ROS preservation. These disease-relevant in vitro phenotypic markers will assist testing of approaches aimed at treating DD and other diseases in which LAMP2 deficiency is implicated. Further studies, with additional lines will be helpful to confirm these early findings.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

 

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