June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Generation and initial characterization of iPSC-derived RPE cells from RP59 donors
Author Affiliations & Notes
  • Lara A Skelton
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
    Ophthalmology, Biochemistry, and Neuroscience Program, SUNY-University at Buffalo, Buffalo, New York, United States
  • Sriganesh Ramachandra Rao
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
    Ophthalmology, Biochemistry, and Neuroscience Program, SUNY-University at Buffalo, Buffalo, New York, United States
  • Eric Soubeyrand
    Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
  • Elisha Monson
    Optometry and Vision Science, The University of Alabama at Birmingham, School of Optometry, Birmingham, Alabama, United States
  • Tariq A Akhtar
    Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
  • Michael B Gorin
    Jules Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Steven J Pittler
    Optometry and Vision Science, The University of Alabama at Birmingham, School of Optometry, Birmingham, Alabama, United States
  • Steven Jay Fliesler
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
    Ophthalmology, Biochemistry, and Neuroscience Program, SUNY-University at Buffalo, Buffalo, New York, United States
  • Footnotes
    Commercial Relationships   Lara Skelton None; Sriganesh Ramachandra Rao None; Eric Soubeyrand None; Elisha Monson None; Tariq Akhtar None; Michael Gorin None; Steven Pittler None; Steven Fliesler None
  • Footnotes
    Support  NIH/NEI (R01EY029341; SJF, SJP) and P30EY003039 (SJP); Knights Templar Eye Foundation Pediatric Ophthalmology Career Starter Research Grant (SRR); VA MERIT Award (1I0 BX002439; LAS), Research Career Scientist Awards (I K6 BX005787; SJF); Harold and Pauline Price Foundation and Unrestricted Grant from Research to Prevent Blindness (MBG).
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3180. doi:
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    • Get Citation

      Lara A Skelton, Sriganesh Ramachandra Rao, Eric Soubeyrand, Elisha Monson, Tariq A Akhtar, Michael B Gorin, Steven J Pittler, Steven Jay Fliesler; Generation and initial characterization of iPSC-derived RPE cells from RP59 donors. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3180.

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

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Abstract

Purpose : RP59 (OMIM #613861) is an autosomal recessive form of retinitis pigmentosa caused by mutations in the gene encoding dehydrodolichyl diphosphate synthase (DHDDS), which is required for the synthesis of dolichol (Dol), which in turn is required for protein N-glycosylation. Here we describe the initial characterization of RPE cells generated from induced pluripotent stem cells (iPSCs) derived from RP59 donor blood cells.

Methods : Blood cells from 3 RP59 donors homozygous for the K42E DHDDS pathogenic variant were obtained, then converted to iPSCs and then to RPE cells (PMID:24873859). Normal human iPSC-derived RPE (Dys100) cells were obtained as previously described (PMID:36540063). All cell lines were genotyped and sequence-verified for presence of the homozygous K42E variants; cells were grown to confluence under identical (low-serum) conditions for ≥4 weeks to achieve differentiated phenotype. Cells were rinsed copiously with PBS, harvested, then subjected to SDS-PAGE/Western analysis (probed with antibodies against CRALBP, BEST-1, RPE65, Ezrin, and transferrin, and suitable fluor-conjugated secondaries), with correlative immunofluorescence microscopy analysis (including ZO-1), and Con A lectin blot analysis, ±PNGase-F treatment. Dol chain length analysis and quantification (HPLC) were performed in parallel, essentially as described previously (PMID:35960639). Statistical analysis: Student’s t-test, significance threshold p<0.05 (N≥3/group).

Results : Genotypically verified RP59-derived and control iPSC-RPE cells had polygonal morphology and phase-bright borders, and expressed characteristic RPE markers (ZO-1, CRALBP, BEST-1, RPE65) and cytoskeletal linker Ezrin. Lectin blot patterns were comparable for RP59 and control iPSC-RPE cells. Transferrin Mr and PNGase-F sensitivity, by SDS-PAGE/Western analysis, also were comparable for RP59-derived vs. control cells. However, Dol chain lengths were shorter than normal in RP59 iPSC-RPE cells, consistent with findings reported for RP59 patient-derived bodily fluids (PMID:24078709).

Conclusions : No glycosylation defects were apparent in RP59 iPSC-RPE cells, despite having shorter than normal Dol chain lengths. These findings cast further doubt on the current classification of RP59 as a congenital disorder of glycosylation (CDG) and suggest involvement of more complex mechanisms by which this genetic defect leads to retinal degeneration.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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