June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Generating and characterizing iPSC-RPE cells for studying disease mechanisms in peroxisomal biogenesis disorders
Author Affiliations & Notes
  • Carly Feldman
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Temesgen Fufa
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Robert B Hufnagel
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Matthew Benson
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Carly Feldman None; Temesgen Fufa None; Robert Hufnagel None; Matthew Benson None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2472 – F0179. doi:
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      Carly Feldman, Temesgen Fufa, Robert B Hufnagel, Matthew Benson; Generating and characterizing iPSC-RPE cells for studying disease mechanisms in peroxisomal biogenesis disorders. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2472 – F0179.

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

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Abstract

Purpose : Peroxisomal biogenesis disorders (PBDs) represent a group of rare diseases that can cause retinal degeneration, sensorineural hearing loss, and other systemic abnormalities, due to defects in peroxisome structure and function. Over two-thirds of all PBDs are caused by biallelic pathogenic variants in PEX1 or PEX6. Despite retinal degeneration manifesting in many PBDs, precisely how peroxisomal dysfunction causes retinal pathology is not well understood. To this effect, we differentiated PEX1 and PEX6 wild-type and knock-out induced pluripotent stem cells (iPSCs) into retinal pigment epithelial (RPE) cells to evaluate the effect of peroxisome dysfunction on this disease-relevant cell type.

Methods : CRISPR/Cas9-mediated genome-editing was used to generate PEX1 and PEX6 gene knockouts in the Gibco Human Episomal iPSC Line (Thermo Fisher Scientific, Inc.). We differentiated RPE from knock-out iPSC lines, normal parental lines, and a positive control line (3D1.8). We characterized and validated the iPSCs with flow cytometry using the pluripotency markers SOX2, NANOG and TRA-1-60 antibodies. At day 40 of RPE differentiation, we purified the cells by cell sorting and characterized the iPSC-derived RPE by morphology, pigmentation, and analysis of proteins associated with differentiated RPE using immunostaining followed by flow cytometry. Lastly, we evaluated peroxisome function by assessing very long-chain fatty acid and phytanic acid content in our iPSC-derived RPE cells.

Results : We successfully characterized and validated PEX1 and PEX6 knock-out iPSCs, parental control iPSCs, and a positive control iPSC line. The iPSCs expressed the appropriate stem cell markers as determined by flow cytometry. In addition, we successfully differentiated each iPSC line into RPE cells. The differentiated RPE cells expressed the appropriate markers indicating maturation. Detailed biochemical and peroxisomal function studies are underway.

Conclusions : We have demonstrated the successful differentiation, characterization, and validation of human-derived iPSC into RPE cells to model the effect of PBDs on a retinal cell type. Future studies will evaluate the requirements of PEX1 or PEX6 in RPE maturation and how abnormalities in peroxisome structure and metabolism influence RPE function.

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

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