June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Comparative study of the effects of nicotinamide oncultured iPSC-derived human RPE cells from normal and Smith-Lemli-Opitz Syndrome (SLOS) donors
Author Affiliations & Notes
  • John A Petrie
    Ophthalmology and Biochemistry, University at Buffalo, Buffalo, New York, United States
    Research Service, VA Western New York Healthcare System Buffalo VA Medical Center, Buffalo, New York, United States
  • Lara Ann Skelton
    Ophthalmology and Biochemistry, University at Buffalo, Buffalo, New York, United States
    Research Service, VA Western New York Healthcare System Buffalo VA Medical Center, Buffalo, New York, United States
  • Sriganesh Ramachandra Rao
    Ophthalmology and Biochemistry, University at Buffalo, Buffalo, New York, United States
    Research Service, VA Western New York Healthcare System Buffalo VA Medical Center, Buffalo, New York, United States
  • Steven J. Fliesler
    Ophthalmology and Biochemistry, University at Buffalo, Buffalo, New York, United States
    Research Service, VA Western New York Healthcare System Buffalo VA Medical Center, Buffalo, New York, United States
  • Footnotes
    Commercial Relationships   John Petrie, None; Lara Skelton, None; Sriganesh Ramachandra Rao, None; Steven Fliesler, None
  • Footnotes
    Support  UB Ophthalmology Summer Student Fellowship (JAP); Institutional IFR Funds (SJF); NIH/NCATS (1UL1 TR001412; SJF); Fight For Sight Postdoctoral Fellowship (SRR); Knights Templar Eye Foundation Pediatric Ophthalmology Career Starter Award (SRR); VAWNYHS facilities and resources (JAP, LAS, SRR, SJF), VA BLR&D Research Career Scientist Award (SJF).
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 2212. doi:
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    • Get Citation

      John A Petrie, Lara Ann Skelton, Sriganesh Ramachandra Rao, Steven J. Fliesler; Comparative study of the effects of nicotinamide oncultured iPSC-derived human RPE cells from normal and Smith-Lemli-Opitz Syndrome (SLOS) donors. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2212.

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

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Abstract

Purpose : To assess the effects of nicotinamide (Nic) ondifferentiation and protein expression profiles of cultured iPSC-derived RPE cells from normal human donors (Dys0100; DYS), vs. mildly (CWI) and severely (A2) affected SLOS donors.

Methods : DYS, CWI and A2 iPSC-RPE cells were derived as previously described (Ferrer M et al., 2014; Ramachandra Rao et al., 2018). Cells (passage #6) were seeded in low-Ca2+ medium on tissue culture plastic, grown to confluence, and switched to high-Ca2+ medium containing either basal (~33 mM; vehicle control (VC)) or 10 mM Nic. Cell morphology was monitored by phase-contrastand confocal fluorescence microscopy. After 1.5 mo., monolayers were harvested in RIPA buffer and processed for Western blot (WB) analysis. Blots were probed with antibodies to RPE65, CRALBP, and Best-1, normalized to b-actin levels. CWI and A2 RPE cells cultured on poly-L-ornithine-coated RINZL plastic coverslips ± Nic, were probed with anti-Occludin MAb (clone OC3F10); F-actin was detected with Alexa Fluor® 568-Phalloidin. Statistical analysis: Student’s t-test, with significance threshold P<0.05.

Results : DYS and CWI cells exhibited relatively normal RPE-like morphology; however, A2 cells appeared markedly unhealthy and hypertrophic. Supplementation of culture media with 10 mM Nic improved iPSC-RPE differentiation, polygonal morphology, monolayer uniformity, phase-bright border and anti-Occludin staining continuity, and F-actin organization, relative to VC. While the effects were modest for DYS and CWI cells, A2 cells showed profound improvement. By WB analysis, RPE65 expression levels increased in all cell lines (+Nic, vs. VC): by ~2.5X (DYS) and ~2.2X (CWI), vs. ~5.8X (A2). Similarly, CRALBP expression increased (+Nic, vs. VC) by ~1.4X (DYS) and ~1.5X (CWI), vs. ~7.4X (A2). Also, Best-1 expression increased (+Nic, vs. VC) by ~2.5X (DYS) and ~3.7X (CWI), vs. ~9X (A2).

Conclusions : Culture medium supplementation with 10 mM Nic promotes up-regulation of RPE cell markers and normal RPE-like morphology and cytology of cultured iPSC-RPE cells, particularly those derived from severely-affected SLOS donors. Nic supplementation may augment therapeutic efficacy of current standard-of-care treatment for SLOS patients.

This is a 2021 ARVO Annual Meeting abstract.

 

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