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.