Abstract
Purpose :
This study aims to determine the ability of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors to selectively eliminate undifferentiated and tumorigenic cells from hPSC cultures differentiating to retinal pigmented epithelial (RPE) cells.
Methods :
We cultured and differentiated hPSCs to neural progenitors and RPE cells, and studied ARPE19 cells. STF31 and FK866, inhibitors of the NAD salvage pathway protein NAMPT, were added in a dose- and time-dependent manner and cell viability assays (Neutral Red and XTT) performed . Teratoma assays are performed in NOD-SCID mice.
Results :
Human PSCs differentiated in vitro to RPE cells exhibit epithelial cell morphology, visible pigment from week 4 of differentiation, and contained RNA (BEST1, MITF) markers typical of RPE cells. The putative hPSC-derived RPE cell populations are ~70% pure, and work is on-going to improve RPE purity for functional assessments and for viability testing. To evaluate selective toxicity of NAMPT inhibitors on cells of the neuroectoderm lineage, human ES cells (H7), iPS cells (KB3), PAX 6-positive neural progenitors and ARPE 19 cells were treated. NAMPT inhibitors were highly toxic to hPSCs (0-5% viable cells) within 48-72 hours. PAX6 positive cells showed some decrease in viability (50-60% viable cells) at 72 hours. ARPE19 cell toxicity was minimal. Teratoma assays following 24-hour treatment of hPSCs did not completely prevent hPSC tumour formation, but longer treatment times appear effective.
Conclusions :
Elimination of tumorigenicity is a prerequisite for clinical intervention. NAMPT shows selective cell toxicity: hPSCs>>neural progenitors> ARPE19 cells. Purified hPSC-derived RPEs are likely to show limited toxicity to NAMPT, suggesting that treatment of these cultures with NAMPT inhibitors prior to transplantation will effectively eliminate all tumorigenic potential from these cultures – thus advancing efforts to treat Aging-related Macular Degeneration in humans.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.