Inhibition of an NAD+ Salvage Pathway Provides Efficient and Selective Toxicity to Human Pluripotent Stem Cells

Abstract

The tumorigenic potential of human pluripotent stem cells (hPSCs) is a major limitation to the widespread use of hPSC derivatives in the clinic. Here, we demonstrate that the small molecule STF-31 is effective at eliminating undifferentiated hPSCs across a broad range of cell culture conditions with important advantages over previously described methods that target metabolic processes. Although STF-31 was originally described as an inhibitor of glucose transporter 1, these data support the reclassification of STF-31 as a specific NAD + salvage pathway inhibitor through the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). These findings demonstrate the importance of an NAD + salvage pathway in hPSC biology and describe how inhibition of NAMPT can effectively eliminate hPSCs from culture. These results will advance and accelerate the development of safe, clinically relevant hPSCderived cell-based therapies. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:483-493 SIGNIFICANCEThe tumorigenic potential of human pluripotent stem cells (hPSCs) is a major limitation to the widespread use of hPSC derivatives in the clinic. This study provides detailed analyses of cellular metabolic flux to define an efficient strategy for selective hPSC elimination that is effective across many culture conditions and does not have cytotoxic effects on hPSC-derived progeny. Of broad significance to the stem cell and regenerative medicine fields, this study also highlights the importance of examining the effect of in vitro culturing parameters when evaluating the efficacy of hPSC-elimination strategies, especially those that target metabolic processes.

Cardiomyocyte Differentiation Promotes Cell Survival During Nicotinamide Phosphoribosyltransferase Inhibition Through Increased Maintenance of Cellular Energy Stores

“…Cell viability under NAMPT inhibition was assessed by neutral red uptake (an indirect assay of ATP levels) and SYTOX cell death assay (dependent on cell membrane permeability). Consistent with our previous studies , continuous NAMPT inhibition is toxic to hiPSC (Fig. a, b).…”

“…Day 10 hiPSC‐CM and hESC‐CM have increased cell survival with NAMPT inhibition; however, spontaneous contraction ceases by 72 hours of treatment and increased cell death is observed by 96 hours (data not shown). The toxicity resulting from continuous NAMPT inhibitor treatment at day 5 and 10 is consistent with our previous report , demonstrating that treatment with 2.5 µM STF‐31 for 24–48 hours did not produce adverse effects on hiPSC‐CM, although measurable toxicity was observed with 72 hours treatment.…”

“…b). At this time point, cell death has been initiated as evidenced by enhanced cleavage of caspases . Unlike hiPSC, day 28 hiPSC‐CM (Fig.…”

Mitochondria in human pluripotent stem cell apoptosis

“…There are conflicting reports that STF-31 kills hPSCs by inhibiting either glucose transporters [96] or nicotinamide phosphoribosyltransferase (NAMPT) [97]. Facilitated glucose transporters are abundant at the hPSC cell membrane, which led to the identification of STF-31 [96].…”

“…STF-31 treatment causes a decrease in NAD + levels before it causes a decrease in glucose uptake. Apoptosis of hPSCs by STF-31 treatment can be rescued by supplementation of nicotinic acid, supporting inhibition of NAMPT as a mechanism for STF-31 killing of hPSCs [97]. …”