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  • Discussion A genome scale location analysis using published

    2018-11-08

    Discussion A genome-scale location analysis using published data (Gifford et al., 2013) was performed to identify surface proteins that have promoters occupied by POU5F1(OCT4)/SOX2/NANOG (OSN). In total, 30 (6%) of the 496 proteins identified on the surface of hPSCs have promoters occupied by OSN (Table S7). An additional 47 were occupied by two factors. Of the 206 hPSC-restricted proteins, 16 (7%) are occupied by OSN. Transcriptional dogma would suggest that promoters occupied by OSN are critical to pluripotency, and the surface data are consistent with this fact, because none of the putative negative markers were occupied by OSN and only five of the hFib restricted were occupied by OSN. Although a direct role in pluripotency has not yet been described for these 16 OSN occupied PSC-restricted proteins, several have been implicated in early developmental processes or mechanisms with relevance to pluripotency, including CXADR (Dorner et al., 2005), GPR98 (McMillan et al., 2002), IL17RD (Torii et al., 2004), LINGO1 (Mi et al., 2004), LRIG1 (Laederich et al., 2004), and LRRN1 (Hossain et al., 2008). In the HSC field, immunophenotyping with a01 has been critical to the evaluation of cell-surface proteins as surrogate markers of potency, function, immunological compatibility, and for the isolation of cells for therapeutic purposes. As we (Gundry et al., 2011, 2012) and others (Kahler et al., 2013; Kang et al., 2011; Manos et al., 2011) have demonstrated, accessible surface proteins are useful as markers for sorting and characterizing pluripotent cells. Application of the positive selection hPSC markers described in this resource provides the field with new targets suitable for improving the efficiency of isolation and characterization of high quality hiPSC colonies and/or those with defined differentiation potential. In addition to the positive PSC markers, these data reveal fibroblast markers (DPP4, NRP1, and COLEC12) useful for negative selection of hiPSCs from reprogrammed fibroblasts. Additional negative markers are likely to emerge as the cell types within the Cell Surface Protein Atlas expands or if less stringent data filtering are used (i.e., only require presence in hFib and absence in hPSCs, regardless of expression among other cell types). Precise mapping (time and quantity) of transcriptional and protein levels of these putative negative markers during reprogramming will ultimately influence the utility of these accessible proteins for selection purposes. The major bottleneck in using surface proteins for isolation and cell characterization is the lack of suitable antibodies or other affinity reagents that recognize native extracellular epitopes, a requirement for live cell selection. The information generated in this study partially overcome this limitation as the CSC technology experimentally verifies N-glycosylated peptides present in extracellular exposed domains of transmembrane and GPI-anchored proteins, which directly facilitates antigen design of surface epitopes useful for antibody production. Therefore, CSC technology lends itself for the immunophenotyping of cells without antibodies. The hPSC surface proteome resource described here can be further exploited to rationally identify accessible and putative drug targets on the surface of hESCs and hiPSCs. This might be especially useful for repurposing known drugs. Although GLUT1 is present on many cell types, hPSCs, unlike many other cells that often contain multiple glucose transporters or rely on oxidative phosphorylation, are believed to be highly reliant on glycolysis, analogous to the Warburg effect in cancer cells (Folmes et al., 2011; Varum et al., 2011). Of the cells tested in this study, the reported GLUT1 inhibitor STF-31 proved to be selectively toxic to hPSCs. Recently, using a small molecule screening approach, Ben-David et al. (2013a) discovered the utility of a small molecule inhibitor of oleic acid biosynthesis, PluriSIn, with selective toxicity for hPSCs. Although it is known that metabolism influences a cell’s decision to proliferate, differentiate, or remain quiescent (Shyh-Chang et al., 2013), these two studies firmly establish that small molecule-based metabolic inhibitors represent viable alternative strategies to suicide genes (Cao et al., 2007; Rong et al., 2012), SSEA-5, lactate, and Claudin-6-based approaches (Ben-David et al., 2013b; Tang et al., 2011; Tohyama et al., 2013). Furthermore, because another reported small molecule inhibitor of GLUT1 that we tested, WZB117, also proves toxic to hPSCs (data not shown), the use of other strategies to specifically inhibit GLUT1 may be broadly applicable for the removal of tumorigenic hPSCs from cultures of differentiating cells. In contrast to the previous reports that required alternative energy sources (Tohyama et al., 2013; Tomizawa et al., 2013), our study achieves selective hPSC elimination without alternative fuel substrates or general glucose starvation, making our findings more broadly useful to the community than previous approaches.