SummaryDetailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.

Background:We hypothesized that C/EBP homologous protein mediates hepatocyte apoptosis in nonalcoholic steatohepatitis. Results: Paradoxically, Chop deletion protects from steatohepatitis by inducing apoptosis in activated macrophages. Conclusion: CHOP-dependent macrophage apoptosis in NASH highlights the cell type-specific complexity of the ER stress response. Significance: Therapeutic manipulation of mediators of ER stress response may have opposite effects in different cell populations; therefore, such studies should be interpreted cautiously.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle "in a dish" for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described. Video LinkThe video component of this article can be found at

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.

Purpose Detailed knowledge of cell surface proteins present during early embryonic development remains limited for most cell lineages. Due to the relevance of cell surface proteins in their functional roles controlling cell signaling and their utility as accessible, non-genetic markers for cell identification and sorting, the goal of this study was to provide new information regarding the cell surface proteins present during early mouse embryonic development. Experimental Design Using the Cell Surface Capture Technology, the cell surface N-glycoproteomes of three cell lines and one in vitro differentiated cell type representing distinct cell fates and stages in mouse embryogenesis were assessed. Results Altogether, more than 600 cell surface N-glycoproteins were identified represented by >5500 N-glycopeptides. Conclusions and Clinical Relevance The development of new, informative cell surface markers for the reliable identification and isolation of functionally defined subsets of cells from early developmental stages will advance the use of stem cell technologies for mechanistic developmental studies, including disease modeling and drug discovery.

SummarySeveral protocols now support efficient differentiation of human pluripotent stem cells to cardiomyocytes (hPSC-CMs) but these still indicate line-to-line variability. As the number of studies implementing this technology expands, accurate assessment of cell identity is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity has not yet been established. Therefore, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in day 25 hPSC-CMs and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges to interpreting data generated by published methods and inform the development of a robust protocol for routine assessment of hPSC-CMs. The data, workflow for antibody evaluation, and standardized protocol described here should benefit investigators new to this field and those with expertise in hPSC-CM differentiation.

Running Title: Standard flow cytometry protocol for assessing stem cell-derived cardiomyocytes SummaryModern differentiation protocols enable efficient, yet imperfect, differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM). As the number of laboratories and studies implementing this technology expands, the accurate assessment of cell identity in differentiation cultures is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity in hPSC-CM cultures has not yet been established. To address this gap, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in hPSC-CM and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges of interpreting data generated by published methods and informed the development of a robust protocol for routine assessment of hPSC-CM. Overall, the new data, workflow for evaluating fitfor-purpose use of antibodies, and standardized protocol described here should benefit investigators new to this field as well as those with expertise in hPSC-CM differentiation.

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