ß-cell-selective inhibition of DNA damage response signaling by nitric oxide is associated with an attenuation in glucose uptake.

Nitric oxide (NO) plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic ß-cells. As a genotoxic agent, NO activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of NO synthase, NO inhibits DDR signaling and DDR-induced apoptosis in a ß-cell-selective manner. DDR signaling inhibition by NO correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, ß-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in ß-cells and non-ß-cells treated with NO or complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and tricarboxylic acid cycle intermediates as well as NADPH are significantly decreased in ß-cells treated with NO or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a ß-cell-selective manner. Our findings indicate that the ß-cell-selective inhibition of DDR signaling by NO is associated with a decrease in ATP to levels that fall significantly below the KM for ATP of glucokinase (glucose uptake) and suggest that this action places the ß-cell in a state of suspended animation where it is metabolically inert until NO is removed, and metabolic function can be restored.

C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.

Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop(+/+)) and knock-out (Chop(-/-)) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop(-/-) mice developed greater liver injury, inflammation, and fibrosis than Chop(+/+) mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop(+/+) and Chop(-/-) were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop(+/+) and Chop(-/-) macrophages, Chop(-/-) macrophages were resistant to its lipotoxicity. Chop(-/-) mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop(-/-) and Chop(+/+) mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.

Mechanisms of resistance to targeted therapies for relapsed or refractory acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive disease of clonal hematopoiesis with a high rate of relapse and refractory disease despite intensive therapy. Traditionally, relapsed or refractory AML has increased therapeutic resistance and poor long-term survival. In recent years, advancements in the mechanistic understanding of leukemogenesis have allowed for the development of targeted therapies. These therapies offer novel alternatives to intensive chemotherapy and have prolonged survival in relapsed or refractory AML. Unfortunately, a significant portion of patients do not respond to these therapies and relapse occurs in most patients who initially responded. This review focuses on the mechanisms of resistance to targeted therapies in relapsed or refractory AML.

Are These Cardiomyocytes? Protocol Development Reveals Impact of Sample Preparation on the Accuracy of Identifying Cardiomyocytes by Flow Cytometry.

Several 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.

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

To address concerns regarding the tumorigenic potential of undifferentiated human pluripotent stem cells (hPSC) that may remain after in vitro differentiation and ultimately limit the broad use of hPSC-derivatives for therapeutics, we recently described a method to selectively eliminate tumorigenic hPSC from their progeny by inhibiting nicotinamide phosphoribosyltransferase (NAMPT). Limited exposure to NAMPT inhibitors selectively removes hPSC from hPSC-derived cardiomyocytes (hPSC-CM) and spares a wide range of differentiated cell types; yet, it remains unclear when and how cells acquire resistance to NAMPT inhibition during differentiation. In this study, we examined the effects of NAMPT inhibition among multiple time points of cardiomyocyte differentiation. Overall, these studies show that in vitro cardiomyogenic commitment and continued culturing provides resistance to NAMPT inhibition and cell survival is associated with the ability to maintain cellular ATP pools despite depletion of NAD levels. Unlike cells at earlier stages of differentiation, day 28 hPSC-CM can survive longer periods of NAMPT inhibition and maintain ATP generation by glycolysis and/or mitochondrial respiration. This is distinct from terminally differentiated fibroblasts, which maintain mitochondrial respiration during NAMPT inhibition. Overall, these results provide new mechanistic insight into how regulation of cellular NAD and energy pools change with hPSC-CM differentiation and further inform how NAMPT inhibition strategies could be implemented within the context of cardiomyocyte differentiation. Stem Cells Translational Medicine 2017;6:1191-1201.

Inhibition of an NAD⁺ salvage pathway provides efficient and selective toxicity to human pluripotent stem cells.

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 hPSC-derived cell-based therapies.

High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry.

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.

N-glycoprotein surfaceomes of four developmentally distinct mouse cell types.

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, nongenetic 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.

A human pluripotent stem cell surface N-glycoproteome resource reveals markers, extracellular epitopes, and drug targets.

Detailed 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.