Energy metabolism is intrinsic to cell viability but surprisingly has been little studied in human embryonic stem cells (hESCs). The current study aims to investigate the effect of environmental O2 tension on carbohydrate utilisation of hESCs. Highly pluripotent hESCs cultured at 5% O2 consumed significantly more glucose, less pyruvate and produced more lactate compared to those maintained at 20% O2. Moreover, hESCs cultured at atmospheric O2 levels expressed significantly less OCT4, SOX2 and NANOG than those maintained at 5% O2. To determine whether this difference in metabolism was a reflection of the pluripotent state, hESCs were cultured at 5% O2 in the absence of FGF2 for 16 hours leading to a significant reduction in the expression of SOX2. In addition, these cells consumed less glucose and produced significantly less lactate compared to those cultured in the presence of FGF2. hESCs maintained at 5% O2 were found to consume significantly less O2 than those cultured in the absence of FGF2, or at 20% O2. GLUT1 expression correlated with glucose consumption and using siRNA and chromatin immunoprecipitation was found to be directly regulated by hypoxia inducible factor (HIF)-2α at 5% O2. In conclusion, highly pluripotent cells associated with hypoxic culture consume low levels of O2, high levels of glucose and produce large amounts of lactate, while at atmospheric conditions glucose consumption and lactate production are reduced and there is an increase in oxidative metabolism. These data suggest that environmental O2 regulates energy metabolism and is intrinsic to the self-renewal of hESCs.
Summary
Cross‐presentation is the presentation by MHC class I of antigenic peptides from exogenous proteins that have been internalized and processed by professional antigen‐presenting cells, e.g. dendritic cells. We have investigated the influence of particle size and antigen load on cross‐presentation following antigen delivery on microspheres (MS). Cross‐presentation from small particles (0·8‐μm) is sensitive to proteasome inhibition and the blockade of endoplasmic reticulum‐resident MHC class I complex export, whereas cross‐presentation from larger particles (aggregated clumps of 0·8‐μm MS) is resistant to these antagonists. This observation may have been overlooked previously, because of the heterogeneity of particle size and MS uptake in unsorted dendritic cell populations. Larger particles carry more antigen, but we show that antigen load does not influence the cross‐presentation pathway used. Whereas early endosome autoantigen 1 (EEA1) could be observed in all phagosomes, we observed endoplasmic reticulum SNARE of molecular weight 24 000 (ERS24) and cathepsin S in association with 3·0‐μm and aggregated 0·8‐μm MS, but not individual 0·8‐μm MS. A potential mechanism underlying our observations may be the activation of β‐catenin by disruption of E‐cadherin‐mediated adhesion. Activated β‐catenin was detected in the cytoplasm of cells after phagocytosis of MS (highest levels for the largest particles). We propose that particle size can direct the use of different pathways for the cross‐presentation of an identical antigen. Furthermore, these pathways have differing yields of MHC class I–peptide complexes, which is an important variable in designing vaccination strategies for maximal antigen expression and CD8+ T‐cell priming.
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