Environmental oxygen tension regulates the energy metabolism and self-renewal of human embryonic stem cells - PubMed (original) (raw)
Environmental oxygen tension regulates the energy metabolism and self-renewal of human embryonic stem cells
Catherine E Forristal et al. PLoS One. 2013.
Abstract
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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Hypoxic culture promotes glucose uptake and lactate production in hESCs.
Glucose, pyruvate and lactate utilisation were non-invasively measured in a defined hESC medium. More glucose was consumed and lactate produced by Hues7 hESCs cultured at 5% O2 than at 20% O2 on (A) day 2 (B) day 3 and (C) day 4 post-passage. In contrast, less pyruvate was consumed by hESCs at 5% O2 compared to 20% O2. The rate of glucose consumption and lactate production was also greater on day 3 post-passage in Shef3 hESCs cultured at 5% O2 compared to those maintained at 20% O2 (D). **P<0.01, ***P<0.001 significantly different to 5% O2 (n = 12–23).
Figure 2. Short term removal of FGF2 at 5% O2 alters hESC metabolism and promotes O2 consumption.
Removal of FGF2 for 16 hours from Hues7 hESCs cultured at 5% O2 (5% O2– FGF2) resulted in a reduction of glucose consumption and lactate production, whereas pyruvate consumption dramatically increased (A). Shef3 hESCs cultured at 5% O2– FGF2 displayed a significant reduction in lactate production (B). **P<0.01, ***P<0.001 significantly different to 5% O2+FGF2 (n = 10–18). Hues7 hESCs cultured at 5% O2 consumed less O2 than when FGF2 was removed for 16 hours (C). hESCs maintained at 20% O2 consumed the greatest amount of O2. Bars with the same superscript are significantly different; a, b, P<0.001, c, P<0.05 (n = 7–8).
Figure 3. hESCs maintained at atmospheric O2 levels express reduced levels of pluripotency markers compared to those cultured at 5% O2.
Hues7 hESCs were cultured at either 5% O2, 5% O2 with FGF2 removed for 16 hours (5% O2– FGF2) or 20% O2. Protein was isolated and OCT4 (A and B), SOX2 (C and D) and NANOG (E and F) quantified using Western blotting. All data has been normalised to β-actin and to 1 for 5% O2. *P<0.05, **P<0.01, ***P<0.001 significantly different from 5% O2 (n = 3–4).
Figure 4. GLUT1 expression parallels glucose utilisation and is directly regulated by HIF-2α under hypoxic conditions.
RT-qPCR was used to quantify GLUT1 mRNA expression in Hues7 hESCs cultured at either 5% O2, or 20% O2 on day three post-passage (A). All data has been normalised to UBC and to 1 for 5% O2. *P<0.05 significantly different to 5% O2 (n = 3). Using siRNA to silence HIF-α subunits in Hues7 hESCs cultured at 5% O2, GLUT1 mRNA was found to be regulated by HIF-2α (B). All data has been normalised to UBC and to 1 for the transfection control. *P<0.05 significantly different to transfection control (n = 6). Using ChIP HIF-2α was found to bind to the proximal promoter of GLUT1 only in hESCs cultured at 5% O2. ChIP assays were performed with either a HIF-2α or IgG control antibody on chromatin isolated from Hues7 hESCs cultured at either 20% O2 or 5% O2. DNA enrichment is expressed as a percentage of input (non-immunoprecipitated chromatin). *P<0.05, **P<0.01, NS indicates no significant difference (n = 5).
References
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