Hypoxic culture promotes glucose uptake and lactate production in hESCs.

<p>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).</p

GLUT1 expression parallels glucose utilisation and is directly regulated by HIF-2α under hypoxic conditions.

<p>RT-qPCR was used to quantify <i>GLUT1</i> 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 <i>UBC</i> 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, <i>GLUT1</i> mRNA was found to be regulated by HIF-2α (B). All data has been normalised to <i>UBC</i> 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).</p

hESCs maintained at atmospheric O₂ levels express reduced levels of pluripotency markers compared to those cultured at 5% O₂.

<p>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).</p