Human embryonic stem (hES) cells are routinely cultured under atmospheric (~20%) oxygen, a concentration known to impair embryo development in vitro and which is likely suboptimal for maintaining hES cells compared with physiological (~5%) oxygen conditions. Studies to date have largely focused on the characterisation of molecular changes in response to oxygen, with little attention paid to cellular physiology and metabolic function. Therefore, we examined the effect of oxygen on hES cell carbohydrate and amino acid utilisation, mitochondrial activity and metabolic gene expression.
MEL2 hES cells were cultured in a defined medium (mTeSRTM1) under either 5% or 20% oxygen. Glucose consumption and lactate production were assessed by the reduction of NADP+ and NAD+ to NADPH and NADH respectively. The utilisation of amino acids was measured by liquid chromatography-mass spectrometry (LC-MS) of spent medium samples. Mitochondrial DNA copy number was determined using a mitochondrial to nuclear DNA ratio kit. Mitochondrial membrane potential was quantified by JC-1 labelling. Gene expression was assessed by real-time PCR.
Culture under atmospheric oxygen substantially decreased MEL2 hES cell glucose consumption and lactate production (P <0.001), and increased total amino acid consumption (P < 0.05) compared with those cultured under physiological oxygen. MEL2 hES cells also displayed increased mitochondrial DNA copy number (P <0.001) and mitochondrial membrane potential (P<0.01). Changes in hES cell metabolism under atmospheric oxygen were associated with a significant increase (P < 0.0??) in mitochondrial gene expression and reduction in glycolytic gene expression, in the absence of alterations in pluripotency and differentiation marker expression.
In summary, the culture of MEL2 hES cells in atmospheric oxygen conditions is associated with significant alterations in cell metabolism and underlying modification of the expression of metabolic genes, in the absence of changes in pluripotency. Thus, regulation of metabolism and mitochondrial activity may underlie hES cell state and stability.