Differentiation of human embryonic stem (hES) cells is associated with an increase in mitochondrial DNA (mtDNA) copy number, oxygen consumption rate, ATP and ROS production, suggesting that pluripotency/differentiation may be significantly influenced by metabolism. Physiological oxygen (O₂; 1-5%) has been reported to improve hES cell pluripotency over atmospheric (20%) O₂ by reducing spontaneous differentiation, increasing pluripotency marker expression and upregulating glycolysis. Therefore the aim of this study was to assess the effect of O₂ on hES cell mitochondrial activity.

hES cell lines (MEL1, MEL2) were cultured in a defined medium (mTeSR^TM1) in either 5% or 20% O₂. mtDNA copy number was determined using a mitochondrial to nuclear DNA ratio kit. Mitochondrial membrane potential was quantified by flow cytometry and JC-1 labelling. Gene expression was assessed by real-time PCR.

Culture of MEL2 hES cells at 20% O₂ increased mtDNA copy number (P <0.001), mitochondrial membrane potential (P<0.01) and the expression of mitochondrially encoded genes ND2 (P<0.05) and ATPase6 (P<0.05). In contrast, mitochondrial activity was not affected by O₂ in MEL1 hES cells. In MEL2 but not MEL1 hES cells, 20% O₂ upregulated the pluripotency marker Nanog (P<0.05). Furthermore, both MEL1 and MEL2 hES cells had increased mRNA expression of the mesoderm lineage marker MIXL1 at 20% oxygen (P<0.05).

Data suggest MEL2 hES cells adapt to atmospheric O₂ concentrations by increasing mitochondrial biogenesis and activity. Differences in the metabolic response to O₂ between the two hES cell lines examined highlight the importance of metabolic analyses, as standard measures of characterisation fail to differentiate the two lines. Furthermore, increased expression of MIXL1 in hES cells suggests an increased susceptibility to differentiation under 20% O₂ conditions.  Regulation of metabolism and mitochondrial biogenesis by O₂ may underlie hES cell state and stability.