The effect of physiological oxygen levels on GABAergic neuronal differentiation from mouse embryonic stem cells

Embryonic stem cells (ESCs) have the ability to generate any kind of cell in the body. They, therefore, have great potential for use in cell therapies for neurodegenerative disorders such as Huntington’s disease. Establishing a culture environment to mimic components of physiological conditions may help to maintain and differentiate ESCs more successfully. One of the important conditions is the level of oxygen. Traditionally, 20% oxygen (O₂) has been used to culture cells, but this is much higher than physiological levels (2% O₂). In this study, we used the mouse ESC line 46C (Sox1- GFP knock-in) to investigate the effect of physiological oxygen on proliferation of mESCs, and their differentiation to neural progenitors (where Sox1 is expressed) and mature GABAergic neurons. mESCs were cultured in either high (20%, H) or low (2%, L) oxygen levels for four days before induction of differentiation, and subsequently differentiated under either high or low oxygen, in a 2x2 factorial design (H-H, H-L, L-H, L-L). mESCs placed in low oxygen levels during the differentiation phase showed less proliferation (a decreased proportion of Ki67⁺ cells), complete loss of the self-renewing population (Oct4⁺ cells), and a decrease in Sox-1⁺ neural precursors. Consistent with this, neurons generated under low levels of oxygen showed a more mature morphology with an increased number of primary neurites and increased levels of GABA neurotransmitter. There was no significant difference in the percentage of neurons generated from either condition. We conclude that mESC culture in low oxygen conditions promotes maturation during neuronal differentiation and helps eliminate the residual Oct4⁺ population. The adoption of low oxygen environments during neuronal differentiation may, therefore, decrease teratoma formation and increase the potential for ESC use in cell therapies for neurodegenerative disease.