RESUMO
The importance of oxygen tension in in vitro cultures and its effect on embryonic stem cell (ESC) differentiation has been widely acknowledged. Research has mainly focussed on ESC maintenance or on one line of differentiation and only few studies have examined the potential relation between oxygen tension during ESC maintenance and differentiation. In this study we investigated the influence of atmospheric (20%) versus physiologic (5%) oxygen tension in ESC cultures and their differentiation within the cardiac and neural embryonic stem cell tests (ESTc, ESTn). Oxygen tension was set at 5% or 20% and cells were kept in these conditions from starting up cell culture until use for differentiation. Under these oxygen tensions, ESC culture showed no differences in proliferation and gene and protein expression levels. Differentiation was either performed in the same or in the alternative oxygen tension compared to ESC culture creating four different experimental conditions. Cardiac differentiation in 5% instead of 20% oxygen resulted in reduced development of spontaneously beating cardiomyocytes and lower expression of cardiac markers Nkx2.5, Myh6 and MF20 (myosin), regardless whether ESC had been cultured in 5% or 20% oxygen tension. As compared to the control (20% oxygen during stem cell maintenance and differentiation), neural differentiation in 5% oxygen with ESC cultured in 20% oxygen led to more cardiac and neural crest cell differentiation. The opposite experimental condition of neural differentiation in 20% oxygen with ESC cultured in 5% oxygen resulted in more glial differentiation. ESC that were maintained and differentiated in 5% oxygen showed an increase in neural crest and oligodendrocytes as compared to 20% oxygen during stem cell maintenance and differentiation. This study showed major effects on ESC differentiation in ESTc and ESTn of oxygen tension, which is an important variable to consider when designing and developing a stem cell-based in vitro system.
