Activation of hypoxic response in human embryonic stem cell-derived embryoid bodies

Oxygen tension can provide an important determinant for differentiation and development of many cells and tissues. Genetic regulation of hemato-endothelial commitment is known to respond to oxygen deprivation via stimulation of hypoxia inducible factors (HIFs). Here, we use a closed bioreactor system to monitor and control the dissolved oxygen during differentiation of human embryonic stem cells (hESCs) via formation of embryoid bodies (hEBs). Exposing hESC-derived EBs to ambient oxygen at or below 5% results in stabilization of HIF-1alpha and increased transcription of hypoxic responsive genes. Interestingly, we find that rather than HIF-1alpha expression being stable over prolonged (7-16 days) culture in hypoxic conditions, HIF-1alpha expression peaks after approximately 48 hours of hypoxic exposure, and then declines to near undetectable levels, despite constant hypoxic exposure. This transient stabilization of HIF-1alpha during hESC-derived EB culture is demonstrated for four distinct stages of differentiation. Furthermore, we demonstrate hEB cell expansion is slowed by hypoxic exposure, with increased apoptosis. However, hEB cell proliferation returns to normal rates upon return to normoxic conditions. Therefore, although hypoxia effectively stimulates hypoxic responsive genes, this single variable was not sufficient to improve development of hemato-endothelial cells from hESCs.