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ORIGINAL RESEARCH ARTICLE

Activation of Hypoxic Response in Human Embryonic Stem Cell–Derived Embryoid Bodies

C. M. Cameron^*, Frances Harding^*,, Wei-Shou Hu^* and Dan S. Kaufman^,1

^* Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455; and Stem Cell Institute and Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455

To whom requests for reprints should be addressed at ¹ University of Minnesota Stem Cell Institute, Translational Research Facility, Room 2–220, 2001 6th Street SE, Mail Code 2873, Minneapolis, MN 55455. E-mail: kaufm020{at}umn.edu

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-1 and increased transcription of hypoxic responsive genes. Interestingly, we find that rather than HIF-1 expression being stable over prolonged (7–16 days) culture in hypoxic conditions, HIF-1 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-1 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.

Key Words: human embryonic stem cell • embryoid body • hematopoiesis • hypoxia • hypoxic inducible factor (HIF)