Human embryonic stem cell neural differentiation and enhanced cell survival promoted by hypoxic preconditioning

Transplantation of neural progenitors derived from human embryonic stem cells (hESCs) provides a potential therapy for ischemic stroke. However, poor graft survival within the host environment has hampered the benefits and applications of cellbased therapies. The present investigation tested a preconditioning strategy to enhance hESC tolerance, thereby improving graft survival and the therapeutic potential of hESC transplantation. UC06 hESCs underwent neural induction and terminal differentiation for up to 30 days, becoming neural lineage cells, exhibiting extensive neurites and axonal projections, generating synapses and action potentials. To induce a cytoprotective phenotype, hESC-derived neurospheres were cultured at 0.1% oxygen for 12 h, dissociated and plated for terminal differentiation under 21% oxygen. Immunocytochemistry and electrophysiology demonstrated the 'hypoxic preconditioning' promoted neuronal differentiation. Western blotting revealed significantly upregulated oxygen-sensitive transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, while producing a biphasic response within HIF targets, including erythropoietin, vascular endothelial growth factor and Bcl-2 family members, during hypoxia and subsequent reoxygenation. This cytoprotective phenotype resulted in a 50% increase in both total and neural precursor cell survival after either hydrogen peroxide insult or oxygen-glucose deprivation. Cellular protection was maintained for at least 5 days and corresponded to upregulation of neuroprotective proteins. These results suggest that hypoxic preconditioning could be used to improve the effectiveness of human neural precursor transplantation therapies.