Thesis

Role if HIF1α in human embryonic stem cell differentiation

Hypoxia stimulates human embryonic stem cell (hESC) self-renewal. It has been shown that hypoxia inducible factor 2α (HIF2α) up regulates OCT4 (POU5F1) transcription, a pluripotency marker gene. Hypoxia also increases the efficiency of reprogramming of differentiated cells to a pluripotent-like state. Combined, these results suggest that hypoxia, or more specifically HIF, would impair the purposeful differentiation of pluripotent stem cells. Here, we unexpectedly show that HIF expression also promotes hESC differentiation. shRNA knockdown of HIF1α or its binding partner, aryl hydrocarbon receptor nuclear translocator (ARNT), inhibits the early induction of developmental genes GATA2, GATA3, GATA6, HAND1 and HOXB3 during retinoic acid -induced differentiation. shRNA knockdown of HIF1α or ARNT also impaired embryoid body formation, one of the commonlyused in vitro differentiation assays in the human embryonic stem cell world. Conversely, ectopic expression of a stabilized HIF1α mutant, HIF1α (P402A/P564A), in normoxia leads to enhanced developmental genes GATA2, GATA3, GATA6, HAND1 and HOXB3 induction during retinoic acid-induced differentiation. Ectopic expression of HIF1α (P402A/P564A) also increased the chromatin modifier gene JMJD3 induction during retinoic acid induced differentiation in normoxia. Combined, the data show a previously unanticipated role for HIF in promoting early hESC differentiation in hypoxia.

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