P57. Self-renewal of hESCs is maintained in hypoxia through cooperation of Notch and Shh pathways

Spontaneous differentiation of human embryonic stem cells (hESCs) is a bottleneck for their use in regenerative applications. Epigenetic programs along with developmental signaling pathways, such as Notch and Sonic Hedgehog (Shh), constitute a network system coordinating the fate of hESCs. The individual elements are in turn regulated by a number of micro-environmental factors, among which oxygen has been shown to play an important role. Previously, we have assessed the effect of short- and long-term hypoxic exposure on the inhibition of hESCs spontaneous differentiation. In the current study, we examined the effect of Notch and Shh pathways on the self-renewal of hESCs exposed to low oxygen (5%) for short and long term, corresponding 4 weeks and 18 months, respectively. Using specific antagonists of Notch and Shh, we demonstrated a significant decrease of pluripotency-associated markers Oct4 and Nanog for each of the pathways. Co-inhibition of the two pathways had an additive effect. The proliferative capacity of hESC colonies and the expression of Notch and Shh downstream genes, Hes and Hey, and Gli1, respectively, were assessed as well. In conclusion, our data highlight a complementary role of Notch and Shh signaling pathways in preventing hESCs spontaneous differentiation in hypoxia.