Notch and hedgehog signaling cooperate to maintain self-renewal of human embryonic stem cells exposed to low oxygen concentration.

Abstract

Expansion and maintenance of human embryonic stem cells (hESCs) in undifferentiated state is influenced by complex signals in the microenvironment, including those contingent upon oxygen availability. Responses mediated by Notch and Hedgehog (Hh) have essential role in the growth and maintenance of hESCs, therefore this study examined their effect on the self-renewal of hESCs exposed to low oxygen. Using potent antagonists γ-secretase inhibitor and cyclopamine, we inhibited Notch and Hh pathways, respectively, in the CLS1 hESC line expanded continuously in a hypoxic atmosphere of 5% oxygen. Immunohistochemical staining and protein assays revealed loss of Oct4 and gain of stage-specific embryonic antigen 1 (SSEA1) markers in the inhibited cells. Semiquantitative real-time RT-PCR, and bromodeoxyuridine and thymidine incorporation assays demonstrated low Oct4 and Nanog mRNA expression, and decreased DNA synthesis, respectively, resulting from the block of each of the pathways. The loss increased significantly with co-inhibition of both pathways. Importantly, Notch and Hh downstream targets, including Hes1, Hey1, GIi1, and Ptc1, were surprisingly suppressed not only by the pathway-specific but also the unrelated inhibitor. These findings demonstrate complementary effect of Notch and Hh signaling in hypoxia enhanced maintenance of hESCs.