Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4

Signal transduction pathways play diverse, context-dependent roles in vertebrate development. In studies of human embryonic stem cells (hESCs), conflicting reports claim Wnt/beta-catenin signaling promotes either self-renewal or differentiation. We use a sensitive reporter to establish that Wnt/beta-catenin signaling is not active during hESC self-renewal. Inhibiting this pathway over multiple passages has no detrimental effect on hESC maintenance, whereas activating signaling results in loss of self-renewal and induction of mesoderm lineage genes. Following exposure to pathway agonists, hESCs exhibit a delay in activation of beta-catenin signaling, which led us to postulate that Wnt/beta-catenin signaling is actively repressed during self-renewal. In support of this hypothesis, we demonstrate that OCT4 represses beta-catenin signaling during self-renewal and that targeted knockdown of OCT4 activates beta-catenin signaling in hESCs. Using a fluorescent reporter of beta-catenin signaling in live hESCs, we observe that the reporter is activated in a very heterogeneous manner in response to stimulation with Wnt ligand. Sorting cells on the basis of their fluorescence reveals that hESCs with elevated beta-catenin signaling express higher levels of differentiation markers. Together these data support a dominant role for Wnt/beta-catenin signaling in the differentiation rather than self-renewal of hESCs.