Wnt/ß-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/ß-catenin signaling promotes either self-renewal or differentiation. We use a sensitive reporter to establish that Wnt/ß-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 ß-catenin signaling, which led us to postulate that Wnt/ß-catenin signaling is actively repressed during self-renewal. In support of this hypothesis, we demonstrate that OCT4 represses ß-catenin signaling during self-renewal and that targeted knockdown of OCT4 activates ß-catenin signaling in hESCs. Using a fluorescent reporter of ß-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 ß-catenin signaling express higher levels of differentiation markers. Together these data support a dominant role for Wnt/ß-catenin signaling in the differentiation rather than self-renewal of hESCs.

An SMA project report: neural cell-based assays derived from human embryonic stem cells.

Human embryonic stem (ES) cells are promising resources for developing new treatments for neurodegenerative diseases. Spinal muscular atrophy (SMA) is one of the leading causes of childhood paralysis and infant mortality. SMA is caused by inactivation of the survival motor neuron-1 (SMN1) gene. The nearly identical SMN2 gene contains a silent polymorphism that disrupts splicing and as a result cannot compensate for loss of SMN1. The SMA Project was established by the National Institute of Neurological Disorders and Stroke (NINDS) as a pilot effort to establish a fully transparent coalition between academics, industry, and government to create a centralized network of shared resources and information to identify and test new SMA therapeutics. As one of the funded projects, the work described here tested the feasibility of generating a SMA cell-based assay using neural lineages derived from human ES cells approved for National Institutes of Health (NIH)-funded research. Minigene cassettes were constructed, employing firefly luciferase or green fluorescent protein (GFP) as reporters for splicing efficiency of SMN1 and/or SMN2 under the control of the SMN1, SMN2, or cytomegalovirus (CMV) promoters. Transient transfection of proliferating neuroprogenitors in a 96-well format with plasmid DNA or adenoviral vectors showed differential levels that correlated with the splicing minigene and the promoter used; luciferase activities with SMN1 splicing minigenes were higher than SMN2, and the CMV promoter generated higher levels of activity than the SMN1 and SMN2 promoters. Our results indicate that human ES cell-derived neuroprogenitors provide a promising new primary cell source for assays of new therapeutics for neurodegenerative diseases.