Dissecting Signaling Pathways That Govern Self-renewal of Rabbit Embryonic Stem Cells*

The pluripotency and self-renewal of embryonic stem cells (ESC) are regulated by a variety of cytokines/growth factors with some species differences. We reported previously that rabbit ESC (rESC) are more similar to primate ESC than to mouse ESC. However, the signaling pathways that regulate rESC self-renewal had not been identified. Here we show that inhibition of the transforming growth factor β (TGFβ), fibroblast growth factor (FGF), and canonical Wnt/β-catenin (Wnt) pathways results in enhanced differentiation of rESC accompanied by down-regulation of Smad2/3 phosphorylation and β-catenin expression and up-regulation of phosphorylation of Smad1 and β-catenin. These results imply that the TGFβ, FGF, and Wnt pathways are required for rESC self-renewal. Inhibition of the MAPK/ERK and PI3K/AKT pathways, which lie downstream of the FGF pathway, led to differentiation of rESC accompanied by down-regulation of phosphorylation of ERK1/2 or AKT, respectively. Long-term self-renewal of rESC could be achieved by adding a mixture of TGFβ ligands (activin A, Nodal, or TGFβ1) plus basic FGF (bFGF) and Noggin in the absence of serum and feeder cells. Our findings also suggest that there is a regulatory network consisting of the FGF, Wnt, and TGFβ pathways that controls rESC pluripotency and self-renewal. We conclude that bFGF controls the stem cell properties of rESC both directly and indirectly through TGFβ or other pathways, whereas the effect of Wnt on rESC might be mediated by the TGFβ pathway.