Control of human pluripotent stem cell fate by engineering signaling pathways

Because human pluripotent stem cells (hPSCs) can be propagated indefinitely while still retaining the capacity to differentiate into all somatic cell types, they hold great promise for a wide range of applications, including establishment of in vitro development model system, drug and toxicity screening, modeling diseases and potentially cellular therapies. In order to realize the potential of hPSCs, spatio-temporal signaling molecules controlling fate decision must be identified to establish robust protocol for generating desired somatic cells which function as their in vivo counterpart.;The Wnt signaling pathway controls myriad biological phenomena throughout human embryo development and adult life. Our approach was to engineer Wnt signaling pathway to better regulate growth and differentiation of hPSCs. We showed that retinoic acid (RA) promoted epithelial differentiation process correlates with membrane beta-catenin translocation. We then engineer a way to modulate beta-catenin translocation by small-molecule inhibitor of tyrosine kinase to effectively direct simple epithelial differentiation of hPSCs.;Besides epithelial cells, we found that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs. Short hairpin RNA (shRNA) knockdown of beta-catenin during the initial stage of hPSC differentiation blocked cardiomyocyte specification while Gsk3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with Gsk3 inhibitors followed by inducible expression of beta-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually pure functional human cardiomyocytes from multiple hPSC lines.;Finally, during cardiac differentiation of hPSCs, stage-specific activation of insulin signaling strongly inhibited cardiac differentiation during a protocol that used TGFbeta superfamily ligands to generate cardiomyocytes. However, insulin did not repress cardiomyocyte differentiation in a defined protocol that employed small molecules of canonical Wnt signaling. By examining the context of insulin inhibition of cardiomyocyte differentiation, we determined that the inhibitory effects by insulin required Wnt/beta-catenin signaling and that cardiomyocyte differentiation in the presence of insulin was rescued by inhibition of Wnt/beta-catenin.;Taken together, these findings provide evidence that Wnt signaling plays critical roles in regulating hPSCs fate decisions and developing robust ways to spatio-temporally control this pathway is critical to produce desired cell type.