Comparison between the activities of canonical Wnt ligands in human pluripotent stem cell differentiation

WNT ligands are key regulators of mammalian gastrulation and anterior-posterior (A–P) patterning. The activities of different Wnt ligands that signal through the canonical pathway are often assumed to be similar due to their high structural homology. To explore potential differences, we generated human pluripotent stem cell (hPSC) lines with inducible expression of WNT3, WNT3A, WNT6, or WNT8A, enabling direct comparisons of ligand activity in multiple differentiation contexts. We find that only WNT3 and WNT3A robustly induce mesodermal markers, while WNT6 and WNT8A do not. In the context of ectodermal differentiation, WNT3, WNT3A, and WNT8 activate a similar set of targets but WNT3 and 3A do so more strongly than WNT8A. WNT6 does not induce this set of targets, however, all four ligands, including WNT6, downregulate a similar set of targets. We also observed ligand-specific differences in spatial signaling range and the ability to induce self-organized patterns of ectodermal fates including neural crest-like domains. These results highlight how signaling strength, spatial range, and diverse target genes of Wnt ligands contribute to distinct developmental outcomes in early human stem cell differentiation.