Purpose: WNT proteins regulate key developmental and physiological processes. Recent studies have found that exosomes, which are small extracellular vesicles, are novel transport carriers of WNT signaling proteins. In the present study, we isolated exosomes from conditioned media of canonical ligand WNT3A-and non-canonical ligand WNT5A-expressing HEK293T cells and analyzed their bioactivity. Methods: Exosomes were isolated by differential centrifugation followed by size exclusion chromatography. Isolated exosomes were subjected to Western blot analysis and nanoparticle tracking analysis for their cargo contents and size distribution, respectively. Results: Most enriched exosomes measured 120-140 nm and expressed canonical exosomal markers, including ALIX, CD63, and CD81. Expression of either WNT3A or WNT5A led to an increase in the total number of secreted exosomes. While WNT3A and active β-catenin were both recovered in exosomes derived from WNT3A-producing cells, only active β-catenin was detected in exosomes recovered from WNT5A-producing cells. Intriguingly, both types of exosomes activated β-catenin-dependent response in recipient cells, as evidenced by accumulation of cytosolic active β-catenin and transcriptional activation of T-cell factor/lymphoid enhancer factor (TCF/LEF) responsive elements. Conclusion: These results suggest that WNT3A- and WNT5A-producing cells share a common mechanism for packaging of active β-catenin in exosomes.
