Boldine promotes stemness of human urine-derived stem cells by activating the Wnt/β-catenin signaling pathway

Human urine-derived stem cells (hUSCs) process self- renewal and multilineage differentiation ability. Due to their noninvasive and easily available clinical source, hUSCs represent a promising alternative source of mesenchymal stem cells (MSCs) for application potential in cytotherapy. However, technical limitations, such as stemness property maintenance, have hindered hUSCs' clinical application. Certain some small molecules have been recognized with advantage in maintaining the stemness of stem cells. In this study, we identified stemness-regulated key targets of hUSCs based on the StemCellNet database, CMAP database and literature mining. Furthermore, we identified a small molecule compound, boldine, which may have the potential to promote the stemness of hUSCs. It promotes cell proliferation, multilineage differentiation and maintains stemness of hUSCs by cell viability assay, single-cell clone formation, osteogenic differentiation and stemness marker expression (OCT-4 and C-MYC). We identified that boldine may be a potential GSK-3 beta inhibitor by molecular docking and confirmed that it can upregulate the level of beta-catenin and promote translocation of beta-catenin into nucleus of hUSCs using Western blotting and immunofluorescence analysis. Our study indicates boldine activates the Wnt/beta-catenin signaling pathway in hUSCs and provides an effective strategy for MSCs research and application of small molecules in maintaining the stemness of hUSCs.