In silico chemical fingerprint screening identifies fisetin as a novel stemotoxic flavonoid.

Despite recent advances in stem cell therapy using human pluripotent stem cells (hPSCs), the risk of teratoma formation due to residual undifferentiated cells remains a significant safety concern. Numerous small molecules, termed stemotoxic agents, have been developed to eliminate these residual cells, such as the promising natural flavonoid quercetin (QC). Application of QC in clinical settings during the differentiation of dopaminergic neurons from hPSCs has demonstrated its safety and efficacy in selectively targeting residual hPSCs without affecting neural stem cells (NSCs). Interestingly, not all flavonoids with structural similarity to QC exhibit stemotoxic activity. This study conducted an in silico analysis of the common chemical fingerprints of QC, luteolin, and apigenin, three flavonoids known to possess stemotoxicity, and subsequently identified fisetin (FST) as a possible candidate among thousands of dietary flavonoids in PubChem. In vitro, FST selectively induced cell death in hPSCs (distinguished by high NANOG and POU5F1 expression) while sparing human dermal fibroblasts and hPSC-derived NSCs. Notably, by eliminating undifferentiated cells, FST treatment enriched PAX6-positive NSCs and DA neurons. These findings suggest that, in addition to QC, FST may serve as an effective stemotoxic flavonoid for promoting DA neurons enrichment and minimizing the risk of residual hPSC contamination.