Long-term prevention of aneuploidy in human pluripotent stem cells by fine-tuning GSK3 activity

Human pluripotent stem cells (hPSCs) are critical cell sources to model human development and hold promise towards regenerative medicine. However, cultured hPSCs quickly acquire chromosome gains or losses (aneuploidy) due to high replicative stress and errors in chromosome segregation, which hampers their use for stem cell therapies. We have recently described that embryo patterning signals control chromosome segregation fidelity during early human lineage specification by modulating the response to replicative stress. Here, we demonstrate that fine-tuned activation of WNT signalling using off-the-shelf GSK3 inhibitors prevents chromosome missegregation and aneuploidy during long term passaging of hPSCs. Lineage-specification and sequencing analyses demonstrate that hPSCs culture with E8 supplemented with a low dosage of GSK3 inhibitors retain primed pluripotency and full potential to the differentiation into the 3 germ layers for over 30 passages. Strikingly, detailed mitotic studies revealed that long-term cultured hPSCs often display ultra-fine chromosome bridges during anaphase due to unreplicated DNA, which can result in hitherto uncharacterised copy-number-variations and other genomic aberrations. We show that culture with low dosage of the GSK3 inhibitor CHIR99021, but not direct attenuation of DNA replication stress using nucleosides, prevents mitotic DNA synthesis and the formation of ultra-fine bridges during long term hPSC passage. Taken together, we propose to enrich E8 culture media with 100 nM CHIR99021 to maintain euploidy in primed hPSCs, and to routinely map for genomic alterations, in addition to aneuploidy, for modelling and regenerative medicine studies.