Method to Synchronize Cell Cycle of Human Pluripotent Stem Cells without Affecting Their Fundamental Characteristics.

Cell cycle progression and cell fate decisions are closely linked in human pluripotent stem cells (hPSCs). However, the study of these interplays at the molecular level remains challenging due to the lack of efficient methods allowing cell cycle synchronization of large quantities of cells. Here, we screened inhibitors of cell cycle progression and identified nocodazole as the most efficient small molecule to synchronize hPSCs in the G2/M phase. Following nocodazole treatment, hPSCs remain pluripotent, retain a normal karyotype and can successfully differentiate into the three germ layers and functional cell types. Moreover, genome-wide transcriptomic analyses on single cells synchronized for their cell cycle and differentiated toward the endoderm lineage validated our findings and showed that nocodazole treatment has no effect on gene expression during the differentiation process. Thus, our synchronization method provides a robust approach to study cell cycle mechanisms in hPSCs.This work was supported by the Wellcome Trust PhD program (PSAG/048 to L.Y. and PSAG/051 to A.O.); the European Research Council advanced grant New-Chol (ERC: 741707 to L.V. and R.A.G.), the Cambridge University Hospitals National Institute for Health Research Biomedical Research Center (to L.V.); an NC3Rs grant (NC/N001540/1 to C.M.M.), an MRC UK-RPM II grant (to R.A.T.), a Grant-in-Aid for JSPS Research Fellow (16J08005 to S.N.), a BHF Senior Research Fellowship (FS/13/29/30024 to S.S.), the Cystic Fibrosis Foundation, the Cystic Fibrosis Trust, a core support grant from the Wellcome and Medical Research Council to the Wellcome – Medical Research Council Cambridge Stem Cell Institute (PSAG028) and a core support grant from the Wellcome Trust to the Wellcome Trust Sanger Institute (WT206194)