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

Abstract

Summary: 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. : In this study, Vallier and colleagues show that small molecule cell cycle inhibitors can be used to successfully synchronize and enrich hPSCs in different cell cycle phases. Particularly, the G2/M inhibitor nocodazole successfully enriched cells in G2/M, G1, and S phases of the cell cycle, without affecting pluripotency and differentiation capacity as shown by molecular and genome-wide single-cell RNA-seq analyses. Keywords: hPSCs, cell cycle, nocodazole, cell cycle synchronization, single-cell RNA-seq, mesoderm, endoderm, ectoderm