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

Abstract

SummaryCell 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.

A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and their Differentiated Derivatives

“…While less understood in humans, the extent of synchrony in the first few PSC divisions is predictive of embryo viability and implantation of in vitro fertilized human embryos (Wong et al, 2010;Meseguer et al, 2011;Desai et al, 2014), and the eventual loss of hiPSC division synchrony is consistent with studies in model organisms that demonstrated this synchronization persists for the first 13 rounds of cell division that give rise to the 8,192 cell embryo (O'Farrell, Stumpff and Tin Su, 2004). Future studies aimed at promoting PSC synchronization or isolating synchronized PSCs will help optimize differentiation and cell therapy strategies as cell division synchrony plays important roles in PSC cell type specification (Sela et al, 2012;Pauklin and Vallier, 2013;Yiangou et al, 2019) and improves the success of stem cell transplantation (Zhang et al, 2005;Desai et al, 2014).…”

“…Tracking individual cells over time revealed that hiPSCs from common ancestors had synchronized timing of cell divisions, consistent with PSC embryogenesis in vivo (O'Farrell, Stumpff and Tin Su, 2004;Deneke et al, 2016). This finding was undiscernible in previous in vitro studies of bulk PSC populations that concluded cell cycle arrest via serum deprivation or chemical inhibition of cell cycle machinery was required to achieve synchronized PSC divisions (Zhang et al, 2005;Yiangou et al, 2019). Though common ancestor cell division synchrony persists over several divisions, we note that, considering all cells in our study were derived from a single cell clone when we first generated the line, it appears that slight drift over time eventually leads to loss of synchrony.…”

“…A common feature of early metazoan embryogenesis, including mice and humans, is synchronized cell division in PSCs in vivo (O'Farrell, Stumpff and Tin Su, 2004;Wong et al, 2010;Meseguer et al, 2011;Desai et al, 2014;Deneke et al, 2016). In contrast, in vitro studies of mammalian PSCs have suggested PSC divisions are not synchronized, unless one manipulates the cell cycle machinery with chemical inhibitors or serum deprivation (Zhang et al, 2005;Yiangou et al, 2019). However, tracking the timing of cell divisions in progeny that arose from a single PSC demonstrated that PSCs arising from the same lineage underwent synchronous cell divisions ( Figure 2D).…”

Analysis of Endothelial-to-Haematopoietic Transition at the Single Cell Level identifies Cell Cycle Regulation as a Driver of Differentiation

“…We next asked whether RUNX1+ haemogenic endothelial cells present at EHT D3 could transit to haematopoietic progenitors if their cell cycle progression was blocked. To address this question, EHT D3 cells were grown for 48 hours in the presence of Nocodazole, a small molecule commonly used to block cell cycle progression (40, 41). The efficacy of Nocodazole treatment was confirmed by EdU incorporation analyses showing an expected enrichment of cells in G2/M ( Fig.…”

Proliferation is a requirement for differentiation of oligodendrocyte progenitor cells during CNS remyelination

“…To test if proliferation is required for OPC differentiation, we blocked cell cycle progression in OPCs in a reversible and non-toxic way during differentiation using nocodazole, a M-phase inhibitor (Yiangou et al, 2019). Treatment with nocodazole for 48h blocked the division of neonatal OPC cultured in a proliferation medium in a dose-dependent manner (Figure 2E,F).…”