Induction of the G2/M transition stabilizes haploid embryonic stem cells

Abstract

The recent successful establishment of mouse parthenogenetic haploid embryonic stem cells ( phESCs) and androgenetic haploid ESCs (ahESCs) has stimulated genetic research not only in vitro but also in vivo because of the germline competence of these cell lines. However, it is difficult to maintain the haploid status over time without a frequent sorting of the G1 phase haploid ESCs by fluorescenceactivated cell sorting (FACS) at short intervals, because haploid cells tend to readily self-diploidize. To overcome this spontaneous diploid conversion, we developed a phESC culture condition using a small molecular inhibitor of Wee1 kinase to regulate the cell cycle by accelerating the G2/M phase transition and preventing re-entry into extra G1/S phase. Here, we demonstrate that, under this condition, phESCs maintained the haploid status for at least 4 weeks without the need for FACS. This method will greatly enhance the availability of these cells for genetic screening.

SIRT1 regulates autophagy and diploidization in parthenogenetic haploid embryonic stem cells

“…It has been reported that the oxidative stress and autophagic cell death induced by H 2 O 2 as a ROS-generating agent were similar with aged-related cellular morphology in ph ESCs at high passage [7,9]. As shown in Fig.…”

“…Diploid status makes mutation screens more complicated and limits efforts to identify the function of specific genes. The mechanism for readily diploidizing of hESCs remains unclear now, but the age-related pathologies might result from cellular stress conditions during high passage [7]. In certain situations, autophagy has often been regarded as a defense mechanism to protect cells under stress conditions [8], but it has been complicated by the identification that whether autophagy or apoptosis mainly occur in diploidizated hESCs at high passage.…”

Single-Cell Dynamic Analysis of Mitosis in Haploid Embryonic Stem Cells Shows the Prolonged Metaphase and Its Association with Self-diploidization

“…Although the cell cycle of diploid cells has been extensively studied, the cell cycle of haploid ESCs is far less understood. Interestingly, a recent study reported that accelerating G2/M transition could partially stabilize mouse haploid ESCs, suggesting an interconnection between the cell cycle and self-diploidization of haploid ESCs (Takahashi et al., 2014). However, whether the M phase itself is associated with the self-diploidization of haploid ESCs is elusive.…”

Haploid embryonic stem cells can be enriched and maintained by simple filtration

“…Diploidization is one specific characteristic of haESCs (1) and may be related to endoreduplication (18) or prolonged metaphase during the cell cycle in haESCs compared that in diploid ESCs (19,20). Chemicals that accelerate S-G2/M phases have been successfully used to stabilize haploid state for several weeks longer (20)(21)(22).…”

“…Diploidization is one specific characteristic of haESCs (1) and may be related to endoreduplication (18) or prolonged metaphase during the cell cycle in haESCs compared that in diploid ESCs (19,20). Chemicals that accelerate S-G2/M phases have been successfully used to stabilize haploid state for several weeks longer (20)(21)(22). However, the detailed differences in mitosis between haploid cells and diploid cells and molecular mechanisms underlying diploidization remain to be uncovered for generating haESCs with more stable haploidy (22,23).…”