Stabilization of mouse haploid embryonic stem cells with combined kinase and signal modulation

Abstract

Mammalian haploid embryonic stem cells (haESCs) provide new possibilities for large-scale genetic screens because they bear only one copy of each chromosome. However, haESCs are prone to spontaneous diploidization through unknown mechanisms. Here, we report that a small molecule combination could restrain mouse haESCs from diploidization by impeding exit from naïve pluripotency and by shortening the S-G2/M phases. Combined with 2i and PD166285, our chemical cocktail could maintain haESCs in the haploid state for at least five weeks without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Taken together, we established an effective chemical approach for long-term maintenance of haESCs, and highlighted that proper cell cycle progression was critical for the maintenance of haploid state.

Uncoupling of DNA Replication and Centrosome Duplication Cycles Is a Primary Cause of Haploid Instability in Mammalian Somatic Cells

“…Because of the rich potential of mammalian haploid cells in bioengineering, many attempts have been made to improve haploid stability. Different studies have reported that diploidization in mouse haploid ES cells is substantially suppressed by optimized inhibition of wee1 kinase, cdk1, Rho-kinase or GSK-3/TGF-β pathways/BMP4 pathway ( Takahashi et al, 2014 ; He et al, 2017 ; Li et al, 2017 ), or by gene manipulations including ectopic expression of aurora B kinase or Dnmt3b ( Guo et al, 2017 ; He et al, 2018 ). Intriguingly, the effects of these treatments on haploid stability have been attributed to the acceleration of G2/M.…”

Uncoupling of DNA replication and centrosome duplication cycles is a primary cause of haploid instability in mammalian somatic cells

“…Because of the rich potential of mammalian haploid cells in bioengineering, many attempts have been made to improve haploid stability. Different studies have reported that diploidization in mouse haploid ES cells is substantially suppressed by optimized inhibition of wee1 kinase, cdk1, Rho-kinase or GSK-3/TGF- pathways/BMP4 pathway (Takahashi et al, 2014;He et al, 2017;Li et al, 2017), or by gene manipulations including ectopic expression of aurora B kinase or Dnmt3b He et al, 2018). Intriguingly, the effects of these treatments on haploid stability have been attributed to the acceleration of G2/M.…”

The milestone of genetic screening: Mammalian haploid cells

“…Similarly, the combination of 2i inhibitors (PD0325901 and CHIR99021), PD166285 and RDF could also inhibit the self-diploidization by shortening the S-G2/M phase ( Fig. 2 A), and simultaneously guarantee the pluripotency of haESCs [38] . Besides, 10-Deacetyl-baccatin-III (DAB) was selected out to enrich the haploid cells in HAP1 or mouse haESCs cell cultures, by promoting mitotic arrest in a ploidy-dependent manner [39] .…”

“…2 Different strategies for sustaining and enriching for haploid cells A. Effective chemical inhibitors to reduce self-diploidization of mouse haESCs [32] , [37] , PD166285 (Wee1 inhibitor) repress diploidization via promoting G2/M transition; RDF (cocktail of Repsox, DMH1 and Forskolin) can shorten mitosis [38] . RO-3306 (CDK1 inhibitor) and Y-27632 (ROCK inhibitor) can significantly reduce the mitotic slippage [32] .…”