Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation

Abstract

Inhibitors of Mek1/2 and Gsk3β, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.

Naive Pluripotent Stem Cells as a Model for Studying Human Developmental Epigenomics: Opportunities and Limitations

“…The difference in DNA methylation stability between hESCs and the embryo might not be restricted to human cells, as two recent reports showed that naive mouse ESCs lose their imprints when cultured in media containing MEK1/2 inhibitor [20,21]. These studies contrast with several previous reports that demonstrated imprint stability, and the reasons for the discrepant findings are not obvious.…”

“…These studies contrast with several previous reports that demonstrated imprint stability, and the reasons for the discrepant findings are not obvious. Loss of imprints in mouse ESCs might be associated with defective developmental potential [20,21], although this needs to be examined more systematically and also in the context of naive hESC differentiation.…”

Epigenetic foundations of pluripotent stem cells that recapitulate in vivo pluripotency

“…Indeed, ectopic expression of Dnmt3a or Dnm3b can rescue the reduced methylation levels at particular regions in female ES cells. 76 74,78 These results suggest that female-specific hypomethylation is in part caused by impaired maintenance of DNA methylation due to two active X chromosomes. It is also interesting that female ES cells tend to lose all or part of one X chromosome during their propagation, while male ES cells occasionally lose Y chromosome, indicating that female ES cells are genetically unstable.…”

“…However, it has not been fully elucidated whether genetic and epigenetic stabilities of 2i-treated ES cells are stable. Recent studies demonstrated that derivation of ES cells directly from blastocysts in 2i/LIF and prolonged culture of ES cells in 2i/LIF result in loss of DNA methylation at imprinted loci 74,75 (Figure 4). Surprisingly, such imprint eroded ES cells compromise autonomous developmental potential by tetraploid embryo complementation and nuclear transfer 74,75 (Figure 4).…”

“…Mechanistically, the inhibition of MEK1/2 is responsible for these opposing effects. In addition, replacement of the MEK1/2 inhibitor with an Src inhibitor (a2i includes Src inhibitor; CGP77675 and CHIR99021) preserves genetic and epigenetic stability as well as autonomous developmental potential 74,75 (Figure 4). Given that many laboratories have implemented 2i culture condition as standard practice since the discovery of 2i, these findings should be taken into account for future experiments using 2i-treated ES cells.…”