Blockage of the Epithelial-to-Mesenchymal Transition Is Required for Embryonic Stem Cell Derivation

Abstract

SummaryPluripotent cells emanate from the inner cell mass (ICM) of the blastocyst and when cultivated under optimal conditions immortalize as embryonic stem cells (ESCs). The fundamental mechanism underlying ESC derivation has, however, remained elusive. Recently, we have devised a highly efficient approach for establishing ESCs, through inhibition of the MEK and TGF-β pathways. This regimen provides a platform for dissecting the molecular mechanism of ESC derivation. Via temporal gene expression analysis, we reveal key genes involved in the ICM to ESC transition. We found that DNA methyltransferases play a pivotal role in efficient ESC generation. We further observed a tight correlation between ESCs and preimplantation epiblast cell-related genes and noticed that fundamental events such as epithelial-to-mesenchymal transition blockage play a key role in launching the ESC self-renewal program. Our study provides a time course transcriptional resource highlighting the dynamics of the gene regulatory network during the ICM to ESC transition.

Enhanced development of mouse single blastomeres into blastocysts via the simultaneous inhibition of TGF‐β and ERK pathways in microdroplet culture

“…Via temporal gene expression analysis, we revealed before the key genes involved in the ICM to ESC transition by blocking both the ERK1/2 and TGFβ signaling pathways. 30 Interestingly, our results depicted the upregulation of 233 well-known relevant pluripotency-related markers, such as Pou5f1 (Oct4), Zfp42(Rex1), Klf4, Ctnnb1 (β-Catenin), Sall4, Nanog, Klf2, Dppa3, Sox2, Esrrb, Utf1, Gbx2, and Nr5a2. In addition, blockage of the ERK and TGF-β pathways resulted in a high expression of epigenetic modifiers and DNA methylation-related genes such as Dnmt3b, Dnmt3l, Chd8, Mtss1, Suz12, Eed, Wdr3, and Mat2b in the transition from ICM to ESCs such as Dnmt3b, Dnmt3l, Chd8, Mtss1, Suz12, Eed, Wdr3, and Mat2b.…”

“…Via temporal gene expression analysis, we revealed before the key genes involved in the ICM to ESC transition by blocking both the ERK1/2 and TGFβ signaling pathways . Interestingly, our results depicted the upregulation of 233 well‐known relevant pluripotency‐related markers, such as Pou5f1 ( Oct4 ), Zfp42 ( Rex1 ), Klf4 , Ctnnb1 (β ‐Catenin ), Sall4 , Nanog , Klf2 , Dppa3 , Sox2 , Esrrb , Utf1 , Gbx2 , and Nr5a2 .…”

“…In addition, blockage of the ERK and TGF-β pathways resulted in a high expression of epigenetic modifiers and DNA methylation-related genes such as Dnmt3b, Dnmt3l, Chd8, Mtss1, Suz12, Eed, Wdr3, and Mat2b in the transition from ICM to ESCs such as Dnmt3b, Dnmt3l, Chd8, Mtss1, Suz12, Eed, Wdr3, and Mat2b. 30 This analysis showed that both the ERK1/2 and TGFβ signaling inhibitions play a pivotal role in supporting the dynamic expression of pluripotency-related markers, epigenetic modifiers, and DNA methylation-related genes.…”

Genomic integrity of ground‐state pluripotency

“…21 Normally, CpG dinucleotides are methylated in LINEs and other retrotransposable sequences to prevent their activation. 23 The results reflected that early changes in global DNA methylation probably occurred in tandem with increased in expression of de novo methyltransferases. 22 We have already shown that the methylation level of the CpG dyads for three classes of REs increased on the transition between the in vivo and in vitro pluripotent cell states.…”

“…The effects of unsilenced retrotransposons can be seen in cancer cells that have unstable genetic profiles. 23 To address the question of which regimen could better conserve genomic integrity during long-term passaging, in this study, the DNA methylation pattern of REs; their activation levels under 2i, R2i, and serum culture conditions; and the genomic integrity of cells were investigated. 23 The results reflected that early changes in global DNA methylation probably occurred in tandem with increased in expression of de novo methyltransferases.…”