A Simple and Robust Method for Establishing Homogeneous Mouse Epiblast Stem Cell Lines by Wnt Inhibition

Sugimoto, Michihiko; Kondo, Masayo; Koga, Yumiko; Shiura, Hirosuke; Ikeda, Rieko; Hirose, Michiko; Ogura, Atsuo; Murakami, Ayumi; Yoshiki, Atsushi; Lopes, Susana M. Chuva de Sousa; Abe, Kuniya

doi:10.1016/j.stemcr.2015.02.014

Cited by 71 publications

(84 citation statements)

References 32 publications

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“…Homogenous primed-state mEpiSC lines can be more efficiently established in the presence of Wnt inhibitors, such as IWP2 (42) and XAV939 (43). Our mass spectrometry-based analysis of protein expression profiles of naïve ELF1 hESCs with inhibition of Wnt/ β-catenin signaling also suggests that blocking of Wnt/β-catenin signaling could lead naïve hESCs to a more primed-like state (Fig.…”

Section: Discussionmentioning

confidence: 73%

Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells

Robitaille

Berndt

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

111

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In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of β-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/β-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/β-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.Wnt/β-catenin signaling | naïve pluripotency | naïve-to-primed transition | human embryonic stem cells | self-renewal

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Section: Discussionmentioning

confidence: 73%

Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells

Robitaille

Berndt

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

111

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“…; Sugimoto et al . ). Both Tankyrase inhibitors such as XAV939 or IWR‐1, or Porcupine inhibitors such as IWP‐2 have been used.…”

Section: Primed Pluripotencymentioning

confidence: 97%

Pluripotency Deconstructed

Kinoshita

Smith

2018

Dev Growth Differ

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Pluripotency denotes the flexible capacity of single cells to give rise to all somatic lineages and typically also the germline. Mouse ES cells and post-implantation epiblast-derived stem cells (EpiSC) are widely used pluripotent cell culture systems. These two in vitro stem cell types have divergent characteristics. They are considered as representative of distinct developmental stages, distinguished by using the terms "na€ ıve" and "primed". A binary description is an over-simplification, however. Here, we discuss an intermediate stage of pluripotency that we term "formative". Formative pluripotency features a gene regulatory network switch from the na€ ıve state and comprises capacitation of enhancers, signaling pathways and epigenetic machinery in order to install competence for lineage specification.

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“…E3.5 and E4.0 embryos were flushed out from the uterus at noon of the third day, cultured in KSOM (Millipore), and fixed at noon (E3.5) or midnight (E4.0). For E6.5 embryos, ExE was dissected as described previously (Sugimoto et al 2015). For some experiments, embryos were immunostained with rabbit anti-Nanog antibody (1/100; Cosmobio, RCAB002pF) or rabbit anti-H3K27me3 antibody (1/500; Millipore, 07449) after completion of the Xist RNA FISH procedure.…”

Section: Collection Of Mouse Oocytes and Preimplantation Embryosmentioning

confidence: 99%

“…The day of plug was counted as E0.5. Dissection of ExE was performed as described previously (Sugimoto et al 2015). RNA-seq libraries were prepared as described previously (Inoue et al 2017b).…”

Section: Rna-seqmentioning

confidence: 99%

Maternal Eed knockout causes loss of H3K27me3 imprinting and random X inactivation in the extraembryonic cells

et al. 2018

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Genomic imprinting is essential for mammalian development. Recent studies have revealed that maternal histone H3 Lys27 trimethylation (H3K27me3) can mediate DNA methylation-independent genomic imprinting. However, the regulatory mechanisms and functions of this new imprinting mechanism are largely unknown. Here we demonstrate that maternal Eed, an essential component of the Polycomb group complex 2 (PRC2), is required for establishing H3K27me3 imprinting. We found that all H3K27me3-imprinted genes, including Xist, lose their imprinted expression in Eed maternal knockout (matKO) embryos, resulting in male-biased lethality. Surprisingly, although maternal X-chromosome inactivation (XmCI) occurs in Eed matKO embryos at preimplantation due to loss of Xist imprinting, it is resolved at peri-implantation. Ultimately, both X chromosomes are reactivated in the embryonic cell lineage prior to random XCI, and only a single X chromosome undergoes random XCI in the extraembryonic cell lineage. Thus, our study not only demonstrates an essential role of Eed in H3K27me3 imprinting establishment but also reveals a unique XCI dynamic in the absence of Xist imprinting.

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A Simple and Robust Method for Establishing Homogeneous Mouse Epiblast Stem Cell Lines by Wnt Inhibition

Cited by 71 publications

References 32 publications

Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells

Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells

Pluripotency Deconstructed

Maternal Eed knockout causes loss of H3K27me3 imprinting and random X inactivation in the extraembryonic cells

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