scholarly journals β-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus

2020 ◽  
Vol 6 (29) ◽  
pp. eaba1593
Author(s):  
Meng Zhang ◽  
Yiwei Lai ◽  
Vladislav Krupalnik ◽  
Pengcheng Guo ◽  
Xiangpeng Guo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Ground State ◽  
Transcription Initiation ◽  
Inner Cell Mass ◽  
Glycogen Synthase ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mitogen Activated Protein Kinase ◽  
Cell Cycle Gene

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.

89 INHIBITION OF MAPKK AND GSK3 SIGNALLING PROMOTES DEVELOPMENT AND EPIBLAST-SPECIFIC EXPRESSION OF PLURIPOTENCY MARKERS IN BOVINE BLASTOCYSTS

2013 ◽  
Vol 25 (1) ◽  
pp. 192
Author(s):  
D. Harris ◽  
B. Huang ◽  
B. Oback
Keyword(s):  
Dimethyl Sulfoxide ◽  
Inner Cell Mass ◽  
Glycogen Synthase ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Mitogen Activated Protein Kinase ◽  
Differential Staining ◽  
Blastocyst Development ◽  
Specific Expression

During blastocyst development, the inner cell mass segregates into the epiblast and the hypoblast. These 2 tissues form morphologically and molecularly distinct cell populations that subsequently develop into the embryo proper and some extraembryonic components, respectively. In mouse, isolated epiblast cells can be directly converted into pluripotent embryonic stem cells, capable of differentiating into all cell types of an adult animal. Epiblast pluripotency is promoted by pharmacological inhibition of mitogen-activated protein kinase kinase (Mapkk). This shields epiblast cells from secreted fibroblast growth factor (Fgf), which would otherwise instruct them to exit pluripotency and differentiate into extraembryonic lineages. Indirect stimulation of the Wnt pathway by inhibiting glycogen synthase kinase 3 (GSK3) further antagonises inductive Fgf/Mapkk signalling. Thus the double inhibition (2i) of Mapkk and Gsk3 effectively promotes pluripotency (Q. L. Ying et al. 2008 Nature 453, 519–523; J. Nichols et al. 2009 Development 136, 3215–3222). We investigated the effect of 2i culture on bovine blastocysts. The IVF embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAPKK (0.4 µM PD0325901) and GSK3 (3 µM CHIR99021) from the zygote (Day 1) stage onward. Compared to vehicle controls, 2i increased the abundance of cumulus cells in bovine IVF cultures, compromising blastocyst formation in cumulus-intact (248/823 = 30% v. 211/824 = 26%, respectively, n = 10; P < 0.05) but not cumulus-free cultures (546/1653 = 33% v. 572/1674 = 34%, respectively, n = 15; P = 0.51). In all subsequent experiments, we therefore cultured cumulus-free zygotes in 2i v. dimethyl sulfoxide until the blastocyst stage. This treatment increased the proportion of hatching (19/433 = 4% v. 7/416 = 2%, respectively, n = 10; P < 0.05) at the expense of early blastocysts (70/433 = 16% v. 93/416 = 22%, respectively, n = 11; P < 0.05). Differential staining of expanded IETS grade 1 and 2 blastocysts showed that 2i culture increased putative inner cell mass, trophectoderm, and total cell nuclei numbers by about 30% compared with controls (57 v. 43, 89 v. 69, and 146 v. 112, respectively; P < 0.01). Accelerated development and increased cell numbers were accompanied by gene expression changes in grade 1 and 2 blastocysts. Under 2i conditions, mRNA abundance of putative epiblast markers NANOG and SOX2 was >3-fold increased (P < 0.0001 and P < 0.01, respectively), and the putative hypoblast marker GATA4 was 2-fold reduced (P < 0.05). Other lineage-related markers (POU5F1, KLF4, DPPA3, and CDX2) showed no significant changes. Using microsurgical blastocyst dissection, we found that the increase in NANOG and SOX2 levels was specific to the inner cell mass-containing portion (7-fold for NANOG and 3-fold for SOX2; P < 0.00005 and P < 0.05, respectively) and not due to ectopic expression in the trophoblast-containing part, which showed similarly low expression levels for both genes. In summary, 2i treatment primed bovine blastocysts for pluripotency in the epiblast. Supported by MSI C10X1002.

scholarly journals Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K- and GSK-3-dependent signalling

2011 ◽  
Vol 39 (2) ◽  
pp. 674-678 ◽  
Author(s):  
Melanie J. Welham ◽  
Emmajayne Kingham ◽  
Yolanda Sanchez-Ripoll ◽  
Benjamin Kumpfmueller ◽  
Michael Storm ◽  
...  
Keyword(s):  
Cell Fate ◽  
Molecular Mechanisms ◽  
Inner Cell Mass ◽  
Glycogen Synthase ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Specific Cell ◽  
Human Escs ◽  
Phosphoinositide 3 Kinase

ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs.

279 INNER CELL MASS-DERIVED BOVINE CELL CULTURES MAINTAIN PLURIPOTENCY UNDER CHEMICALLY DEFINED CONDITIONS OF DUAL KINASE INHIBITION

2013 ◽  
Vol 25 (1) ◽  
pp. 287
Author(s):  
V. Verma ◽  
B. Huang ◽  
P. K. Kallingappa ◽  
B. Oback
Keyword(s):  
Inner Cell Mass ◽  
Glycogen Synthase ◽  
Primary Cultures ◽  
Cell Mass ◽  
Embryoid Bodies ◽  
Mitogen Activated Protein Kinase ◽  
Diagnostic Feature ◽  
Pulse Labelling ◽  
Pluripotent Cells ◽  
Kinase Inhibition

Authentic embryonic pluripotent stem cells (ePSC), capable of giving rise to all cell types of an adult animal, are only available in mouse and rat. Here, we report the generation of bovine ePSC-like cells under minimal conditions. Inner cell masses were immunosurgically isolated from IVF bovine blastocysts and explanted on laminin/gelatine-coated substrates. Explants were cultured feeder-free under low oxygen (7%) in a chemically defined medium containing inhibitors of mitogen-activated protein kinase kinase (MAPKK) and glycogen synthase kinase-3 signalling (GSK3). Dual kinase inhibition (2i) was necessary to sustain expression of epiblast-specific pluripotency markers SOX2 and NANOG in the central colony, which comprised a multi-layered clump of tightly packed cells that was clearly demarcated from the surrounding monolayer outgrowth. In 2i, explanted inner cell mass (ICM) expanded from 51 ± 4 to 1102 ± 55 cells per colony and surrounding outgrowth within 6 days of culture, equivalent to ~4 to 5 population doublings, before passaging. Moreover, 2i suppressed apoptosis after mechanical passaging and the cell number per colony and outgrowth remained constant for up to 8 passages every 4 to 5 days, after which cultures were discontinued. As a proxy for cell proliferation, we quantified DNA synthesis following different 5-ethynyl-2′-deoxyuridine (EdU)-incorporation protocols. EdU pulse-labelling for 30 min revealed that in steady state, 20 to 30% of cells were in S-phase in primary and passaged colonies, respectively. After cumulative labelling for 24 h, almost all primary and passaged cells were cycling. Throughout this passaging regime, ICM-derived cell lines expressed a repertoire of core pluripotency-related factors (CDH1, OCT4, SALL4, SOX2, TCF3), including markers enriched in naïve pluripotent cells (DPPA3, KLF4, LIN28, NANOG, SOCS3, STAT3) and primordial germ cells (IIFITM3). Genes that are downregulated in primed pluripotent cells were either undetectable (FGF5, T-BRACHYURY) or downregulated (LEFTY) after passaging. These mRNA results were confirmed on the protein level, where OCT4, KLF4, SOX2, and NANOG, as well as SSEA-3/4 and TRA-1-60/-81, but not SSEA-1, remained widely expressed. A diagnostic feature of murine ePSC is the simultaneous presence of 2 active X chromosomes (Xa Xa) and OCT4. We derived cultures from ICM of female blastocysts, produced through IVF with sexed semen, and stained primary cultures on Day 6 with an antibody against trimethylated histone (H) 3 lysine (K) 27 (H3K27me3). Nuclear foci of intense H3K27me3 immunoreactivity were absent in most OCT4-positive cells (660/724 = 92%), indicating presence of Xa Xa. In suspension culture, bovine ePSC-like cells formed cystic embryoid bodies expressing ectoderm (TUBB3, GFAP, NES), endoderm (AFP), and mesoderm (SPP1) markers. Bovine ePSC-like cells after 3 passages showed a normal chromosome number in the majority of spreads (17/18 = 94%). Our short-term culture system provides a chemically defined screening platform for factors that maintain long-term proliferation and pluripotency of ePSC in cattle. Supported by MSI C10X1002.

The ground state of pluripotency

2010 ◽  
Vol 38 (4) ◽  
pp. 1027-1032 ◽  
Author(s):  
Jason Wray ◽  
Tuzer Kalkan ◽  
Austin G. Smith
Keyword(s):  
Ground State ◽  
Cell Biology ◽  
Kinase Inhibitors ◽  
Glycogen Synthase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mitogen Activated Protein Kinase ◽  
Mammalian Development ◽  
Mitogen Activated Protein ◽  
The Stability

Pluripotency is defined as the capacity of individual cells to initiate all lineages of the mature organism in response to signals from the embryo or cell culture environment. A pluripotent cell has no predetermined programme; it is a blank slate. This is the foundation of mammalian development and of ES (embryonic stem) cell biology. What are the design principles of this naïve cell state? How is pluripotency acquired and maintained? Suppressing activation of ERKs (extracellular-signal-regulated kinases) is critical to establishing and sustaining ES cells. Inhibition of GSK3 (glycogen synthase kinase 3) reinforces this effect. We review the effect of selective kinase inhibitors on pluripotent cells and consider how these effects are mediated. We propose that ES cells represent a ground state, meaning a basal proliferative state that is free of epigenetic restriction and has minimal requirements for extrinsic stimuli. The stability of this state is reflected in the homogeneity of ES cell populations cultured in the presence of small-molecule inhibitors of MEK (mitogen-activated protein kinase/ERK kinase) and GSK3.

Ethanol alters cell cycle gene expression in human embryonic stem cells

2016 ◽  
Vol 01 (03) ◽  
pp. 201-208 ◽  
Author(s):  
Malini Krishnamoorthy ◽  
Brian Gerwe ◽  
Jamie Heimburg-Molinaro ◽  
Rachel Nash ◽  
Jagan Arumugham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Cell Cycle Gene ◽  
Cell Cycle Gene Expression

scholarly journals Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos

2021 ◽  
Author(s):  
Chuan Chen ◽  
Wenqiang Liu ◽  
Jiayin Guo ◽  
Yuanyuan Liu ◽  
Xuelian Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Rrna Synthesis ◽  
Binding Ability ◽  
Transcriptome Regulation ◽  
Early Embryos ◽  
Intrinsic Mechanism ◽  
Regulatory Rnas

AbstractN6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells (ESCs), which highly depends on the m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell (2C) transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on 2C-related retrotransposons is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.

scholarly journals Differentiation of Human Embryonic Stem Cells into Neuron, Cholinergic, and Glial Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Kimia Hosseini ◽  
Emilia Lekholm ◽  
Aikeremu Ahemaiti ◽  
Robert Fredriksson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Glial Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cholinergic Neurons ◽  
Neuronal Cultures ◽  
Short Period

Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 815-824 ◽  
Author(s):  
M.B. Rogers ◽  
B.A. Hosler ◽  
L.J. Gudas
Keyword(s):  
Retinoic Acid ◽  
Germ Cell ◽  
Cell Fate ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Specific Expression

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

2. Embryonic stem cells

2021 ◽  
pp. 21-37
Author(s):  
Jonathan Slack
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Practical Importance ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Human Embryos ◽  
Mouse Es Cells ◽  
Stage Embryo

‘Embryonic stem cells’ focuses on embryonic stem (ES) cells, which are grown in tissue culture from the inner cell mass of a mammalian blastocyst-stage embryo. Human ES cells offer a potential route to making the kinds of cells needed for cell therapy. ES cells were originally prepared from mouse embryos. Although somewhat different, cells grown from inner cell masses of human embryos share many properties with mouse ES cells, such as being able to grow without limit and to generate differentiated cell types. Mouse ES cells have so far been of greater practical importance than those of humans because they have enabled a substantial research industry based on the creation of genetically modified mice.

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