An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a ?race for fates? in which the neuroectodermal fate has an advantage.

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Human embryonic stem cells: challenges and opportunities

Reproduction Fertility and Development ◽

10.1071/rd06113 ◽

2006 ◽

Vol 18 (8) ◽

pp. 839 ◽

Cited By ~ 3

Author(s):

Steven L. Stice ◽

Nolan L. Boyd ◽

Sujoy K. Dhara ◽

Brian A. Gerwe ◽

David W. Machacek ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Cell Line ◽

Cell Fate ◽

Neural Development ◽

Es Cells ◽

Embryonic Stem ◽

Normal Karyotype ◽

Es Cell ◽

Cell Therapies

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

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Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

10.1101/299073 ◽

2018 ◽

Cited By ~ 4

Author(s):

Daniel Strebinger ◽

Cédric Deluz ◽

Elias T. Friman ◽

Subashika Govindan ◽

Andrea B. Alber ◽

...

Keyword(s):

Transcription Factors ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Directed Differentiation ◽

Es Cell ◽

Endogenous Fluctuations ◽

Cell Fate Decisions ◽

Oct4 Protein

AbstractSOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

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An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

Biology Open ◽

10.1242/bio.20148409 ◽

2014 ◽

Vol 3 (7) ◽

pp. 614-626 ◽

Cited By ~ 40

Author(s):

D. A. Turner ◽

J. Trott ◽

P. Hayward ◽

P. Rue ◽

A. Martinez Arias

Keyword(s):

Cell Fate ◽

Es Cells ◽

Cell Fate Decisions ◽

Mouse Es Cells ◽

Extracellular Signalling

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Inositol transport in mouse embryonic stem cells

Reproduction Fertility and Development ◽

10.1071/rd05021 ◽

2005 ◽

Vol 17 (6) ◽

pp. 633 ◽

Cited By ~ 2

Author(s):

B. D. Higgins ◽

M. T. Kane

Keyword(s):

Glucose Transporter ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Kinetics Parameters ◽

Hypertonic Medium ◽

Transporter Protein ◽

Mouse Es Cells ◽

Sodium Dependent ◽

Inositol Uptake

The uptake of myo-inositol by mouse embryonic stem (ES) cells was measured using [2-3H]myo-inositol. Uptake of myo-inositol by ES cells occurred in a mainly saturable, sodium-, time- and temperature-dependent manner, which was inhibited by glucose, phloridzin and ouabain. Self inhibition by inositol was much greater than inhibition by glucose indicating that transport was not occurring via a sodium-dependent glucose transporter. Uptake rate was much greater than efflux rate indicating a mainly unidirectional transport mechanism. Estimated kinetics parameters for sodium-dependent inositol uptake were a Km of 65.1 ± 11.8 μ mol L−1 and a Vmax of 5.0 ± 0.59 pmol μ g protein−1 h−1. Inositol uptake was also sensitive to osmolality; uptake increased in response to incubation in hypertonic medium indicating a possible role for inositol as an osmolyte in ES cells. These characteristics indicate that myo-inositol transport in mouse ES cells occurs by a sodium-dependent myo-inositol transporter protein.

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Cleavage of histone H2A during embryonic stem cell differentiation destabilizes nucleosomes to counteract gene activation

10.1101/2021.08.10.455684 ◽

2021 ◽

Author(s):

Mariel Coradin ◽

Joseph Cesare ◽

Yemin Lan ◽

Zhexin Zhu ◽

Peder J. Lund ◽

...

Keyword(s):

Cell Fate ◽

Es Cells ◽

Expression Patterns ◽

Gene Activation ◽

Embryonic Stem ◽

Maximum Level ◽

Stem Cell Differentiation ◽

Embryonic Stem Cell Differentiation ◽

Cell Fate Decisions ◽

Lysosomal Protease

Histone proteolysis is a poorly understood phenomenon in which the N-terminal tails of histones are irreversibly cleaved by intracellular proteases. During development, histone post-translational modifications are known to orchestrate gene expression patterns that ultimately drive cell fate decisions. Therefore, deciphering the mechanisms of histone proteolysis is necessary to enhance the understanding of cellular differentiation. Here we show that H2A is cleaved by the lysosomal protease Cathepsin L during ESCs differentiation. Using quantitative mass spectrometry (MS), we identified L23 to be the primary cleavage site that gives rise to the clipped form of H2A (cH2A), which reaches a maximum level of ~1% of total H2A after four days of differentiation. Using ChIP-seq, we found that preventing proteolysis leads to an increase in acetylated H2A at promoter regions in differentiated ES cells. We also report the identification of novel readers of acetylated H2A in pluripotent ES cells, including members of the PBAF remodeling complex, which can recognize different acetylated forms of H2A. Finally, we show that H2A proteolysis abolishes this recognition. Altogether, our data suggests that proteolysis serves as an efficient mechanism to silence pluripotency genes and destabilize the nucleosome core particle.

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The activation of a Suv39h1-repressive antisense lncRNA by OCT4 couples the control of H3K9 methylation to pluripotency

10.1101/2021.09.07.459323 ◽

2021 ◽

Author(s):

Laure D. Bernard ◽

Agnès Dubois ◽

Victor Heurtier ◽

Almira Chervova ◽

Alexandra Tachtsidi ◽

...

Keyword(s):

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

H3k9 Methylation ◽

Targeted Deletion ◽

Mouse Es Cells ◽

Non Coding Rna ◽

Fate Restriction ◽

In Cis ◽

Long Non Coding Rna

Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. For instance, in pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. Conversely, H3K9 methylation represents an epigenetic barrier for reprogramming somatic cells back to pluripotency. How global H3K9 methylation levels are coupled with the acquisition and loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of an uncharacterized antisense long non-coding RNA to Suv39h1, which we name Suv39h1as. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, leading to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the global levels of H3K9 methylation to pluripotency in mouse ES cells.

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Interactions between Nodal and Wnt signalling Drive Robust Symmetry-Breaking and Axial Organisation in Gastruloids (Embryonic Organoids)

10.1101/051722 ◽

2016 ◽

Cited By ~ 2

Author(s):

D.A. Turner ◽

C.R. Glodowski ◽

L. Alonso-Crisostomo ◽

P. Baillie-Johnson ◽

P.C. Hayward ◽

...

Keyword(s):

Pattern Formation ◽

Symmetry Breaking ◽

Cell Fate ◽

Embryonic Stem ◽

Early Embryo ◽

Model System ◽

Wnt Signalling ◽

Signalling Pathways ◽

Cell Fate Decisions ◽

Three Body

AbstractGeneration of asymmetry within the early embryo is a critical step in the establishment of the three body axes, providing a reference for the patterning of the organism. To study the establishment of asymmetry and the development of the anteroposterior axis (AP) in culture, we utilised our ‘Gastruloid’ model system. ‘Gastruloids’, highly reproducible embryonic organoids formed from aggregates of mouse embryonic stem cells, display symmetry-breaking, polarised gene expression and axial development, mirroring the processes on a time-scale similar to that of the mouse embyro. Using Gastruloids formed from mouse ESCs containing reporters for Wnt, FGF and Nodal signalling, we were able to quantitatively assess the contribution of these signalling pathways to the establishment of asymmetry through single time-point and live-cell fluorescence microscopy.During the first 24-48h of culture, interactions between the Wnt/β-Catenin and Nodal/TGF/β signalling pathways promote the initial symmetry-breaking event, manifested through polarised Brachyury (T/Bra) expression. Neither BMP nor FGF signalling is required for the establishment of asymmetry, however Wnt signalling is essential for the amplification and stability of the initial patterning event. Additionally, low, endogenous levels of FGF (24-48h) has a role in the amplification of the established pattern at later time-points.Our results confirm that Gastruloids behave like epiblast cells in the embryo, leading us to translate the processes and signalling involved in pattern formation of Gastruloids in culture to the development of the embryo, firmly establishing Gastruloids as a highly reproducible, robust model system for studying cell fate decisions and early pattern formation in culture.

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Transcriptional heterogeneity in mouse embryonic stem cells

Reproduction Fertility and Development ◽

10.1071/rd08219 ◽

2009 ◽

Vol 21 (1) ◽

pp. 67 ◽

Cited By ~ 16

Author(s):

Tetsuya S. Tanaka

Keyword(s):

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Cell Types ◽

The Body ◽

Cell Subpopulation ◽

Es Cell ◽

Differentiation Potential ◽

Mouse Es Cells

The embryonic stem (ES) cell is a stem cell derived from early embryos that can indefinitely repeat self-renewing cell division cycles as an undifferentiated cell in vitro and give rise to all specialised cell types in the body. However, manipulating ES cell differentiation in vitro is a challenge due to, at least in part, heterogeneous gene induction. Recent experimental evidence has demonstrated that undifferentiated mouse ES cells maintained in culture exhibit heterogeneous expression of Dppa3, Nanog, Rex1, Pecam1 and Zscan4 as well as genes (Brachyury/T, Rhox6/9 and Twist2) normally expressed in specialised cell types. The Nanog-negative, Rex1-negative or T-positive ES cell subpopulation has a unique differentiation potential. Thus, studying the mechanism that generates ES cell subpopulations will improve manipulation of ES cell fate and help our understanding of the nature of embryonic development.

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Phenotypic Complementation Establishes Requirements for Specific POU Domain and Generic Transactivation Function of Oct-3/4 in Embryonic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.22.5.1526-1536.2002 ◽

2002 ◽

Vol 22 (5) ◽

pp. 1526-1536 ◽

Cited By ~ 203

Author(s):

Hitoshi Niwa ◽

Shinji Masui ◽

Ian Chambers ◽

Austin G. Smith ◽

Jun-ichi Miyazaki

Keyword(s):

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Cell Phenotype ◽

Dependent Manner ◽

Transactivation Domain ◽

Es Cell ◽

Self Renewal ◽

Pou Domain ◽

Cell Propagation

ABSTRACT Transcription factors of the POU family govern cell fate through combinatorial interactions with coactivators and corepressors. The POU factor Oct-3/4 can define differentiation, dedifferentation, or self-renewal of pluripotent embryonic stem (ES) cells in a sensitive, dose-dependent manner (H. Niwa, J.-I. Miyazali, and A. G. Smith, Nat. Genet. 24:372-376, 2000). Here we have developed a complementation assay based on the ability of Oct-3/4 transgenes to rescue self-renewal in conditionally null ES cells and used this to define which domains of Oct-3/4 are required to sustain the undifferentiated stem cell phenotype. Surprisingly, we found that molecules lacking either the N-terminal or C-terminal transactivation domain, though not both, can effectively replace full-length Oct-3/4. Furthermore, a fusion of the heterologous transactivation domain of Oct-2 to the Oct-3/4 POU domain can also sustain self-renewal. Thus, the unique function of Oct-3/4 in ES cell propagation resides in combination of the specific POU domain with a generic proline-rich transactivation domain. Interestingly, however, Oct-3/4 target gene expression elicited by the N- and C-terminal transactivation domains is not identical, indicating that at least one class of genes activated by Oct-3/4 is not required for ES cell propagation.

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Abscission couples cell division to embryonic stem cell fate

10.1101/798165 ◽

2019 ◽

Cited By ~ 3

Author(s):

Agathe Chaigne ◽

Celine Labouesse ◽

Meghan Agnew ◽

Edouard Hannezo ◽

Kevin J Chalut ◽

...

Keyword(s):

Stem Cells ◽

Cell Division ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Strongly Correlated ◽

Cellular Mechanisms ◽

Mouse Es Cells ◽

Cytoplasmic Bridges

Cell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cells, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here we show that exit from naïve pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naïve pluripotency. Finally, interfering with abscission impairs exit from naïve pluripotency. Altogether, our data indicate that a switch in the division machinery leading to faster abscission is crucial for pluripotency exit. Our study identifies abscission as a key step coupling cell division to fate transitions.

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