TGF-β-Superfamily Signaling Regulates Embryonic Stem Cell Heterogeneity: Self-Renewal as a Dynamic and Regulated Equilibrium

Department of Pathology and Laboratory, Institute for Reproductive Health and Regenerative Medicine, University of Kansas Medical Center.
Stem Cells (Impact Factor: 6.52). 01/2013; 31(1). DOI: 10.1002/stem.1252
Source: PubMed


Embryonic stem cells dynamically fluctuate between phenotypic states, as defined by expression levels of genes such as Nanog, while remaining pluripotent. The dynamic phenotype of stem cells is in part determined by gene expression control and dictated by various signaling pathways and transcriptional regulators. We sought to define the activities of two TGF-beta-related signaling pathways, Bone morphogenetic protein (BMP) and Nodal signaling, in modulating mouse embryonic stem cell heterogeneity in undifferentiated culture conditions. Both BMP and Nodal signaling pathways were seen to be active in distinct Nanog subpopulations, with subtle quantitative differences in activity. Pharmacological and genetic modulation of BMP or Nodal signaling strongly influenced the heterogeneous state of undifferentiated ES cells, as assessed by dynamic expression of Nanog reporters. Inhibition of Nodal signaling enhanced BMP activity, which through the downstream target Id factors, enhanced the capacity of ES cells to remain in the Nanog-high epigenetic state. The combined inhibition of Nodal and BMP signaling resulted the accumulation of Nanog-negative cells, even in the presence of LIF, uncovering a shared role for BMP and Nodal signaling in maintaining Nanog expression and repression of differentiation. These results demonstrate a complex requirement for both arms of TGF-beta-related signaling to influence the dynamic cellular phenotype of undifferentiated ES cells in serum-based media, and that differing subpopulations of ES cells in heterogeneous culture have distinct responses to these signaling pathways. Several pathways, including BMP, Nodal, and FGF signaling, have important regulatory function in defining the steady-state distribution of heterogeneity of stem cells.

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    • "In mESCs, pluripotency is maintained by a core network of regulatory transcription factors, including Pou5f1, Sox2, and Nanog (Kashyap et al., 2009; Kim et al., 2008; Marson et al., 2008; Navarro et al., 2012); the balance between self-renewal and differentiation is regulated by protein-encoding genes that include Id1 and Dusp9, both downstream targets of the bone morphogenetic protein (BMP) signaling pathway (Li and Chen, 2013). Moreover, it has been shown that both the BMP and TGFb (via NODAL) SMAD-mediated signaling pathways are involved in maintaining heterogeneity of NANOG in naive mESCs (Galvin-Burgess et al., 2013). Conversely, NANOG may attenuate BMP signaling via a feedback loop that involves titration of phosphorylated (P)SMAD1 by direct NANOG-SMAD1 interaction (Suzuki et al., 2006). "
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    ABSTRACT: Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases Tet1/2 and low levels of DNA methyltransferases Dnmt3a/b. Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double Smad1;Smad5 knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation.
    Full-text · Article · Jan 2016 · Stem Cell Reports
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    • "The effect on NECT cannot be explained in terms of effects on pluripotency since Activin/Nodal promotes exit from pluripotency (Galvin-Burgess et al., 2013) (Fig. 3) and therefore must reflect an active suppression of the NECT fate; the effects of SB43 on Sox1::GFP confirm this possibility (Fig. 2A,C). On the other hand, the reduction of T::GFP expression upon exposure to Activin (Fig. 2D) is surprising and might reflect a delayed differentiation associated with the requirement for Activin/Nodal in the maintenance of the EpiSC state (Vallier et al., 2009; Galvin-Burgess et al., 2013) i.e. the exposure to Activin induces a slow transition through an Epiblast-like state and a delay in cell fate decisions through a pause in an Epi-like state. "
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    ABSTRACT: 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 analyzed 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” at the level of single cells in which the neuroectodermal fate has an advantage.
    Full-text · Article · Mar 2014 · Biology Open
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    ABSTRACT: Heterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells. Although signaling pathways remain active throughout the cell cycle, their contribution to heterogeneous gene expression is restricted to G1. Surprisingly, we identify dramatic changes in the levels of global 5-hydroxymethylcytosine, an unanticipated source of epigenetic heterogeneity that is tightly linked to cell-cycle progression and the expression of developmental regulators. When we evaluated gene expression in differentiating cells, we found that cell-cycle regulation of developmental regulators was maintained during lineage specification. Cell-cycle regulation of developmentally regulated transcription factors is therefore an inherent feature of the mechanisms underpinning differentiation.
    Full-text · Article · Dec 2013 · Stem Cell Reports
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