Maintenance Of ES Cells Research Articles

SETDB1 acts as a topological accessory to Cohesin via an H3K9me3-independent, genomic shunt for regulating cell fates.

SETDB1 is a key regulator of lineage-specific genes and endogenous retroviral elements (ERVs) through its deposition of repressive H3K9me3 mark. Apart from its H3K9me3 regulatory role, SETDB1 has seldom been studied in terms of its other potential regulatory roles. To investigate this, a genomic survey of SETDB1 binding in mouse embryonic stem cells across multiple libraries was conducted, leading to the unexpected discovery of regions bereft of common repressive histone marks (H3K9me3, H3K27me3). These regions were enriched with the CTCF motif that is often associated with the topological regulator Cohesin. Further profiling of these non-H3K9me3 regions led to the discovery of a cluster of non-repeat loci that were co-bound by SETDB1 and Cohesin. These regions, which we named DiSCs (domains involving SETDB1 and Cohesin) were seen to be proximal to the gene promoters involved in embryonic stem cell pluripotency and lineage development. Importantly, it was found that SETDB1-Cohesin co-regulate target gene expression and genome topology at these DiSCs. Depletion of SETDB1 led to localized dysregulation of Cohesin binding thereby locally disrupting topological structures. Dysregulated gene expression trends revealed the importance of this cluster in ES cell maintenance as well as at gene ‘islands’ that drive differentiation to other lineages. The ‘unearthing’ of the DiSCs thus unravels a unique topological and transcriptional axis of control regulated chiefly by SETDB1.

Maintenance and differentiation of human ES cells on polyvinylidene fluoride scaffolds immobilized with a vitronectin-derived peptide.

Polyvinylidene fluoride (PVDF) is biocompatible, easy to fabricate, and has piezoelectric properties; it has been used for many biomedical applications including stem cell engineering. However, long-term cultivation of human embryonic stem cells (hESCs) and their differentiation toward cardiac lineages on PVDF have not been investigated. Herein, PVDF nanoscaled membrane scaffolds were fabricated by electrospinning; a vitronectin-derived peptide-mussel adhesive protein fusion (VNm) was immobilized on the scaffolds. hESCs cultured on the VNm-coated PVDF scaffold (VNm-PVDF scaffold) were stably expanded for more than 10 passages while maintaining the expression of pluripotency markers and genomic integrity. Under cardiac differentiation conditions, hESCs on the VNm-PVDF scaffold generated more spontaneously beating colonies and showed the upregulation of cardiac-related genes, compared with those cultured on Matrigel and VNm alone. Thus, VNm-PVDF scaffolds may be suitable for the long-term culture of hESCs and their differentiation into cardiac cells, thus expanding their application in regenerative medicine.

TERRA regulate the transcriptional landscape of pluripotent cells through TRF1-dependent recruitment of PRC2.

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

Serum Inter-α-inhibitor Activates the Yes Tyrosine Kinase and YAP/TEAD Transcriptional Complex in Mouse Embryonic Stem Cells

We have previously demonstrated that the Src family kinase Yes, the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition, we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation, YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI, is demonstrated to be responsible for this effect. Moreover, IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion, we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal.

YAP/TAZ Incorporation in the β-Catenin Destruction Complex Orchestrates the Wnt Response

The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the β-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for β-TrCP recruitment to the complex and β-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/β-catenin signaling. In line, the β-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.

Abstract 2293: KLF5 is essential for oncogenesis of intestinal tumors and control of intestinal stem cells.

Abstract Genetic changes in intestinal epithelial stem cells are more important for oncogenesis of intestinal tumors than such changes in non-stem cells. A large-scale sequencing analysis of human colorectal carcinoma (CRC) revealed that Wnt/β-catenin signal activation is the most frequent genomic changes (92-97%) in human colorectal carcinoma (CRC). However, fundamental significance of its signal activation of intestinal stem cells is still remains unsolved. Kruppel-like factor 5 (KLF5) is one of KLF zinc finger transcription factor with diverse biological functions. KLF5 has stronger expression in the bottom of intestinal crypts, where the stem cells reside, and weaker expression in villi as the epithelial cells differentiate into more mature cells, which is complementary pattern of KLF4. KLF5 is implicated in the renewal and maintenance of ES cells, and has the potential to induce iPS cells, although weaker than KLF4. In this study, we found that inducible deletion of KLF5, specifically in Lgr5+ intestinal epithelial stem cells, suppressed the proliferation and nuclear localization of β-catenin, and generated some apoptotic cells in the intestinal crypts of mice. Furthermore, we demonstrated that inducible deletion of KLF5 in all intestinal epithelia caused mislocalization of Paneth cells, which constitute the stem cell niche. These data indicate that KLF5, rather than KLF4, is required for the maintenance and differentiation of Lgr5+ intestinal stem cells. Moreover, we detected frequent genome amplification at the KLF5 locus in human colorectal cancers (CRCs), and enhanced expression of KLF5 in CRC samples. In mice, Lgr5+ stem cell-specific induction of an active β-catenin mutant generated severe lethal adenomas and carcinomas. By contrast, when the induction of active β-catenin was performed simultaneously with deletion of KLF5 in stem cells, oncogenesis was almost completely suppressed. The simultaneous deletion of KLF5 under the induction of the active mutant of β-catenin in the stem cells suppressed the transcription of HDAC1and Sphk1, which promotes CRCs, and upregulated the transcription of Aim2 and Foxo3, tumor suppressors in CRCs, in both the FACS sorted stem cells and the transit amplifying cells. These results indicate that oncogenesis of intestinal tumors induced by Wnt/β-catenin signal activation (92-97% in CRCs) in stem cells requires KLF5, as in the case of Myc, and that KLF5 is also an essential controlling factor for homeostatic turnover and maintenance, niche formation, and differentiation of the intestinal epithelial stem cells. These suggest that KLF5 is a promising target oncogene for CRC therapy directed at colon cancer stem cells. Citation Format: Takeo Nakaya, Masahiko Kuroda, Ryozo Nagai. KLF5 is essential for oncogenesis of intestinal tumors and control of intestinal stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2293. doi:10.1158/1538-7445.AM2013-2293

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in (1,2)). To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells (3). The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region (4,5). Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation (6,7). As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity (5), and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level (3,8). Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal (3,8). Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

Abstract IA21: Role of Tet1-catalyzed 5mC hydroxylation in transcriptional regulation and ES cell maintenance

Abstract Epigenetic modifications play important roles in diverse biological processes that range from regulation of gene expression, embryonic development, stem cell reprogramming, and human diseases such as cancers. One of the epigenetic modifications is DNA methylation. Although enzymes responsible for DNA methylation have been well characterized, enzymes responsible for active DNA demethylation in mammalian cells have remained elusive. Recent studies have demonstrated that a novel family of proteins— Tet1–3—have the capacity to convert 5mC to 5hmC, raising the possibility that Tet proteins may catalyze the initial step leading to DNA demethylation. Interestingly, Tet1 plays an important role in stem cell self-renewal and maintenance. To understand how Tet1 contributes to these processes, we profiled the genome-wide location of Tet1 and 5hmC in ES cells. This study revealed that Tet1 has a dual function in activating pluripotency stem cell genes and silencing lineage-specific differentiation genes. Further studies revealed that Tet1 contributes to gene silencing by facilitating recruitment of PRC2. Thus, our studies establish a link between Tet1 and PRC2 and revealed a dual function for Tet1 and 5hmC in transcriptional regulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr IA21.

Chromatin regulation landscape of embryonic stem cell identity

ES cells (embryonic stem cells) derived from the ICM (inner cell mass) of blastocysts are pluripotent and are capable of giving rise to most cell types. The ES cell identity is mainly maintained by the Oct4 (octamer-binding transcription factor 4) and Nanog transcriptional networks. Recently, a tremendous amount of work has focused on deciphering how ES cell identity is regulated epigenetically. It has been shown that histone methylation/demethylation, histone acetylation/deacetylation, histone variants and chromatin remodelling play crucial roles in ES cell maintenance and differentiation. Moreover, perturbation of those chromatin regulators results in loss of ES cell identity or aberrant differentiation. Therefore, it is important to fully understand the chromatin regulation landscape of ES cells. The knowledge gained will help us to harness the unique characteristics of ES cells for stem cell-related therapy and regenerative medicine. In the present review, we will discuss recent proceedings that provide novel insights into chromatin regulation of ES cell identity.

An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency

Mammalian SWI/SNF [also called BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodeling complexes are essential for formation of the totipotent and pluripotent cells of the early embryo. In addition, subunits of this complex have been recovered in screens for genes required for nuclear reprogramming in Xenopus and mouse embryonic stem cell (ES) morphology. However, the mechanism underlying the roles of these complexes is unclear. Here, we show that BAF complexes are required for the self-renewal and pluripotency of mouse ES cells but not for the proliferation of fibroblasts or other cells. Proteomic studies reveal that ES cells express distinctive complexes (esBAF) defined by the presence of Brg (Brahma-related gene), BAF155, and BAF60A, and the absence of Brm (Brahma), BAF170, and BAF60C. We show that this specialized subunit composition is required for ES cell maintenance and pluripotency. Our proteomic analysis also reveals that esBAF complexes interact directly with key regulators of pluripotency, suggesting that esBAF complexes are specialized to interact with ES cell-specific regulators, providing a potential explanation for the requirement of BAF complexes in pluripotency.

A Matter of Some Importins: Nuclear Transport Factors in ES Cell Maintenance and Differentiation

A recent paper in Nature Cell Biology reports an unexpected role for nuclear transport proteins in triggering the differentiation of ES cells. The authors show how switching of importin-alpha subtypes exerts a selective gate-keeping function in the nuclear import of key transcription factors that regulate stem cell maintenance and differentiation.

Basic Fibroblast Growth Factor Activates ERK and Induces c-Fos in Human Embryonic Stem Cell Line MizhES1

Human embryonic stem (hES) cells can be maintained in a proliferative undifferentiated state in vitro by growing them on feeder layers of mouse embryonic fibroblast (MEF) cells along with basic fibroblast growth factor (bFGF/FGF-2). To understand the molecular mechanisms involved in the requirement of bFGF in human ES cells, we investigated expression of FGF receptors and intracellular signaling events in response to bFGF in human ES cell line MizhES1. On the basis of the results of RT-PCR, clear expression of FGF receptors FGFR1, FGR2, and FGFR3 was noticed. Because MAPK, PI3K, and PKC pathways are well-known pathways triggered by bFGF in other cells, these pathways were investigated after stimulation with bFGF. bFGF did not induce activation of PI3K or PKC, but induced activation of ERK (extracellular signal-regulated kinase). To monitor the consequences of ERK activation, we examined expression of the immediate early gene c-fos, one downstream target of the MEK1/ERK pathway. mRNA and protein levels of the c-fos gene were increased by bFGF. Induction of c-Fos was dependent on MEKl. Therefore, it is likely that bFGF contributes to maintenance of human ES cells, at least in part, through the MEK1/ERK pathway.

A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

One of the most important issues in stem cell research is to understand the regulatory mechanisms responsible for their differentiation. An extensive understanding of mechanism underlying the process of differentiation is crucial in order to prompt stem cells to perform a particular function after differentiation. To elucidate the molecular mechanisms responsible for the hematopoietic differentiation of embryonic stem cells (ESCs), we investigated murine ES cells for the presence of hematopoietic lineage markers as well as Wnt signaling pathway during treatments with different cytokines alone or in combination with another. Here we report that Wnt/β-catenin signaling is down-regulated in hematopoietic differentiation of murine ES cells. We also found that differentiation induced by the interleukin-3, interleukin-6, and erythropoietin combinations resulted in high expression of CD3e, CD11b, CD45R/B220, Ly-6G, and TER-119 in differentiated ES cells. A high expression of β-catenin was observed in two undifferentiated ES cell lines. Gene and protein expression analysis revealed that the members downstream of Wnt in this signaling pathway including β-catenin, GSK-3β, Axin, and TCF4 were significantly down-regulated as ES cells differentiated into hematopoietic progenitors. Our results show that the Wnt/β-catenin signaling pathway plays a role in the hematopoietic differentiation of murine ESCs and also may support β-catenin as a crucial factor in the maintenance of ES cells in their undifferentiated state.

LIF: a molecule with divergent actions on myeloid leukaemic cells and embryonic stem cells

We have previously characterized, purified and cloned a novel murine and human regulator [leukaemia inhibitory factor, LIF] which induces the differentiation of certain murine and human myeloid leukaemic cells. Recently we have shown that there are specific LIF receptors on murine embryonic stem [ES] and embryonal carcinoma [EC] cells and that purified recombinant LIF can substitute for feeder cells and crude sources of differentiation inhibiting activity [DIA] [such as BRL-cell-conditioned medium] in the maintenance of ES cells in a pluripotential state in vitro. Furthermore, ES cells maintained in culture in recombinant LIF for a prolonged period can give rise to germline chimaeric mice. Thus, based on a number of biochemical and biological similarities, it is likely that LIF and DIA are the same molecule. The identification of LIF as a molecule, necessary and sufficient for the maintenance of ES cells in culture, should have a profound impact on the use of these cells for genetic manipulations.