Genetic Exploration of the Exit from Self-Renewal Using Haploid Embryonic Stem Cells

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Porphyrin Homeostasis Maintained by ABCG2 Regulates Self-Renewal of Embryonic Stem Cells

Background

Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined.

Methodology/Principal Findings

Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of γH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment.

Conclusions/Significance

The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and γH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells.     

Inhibition of Histone Deacetylase Impacts Cancer Stem Cells and Induces Epithelial-Mesenchyme Transition of Head and Neck Cancer

The genome is organized and packed into the nucleus through interactions with core histone proteins. Emerging evidence suggests that tumors are highly responsive to epigenetic alterations that induce chromatin-based events and dynamically influence tumor behavior. We examined chromatin organization in head and neck squamous cell carcinoma (HNSCC) using acetylation levels of histone 3 as a marker of chromatin compaction. Compared to control oral keratinocytes, we found that HNSCC cells are hypoacetylated and that microenvironmental cues (e.g., microvasculature endothelial cells) induce tumor acetylation. Furthermore, we found that chemical inhibition of histone deacetylases (HDAC) reduces the number of cancer stem cells (CSC) and inhibits clonogenic sphere formation. Paradoxically, inhibition of HDAC also induced epithelial-mesenchymal transition (EMT) in HNSCC cells, accumulation of BMI-1, an oncogene associated with tumor aggressiveness, and expression of the vimentin mesenchymal marker. Importantly, we observed co-expression of vimentin and acetylated histone 3 at the invasion front of human HNSCC tumor tissues. Collectively, these findings suggest that environmental cues, such as endothelial cell-secreted factors, modulate tumor plasticity by limiting the population of CSC and inducing EMT. Therefore, inhibition of HDAC may constitute a novel strategy to disrupt the population of CSC in head and neck tumors to create a homogeneous population of cancer cells with biologically defined signatures and predictable behavior.     

LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

LSD1 is essential for the maintenance of pluripotency of embryonic stem (ES) or embryonic carcinoma/teratocarcinoma (EC) cells. We have previously developed novel LSD1 inhibitors that selectively inhibit ES/EC cells. However, the critical targets of LSD1 remain unclear. Here, we found that LSD1 interacts with histone deacetylase 1 (HDAC1) to regulate the proliferation of ES/EC cells through acetylation of histone H4 at lysine 16 (H4K16), which we show is a critical substrate of HDAC1. The LSD1 demethylase and HDAC1 deacetylase activities were both inactivated if one of them in the complex was chemically inhibited in ES/EC cells or in reconstituted protein complexes. Loss of HDAC1 phenocopied the selective growth-inhibitory effects and increased the levels of H3K4 methylation and H4K16 acetylation of LSD1 inactivation on ES/EC cells. Reduction of acetylated H4K16 by ablation of the acetyltransferase males absent on the first (MOF) is sufficient to rescue the growth inhibition induced by LSD1 inactivation. While LSD1 or HDAC1 inactivation caused the downregulation of Sox2 and Oct4 and induction of differentiation genes, such as FOXA2 or BMP2, depletion of MOF restored the levels of Sox2, Oct4, and FoxA2 in LSD1-deficient cells. Our studies reveal a novel mechanism by which LSD1 acts through the HDAC1- and MOF-mediated regulation of H4K16 acetylation to maintain the pluripotency of ES/EC cells.     

Histone Deacetylase Inhibition Induces Apoptosis in Neuroblastoma

Histone deacetylase inhibitors constitute a promising new treatment for cancer due to their novel site of action and low toxicity. Almost all histone deacetylase inhibitors currently in clinical development have anti-proliferate activities against cells in cultures, and specially cause cell cycle arrest, differentiation and apoptosis. Interestingly, despite their rapid advance into clinical use, the cellular responses leading to these effects remain unclear. We recently reported that histone deacetylase inhibitor treatment induces apoptosis of neuroblastoma cells by increasing the acetylation of Ku70 in the cytoplasm, resulting in the release of Bax from Ku70. Subsequently, Bax releases cytochrome c from mitochondria causing apoptosis. Here we will discuss these findings and the implications of our model for the further clinical development of histone deacetylase inhibitors in the treatment of cancer.     

Conserved and Divergent Roles of FGF Signaling in Mouse Epiblast Stem Cells and Human Embryonic Stem Cells

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Highlights? Nanog is a direct target of Activin and SMAD2/3 but not FGF-ERK in EpiSCs ? FGF signaling inhibits neuroectodermal commitment of EpiSCs and hESCs ? FGF inhibition relieves Klf2 repression and reverts EpiSCs to an ESC-like state ? mESCs transition to an EpiSC-like state with LIF inhibition and FGF activation     

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

Embryonic stem cells (ESCs) can generate all of the cell types found in the adult organism. Remarkably, they retain this ability even after many cell divisions in vitro, as long as the culture conditions prevent differentiation of the cells. Wnt signaling and β-catenin have been shown to cause strong effects on ESCs both in terms of stimulating the expansion of stem cells and stimulating differentiation toward lineage committed cell types. The varied effects of Wnt signaling in ESCs, alongside the sometimes unconventional mechanisms underlying the effects, have generated a fair amount of controversy and intrigue regarding the role of Wnt signaling in pluripotent stem cells. Insights into the mechanisms of Wnt function in stem cells can be gained by examination of the causes for seemingly opposing effects of Wnt signaling on self-renewal versus differentiation.For a single-cell embryo to eventually form an adult organism of trillions of cells, some cells in the early mammalian embryo must be able to generate all cell lineages in the animal. The potential to make all adult cell types defines the property of pluripotency, and it is maintained in proliferating cells through a process called self-renewal. As cells become specified to contribute to particular lineages, they typically lose the ability to make cell types from distinct lineages (Waddington 1957; Hochedlinger and Plath 2009). As such, pluripotency is lost during the initial steps of lineage commitment that occur during gastrulation (Beddington 1982, 1983; Lawson and Pedersen 1987; Lawson et al. 1991), which is a process that coordinates the generation of adult cell lineages with the elaboration of a basic three-dimensional body structure (Heisenberg and Solnica-Krezel 2008). In the mouse, pluripotency can be tested with various experiments; the gold standard is the injection of cells into a blastocyst-staged embryo followed by contribution to a diversity of cell types in the chimeric animal or chimeric embryo after gastrulation. Cells are typically considered to have been pluripotent only if they contributed to all three germ layers (endoderm, mesoderm, and ectoderm).Embryonic stem cells (ESCs) are generated in vitro by outgrowths from a preimplantation-staged embryo, frequently a blastocyst. Pluripotent cells from the inner cell mass (ICM) of the blastocyst proliferate to form colonies, which can be expanded into ESC cultures. When culture conditions for in vitro propagation of mouse ESCs (mESCs) were first discovered more than 30 years ago (Evans and Kaufman 1981; Martin 1981), the critical achievement was finding conditions supporting indefinite ESC self-renewal, that is, maintenance of pluripotency following cell division. Compared with the other cell systems discussed below in this article, mESCs ostensibly display the greatest capacity for self-renewal and the highest ability to maintain pluripotency. As such, mESCs are typically thought to represent a primitive, or “naive,” cellular state in the early embryo.Several culture conditions can support self-renewal of mESCs. Initially, ESCs were grown in serum containing media atop a layer of mitotically inactivated fibroblasts, called feeder cells (Evans and Kaufman 1981). Feeder cells secrete the LIF cytokine, which binds a transmembrane receptor complex consisting of LIFR and gp130 proteins (Gearing et al. 1991; Gearing and Bruce 1992; Davis et al. 1993). LIF binding activates Jak/Stat signaling and Stat3 phosphorylation, which promotes ESC self-renewal (Niwa et al. 1998; Matsuda et al. 1999). Convincing proof of LIF’s importance for self-renewal in vitro was shown when recombinant LIF protein was shown to be sufficient to replace feeder cells in ESC cultures (Smith et al. 1988; Williams et al. 1988; Nichols et al. 1990).Essentially the same feeder cells can be used for both mESCs and human ESCs (hESCs); however, discrete activities of the feeders in terms of the cytokines they release are needed to effect optimal self-renewal for each cell. The LIF cytokine important for mESC self-renewal did not stimulate hESC self-renewal (Thomson et al. 1998). Instead, ERK signaling downstream from Fgf2 must accompany a feeder layer in serum-containing media for optimal hESC self-renewal (Xu et al. 2005). Interestingly, recombinant Fgf2 by itself could not replace feeders, and Fgf2 has been suggested to work in part by stimulating feeders to produce Activin/Nodal ligands; the combination of Fgf2 and Nodal/Activin is sufficient to support hESC self-renewal in serum-free chemically defined culture conditions (Vallier et al. 2004, 2009; James et al. 2005).Clear differences exist between mESCs and hESCs. The colonies adopt different morphologies, they require distinct culture conditions for self-renewal, and they have significantly different gene expression signatures (Brons et al. 2007; Tesar et al. 2007). Mouse EpiSCs are made from the epiblast of postimplantation-staged embryos between embryonic days 5.5 (E5.5) and E6.5 of embryogenesis (Brons et al. 2007; Tesar et al. 2007; Han et al. 2010). Lineage specification of pluripotent epiblast cells begins soon after formation of a cup-like structure, and at E6.5, the cells in the epiblast begin to be specified to primary cell lineages during gastrulation. The in vivo cellular environment for ICM cells and postimplantation epiblast cells is considerably different, and it is not surprising that EpiSCs and mESCs display many different characteristics (Xu et al. 2010). However, it was somewhat surprising that EpiSCs share many characteristics with hESCs, including a common colony morphology, Fgf2 + Activin A culture conditions, and gene expression signatures (Brons et al. 2007; Tesar et al. 2007). Like mESCs and hESCs, EpiSCs pass pluripotency tests for in vitro differentiation and teratoma formation. Whereas mESC can efficiently convert (i.e., differentiate) into EpiSC-like cells when switched to Fgf2/Activin A media (Hanna et al. 2009; Greber et al. 2010), EpiSCs required genetic manipulation or reprogramming for efficient conversion to mES-like cells (Guo et al. 2009; Hanna et al. 2009; Greber et al. 2010; Guo and Smith 2010). Many investigators consider hESCs and mouse EpiSCs to be primed for differentiation as they reside in a less primitive differentiation state relative to the naive state of pluripotency in mESCs.

Table 1.

Pluripotent stem cell states: Naive and primed

High-Resolution Mapping of H1 Linker Histone Variants in Embryonic Stem Cells

H1 linker histones facilitate higher-order chromatin folding and are essential for mammalian development. To achieve high-resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H1⁰ displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d, and H1e causes pericentric chromocenter clustering and de-repression of major satellites. These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.     

Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.     

Construction and Validation of a Regulatory Network for Pluripotency and Self-Renewal of Mouse Embryonic Stem Cells

A 30-node signed and directed network responsible for self-renewal and pluripotency of mouse embryonic stem cells (mESCs) was extracted from several ChIP-Seq and knockdown followed by expression prior studies. The underlying regulatory logic among network components was then learned using the initial network topology and single cell gene expression measurements from mESCs cultured in serum/LIF or serum-free 2i/LIF conditions. Comparing the learned network regulatory logic derived from cells cultured in serum/LIF vs. 2i/LIF revealed differential roles for Nanog, Oct4/Pou5f1, Sox2, Esrrb and Tcf3. Overall, gene expression in the serum/LIF condition was more variable than in the 2i/LIF but mostly consistent across the two conditions. Expression levels for most genes in single cells were bimodal across the entire population and this motivated a Boolean modeling approach. In silico predictions derived from removal of nodes from the Boolean dynamical model were validated with experimental single and combinatorial RNA interference (RNAi) knockdowns of selected network components. Quantitative post-RNAi expression level measurements of remaining network components showed good agreement with the in silico predictions. Computational removal of nodes from the Boolean network model was also used to predict lineage specification outcomes. In summary, data integration, modeling, and targeted experiments were used to improve our understanding of the regulatory topology that controls mESC fate decisions as well as to develop robust directed lineage specification protocols.     

胚胎干细胞体外分化为造血干细胞

造血干细胞移植已成为治疗白血病、再生障碍性贫血、重症免疫缺陷征、地中海贫血、急性放射病、某些恶性实体瘤和淋巴瘤等造血及免疫系统功能障碍性疾病的成熟技术和重要手段,另外这一技术还被尝试用于治疗艾滋病,已取得积极的效果。但是由于移植需要配型相同的供体,并且过程复杂,使得造血干细胞移植因缺少配型相同的供体来源以及费用昂贵而不能被广泛应用。胚胎干细胞是一种能够在体外保持未分化状态并且能进行无限增殖的细胞,在适合条件下能够分化为体内各种类型的细胞,研究胚胎干细胞分化为造血干细胞,不仅可作为研究动物的早期造血发生的模型,而且可以增加造血干细胞的来源,还可以通过基因剔除、治疗性克隆等方法来解决移植排斥的问题,从而为造血干细胞移植的发展扫除了障碍,因此有着重要的研究价值和应用前景。现对胚胎干细胞体外分化为造血干细胞的诱导方法,诱导过程中的调控机制,并对胚胎干细胞分化为造血干细胞的存在问题和发展前景进行讨论。