Keywords

Introduction

During the last few decades, nitric oxide (NO) has been proposed as a signaling molecule that regulates cell survival and proliferation in diverse cell types. It has also been shown to affect gene expression at the level of transcription and translation. NO is produced by isoforms of NO synthases (NOSs) that have been found to be expressed in embryonic tissues and recently, in stem cells. Therefore, it is feasible that NO might contribute to the regulation of some features of ESC biology (Tejedo et al., 2010).

Studies on development in diverse organisms have associated NO with a role in egg activation at fertilization, and it is also involved in developmental processes in the embryo (Gouge et al., 1998). One of the first achievements in obtaining differentiated ES cells using NO occurred when Kanno reported that cardiomyocytes could be generated from ES cells upon exposure to high concentrations of chemical NO donors (Kanno et al., 2004). This success revealed the potential importance of this molecule for the differentiation of ES cells and its impact in regenerative medicine. Recently, NO donors and soluble guanylate cyclase (sGC) activators have been shown to play a role in the differentiation of ES cells into myocardial cells (Mujoo et al., 2008).

An important feature of NO is that it has an enormous range of important functions and multiple physiological roles as a biological signaling molecule. Similarly, NO is essential for the survival of neuronal cell lines and primary neurons in culture after various death challenges. NO can also negatively regulate the proliferation of neuronal cell precursors and promote their differentiation by downregulating the oncogene n-Myc in neuroblastoma cell lines (SK-N-BE). Additional studies link NO generation with differentiation and development, suggesting an evolutionarily conserved role for NO in these processes (Tapia-Limonchi et al., 2011).

In this chapter, the latest research on the impact of NO on ES cell biology and its role in differentiation procedures will be introduced. This chapter will also try to explain how the main signaling pathways activated by NO mediate cell functions that could commit cells toward specific tissue lineages. In addition, advances in understanding the role of NO in the regulation of apoptosis and gene expression through chromatin remodeling mechanisms and the post-translational modification of transcriptional regulators will be discussed.

Nitric Oxide as a Signaling Molecule in Development

The Role of Nitric Oxide During Development

During organ and tissue development, a tightly controlled program of cell proliferation in coordination with growth arrest and differentiation is required. Despite possessing identical genetic material, the cells of each individual organism exhibit clear distinctions in their morphologies and cellular functions. Development in multicellular organisms and embryonic stem cells is marked by an intricate process of cell differentiation in which cells gradually lose their innate plasticity to take on specialized functions. Some signals activate and regulate signaling pathways by inducing discrete reactions. By looking at the progression of such signaling pathways, it may be possible to predict which cell type will be the outcome of the differentiation process.

It has been suggested that NO may act as an essential negative regulator of cell proliferation during tissue differentiation and organ development in Xenopus and Drosophila, controlling the balance between cell proliferation and differentiation in the developing tissue. It may also affect specific traits that characterize the differentiated tissue, thus coupling the exit from the cell cycle to the acquisition of the differentiated phenotype (Enikolopov et al., 1999).

Similarly, studies in mice have demonstrated that iNOS and eNOS isoforms are expressed early during mouse/rat embryonic heart development and that starting from E14.5 they are strongly downregulated (Bloch et al., 1999). These results implicate NO in organogenesis and development and support the hypothesis that NO is involved in the differentiation of ES and iPS cells.

Signaling in ES Cells Regulated by Nitric Oxide

Nitric oxide (NO) is a versatile diffusible second messenger implicated in numerous physiological functions in mammals, ranging from the dilation of blood vessels and muscle relaxation to immune responses and the potentiation of synaptic transmission (Enikolopov et al., 1999). NO actively participates in NO/cyclic guanosine monophosphate (cGMP) signaling to play a crucial role in cardiovascular, gastrointestinal, platelet, and neuronal functions. Recently, the hypothesis that NO/cGMP signaling plays a significant role in embryonic development and cell differentiation has gained stronger support. This idea is supported by studies in triple NOS knockout mice, which completely lack endogenous production of NO; in these mice, the survival rate and the number of offspring are significantly reduced (Tsutsui et al., 2006).

Several reports from different groups indicate that alterations in intracellular NO/cGMP levels affect the differentiation of bone-marrow-derived progenitor and embryonic stem (ES) cells. Dynamic expression of NOS isoforms and sGC subunits during the differentiation of mouse and human embryonic cells has been demonstrated. Undifferentiated ES cells do not express enzymatically active sGC, but as cell differentiation progresses, the α1/β1 sGC mRNA and protein levels increase, which coincides with the augmentation of NO-inducible intracellular cGMP levels (Mujoo et al., 2006; Sharin et al., 2010). In addition, components of NO signaling have been implicated in the differentiation of hESCs to neural cells (Tao Li et al., 2010).

There is some evidence that NO/cGMP has a pro-differentiation effect through cross-talk with other signaling pathways, e.g., both retinoic acid and cGMP are known for their positive effects on hESC differentiation (Tao Li et al., 2010). Similarly, NO contributes to oxytocin-induced cardiomyocyte differentiation through a pathway involving eNOS mRNA upregulation and iNOS- and NO-dependent sGC activity (Danalache et al., 2007). However, exposure to NO does not induce Stat3 phosphorylation in ES cells, indicating that the mechanism of action of NO is independent of LIF/Stat3 signaling. NO-induced activation of c-Src and Akt has also been observed in ESCs (Tejedo et al., 2010) (Fig. 36.1).

Fig. 36.1
figure 36_1_272396_1_En

Nitric oxide signaling in ES cells. Undifferentiated ES cells express NOS-1 and NOS-3 and produce low concentrations of NO. The expression of the NO receptor gene, sGC, was very low, and therefore, cGMP synthesis and PKG-1 are inhibited. This results in changes in gene expression that promote pluripotency and self-renewal, as well as cell survival; the same effect is obtained when cells are treated with exogenous sources of NO at low doses. As differentiation progresses, NOS-2 expression and activity increases, inducing the activation of sGC, subsequent synthesis of cGMP and the downstream NO signaling cascade. Similar activation occurs when ES cells are treated with high doses of NO, leading to the activation of apoptosis, mitochondrial metabolism and tissue-specific gene regulation. These effects are dependent on culture conditions and differentiation approaches (Tejedo et al., 2010; Mora-Castilla et al., 2010)

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Dose-Dependent Effects of Nitric Oxide on ES Cells

NO is a molecule with dual effects on the biology of embryonic stem cells. The activation of NOS and sGC isoforms during early embryo development, leading to the delivery of NO at variable doses can promote cell proliferation, cell movement or cell differentiation. Direct effects are those chemical reactions that occur fast enough to allow NO to react directly with a biological molecule. Indirect effects result from the production of reactive nitrogen species (RNS), leading to nitrosative and oxidative stress (Thomas et al., 2008). Concentration-dependent reactions induce different post-translational modifications, such as phosphorylation, acetylation, ubiquitination, nitration and nitrosylation, on different proteins. Nitration events can occur when RNS are produced from high concentrations of NO, which reacts with O2 to form peroxynitrite (ONOO–). Nitrosylation is the binding of NO in the thiol group of a cysteine, and denitrosylation is the removal of a NO group from a protein or peptide. Nitrosylation of critical cysteines (S-nitrosylation) is, at least in part, non-enzymatically mediated and regulates protein function, allowing cells to dynamically modify signaling in response to physiological stimuli (Mannick, 2006). It has been proposed that high concentrations of NO can compromise redox potential in the cell, allowing the generation of RN and RO species, which can lead to Tyr-nitration events. To date, there have been no published reports of protein nitration in embryonic stem cell models. Low concentrations of NO can modify protein functions through S-nitrosylation, which has attracted the attention of several research groups in recent years due to its reversible and spontaneous nature. Relevant cellular functions have been found to be regulated through the S-nitrosylation of proteins. For example, the nitrosylation of caspase 3 plays an important role in regulating mitochondrial caspase function and has been implicated in the transnitrosylation of Xiap, thereby inhibiting apoptotic activity (Nakamura et al., 2010). These post-translational mechanisms that depend on the NO concentration could be critical for the balance between self-renewal and differentiation in embryonic stem cells.

The regulation of NO production in vivo leads to an increase in stem and progenitor cells in the bone marrow. In addition, the mobilization of stem cells from the bone marrow seems to be regulated by NOS-3, and NOS-3 may also be important for the hemangioblast activity of adult stem cells (Mujoo et al., 2006). Undifferentiated mouse and human embryonic stem cells express NOS and NO signaling components, indicating the presence of endogenous NO with unknown function.

Undifferentiated ES cells express high levels of NOS-3 (eNOS). During differentiation in attached and hanging drop suspension cultures, NOS-3 levels decrease, whereas NOS-2 (iNOS), NOS-1 (nNOS), sGCα1, sGCβ1, and PKG levels increase significantly (Krumenacker et al., 2006; Tejedo et al., 2010). This suggests that, although cGMP-mediated NO activity may not be functional in undifferentiated ES cells, the key components are differentially expressed during early differentiation events in ES cells (Krumenacker et al., 2006).

The exposure of adherent cultures of ES cells to exogenous sources of NO (less than 20 μM of NO from donor DETA-NO) in differentiating conditions, i.e., in the absence of LIF and bFGF, maintains high mRNA and protein expression of the pluripotency markers Oct4, Sox2 and Nanog. Conversely, the expression levels of early differentiation markers, such as Brachyury, Gata4 and FGF5, were decreased. Low concentrations of NO notably decreased the apoptosis induced by the beginning of the differentiation process, increasing the expression of Bcl2 and inhibiting caspase activity (Tejedo et al., 2010). Therefore, low concentrations of NO delay differentiation and help ES cells to maintain self-renewal and pluripotency. This model involved culturing cells from the first day of culture with a constant concentration of NO. Interestingly, different results were obtained when concentrations of NO donors under 100 μM were used in hanging drop differentiation protocols. The expression of NO-sGC signaling components varied, and cardiac markers, such as Nkx2.5, were increased (Krumenacker et al., 2006; Mujoo et al., 2006). Different differentiation conditions were used in this study, supporting the versatility of NO with different approaches.

High concentrations of NO (up to 1 mM of NO donors for periods up to 19 h) have been used in differentiation protocols, despite its lethal and apoptotic effects. Our group has recently shown that treatment with high levels (500 μM) of DETA-NO downregulates the expression of Nanog and Oct4. This treatment further increased the H3K9me3 mark and the expression of endoderm markers, such as FoxA2, Gata4, Hnf1-β and Sox17 (Mora-Castilla et al., 2010). Another approach using high concentrations of NO was proposed by Spallota et al. Mouse ES cells were first cultured in low serum medium supplemented with LIF and then shifted to complete medium containing 500 μM DETA/NO without LIF. Deacetylase activity was detected in ES nuclear extracts after 1 and 3 h of treatment, and a sharp increase in several mesodermal markers, including vascular and muscular cell markers, such as Flk-1, SM22α and Desmin, was seen after 20 h of treatment with the same donor (Spallotta et al., 2010).

Stem Cell Differentiation Induced by Nitric Oxide

Although the use of ES cells to generate cells suitable for tissue replacement therapy has gained interest in recent years, accurate methods for producing such cells have yet to be developed. Studies aimed at understanding the behavior of ES cells in the context of other cellular models, such as iPS, will be required to develop better and more efficient differentiation protocols for regenerative medicine and patient-directed cell therapy. To that end, the discovery and characterization of new small molecules that act on specific cell signaling pathways involved in embryonic development can offer valuable information about their development and cell differentiation.

As has been shown previously, NO is a molecule with different effects over a broad range of concentrations. NO acts as a second messenger to regulate important processes such as growth, survival, proliferation, migration, axon guidance, differentiation, and other processes through a variety of downstream signaling cascades.

Diverse protocols have been developed to improve the differentiation of ES cells toward defined cell lineages.

Nitric Oxide-Induced Differentiation of ES Cells into Cardiomyocites and Vascular Tissues

The first report of an efficient differentiation protocol using mouse ES cells treated with NO was by Kanno in 2004. At this time, EB-derived beating foci were counted by microscopy on D3, D5, D7, D10, and D14 after plating. Exposure of these cells to 200 μM SNAP and other NO donors increased the percentage of beating EB outgrowths to 45% compared with 15% in control cells at D14. Moreover, the nonspecific NOS inhibitor l-nitroarginine methyl ester (l-NAME) decreased the incidence of beating foci compared with control cells. iNOS-overexpressing cells showed a 6-fold increase in the number of troponin T-positive cells. Additionally, FC analysis was performed with two different anti-MHC antibodies, including anti-α (adult type) MHC and anti-β (fetal type) MHC. Of the iNOS-transfected cells, 43.9% were positive for β-MHC staining and 47.9% were positive for α-MHC. This heterogeneity is consistent with the known transition from the β to the α isoform of MHC during cardiac development (Kanno et al., 2004). Therefore, these results showed how endogenous or exogenous NO can induce the differentiation of ES cells. In addition, this process appears to mimic the normal sequence seen in cardiac development. Other groups have since revealed the role of NO and cGMP signaling components in cardiac development, as well as certain molecules that could regulate early cardiomyogenesis in ES cells through NO-dependent pathways (Gassanov et al., 2007; Krumenacker et al., 2006; Miao et al., 2010).

The regenerative potential of NO has recently been demonstrated by the regeneration of muscular and vascular structures through the injection of NO-treated ES cells into the cardiac left ventricle of mice with hind limb ischemia. This study established a key role for NO in the modulation of class IIa HDACs in ES mesodermal commitment and enhanced regenerative potential in vivo (Spallotta et al., 2010).

Vasculogenesis and angiogenesis are the main processes responsible for embryonic vascular development. The formation of new vascular tubes involves endothelial cell proliferation and migration as well as the inhibition of apoptosis; these are processes in which NO actively participates (Huang et al., 2010). Although differentiation protocols for generating endothelial cells (ECs) from ES cells do not include NO-like active small molecules, the expression of endothelial NO synthase in these cell types is considered to be a characteristic of mature ECs (McCloskey et al., 2006). Nitric oxide can participate in the formation of vascular networks; this effect appears to involve β1 integrins, which have been identified as important molecules for endothelial cell maturation, migration and sprouting that are required for adequate vascular formation. β1 integrin–/– mouse ES cells show a high rate of apoptosis that is promoted by an increase in eNOS/NO activity, although wild type ECs were not affected in the presence of the eNOS inhibitor l-NAME, suggesting that NO may contribute indirectly to vasculogenesis and the formation of vascular networks (Malan et al., 2010). In summary, recent studies demonstrate the involvement and the potential use of nitric oxide as an inducer of differentiation in the regeneration of cardiac muscle and vascular tissues and in vivo. This role for NO may suggest useful approaches for future treatments of heart and vascular diseases.

Nitric Oxide-Induced Differentiation of ES Cells into Neuronal Tissue

The participation of NO in the formation of the nervous system may be related to its involvement in neuronal programmed cell death, the organization of axonal projection patterns, or the control of cell proliferation. In neuronal cell lines, NOS-1 and NOS-2 isoforms are induced by differentiating agents, and in some cases, the anti-proliferative action of NO was shown to be a prerequisite for differentiation. In neurons, NO production is sensitive to a number of stimuli. Neurotrophic factors such as NGF may regulate NO synthesis by nNOS through mechanisms involving TrkA receptors (Madhusoodanan and Murad, 2007).

Despite the accumulating evidence that NO plays a role in neuronal differentiation, there is still a controversial debate about which NOS isoform is responsible for NO synthesis during the early stages of neuronal differentiation. The evidence for preferential expression of NOS-2 (iNOS) during early embryonic stages and its suspected role during subsequent steps of neuronal differentiation has been shown previously. The specific inhibition of NOS-2 is accompanied by reduced neurite outgrowth formation in cortical neurons and neurally-selected ES cells. Furthermore, reduced network formation in ES cell-derived neuronal cells is reduced under these experimental conditions. These data emphasize the capacity of NO generated from the NOS-2 isoform to promote differentiation; comparable treatment with NO donors has the same effect. This NO effect is mediated independently of the sGC/cGMP pathway, possibly by controlling intracellular calcium homeostasis (Arnhold et al., 2002). The involvement of NOS-2 in neuronal differentiation has been reinforced by the existence of a regulatory locus, anti-NOS2A, which is transcribed into a noncoding NOS. This anti-NOS2A RNA also plays role in the regulation of NOS gene expression and is highly expressed in undifferentiated hESCs. Its expression significantly decreases during neuronal differentiation of hESCs, leading to an increase of NOS-2 expression (Korneev et al., 2008).

A new approach for differentiating hES cells into neural cells was recently published by Ferid Murad’s group in Texas, USA. The aim of this study was to demonstrate that NO/cGMP signaling molecules regulate neural lineage commitment and govern neural precursor differentiation. Spontaneous differentiation of hES cells in differentiation medium leads to co-expression of nestin and NOS-1, but FACS analysis demonstrated a further increase in NOS-1 positive cells after treatment with retinoic acid and 8-brom cGMP (10−5 M). These results suggest that NO/cGMP signaling contributes to the differentiation of neural precursors from hESC and enhances the differentiation of precursors toward functional neurons (Tao Li et al., 2010).

The Involvement of Nitric Oxide in Cell Differentiation Toward Other Lineages

Although the role of NO in other tissues and organs during embryonic development (endodermic lineage) is unknown, the participation of NO in the differentiation of ES cells into other cell types suggests other properties of the molecule and the possibility that it could interact with other reagents. For example, oxidative stress and the generation of RN and RO species could activate apoptosis, which can be used to select for cell populations committed toward an appropriate cell lineage. A protocol for differentiating ES cells into definitive endoderm has been developed by our laboratory. The protocol consists of a ‘preconditioning step’ in which cells are grown in LIF-starved conditions for 3 days, followed by a ‘selection step’ involving NO treatment for 19 h, a ‘pre-maturation step’ with VPA in 15% FBS for 24 h and a ‘maturation step’ with VPA in 2% FBS for 5 days. In this case, NO downregulates Nanog and eliminates cells that have not committed to differentiation. The selected adherent cells continue to differentiate with VPA, which is a factor used for endoderm differentiation and maturation during pancreatic development. The fact that Brachyury expression was inhibited and Pdx1 and Gata4 were increased by NO treatment indicates that NO favored differentiation to endoderm (Mora-Castilla et al., 2010).

NO is a known modulator of hematopoiesis, and its role in ES cell differentiation toward hematopoietic tissue has been studied. The exposure of embryoid body-derived ES cells to biomechanical stimulus, such as wall shear stress for 48 h, caused an increase in the proportion of cells positive for CD31 (PECAM1), a marker of the endothelial and hematopoietic lineages. Shear stress increased the frequency of hematopoietic colony forming units (c.f.u.) compared with static (no shear stress) conditions. The inhibition of NO production via the nitric oxide synthase inhibitor nitro-l-arginine methyl ester (l-NAME) resulted in a significant reduction of the shear-stress-induced enrichment in c.f.u. Similar results were obtained using in vivo assays. These data indicate that NO production is required for the shear-stress-mediated stimulation of hematopoietic progenitors, thereby establishing that the NO pathway is an important mediator of the effect of shear stress on hematopoiesis (Adamo et al., 2009) (Fig. 36.2).

Fig. 36.2
figure 36_2_272396_1_En

Nitric oxide induces the differentiation of ES cells to phenotypes derived from three germ layers. Exogenous sources of high doses of NO, along with the NO produced by NO synthase isoforms, activates diverse cellular programs. The activated apoptotic machinery can select between cells that have already committed to a particular phenotype and those that have not yet committed. Concurrently, a transcriptional network of tissue-specific programs is activated. These two mechanisms can be combined to generate efficient selection strategies for differentiation protocols (Kanno et al., 2004; Li et al., 2010; Mora-Castilla et al., 2010)

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Nitric Oxide and Gene Regulation in ES Cells

A small core set of transcription factors work together to maintain the pluripotent state of ESCs. These transcriptional regulators, including Oct4, Sox2, and Nanog, stimulate the expression of genes controlling self-renewal while repressing genes that drive differentiation. Additional mechanisms of transcriptional regulation are also required to maintain ES cells or switch on differentiation. ES cells have unusually high levels of acetylated H3K9 and methylated H3K4; these markers of open chromatin are also combined with H3K27 trimethylation at some non-expressed genes. Therefore, the pluripotency of ES cells is characterized by a specific epigenetic profile where lineage-specific genes may be accessible but, if so, they carry repressive H3K27 trimethylation modifications. H3K27 methylation is functionally important for preventing the expression of these genes in ES cells (Azuara et al., 2006). A substantial amount of information about the transcriptional effects of NO in different cell types and tissues has been generated in recent years. NO can modulate gene expression through the activation of multiple transcription factors, including CREB, NFκB, and nMyc; post-translational modifications of transcriptional regulators or changes in chromatin structure, such as CpG DNA de/methylation and/or the methylation and acetylation of histones. S-nitrosylation mediates the NO-dependent regulation of various zinc-finger-containing transcription factors, including Egr1 and NFκB. S-nitrosylation of zinc-sulfur clusters of these TFs disrupts zinc binding, inhibiting their association with DNA. Other studies have suggested that NO signaling can inhibit AP1 binding to DNA, through the modification of its two components, c-fos and c-jun (Nott and Riccio, 2009).

In mouse ES cells cultured in the absence of LIF, low concentrations of NO donors can modify the transcriptional regulatory network responsible for maintaining self-renewal. In addition to the master pluripotency markers Oct4, Sox2 and Nanog, nitric oxide increased the expression of proteins such as Zic3, Zic1, FoxD3, Ronin (Thap11), Creb1, cMyb, Sin3A, Sall4 and Dax1 (Nr0b1). In contrast, expression of cMyc, Esrrb and Rex1 (Zfp42) was decreased, whereas the expression of Dppa3, Zfx and nMyc remained unchanged (Tapia-Limonchi et al., 2011). Treatment with high concentrations of NO donors can inhibit the expression of Nanog and Oct4 in mES cells. The suppressive activity of NO on the Nanog gene depends on the activation of the p53 repressor protein. The repression of the self-renewal machinery activates a differentiation program stimulating the expression of transcription factors such as Brachyury, Gata4, Pdx1, and FoxA2. Depletion of p53 significantly inhibits the effect of NO. High levels of NO repress two of the most important pluripotency genes, Nanog and Oct4, enhance the transactivation activity of p53, remodel chromatin and promote the expression of the early endoderm genes Pdx1 and Gata4 (Mora-Castilla et al., 2010).

Recently, the chromatin-modifying enzyme, HDAC2, was identified as a key nuclear target of NO. S-nitrosylation of HDAC2 occurs at two cysteine residues, Cys262 and Cys274, and does not affect its deacetylase activity in neurons. Instead, HDAC2 S-nitrosylation induces its dissociation from CREB-regulated gene promoters (Nott and Riccio, 2009). NO can promote ES differentiation in the presence or absence of LIF, as indicated by its ability to induce mesodermal markers. However, it is only upon LIF withdrawal that NO activates class IIa HDACs; under these conditions, mesodermal proteins are strongly expressed. These events are associated with NO-dependent changes in the chromatin domains of specific mesodermal gene promoters. The ability of NO to induce mesodermal genes in the presence of LIF may rely on its ability to regulate other components of the chromatin remodeling machinery (Spallotta et al., 2010). Additional studies on gene regulation by NO in ES cells are necessary to design and improve protocols to induce differentiation and commitment to specific cell types. Genome-wide transcriptional or regulatory analyses provide valuable knowledge on the effects of nitric oxide on the modulation of ES cell functions. Our studies in this context show that low concentrations of NO have a drastic effect on the transcriptome of differentiating ES cells. Several genes involved in apoptosis, cell proliferation, cell cycle and gene regulation showed expression profiles with notable differences compared with undifferentiated cells and cells differentiated in the absence of LIF (Tapia-Limonchi et al., 2011). Further experiments will be required to elucidate the impact of NO treatment on ES cells and the ways in which NO could affect the post-translational modification of transcription factors and their subsequent effects on gene regulation and differentiation (Fig. 36.3).

Fig. 36.3
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Regulation of gene expression can be affected by nitric oxide. NO is able to modify gene expression in ES cells through two recognized mechanisms. First, changes in chromatin due to DNA methylation at CpG sites and histone modification are downstream targets of NO signaling. Second, post-translational modifications induced by molecular NO, including S-nitrosylation of Cys and nitration of Tyr, produce conformational changes in the protein structure of transcription factors and transcriptional regulator complexes, which affects the expression of genes involved in differentiation pathways (Nott and Riccio, 2009; Mannick, 2006)

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Other Cell Functions Affected by Nitric Oxide in ES Cells

The preservation of the undifferentiated status of an ES cell population requires the maintenance of self-renewal, the inhibition of differentiation and the regulation of apoptosis. ESCs lack the cell cycle G1/S checkpoint or cellular senescence after DNA damage and retain a diploid karyotype. They proliferate without apparent limit and can be propagated clonally. Therefore, it is crucial for ESCs to develop stringent mechanisms to maintain genetic stability during self-renewal. The rate of spontaneous mutations in ESCs is significantly lower than that in somatic cells. The accumulation of unrepaired DNA damage in ESCs could not only promote tumorigenesis in multiple cell lineages, but also pass these mutations to the progeny, leading to genetic instability in differentiated cells (Zhao and Xu, 2010).

As mentioned previously, NO can produce diverse effects depending on its concentration and availability. Long-lasting overproduction of NO acts as a pro-apoptotic mediator by activating caspase family proteases after the release of mitochondrial cytochrome c into the cytosol, upregulating p53 expression, and finally, regulating the expression of apoptosis-associated proteins, including the Bcl protein family. However, low or physiological concentrations of NO protect cells from apoptosis that is induced by the withdrawal of the trophic factors, Fas, TNFα/ActD, and LPS. This antiapoptotic mechanism is mediated by the transcription of protective genes, including heat shock proteins, hemeoxygenase, and cyclooxygenase-2, and the direct inhibition of the apoptotic executive effectors, the caspase family proteases, by S-nitrosylation at their catalytic site (Choi et al., 2002). The potential use of NO as a selection molecule in differentiation protocols is supported by the ability of NO to influence cardiac differentiation by both inducing a switch toward a cardiac phenotype and inducing apoptosis in cells not committed to cardiac differentiation (Kanno et al., 2004). In our model of mES cell differentiation directed toward endodermic cells, exposure to high levels of the chemical NO donor also triggers apoptotic events. In fact, 30–40% of cells are Annexin V positive, which is a sign of apoptosis. Other apoptosis markers, such as PARP degradation and cleaved caspase-3, are also apparent in these cells. These signs of apoptosis are accompanied by a rise in p53 protein levels. p53 phosphorylation on Ser 15 and Ser 392 has been reported to be induced by oxidative stress and is involved in p53-dependent apoptosis. In cells that remain adherent after NO treatment, p53pSer15,pSer315,pSer392 is located in the nucleus (Mora-Castilla et al., 2010). Alternatively, low levels of NO have a protective effect against apoptosis induced by the withdrawal of LIF in mES cells, decreasing DNA fragmentation, inhibiting caspase 3 activity, and inhibiting its principal target, PARP1, by cleavage (Tejedo et al., 2010). Transcriptional analysis has demonstrated how low NO treatment can inhibit the activation of the mitochondrial apoptotic pathway via the downregulation of cytochrome C, caspases 7 and 9, and Parp1. Importantly, Bcl2 and related proteins, which have been reported to be important regulators of apoptosis and, therefore, regulators of self-renewal in ES cells, are upregulated in cells treated with low NO (Yamane et al., 2005). Finally, information generated from hypoxia and mitochondrial metabolism studies in ES cells has implications for differentiation. Von hippen Lindau (Vhl), which regulates HIF function, is downregulated during treatment with 2 μM of DETA-NO, as are many other targets of the hypoxia response. Recently, the gaseous messenger, nitric oxide (NO), has been implicated in mitochondrial biogenesis in a number of cells via stimulation of guanylate cyclase, generation of cyclic GMP, and activation of PGC1-α. This supports our results regarding the increase in gene expression of PGC1-α and its target, NRF1. Elucidation of the relationship between these factors and NO signaling may be relevant to understanding their potential use for improving NO treatment protocols for ES cells.