Proliferation Of Mouse Embryonic Stem Cells Research Articles

Leucine and Arginine Availability Modulate Mouse Embryonic Stem Cell Proliferation and Metabolism.

Amino acids are crucial nutrients involved in several cellular and physiological processes, including fertilization and early embryo development. In particular, Leucine and Arginine have been shown to stimulate implantation, as lack of both in a blastocyst culture system is able to induce a dormant state in embryos. The aim of this work was to evaluate the effects of Leucine and Arginine withdrawal on pluripotent mouse embryonic stem cell status, notably, their growth, self-renewal, as well as glycolytic and oxidative metabolism. Our results show that the absence of both Leucine and Arginine does not affect mouse embryonic stem cell pluripotency, while reducing cell proliferation through cell-cycle arrest. Importantly, these effects are not related to Leukemia Inhibitory Factor (LIF) and are reversible when both amino acids are reconstituted in the culture media. Moreover, a lack of these amino acids is related to a reduction in glycolytic and oxidative metabolism and decreased protein translation in mouse embryonic stem cells (mESCs), while maintaining their pluripotent status.

The study of exposure times and dose-escalation of tick saliva on mouse embryonic stem cell proliferation

The saliva of ticks contains numerous bioactive molecules with anti-hemostatic and immunomodulatory properties. Due to their abilities of self-renewal and pluripotency, stem cells hold considerable promise in the regenerative medicine and biomedical fields. The present study examines the viability and proliferation of mouse embryonic stem cells (mESCs) following the addition of tick salivary gland extracts obtained from three tick species (Dermacentor marginatus, Rhipicephalus bursa and Hyalomma marginatum) to the mESC medium in different quantities (0.2, 2, 20, 40, 80, and 160 µg/ml). On days 2, 5 and 7 of the treatment, the vitality and proliferation of the cells were determined with CellTiter-Glo and morphological tests. The results showed that the culture supplemented with D. marginatus salivary gland extract at a concentration of 80 µg/ml positively affected the proliferation rate of mESC. It was further shown that all concentrations of the salivary gland extracts derived from H. marginatum and R. bursa had a negative effect on the proliferation rate of mESC when compared to the controls.

The effect of BMP4 on mouse embryonic stem cell proliferation and differentiation into primordial germ cells

The effect of BMP4 on mouse embryonic stem cell proliferation and differentiation into primordial germ cells

Interaction Between Sympk and Oct4 Promotes Mouse Embryonic Stem Cell Proliferation.

The scaffold protein Symplekin (Sympk) is involved in cytoplasmic RNA polyadenylation, transcriptional modulation, and the regulation of epithelial differentiation and proliferation via tight junctions. It is highly expressed in embryonic stem cells (ESCs), in which its role remains unknown. In this study, we found Sympk overexpression in mouse ESCs significantly increased colony formation, and Sympk deletion via CRISPR/Cas9 decreased colony formation. Sympk promoted ESC growth and its overexpression sustained ESC pluripotency, as assessed by teratoma and chimeric mouse formation. Genomic stability was preserved in these cells after long-term passage. The domain of unknown function 3453 (DUF3453) in Sympk was required for its interaction with the key pluripotent factor Oct4, and its depletion led to impaired colony formation. Sympk activated proliferation-related genes and suppressed differentiation-related genes. Our results indicate that Sympk interacts with Oct4 to promote self-renewal and pluripotency in ESCs and preserves genome integrity; accordingly, it has potential value for stem cell therapies. Stem Cells 2019;37:743-753.

Bidirectional Regulation of Mouse Embryonic Stem Cell Proliferation by Nicotine Is Mediated Through Wnt Signaling Pathway.

Introduction:Nicotine is a key biologically active compound of cigarettes. Although nicotine is a risk factor for various health issues, it may also be beneficial when treated at moderate concentrations. Nicotine has been shown to bidirectionally regulate stem cell proliferation and differentiation depending on the doses applied. It is not clear whether or how nicotine regulates mouse embryonic stem cell (mESC) survival and proliferation.Methods:Mouse embryonic stem cells were cultured in the presence of 0.01, 0.1, 1, or 10 μM nicotine. The effects of nicotine on cell survival and proliferation were examined. The signaling pathway that mediated these effects was analyzed.Results:Cell viability was not affected by nicotine at all 4 concentrations examined. The proliferation of mESCs was promoted by 0.01 and 0.1 μM nicotine and suppressed by 1 and 10 μM. This dose-dependent regulation was mediated through the Wnt/β-catenin pathway. Modulation of Wnt/β-catenin activity either worsens or reverses the effects of nicotine.Conclusions:We have identified a bidirectional function of nicotine on mESC proliferation through regulation of the Wnt/β-catenin pathway and this is associated with different doses. This study suggests that concentration of nicotine is a crucial aspect for consideration when designing research or therapeutic strategies.

An extended model for culture-dependent heterogenous gene expression and proliferation dynamics in mouse embryonic stem cells

During development, pluripotency is a transient state describing a cell’s ability to give rise to all three germ layers and germline. Recent studies have shown that, in vitro, pluripotency is highly dynamic: exogenous stimuli provided to cultures of mouse embryonic stem cells, isolated from pre-implantation blastocysts, significantly affect the spectrum of pluripotency. 2i/LIF, a recently defined serum-free medium, forces mouse embryonic stem cells into a ground-state of pluripotency, while serum/LIF cultures promote the co-existence of ground-like and primed-like mouse embryonic stem cell subpopulations. The latter heterogeneity correlates with temporal fluctuations of pluripotency markers, including the master regulator Nanog, in single cells. We propose a mathematical model of Nanog dynamics in both media, accounting for recent experimental data showing the persistence of a small Nanog Low subpopulation in ground-state pluripotency mouse embryonic stem cell cultures. The model integrates into the core pluripotency Gene Regulatory Network both inhibitors present in 2i/LIF (PD and Chiron), and feedback interactions with genes found to be differentially expressed in the two media. Our simulations and bifurcation analysis show that, in ground-state cultures, Nanog dynamics result from the combination of reduced noise in gene expression and the shift of the system towards a monostable, but still excitable, regulation. Experimental data and agent-based modelling simulations indicate that mouse embryonic stem cell proliferation dynamics vary in the two media, and cannot be reproduced by accounting only for Nanog-dependent cell-cycle regulation. We further demonstrate that both PD and Chiron play a key role in regulating heterogeneity in transcription factor expression and, ultimately, mouse embryonic stem cell fate decision.

An Efficient Transfection Method for Differentiation and Cell Proliferation of Mouse Embryonic Stem Cells.

Embryonic stem (ES) cells are an important source of stem cells in tissue engineering and regenerative medicine because of their high self-renewal capacities and differentiation potentials. However, the detailed molecular mechanisms controlling the differentiation and renewal programs in ES cells remained unclear. One of the difficulties in understanding these mechanisms substantially results from the low efficacies of gene manipulation by delivering exogenous gene expression or knockdown of endogenous gene expression with small interfering RNA (siRNA) in ES cells. Here we describe an optimized protocol for efficiently transfecting mouse ES cells by Effectene, a liposome-based method. The high transfection efficiency in mouse ES cells is demonstrated in this chapter by (1) achieving a percentage of enhanced green fluorescence protein (EGFP) expression in >98% embryoid bodies after introducing plasmids encoding the protein; (2) decreased SOX-2 and Oct-3/4 expression and subsequent morphological evidences of cell differentiation after introducing siRNA expression for suppressing SOX-2 and Oct-3/4, which are known to be essential for maintenance of stem cell properties in mouse ES cells; and (3) overexpression or attenuated expression of 14-3-3σ to regulate cell proliferation of mouse ES cells.

Critical role of Rgs19 in mouse embryonic stem cell proliferation and differentiation

Mouse embryonic stem cells (ESCs) are self-renewing, pluripotent, and have the ability to differentiate into the three germ layers required to form all embryonic tissues. These properties are maintained by both intrinsic and extrinsic factors. Many studies have contributed to the understanding of the molecular signal transduction required for pluripotency and controlled differentiation. Such an understanding is important in the potential application of stem cells to cell therapy for disease, and thus there is an interest in understanding the cell cycle regulation, pluripotency, and differentiation of ESCs. The regulator of G protein signaling (RGS) family consists of over 20 members. Rgs19, one such protein, specifically interacts with Gαi to enhance its GTPase activity. Growth factor receptors use Gi proteins for signal transduction, and Rgs19 may thus be involved in the regulation of cell proliferation. In a previous gain-of-function study, Rgs19 overexpression was found to enhance proliferation in various cell types. Our data demonstrate a role for Rgs19 in the regulation of ESC differentiation. Based on the presence of Rgs19 in ESCs, the morphological and molecular properties of wild-type and Rgs19 +/− ESCs during LIF withdrawal, in vitro differentiation, and teratoma formation were compared. Our findings provide insight for the first time into the mechanisms involved in Rgs19 regulation of mouse ESC proliferation and differentiation.

Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H2O2), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells.

MicroRNA-221 is required for proliferation of mouse embryonic stem cells via P57 targeting.

Factors responsible for the rapid proliferative properties of embryonic stem (ES) cells are largely unknown. MicroRNA-221/222 (miR-221/222) regulate proliferation in many somatic cells, however, their roles in proliferation of ES cells are unclear. In this study, E14 mouse ES cells proliferation was determined by total cell counting, Cell Counting Kit (CCK-8), size of colonies and cell cycle analysis, while apoptosis and necrosis using Annexin V and propidium iodide staining. miR-221 inhibitor decreased proliferation of ES cells without inducing apoptosis and necrosis. miR-221 mimic, miR-222 mimic and miR-222 inhibitor did not affect ES cells proliferation. The expression level of miR-221 remained unchanged upon embryoid body (EB) formation. ES cells with miR-221 inhibition maintained an undifferentiated state, as indicated by unchanged alkaline phosphatase enzyme activity and Sox2, Nanong, and Oct4 expressions. P57 was post-transcriptionally regulated by miR-221 in ES cells. P57 knockdown completely abolished the inhibition effects of ES cells proliferation observed in miR-221 reduction, further indicating that miR-221 inhibition is likely to mediate its antiproliferative effects via P57 expression. To exclude that the function of miR-221 in ES cells is E14 specific, the effects of miR-221 mimic and inhibitor in size of colonies and cell cycle of R1 mouse ES cells were also determined and similar effects in inhibiting proliferation were achieved with miR-221 inhibition. Therefore, miR-221 is required for mouse ES cells proliferation via P57 targeting. This study indicates that miR-221 is among the regulators that control ES cells proliferation and might be used to influence the fate of ES cells.

Modulation of mouse embryonic stem cell proliferation and neural differentiation by the P2X7 receptor.

BackgroundNovel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.Principal FindingsP2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.ConclusionsIn embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.

Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells

Embryonic and induced pluripotent stem cells have the ability to differentiate into any somatic cell type, and thus have potential to treat a number of diseases that are currently incurable. Application of these cells for clinical or industrial uses would require an increase in production to yield adequate numbers of viable cells. However, the relatively high costs of cytokines and growth factors required for maintenance of stem cells in the undifferentiated state have the potential to limit translational research. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a key regulator in the maintenance of naïve states for both human and mouse stem cells. In this study, we describe a new recombinant human LIF (rhLIF) using a plant-based (rice) expression system. We found that rice-derived rhLIF possessed the same specific activity as commercial Escherichia coli-derived LIF and was capable of supporting mouse embryonic stem cell proliferation in the undifferentiated state as evidenced from pluripotency marker level analysis. Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market.

14-3-3σ Regulates β-Catenin-Mediated Mouse Embryonic Stem Cell Proliferation by Sequestering GSK-3β

BackgroundPluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear.Methodology and Principal FindingsIn this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding.SignificanceOur findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.

Involvement of caveolin‐1 in fibronectin‐induced mouse embryonic stem cell proliferation: Role of FAK, RhoA, PI3K/Akt, and ERK 1/2 pathways

Fibronectin (FN) is the foremost proliferation-associated extracellular matrix component promoting cell adhesion, migration, and survival. We examined the effect of FN on cell proliferation and the related signaling pathways in mouse embryonic stem (ES) cells. FN increased integrin β1, Src, focal adhesion kinase (FAK), and caveolin-1 phosphorylation levels in a time-dependent manner. Phosphorylation of Src, FAK, and caveolin-1 was attenuated by integrin β1 neutralizing antibody. Integrin β1, Src, and FAK coimmunoprecipitated with caveolin-1 in the presence of FN. In addition, FN increased RhoA and Rho kinase activation, which were completely blocked by PP2, FAK small interfering RNA (siRNA), caveolin-1 siRNA, or the caveolar disruptor methyl-β-cyclodextrin (MβCD). FN also increased phosphorylation of Akt and ERK 1/2, which were significantly blocked by either FAK siRNA, caveolin-1 siRNA, MβCD, GGTI-286 (RhoA inhibitor), or Y-27632 (Rho kinase inhibitor). FN-induced increase of protooncogenes (c-fos, c-myc, and c-Jun) and cell-cycle regulatory proteins (cyclin D1/CDK4 and cyclin E/CDK2) expression levels were attenuated by FAK siRNA or caveolin-1 siRNA. Furthermore, inhibition of each pathway such as integrin β1, Src, FAK, caveolin-1, RhoA, Akt, and ERK 1/2 blocked FN-induced [(3)H]-thymidine incorporation. We conclude that FN stimulates mouse ES cell proliferation via RhoA-PI3K/Akt-ERK 1/2 pathway through caveolin-1 phosphorylation.

Effects of Oxidative Stress on Mouse Embryonic Stem Cell Proliferation, Apoptosis, Senescence, and Self-Renewal

Oxidative stress, associated with either normal metabolism or disease conditions, affects many cellular activities. Most of our knowledge in this field is derived from fully differentiated cells. Embryonic stem cells (ESCs) have attracted enormous attention for their potential applications in cell therapy, but little is known about how the unique properties of ESCs are affected by oxidative stress. We have investigated the effects of oxidative stress induced by H(2)O(2) on several cellular activities of mouse ESCs. Like differentiated cells, ESCs are sensitive to H(2)O(2)-induced apoptosis when continuously exposed to H(2)O(2) at the concentrations above 150 microM. However, unlike differentiated cells, ESCs are resistant to oxidative stress induced senescence. This is demonstrated by the results that when subjected to a short-term sublethal concentration and duration of H(2)O(2) treatment, fibroblasts enter the senescent state with enlarged flattened cell morphology concurrent with increased expression of senescence marker p21. On the contrary, ESCs neither show any sign of senescence nor express p21. Instead, ESCs enter a transient cell cycle arrest state, but they have remarkable recovery capacity to resume the normal cell proliferation rate without losing the ability of self-renewal and pluripotency. Our results further revealed that H(2)O(2) inhibits cell adhesion and the expression of cyclin D1, which are early events proceeding apoptosis and cell cycle arrest. In conclusion, our data suggest that ESCs are sensitive to H(2)O(2) toxicity, but may have unique mechanisms that prevent H(2)O(2)-induced senescence and protect self-renewal capacity.

Effect of leukotriene D4 on mouse embryonic stem cell migration and proliferation: Involvement of PI3K/Akt as well as GSK‐3β/β‐catenin signaling pathways

The actual leukotriene D(4) (LTD(4)) signaling pathways that regulate cell proliferation have not been elucidated thoroughly although fatty acid and its metabolites play a key role in regulations of embryonic functions. Thus, this study investigated the response of mouse embryonic stem (ES) cells exposed to LTD(4) and elucidated the signaling pathways as well. LTD(4) increased DNA synthesis in concentration-dependent (≥10(-7) M) and time-dependent (≥12 h) manners, as determined by [(3)H] thymidine incorporation and increased cell number. LTD(4) induced the phosphorylation of signal transducer and activator of transcription-3 (STAT3) and the increase of intracellular Ca(2+) levels via cysteinyl leukotriene (CysLT) 1 and 2 receptors. LTD(4) increased Akt activation and calcineurin expression, which were blocked by STAT3 inhibitor and calcium chelators. LTD(4)-induced glycogen synthase kinase (GSK)-3β phosphorylation was decreased by LY294002, Akt inhibitor, and cyclosporine A. LTD(4) inhibited the phosphorylation of β-catenin. In addition, LTD(4)-stimulated migration through increased activation of focal adhesion kinase (FAK) and paxillin which were blocked by Akt inhibitor and cyclosporine A. LTD(4)-induced increases in protooncogene and cell cycle regulatory proteins were blocked by cyclosporine A, FAK siRNA, and β-catenin siRNA. In conclusion, LTD(4)-stimulated mouse ES cell proliferation and migration via STAT3, phosphoinositide 3-kinases (PI3K)/Akt, Ca(2+)-calcineurin, and GSK-3β/β-catenin pathway.

Fibronectin synthesis by high glucose level mediated proliferation of mouse embryonic stem cells: Involvement of ANG II and TGF‐β1

The role of individual supplements necessary for the long-term self-renewal of embryonic stem (ES) cells is poorly characterized in feeder/serum-free culture systems. This study sought to characterize the relationship between the effects of glucose on ES cell proliferation and fibronectin (FN) synthesis, and to assess the mechanisms responsible for these cellular effects of glucose. Treatment of the two ES cells (ES-E14TG2a and ES-R1) with 25 mM glucose (high glucose) increased the expression levels of FN mRNA and protein. In addition, high glucose and ANG II synergistically increased FN expression level, which coincident with data showing that high glucose increased the mRNA expression of angiotensin II (ANG II) type 1 receptor (AT(1)R), angiotensinogen, and FN, but not ANG II type 2 receptor. High glucose also increased the intracellular calcium (Ca(2+)) concentration and pan-protein kinase C (PKC) phosphorylation. Inhibition of the Ca(2+)/PKC pathway blocked high glucose-induced FN expression. High glucose or ANG II also synergistically increased transforming growth factor-beta1 (TGF-beta(1)) expression, while pretreatment with losartan abolished the high glucose-induced increase in TGF-beta(1) production. Moreover, TGF-beta(1)-specific small interfering RNA inhibited high glucose-induced FN expression and c-Jun N-terminal kinase (JNK) activation. The JNK inhibitor SP600125 blocked high glucose-induced FN expression and inhibited cell cycle regulatory protein expression induced by high glucose or TGF-beta(1). In this study, inhibition of AT(1)R, Ca(2+)/PKC, TGF-beta(1), JNK, FN receptor blocked the high glucose-induced DNA synthesis, increased the cell population in S phase, and the number of cells. It is concluded that high glucose increases FN synthesis through the ANG II or TGF-beta1 pathways, which in part mediates proliferation of mouse ES cells.

PPARγ regulates LIF-induced growth and self-renewal of mouse ES cells through Tyk2-Stat3 pathway

Embryonic stem (ES) cells are genetically normal, pluripotent cells, capable of self-renewal and multi-lineage differentiation. Leukemia inhibitory factor (LIF) is a growth factor that can maintain the pluripotency of mouse ES cells in culture. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor transcription factors that regulate growth and differentiation of many cell types. We have shown earlier that 15-Deoxy-∆12,14-Prostaglandin J2 (15d-PGJ2), a natural ligand for PPARγ, inhibits LIF-induced proliferation of mouse ES cells in culture. In this study we demonstrate that the PPARγ antagonist Bisphenol A diglycidyl ether (BADGE) and 2-Chloro-5-nitro-N-(4-pyridyl)benzamide (T0070907) reverse the inhibition of ES cell proliferation by PPARγ agonists. Stable transfection of ES cells with a dominant negative PPARγ1 mutant also reduced the inhibition of proliferation by PPARγ agonists. While 15d-PGJ2 and ciglitazone-induced growth-arrest in ES cells by blocking LIF signaling, PPARγ antagonists and dominant negative PPARγ1 mutant reversed proliferation by restoring LIF-induced Tyk2-Stat3 signaling. These results suggest that PPARγ regulates LIF-induced growth and self-renewal of mouse ES cells through Tyk2-Stat3 pathway.

Arachidonic acid potentiates hypoxia-induced VEGF expression in mouse embryonic stem cells: involvement of Notch, Wnt, and HIF-1α

Recent investigations suggest that hypoxia increases the release of fatty acids, which participate in the regulation of cytokine synthesis and cell growth. Therefore, in this study, we examined the effect of arachidonic acid (AA) on hypoxia-induced vascular endothelial growth factor (VEGF) expression and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia increased the level of [(3)H]AA release and VEGF expression. AA treatment concurrent with hypoxia further increased the PGE(2) production and VEGF expression level, which was inhibited by the suppression of cPLA(2) and cyclooxygenase 2 (COX-2) pathways. Hypoxia increased the level of Notch-1 and Wnt-1/beta-catenin expression, which was blocked by the inhibition of COX-2, and inhibition of Notch-1 by gamma-secretase inhibitor blocked Wnt-1 activation. Moreover, the hypoxia-induced increase of hypoxia-inducible factor 1alpha (HIF-1alpha) expression induced Notch-1 activation and was regulated by Wnt-1 activation. The expression of each signaling molecule induced an increase in VEGF expression that was greater in hypoxia with AA than in hypoxia alone. The inhibition of VEGF expression using VEGF-targeted small interfering RNA decreased the hypoxia-induced increase in cell cycle regulatory protein expression, DNA synthesis, and cell number, suggesting that hypoxia-induced VEGF expression stimulates proliferation of mouse ES cells. In conclusion, AA potentiates hypoxia-induced VEGF expression in mouse ES cells through the Notch-1, Wnt-1, and HIF-1alpha pathways.

Oxidative stress on mouse embryonic stem cell proliferation, viability, pluripotency and senescence

Normal cellular activities require a balanced redox environment. Disturbances in this environment may result in oxidative stress, which affects many cellular activities and is considered to be a prominent cause of aging and age‐related diseases. Most of our knowledge in this field is from fully differentiated cells. Embryonic stem cells (ESCs) have attracted enormous attention for their potential applications in cell therapy due to their ability to differentiate to various types of cells and their unlimited capacity for self‐renewal. ESCs are constantly exposed to potential damage by oxidative stress associated with the long‐term in vitro differentiation process and maintenance, but little is known about how they respond to oxidative stress. In this study, we investigated the effects of oxidative stress induced by H2O2 on mouse ESCs and ESC‐differentiated cells. Our results indicated that H2O2 at the concentrations from 10 nM ‐200 µM can inhibit cell proliferation and cause apoptosis depending on the concentration and the duration of the treatment. However, the pluripotency of the ESCs were not significantly affected. Our data suggested ESCs were resistant to the oxidative stress that induced senescence of fibroblasts, however, ESCs‐differentiated cells became susceptible. Our results suggested that ESCs may have superior capacity against oxidative stress than differentiated cells.