BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages

BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.     

Differentiation is coupled to changes in the cell cycle regulatory apparatus of human embryonic stem cells

Mouse embryonic stem cells (mESC) exhibit cell cycle properties entirely distinct from those of somatic cells. Here we investigated the cell cycle characteristics of human embryonic stem cells (hESC). HESC could be sorted into populations based on the expression level of the cell surface stem cell marker GCTM-2. Compared to mESC, a significantly higher proportion of hESC (GCTM-2⁺ Oct-4⁺ cells) resided in G₁ and retained G₁-phase-specific hypophosphorylated retinoblastoma protein (pRb). We showed that suppression of traverse through G₁ is sufficient to promote hESC differentiation. Like mESC, hESC expressed cyclin E constitutively, were negative for D-type cyclins, and did not respond to CDK-4 inhibition. By contrast, cyclin A expression was periodic in hESC and coincided with S and G₂/M phase progression. FGF-2 acted solely to sustain hESC pluripotency rather than to promote cell cycle progression or inhibit apoptosis. Differentiation increased G₁-phase content, reinstated cyclin D activity, and restored the proliferative response to FGF-2. Treatment with CDK-2 inhibitor delayed hESC in G₁ and S phase, resulting in accumulation of cells with hypophosphorylated pRb, GCTM-2, and Oct-4 and, interestingly, a second pRb⁺ GCTM-2⁺ subpopulation lacking Oct-4. We discuss evidence for a G₁-specific, pRb-dependent restriction checkpoint in hESC closely associated with the regulation of pluripotency.     

Paracrine effects of embryo-derived FGF4 and BMP4 during pig trophoblast elongation

The crosstalk between the epiblast and the trophoblast is critical in supporting the early stages of conceptus development. FGF4 and BMP4 are inductive signals that participate in the communication between the epiblast and the extraembryonic ectoderm (ExE) of the developing mouse embryo. Importantly, however, it is unknown whether a similar crosstalk operates in species that lack a discernible ExE and develop a mammotypical embryonic disc (ED). Here we investigated the crosstalk between the epiblast and the trophectoderm (TE) during pig embryo elongation. FGF4 ligand and FGFR2 were detected primarily on the plasma membrane of TE cells of peri-elongation embryos. The binding of this growth factor to its receptor triggered a signal transduction response evidenced by an increase in phosphorylated MAPK/ERK. Particular enrichment was detected in the periphery of the ED in early ovoid embryos, indicating that active FGF signalling was operating during this stage. Gene expression analysis shows that CDX2 and ELF5, two genes expressed in the mouse ExE, are only co-expressed in the Rauber's layer, but not in the pig mural TE. Interestingly, these genes were detected in the nascent mesoderm of early gastrulating embryos. Analysis of BMP4 expression by in situ hybridisation shows that this growth factor is produced by nascent mesoderm cells. A functional test in differentiating epiblast shows that CDX2 and ELF5 are activated in response to BMP4. Furthermore, the effects of BMP4 were also demonstrated in the neighbouring TE cells, as demonstrated by an increase in phosphorylated SMAD1/5/8. These results show that BMP4 produced in the extraembryonic mesoderm is directly influencing the SMAD response in the TE of elongating embryos. These results demonstrate that paracrine signals from the embryo, represented by FGF4 and BMP4, induce a response in the TE prior to the extensive elongation. The study also confirms that expression of CDX2 and ELF5 is not conserved in the mural TE, indicating that although the signals that coordinate conceptus growth are similar between rodents and pigs, the gene regulatory network of the trophoblast lineage is not conserved in these species.     

Combinatorial signals of activin/nodal and bone morphogenic protein regulate the early lineage segregation of human embryonic stem cells

Cell fate commitment of pre-implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. However, the intercellular signals that control fate determination of these cells remain obscure. Human embryonic stem cells (hESCs) provide a unique model for studying human early embryonic development. We have previously shown that Activin/Nodal signaling contributes to maintaining pluripotency of hESCs, which are derivatives of the inner cell mass. Here we further demonstrate that the inhibition of Activin/Nodal signaling results in the loss of hESC pluripotency and trophoblast differentiation, similar to BMP4-induced trophoblast differentiation from hESCs. We also show that the trophoblast induction effect of BMP4 correlates with and depends on the inhibition of Activin/Nodal signaling. However, the activation of BMP signaling is still required for trophoblast differentiation when Activin/Nodal signaling is inhibited. These data reveal that the early lineage segregation of hESCs is determined by the combinatorial signals of Activin/Nodal and BMP.     

Population based model of human embryonic stem cell (hESC) differentiation during endoderm induction

The mechanisms by which human embryonic stem cells (hESC) differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC, performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2) and bone morphogenetic protein 4 (BMP4)). The differentiating cell population is analyzed daily for cellular growth, cell death, and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells, wherefrom it evolves in time by assigning each cell a propensity to proliferate, die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated, and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm, and that during induction proliferation of the endoderm germ layer is promoted. Furthermore, our model suggests that CXCR4 is expressed in mesendoderm and endoderm, but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional, mature cells from their progenitors. While applied to initial endoderm commitment of hESC, the model is general enough to be applicable either to a system of adult stem cells or later stages of ESC differentiation.     

Localized decrease of beta-catenin contributes to the differentiation of human embryonic stem cells

Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, β-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of β-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down β-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of β-catenin contributes to the spatial pattern of differentiation in hESC colonies.     

Influence de l'oxygene sur les transferts d'electrons de la photosynthese. I. Influence de diverses concentrations d'oxygene sur quel ques reactions de Hill

Influence of oxygen on the electron transfers of photosynthesis. I. Influence of some oxygen concentrations on some Hill reactions

The influence of O₂ concentrations on the Hill reactions in the presence of p-benzoquinone, ferricyanide, NADP⁺, NADP⁺ plus ferredoxin has been studied with isolated spinach chloroplasts.

Because of the partial reoxidation of the hydroquinone, which is depending upon the O₂ concentration, it does not seem possible to localize a site of action for O₂.

With ferricyanide the influence of O₂ is weak. However, the rate of ferricyanide reduction is increased in the presence of O₂. The observed stimulation is greater for 21% O₂ than for 70% O₂. Bicarbonate stimulates the ferricyanide reduction and decreases the stimulating effect of 21% O₂.

O₂ decreases the rate of NADP⁺ reduction. Ferredoxin as well as bicarbonate stimulate the NADP⁺ reduction and reduce the O₂ inhibition.

These results seem to indicate that O₂ may enter the electron transport chain at a site situated near Photosystem I and before the ferredoxin's site.

The inhibitory effect of O₂ on the Hill reactions with p-benzoquinone and NADP⁺ is depending upon the plants' growth conditions. It is greater with plants grown under weak light.     

Consumption of O2 in the light by Chlorella pyrenoidosa and Chlamydomonas reinhardtii

A mass spectrometer has been used to measure O₂ production and consumption and net CO₂ exchange near the CO₂ compensation point in Chlorella pyrenoidosa and Chlamydomonas reinhardtii grown in air and in 2% (v/v) and 5% (v/v) CO₂ in air. Some experiments were run in presence of -hydroxy-2-pyridinemethanesulfonic acid and, for others, data are presented from parallel assays for enzymic glycolate oxidation. Although the glycolate-oxidising enzyme was present in cells grown with and without CO₂ enrichment, the activities measured were not compatible with direct measurements of O₂ consumption in the light. This and other evidence argues against glycolate being the primary substrate for light-induced O₂ consumption by these organisms.     

臭氧水对韭蛆的防治效果及对韭菜生长的影响

【目的】为科学合理地浇灌臭氧(O₃)水防治韭菜迟眼蕈蚊Bradysia odoriphaga幼虫(俗称韭蛆),明确最佳浇灌时期和使用条件。【方法】在平地覆膜环境中浇灌不同浓度(5, 10, 20和30 mg/L)O₃水,调查对韭蛆的防治效果;在平地覆膜环境中不同日光强度、3种不同灌溉环境［平地覆膜、小拱棚和小拱棚+草垫］、2种不同灌溉环境［平地覆膜、小拱棚］的不同浇灌频率等条件下分别浇灌30 mg/L O₃水,调查对韭蛆的防治效果;调查平地覆膜环境中浇灌30 mg/L O₃水对韭菜产量和根系生长的影响;分析日光强度对不同灌溉环境中土壤温度及浇灌O₃水对韭蛆的防治效果的影响。【结果】平地覆膜条件下,O₃水防治韭蛆的最佳浓度为20~30 mg/L。在平地覆膜条件下浇灌30 mg/L O₃水处理中,对韭蛆的防治效果与浇灌当天的日光强度呈正相关;当日光强度超过60 000 lx时,第1天韭蛆的死亡率高达100%;日光强度低于10 000 lx时,对韭蛆无显著的防治效果;日光强度处于中间水平时,前期对韭蛆的防治效果较差,而后期防治效果逐渐增强。土壤升温效果与日光强度呈正相关,也与灌溉环境显著相关,不同灌溉环境下土壤5 cm深处的温度为：平地覆膜>小拱棚＞小拱棚+草垫。小拱棚内浇灌O₃水对韭蛆的防治效果显著高于平地覆膜组和小拱棚+草垫组。采取连续多次浇灌O₃水有利于提高O₃水对韭蛆的防治效果,而且小拱棚内韭蛆的防治效果更显著。浇灌O₃水时,进水口离田埂末端的距离不能超过40 m。平地覆膜环境中浇灌30 mg/L O₃水后,韭菜的株高、重量和须根数量显著高于对照组(浇灌清水);韭菜植株分蘖数与对照组差异不显著。【结论】若能科学合理地浇灌O₃水,既能达到防治韭蛆的理想效果,又能促进韭菜生长。     

The methyl viologen-catalyzed mehler reaction and catalase activity in blue-green algae and Chlamydomonas reinhardi

1. The methyl viologen-catalyzed Mehler reaction was investigated in intact cells of five species of blue-green algae and Chlamydomonas reinhardi.

2. In the presence of methyl viologen, all the blue-green algae except Anabaena flos-aquae show a light-dependent O₂ consumption as well as a post-illumination O₂ evolution. The rate of O₂ consumption is stimulated by 1 mM KCN, an inhibitor of catalase, but the dark O₂ evolution becomes suppressed.

3. A. flos-aquae shows a light-dependent methyl viologen-catalyzed O₂ uptake which is not affected by 1 mM KCN. Furthermore, there is no release of O₂ in the dark following illumination.

4. With C. reinhardi, the cells do not show any net O₂ exchange during or after illumination. Addition of 1 mM KCN, however, results in an immediate O₂ uptake in the light.

5. Based on the mechanism postulated for the Mehler reaction in isolated chloroplasts, it was deduced that the differences in the kinetics of the O₂ exchange catalyzed by methyl viologen reflect differences in the endogenous catalase activity in these algae. Cells of A. flos-aquae are deficient in catalase activity whereas those of the other blue-green algae possess catalase, although at low activity. C. reinhardi, on the other hand, has high catalase activity in vivo.

6. These findings are corroborated by results obtained from O₂ electrode measurements of catalase activity in cell-free extracts of these algae.

7. The possible roles of catalase in algae and the implications of these results are also discussed.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies

The vasculature develops primarily through two processes, vasculogenesis and angiogenesis. Although much work has been published on angiogenesis, less is known of the mechanisms regulating the de novo formation of the vasculature commonly called vasculogenesis. Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health-registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium-2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P<0.05). Analysis of the expression of 45 genes by quantitative real time-polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (alpha-smooth muscle actin [alpha SMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.     

Characterization of oxygen-tolerant Chinese hamster ovary cells: II. Energy metabolism and antioxidant status

Further characteristics of an oxygen-tolerant variant of Chinese hamster ovary cells (CHO-99) capable of stable proliferation at 99% O₂/1% CO₂, an O₂ level that is lethal to the parental line (CHO-20), are described. Previous work has revealed that CHO-99 cells have 2- to 4-fold increased activities of superoxide dismutases, catalase and glutathione peroxidase, and substantially increased relative volumes of mitochondria and peroxisomes. To document possible additional mechanisms of O₂ tolerance we compared CHO-20 cells growing at 20% O₂ (normoxia) and CHO-99 cells at 99% O₂ (normobaric hyperoxia). We show the following: (1) the estimated total (oxidative and glycolytic) ATP production in CHO-99 cells was 36% decreased. ATP production through oxidative phosphorylation was 52% lower in CHO-99 cells, while the relative contribution from glycolysis was increased from 6% to 30%. The ATP content was 29% lower in CHO-99 cells, the adenylate energy charge being also significantly decreased, indicating that energy production through oxidative phosphorylation is compromised in CHO-99 cells. Cyanide-resistant respiration was 4-fold higher in CHO-99 cells, probably reflecting, at least partly, the increased peroxisomal activity in these cells. (2) The level of reduced glutathione was several fold increased in CHO-99 cells, oxidized glutathione being unaltered; (NADPH + NADP⁺) levels were elevated 2.7-fold, while the ratio of NADPH to NADP⁺ was increased almost two-fold. These changes were associated with a 50% increased metabolism of glucose through the hexose monophosphate pathway. (3) No evidence was obtained for an increased steady-state level of endogenous lipid peroxidation in CHO-99 cells, in spite of a 50% increased content of polyunsaturated fatty acids in the phospholipid fraction.     

Influence de l'oxygene sur les transferts d'electrons de la photosynthese. II. Influence de tres faibles concentrations en oxygene sur la reduction du NADP par les chloroplastes isoles

Influence of oxygen on the electron transfers of photosynthesis. II. Influence of very low oxygen concentration on the NADP⁺ reduction by isolated chloroplasts

The influence of very low O₂ concentration on the NADP⁺ reduction by isolated spinach chloroplasts has been studied.

The results show that in the presence of very low O₂ concentration (< 0.3%) NADP⁺ reduction is partially inhibited. This inhibition may be partially reversed under some conditions, especially when, in spite of the presence of an O₂ trap (glucose plus glucose oxidase (EC 1.1.3.4)) an O₂ evolution is observed.     

Mechanism of O2 generation in reduction and oxidation cycle of ubiquinones in a model of mitochondrial electron transport systems

O⁻₂ generation in mitochondrial electron transport systems, especially the NADPH-coenzyme Q₁₀ oxidoreductase system, was examined using a model system, NADPH-coenzyme Q₁-NADPH-dependent cytochrome P-450 reductase. One electron reduction of coenzyme Q₁ produces coenzyme Q₁ and O⁻₂ during enzyme-catalyzed reduction and O₂ + coenzyme Q₁ are in equilibrium with O⁻₂ + coenzyme Q₁ in the presence of enough O₂. The coenzyme Q₁ produced can be completely eliminated by superoxide dismutase, identical to bound coenzyme Q₁₀ radical produced in a succinate/fumarate couple-KCN-submitochondrial system in the presence of O₂. Superoxide dismutase promotes electron transfer from reduced enzyme to coenzyme Q₁ by the rapid dismutation of O₂⁻ generated, thereby preventing the reduction of coenzyme Q₁ by O⁻₂. The enzymatic reduction of coenzyme Q₁ to coenzyme Q₁H₂ via coenzyme Q₁ is smoothly achieved under anaerobic conditions. The rate of coenzyme Q₁H₂ autoxidation is extremely slow, i.e., second-order constant for [O₂][coenzyme Q₁H₂] = 1.5 M⁻¹ · s⁻¹ at 258 μM O₂, pH 7.5 and 25°C.     

CO2加富对UV-B辐射胁迫下亚心形扁藻光合作用和膜脂过氧化以及抗氧化酶活性的影响

在CO2浓度分别为当今CO2浓度(360 μL/L)和加富浓度(5 000 μL/L)条件下,研究了UV-B胁迫对亚心形扁藻(Platymonas subcordiformis(Wille)Hazen)的光合作用、膜脂过氧化和抗氧化酶活性的影响.实验结果表明:(1)UV-B单独作用下,亚心形扁藻的干重、光合速率、叶绿素a(Chl a)和类胡萝卜素(Car.)含量显著降低,CO2加富单独作用下,亚心形扁藻的干重和光合速率显著升高,叶绿素a和类胡萝卜素含量与对照相比没有显著变化,而UV-B与CO2共同作用则使亚心形扁藻的干重和光合速率与对照相比没有显著变化,叶绿素a和类胡萝卜素含量显著降低.(2)UV-B单独作用和CO2加富单独作用都使可溶性蛋白含量显著降低,UV-B与CO2共同作用下的可溶性蛋白含量比UV-B单独作用的要高.高CO2对藻的可溶性蛋白含量的变化在很大程度上归因于Rubisco蛋白的降低.(3)UV-B单独作用下,O-.2产生速率、H2O2含量和MDA含量显著升高,而CO2加富单独作用下,O-.2产生速率、H2O2含量和MDA含量显著降低,与UV-B单独作用相比,UV-B与CO2共同作用使O-.2产生速率、H2O2含量和MDA含量显著降低.说明CO2加富可以减少活性氧对亚心形扁藻的氧化胁迫,同时减少UV-B对亚心形扁藻的膜脂过氧化伤害.(4)UV-B单独作用下,SOD、POD、CAT、GR和GPx活性显著升高,高CO2单独作用使SOD、POD和GR活性显著降低,而CAT和GPx活性与对照相比稍有所降低,但降低不明显,而UV-B与CO2共同作用则使SOD、POD、CAT、GR和GPx活性比UV-B单独作用少得多.结果表明,高CO2对UV-B胁迫所造成的氧化胁迫具有一定的改善作用,因此CO2浓度升高可能对增强海洋微藻的抗逆能力有利.