Tumorigenesis in cells derived from induced pluripotent stem cells

Induced pluripotent stem (iPS) cells are an attractive source for potential cell-replacement therapy. However, transplantation of differentiated products harbors the risk of teratoma formation, presenting a serious health risk. Thus, we characterized Nanog-expressing (undifferentiated) cells remaining after induction of differentiation by cytological examination. To induce differentiation of iPS cells, we generated embryoid bodies (EBs) derived from iPS cells carrying a Nanog–green fluorescent protein (GFP) reporter and then injected GFP-positive and GFP-negative EBs into nude mice. GFP-positive EB transplantation resulted in the formation of immature teratoma grade 3, but no tumors were induced by GFP-negative EB. GFP-positive cells revealed significantly lower cytoplasmic area and higher nucleus/cytoplasm ratio than those of GFP-negative cells. Our results suggest that morphological analysis might be a useful method for distinguishing between tumorigenic and nontumorigenic iPS cells.     

Effect of tetrahydrocannabinol and ethidium bromide on DNA metabolism and embryogenesis in Volvox.

The effects of delta 9-tetrahydrocannabinol (THC) and ethidium bromide (EB) on the developmental life cycle and DNA metabolism of Volvox carteri have been investigated. THC, previously shown to interfere specifically with cytoplasmic DNA (cDNA) in this organism, was used at different concentrations and at different times during the life cycle. The morphological consequences observed were found to be dependent on the nature and time of treatment. This study also indicates that ethidium bromide induces degradatin of cDNA similar to that mediated by THC. However, unlike THC, it also causes the cessation of nuclear DNA synthesis. The consequences of EB treatment on morphological development are different from those observed with THC. A correlatin of these observations with the biochemical results presented suggests possible models in which the amounts and proportions of nuclear and cytoplasmic DNA play a role in the regulation of embryogenesis in this organism.     

End-binding protein 1 stimulates paclitaxel sensitivity in breast cancer by promoting its actions toward microtubule assembly and stability

Paclitaxel is a microtubule-targeting agent widely used for the treatment of many solid tumors. However, patients show variable sensitivity to this drug, and effective diagnostic tests predicting drug sensitivity remain to be investigated. Herein, we show that the expression of end-binding protein 1 (EB1), a regulator of microtubule dynamics involved in multiple cellular activities, in breast tumor tissues correlates with the pathological response of tumors to paclitaxel-based chemotherapy. In vitro cell proliferation assays reveal that EB1 stimulates paclitaxel sensitivity in breast cancer cell lines. Our data further demonstrate that EB1 increases the activity of paclitaxel to cause mitotic arrest and apoptosis in cancer cells. In addition, microtubule binding affinity analysis and polymerization/depolymerization assays show that EB1 enhances paclitaxel binding to microtubules and stimulates the ability of paclitaxel to promote microtubule assembly and stabilization. These findings thus reveal EB1 as a critical regulator of paclitaxel sensitivity and have important implications in breast cancer chemotherapy.     

应用蛋白印迹法检测鼻咽癌病人血清中抗Epstein—Barr病毒IgA/EA抗体

曾毅等建立了一系列检测EB病毒IgA/VCA和IgA/EA抗体的鼻咽癌早期诊断方法,取得了满意的结果。为了进一步提高对鼻咽癌诊断更为特异的IgA/EA抗体的检出率,我们建立了检测EB病毒IgA/EA抗体的蛋白印迹法。方法敏感特异,结果令人满意。 本法中所用的两个质粒系由本实验室与西德Pettenkofer研究所Wolf教授的实验室合作构建。pUCARG1140和pUC9MBcE3.2质粒均为表达质粒,前者携带着来源于EB病毒Bam     

Influence of cysteine deprivation on chlamydial differentiation from reproductive to infective life-cycle forms

The effects of omission of individual amino acids from growth medium on the differentiation of Chlamydia trachomatis DK-20 (serotype E) during infection of cycloheximide-treated McCoy cells are described. As judged by inclusion body staining with acridine orange, omission of cysteine from the medium severely retarded differentiation of reproductive reticulate body (RB) to infective elementary body (EB) forms. The effect appeared specific to cysteine in that omission of other amino acids had little or no effect on differentiation once RBs appeared. On restoration of cysteine, culture infectivity increased and inclusions contained organisms which, by cytochemical and morphological criteria, were differentiating to infective forms, indicating that cysteine deprivation did not irreversibly inhibit differentiation. Impairment of RB to EB differentiation in cysteine-less medium was also observed for three strains of Chlamydia psittaci and 10 other strains of C. trachomatis. It is suggested that the effect arises via the biosynthetic requirement for cysteine for provision of three cysteine-rich proteins, whose synthesis and insertion into the outer membrane have previously been shown to accompany RB to EB differentiation of C. psittaci 6BC and C. trachomatis 434 (serotype L2). Synthesis of cysteine-rich outer membrane proteins during differentiation may thus be common to all chlamydiae.     

The Physical and Morphological Characteristics of Mango Powder (<Emphasis Type="Italic">Mangifera indica</Emphasis> L. cv Tommy Atkins) Produced by Foam Mat Drying

The thermal behavior, moisture adsorption properties and structural and morphological characteristics of mango powders were evaluated. The powders were obtained by foam mat drying methodology using albumin (ALB), mixture (EB) of monoglycerides of fatty acids, sorbitan monostearate and polyoxyethylene sorbitan monostearate and a combination of the two (EB-ALB) as foaming agents. The evaluation was done by differential scanning calorimetry (DSC), X-ray diffraction (XRD) and scanning electron microscopy (SEM), and the moisture adsorption isotherms were also determined. The powder with EB had a glass transition temperature (T_g) of ?4.2 °C. The denaturation temperature of pure albumin (82.2 °C) increased in the powders with ALB to 117 °C and in those with EB-ALB to 102 °C, due to the thermal stability provided by the pulp sugars. The moisture sorption isotherm of the EB-powder showed a higher water equilibrium content than the other powders. All the powders were in the amorphous state. The morphology of the powder with EB showed corrugated particles, whereas those with ALB and EB-ALB showed particles with a less porous aspect and more compact surfaces than the powders with EB.     

During seminiferous tubule maturation testosterone and synergistic action of FSH with estradiol support germ cell survival while estradiol alone has pro-apoptotic effect

During establishment of spermatogenesis at the prepubertal age, an early germ cells apoptotic wave occurs likely aimed to remove abnormal germ cells and to maintain a proper cell number ratio between maturating germ cells and Sertoli cells. Here we assessed Sertoli and germ cell apoptosis in relation to morphological parameters of Sertoli cell maturation in neonatal rats under the influence of testosterone, estradiol and FSH given alone or in combinations. From postnatal day (PND) 5th to 15th male rats were daily injected with: 1) 2.5 mg of testosterone propionate (TP), or 2) 12.5 microg of 17beta-estradiol benzoate (EB), or 3) TP+EB, or 4) 7.5 IU of human purified FSH (hFSH), or 5) hFSH+EB or solvents (control-C). Autopsy was performed on PND 16th. Sertoli cell nuclei area and incidence of seminiferous tubule lumen formation (LF) were taken as markers of Sertoli cell maturation. Sertoli and germ cell apoptosis was assessed using TUNEL method. In comparison with C, the area of Sertoli cell nuclei was significantly reduced after EB (25.7+/-2.0 vs. 30.9+/-1.6 microm2 for C, p<0.001) and increased after hFSH+EB (33.1+/-2.3 microm2, p<0.05). Incidence of LF was completely arrested by steroid hormone treatments given separately, significantly inhibited after TP+EB (median: 0.0%, vs. 2.0% for C p<0.05) and significantly enhanced after hFSH+EB (median: 51.0%, p<0.001). hFSH alone did not influence LF. Incidence of TUNEL positive Sertoli cells significantly increased after EB (median: 2.9% vs. 0.5% for C, p<0.05) or TP+EB (median: 2.2%, p<0.01) and was not affected by other treatments. Incidence of TUNEL positive germ cells increased significantly after EB alone (median: 4.4% vs. 2.5%, for C, p<0.01 ) and was significantly decreased by hFSH+EB (median: 0.5%, p<0.01). CONCLUSIONS: 1) Administration of testosterone or estradiol to immature rats inhibits Sertoli cell maturation. 2) Estradiol stimulates Sertoli and germ cell apoptosis while testosterone has no effect. 3) Testosterone eliminates estradiol--induced germ cell apoptosis when both hormones act in concert. 4) FSH in concert with estradiol, but neither one of the hormone alone, accelerate Sertoli cell differentiation and effectively inhibit germ cell apoptosis. 5) During seminiferous tubule maturation testosterone and the synergistic action of FSH with estradiol support germ cell survival while estradiol alone has an inhibitory, pro-apoptotic effect.     

Efficacy of Vitamin D compounds to modulate estrogen receptor negative breast cancer growth and invasion

In estrogen receptor (ER) positive breast cancer cells such as MCF-7 cells, the anti-tumor effects of 1,25(OH)(2)D(3) (1,25D(3)) may be secondary to disruption of estrogen mediated survival signals. If so, then sensitivity to 1,25D(3) mediated growth arrest could be reduced in estrogen independent breast cancer cells. The aim of these studies was to determine the effects of 1,25D(3) and EB1089 on the ER negative, invasive human breast cancer cell line SUM-159PT. 1,25D(3) and EB1089 reduced SUM-159PT cell growth subsequent to elevation of p27 and p21 levels. 1,25D(3) mediated apoptosis of SUM-159PT cells was associated with an enrichment of membrane bound bax, a redistribution of cytochome c from the mitochondria to the cytosol and PARP cleavage. 1,25D(3) and EB1089 also inhibited SUM-159PT cell invasion through an 8 microM Matrigel membrane. In pre-clinical studies, EB1089 dramatically reduced the growth of SUM-159PT xenografts in nude mice. The decreased size of tumors from EB1089 treated mice was associated with decreased proliferation and increased DNA fragmentation. Our data support the concept that Vitamin D(3) compounds trigger apoptosis by mechanisms independent of estrogen signaling. These studies indicate that Vitamin D(3) based therapeutics may be beneficial, alone or in conjunction with other agents, for the treatment of estrogen independent breast cancer.     

Local intracerebral implants of estrogen masculinize some aspects of the zebra finch song system

The song system of zebra finches is sexually dimorphic: the volumes of the song control nuclei and the neurons within these nuclei are larger in males. The song system of hatching female zebra finches is masculinized by systemic treatment with estrogen. We investigated the locus of this estrogen action by using microimplants of estradiol benzoate (EB). We implanted female zebra finch nestlings 10–13 days old with Silastic pellets containing approximately 2 μg EB at one of several sites: near the higher vocal center (HVC), in the brain distant from HVC, or in the periphery either under the skin of the breast or in the peritoneal cavity. Controls were either unimplanted or implanted near HVC with Silastic pellets without hormone. The brains were fixed by perfusion at 60 days, and the volumes of the song control regions as well as the sizes of individual neurons were measured. Neurons in HVC were lerger (more masculine) in the HVC-implanted group than in other groups, which did not differ among themselves. The size of neurons in the robust nucleus of the archistriatum (RA) and the lateral magnocellular nucleus ofthe neostriatum (lMAN) were inversely correlated with the distance of the EB pellet to HVC; neurons in RA and lMAN were larger when the EB pellets were closer to HVC. This result suggests that implants near HVC were at or near a site of estrogen action. To our knowledge, this is the first demonstration that localized brain implants of estrogen cause morphological masculinization in any species. 1994 John Wiley & Sons, Inc.     

Observation of the biological characterizations of nasopharyngeal epithelial cells by EB virus infection in early phase of immortalization]

The multi-stage cell model of the nasopharyngeal carcinoma development in vitro by Epstein-Barr virus transformation is beneficial for the elucidation of the mechanism of nasopharyngeal cancer. To observe the biological changes of primary human nasopharyngeal epithelial cells in early phase of immortalization, in this study, we have detected the morphological changes and the expression profile of senescence-associated beta-galactosidase (SA-beta-Gal) in primary culture. In addition, the expression of EB virus latent membrane protein 1 (LMP1) and the growth curve of primary cells were also detected. Our results showed a low percentage of cells infected with EB virus expressing SA-beta-Gal activity at the late primary culture. In morphology, the cells also formed multilayer foci, and the cell population doubling time was showed. These results demonstrated that the nasopharyngeal epithelial cells by EB virus infection have passed through the senescence and entered the early phase of immortalization. These cells have some of the transformed characteristics. Our results provided the data for further study on the mechanism of immortalization and the establishment of human nasopharyngeal epithelial cell line.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Modification of in vitro mouse embryogenesis by X-rays and fluorochromes

In view of planned studies using single particle irradiation at the Institute for Medical Radiobiology (IMR), confocal microscopy will become an important tool to visualise subtle changes in embryo morphology. Therefore, the influence of X-rays and that of three different fluorochromes on the in vitro development of murine 2-cell stage embryos was investigated. Embryos of B₆C₃F₁ mice were cultured at 32 h post-conception (pc) and treated with X-rays, acridine orange (AO), ethidium bromide (EB) or propidium iodide (PI), respectively, in various doses (0.0–3.0 Gy) or concentrations (AO: 0.05–2.00 µg/ml; EB and PI: 0.50–10.00 µg/ml). Additional experiments using combinations of AO and irradiation were performed. The embryos were cultivated for a total of 7 days and checked for their vital status by morphological endpoints such as the percentage of embryos that reached the blastocyst stage and the hatching rate. After treatment of the 2-cell embryos with AO, EB and PI, the dose-effect curves with the endpoint ‘hatching rate’ showed a 23-fold reduction of the ED₅₀ for AO (0.23 µg/ ml) compared with EB (5.30 µg/ml). Despite the higher toxicity, AO had much better staining qualities in subtoxic concentrations than EB. PI showed no toxicity in these experiments and was used as an inverse control for embryo vitality. No synergistic modification of the radiogenic effects could be seen in combination experiments (AO+X-rays).     

A Morphometric Screen Identifies Specific Roles for Microtubule-Regulating Genes in Neuronal Development of P19 Stem Cells

The first morphological change after neuronal differentiation is the microtubule-dependent initiation of thin cell protrusions called neurites. Here we performed a siRNA-based morphometric screen in P19 stem cells to evaluate the role of 408 microtubule-regulating genes during this early neuromorphogenesis step. This screen uncovered several novel regulatory factors, including specific complex subunits of the microtubule motor dynein involved in neurite initiation and a novel role for the microtubule end-binding protein EB2 in attenuation of neurite outgrowth. Epistasis analysis suggests that competition between EB1 and EB2 regulates neurite length, which links its expression to neurite outgrowth. We propose a model that explains how microtubule regulators can mediate cellular morphogenesis during the early steps of neuronal development by controlling microtubule stabilization and organizing dynein-generated forces.     

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.     

体外诱导鸡胚胎生殖细胞分化为神经干细胞

本研究探讨体外诱导鸡胚胎生殖细胞(EGCs)分化为神经干细胞(NSCs)的可能性.EGCs经类胚体(EB)阶段,以维生素A酸(RA)等进行诱导,在NSCs选择性培养基中筛培养扩增7 d,观察形态变化;采用RT-PCR法检测nestin基因表达及免疫细胞化学法检测nestin等NSCs特异性标志物,并对其扩增及分化能力进行观察.结果显示:EGCs经初级诱导,NSCs选择性培养基筛选培养7 d后,形成大量神经球样结构,可扩增传代;绝大部分神经球样结构呈nestin抗原阳性,表达nestin基因,且可分化为神经上皮样及少突胶质细胞.研究结果表明:RA等诱导的EGCs,经选择性培养基筛选培养可获得NSCs,有望为眼部神经变性疾病的治疗提供新的技术参考.     

Growth and differentiation of embryoid bodies derived from human embryonic stem cells: effect of glucose and basic fibroblast growth factor

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.     

Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 in Chlamydomonas flagella

The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein–tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia.