INFLUENCE OF LOW-FREQUENCY ELECTRIC FIELDS ON PROLIFERATION AND IL-8 RELEASE OF HL-60 CELLS

The influence of capacitively coupled extremely low-frequency (ELF) electric fields on proliferation and on interleukin (IL)-8 release of exponentially growing HL-60 cells was examined. The cell suspensions were treated with the field component of interferential current (IFC) using different exposure protocols. Modulation frequencies of 10 and 100 Hz were applied with field strengths between 0.075 and 11.54 V_pp/cm for 48 hr using a 5-min exposure time at every 3 hr. At a field strength of 1 V_pp/cm, the influence of the time between two exposure sessions was examined for different modulation frequencies. All exposure protocols applied have no effect on cell proliferation (p>0.05), but statistical significant reduction (p<0.05) of the IL-8 release at selected modulation frequencies and interval times could be observed.     

Response of cytosolic calcium,cyclic AMP,and cyclic GMP in Dimethylsulfoxide-differentiated HL-60 cells to modulated low frequency electric currents

The action of interferential current (IFC), an amplitude-modulated 4000 kHz current used in therapeutic applications, upon intracellular calcium, adenosine 3′:5′-cyclic monophosphate (cAMP), and guanosine 3′:5′-cyclic monophosphate (cGMP) was investigated. Human promyelocytes (HL-60) were differentiated to granulocytes by dimethylsulfoxide (DMSO) treatment and exposed for 5 min at 25, 250, and 2500 μA/cm² current density. No significant changes in cytosolic free calcium were detected as a function of modulation frequency of the IFC. However, intracellular cAMP reacted in a complex way to modulation frequency, resulting in stimulations and depressions within the range of frequencies studied (0–125 Hz). The “windows” of modulation frequency, where statistically significant increases or decreases in cAMP were noted, coincided with those published earlier for mouse fibroblasts. Cellular cGMP content was always lowered by IFC treatment. Furthermore, no significant influence of IFC current density upon the three second messengers was noted. These results, which also include data relating to treatment with sinusoidal 50 Hz current, contribute to a more detailed understanding of the primary biophysical mechanisms of signal transduction by time-varying electric fields. Bioelectromagnetics 19:452–458, 1998. © 1998 Wiley-Liss, Inc.     

Aza-induced cardiomyocyte differentiation of P19 EC-cells by epigenetic co-regulation and ERK signaling

Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI⁺ cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and α-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and α-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling.     

INHIBITION OF EHRLICH TUMOR GROWTH IN MICE BY ELECTRIC INTERFERENCE THERAPY (IN VIVO STUDIES)

A study of solid tumor growth retardation by employing extremely low frequency (ELF) electric fields has been carried out. ELF electric fields were generated in tumor tissue in mice by the interference of two high frequency sinusoidal waves with the beat frequency centered at the tumor core. The results indicated a pronounced decrease in tumor growth rate in animals exposed to a 5-Hz interferential frequency for 1 hr daily. The 1 hr/day treatment produced a greater retardation effect than the 1 hr/week treatment. This indicates that treatment duration at the applied field frequency appears to play an important role in tumor growth delay. The dielectric properties of the tumor cells showed higher permittivity and conductivity values than homologous normal tissue. The permittivity of tumor cells treated daily with 5 Hz reaches nearly the same value as control tissue. Moreover, histological studies show that tumor tissues treated daily with the same frequency undergo partial regression and shrinkage of the aggregates of neoplastic cells leaving very little of them. We conclude that this new interferential technique is promising for tumor treatment in which a resonating electric field affects cell-to-cell communication.     

Response of cyclic AMP by DMSO differentiated HL-60 cells exposed to electric interferential current after prestimulation

The action of interferential current (IFC) upon intracellular content of cyclic adenosine monophosphate (cAMP) after prestimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) has been investigated. Human promyelocytes (HL-60) differentiated to granulocytes by dimethylsulphoxide (DMSO) have been treated with different concentrations of fMLP. This enhances their cAMP content. The half maximal effective concentration (EC50) was about 4 nM. Exposure to IFC with modulation frequencies of 35 and 125 Hz (5 min, 250 microA/cm2) after prestimulation with various concentrations of fMLP had no enhancing effect at low or high concentrations of fMLP. In contrast, at medium concentrations in the range of about 100 pM fMLP, a significant enhancement of cAMP could be observed. This synergistic effect of fMLP and IFC has been examined in detail by varying the modulation frequency, current density, and exposure time. Enhancement of cAMP content could be observed at certain modulation frequencies and exposure times suggesting a window effect, whereas for the current density in the range measured (8.5 microA/cm2-2.5 mA/cm2) a constant enhancement could be observed. The synergistic effect of fMLP and IFC could only be observed in the treatment sequence of fMLP followed by IFC; an inverse sequence had no effect on the cAMP content. .     

Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells

The effects of electromagnetic fields (EMFs) on the differentiation of cardiomyocytes in embryoid bodies derived from pluripotent embryonic stem (ES) cells were investigated. A single direct current (DC) field pulse was applied to 4-day-old embryoid bodies. The electrical field induced a hyperpolarization of the anode-facing side of embryoid bodies and a depolarization at the cathode-facing side. Significant effects of a single electrical field pulse applied for 90 s on cardiomyocyte differentiation were achieved with field strengths of 250 and 500 V/m, which increased both the number of embryoid bodies differentiating beating foci of cardiomyocytes and the size of the beating foci. The 500-V/m electrical field increased intracellular reactive oxygen species (ROS), but not [Ca(2+)](i) and activated nuclear factor kappa B (NF-kappaB). A comparable increase in the number of beating embryoid bodies was achieved by an incubation for 1 h with H(2)O(2) (1-10 nM), indicating that the electrical field effect was transduced via the intracellular generation of ROS. Because the radical scavengers dehydroascorbate and pyrrolidinedithiocarbamate (APDC) and the NF-kappaB antagonist N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibited cardiac differentiation, we assume that ROS and NF-kappaB may play a role in early cardiac development.     

Differential expression of a novel murine non-receptor protein tyrosine phosphatase during differentiation of P19 embryonal carcinoma cells.

Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.     

猪羊水干细胞特异向跳动心肌细胞诱导分化

为了筛选并建立一种由猪羊水干细胞向心肌细胞分化的有效方法,以猪羊水干细胞为研究对象,以5-氮胞苷 (5-aza) 和维生素C (Vc) 为诱导剂,对猪羊水干细胞形成的类胚体 (EBs) 进行诱导分化。应用免疫荧光、RT-PCR、透射电镜技术检测跳动细胞团中心肌特异性标记的表达情况。结果显示,在猪羊水干细胞形成的类胚体中加入心肌细胞诱导剂,10 d后即见到节律性跳动的细胞团,t检验发现0.1 mmol/L Vc加5 μmol/L 5-aza联合诱导组的诱导效率最高,达33％。免疫荧光结果显示跳动心肌细胞团表达细胞骨架蛋白α-actin和肌钙蛋白Tnni3。RT-PCR检测跳动心肌细胞团,发现心肌细胞特异性标记分子TbX5、Gata4、α-MHC、Tnni3均呈阳性表达。借助透射电镜观察诱导后的跳动样细胞团,能清晰可见其中的肌丝、糖原粒、糖原池等结构。说明5-氮胞苷和维生素C可以促进猪羊水干细胞向心肌细胞的诱导分化。     

Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.     

Wnt11 facilitates embryonic stem cell differentiation to Nkx2.5-positive cardiomyocytes

Wnt signaling plays a crucial role in the control of morphogenesis in several tissues. Herein, we describe the role of Wnt11 during cardiac differentiation of embryonic stem cells. First, we examined the expression profile of Wnt11 during the course of differentiation in embryoid bodies, and then compared its expression in retinoic acid-treated embryoid bodies with that in untreated. In differentiating embryoid bodies, Wnt11 expression rose along with that of Nkx2.5 expression and continued to increase. When the embryoid bodies were treated with retinoic acid, Wnt11 expression decreased in parallel with the decreased expression of cardiac genes. Further, treatment of embryoid bodies with medium containing Wnt11 increased the expression of cardiac marker genes. Based on these results, we propose that Wnt11 plays an important role for cardiac development by embryoid bodies, and may be a key regulator of cardiac muscle cell proliferation and differentiation during heart development.     

Apelin enhances directed cardiac differentiation of mouse and human embryonic stem cells

Apelin is a peptide ligand for an orphan G-protein coupled receptor (APJ receptor) and serves as a critical gradient for migration of mesodermal cells fated to contribute to the myocardial lineage. The present study was designed to establish a robust cardiac differentiation protocol, specifically, to evaluate the effect of apelin on directed differentiation of mouse and human embryonic stem cells (mESCs and hESCs) into cardiac lineage. Different concentrations of apelin (50, 100, 500 nM) were evaluated to determine its differentiation potential. The optimized dose of apelin was then combined with mesodermal differentiation factors, including BMP-4, activin-A, and bFGF, in a developmentally specific temporal sequence to examine the synergistic effects on cardiac differentiation. Cellular, molecular, and physiologic characteristics of the apelin-induced contractile embryoid bodies (EBs) were analyzed. It was found that 100 nM apelin resulted in highest percentage of contractile EB for mESCs while 500 nM had the highest effects on hESCs. Functionally, the contractile frequency of mESCs-derived EBs (mEBs) responded appropriately to increasing concentration of isoprenaline and diltiazem. Positive phenotype of cardiac specific markers was confirmed in the apelin-treated groups. The protocol, consisting of apelin and mesodermal differentiation factors, induced contractility in significantly higher percentage of hESC-derived EBs (hEBs), up-regulated cardiac-specific genes and cell surface markers, and increased the contractile force. In conclusion, we have demonstrated that the treatment of apelin enhanced cardiac differentiation of mouse and human ESCs and exhibited synergistic effects with mesodermal differentiation factors.     

Modulation of cytokine production by interferential current in differentiated HL-60 cells

The influence of interferential current (IFC) on the release of four cytokines was investigated. IFC is an amplitude-modulated 4 kHz current used in therapeutic applications. Human promyelocytes (HL-60) were differentiated to monocytes/macrophages by treatment with calcitriol. Release of tumor necrosis factor alpha (TNFalpha) and interleukines 1beta, 6, and 8 (IL-1beta, IL-6, and IL-8) into the supernatant was measured after exposure to IFC at different modulation frequencies. TNFalpha release was stimulated about twofold by 4 kHz sine waves alone. The influences of exposure time (5-30 min) and current density (2.5-2500 microA/c m(2)) were tested. A maximum field effect was found at an exposure time of 15 min and a current density of 250 microA/cm(2). With these exposure conditions (15 min and 250 microA/cm(2) ), cells were treated at different modulation frequencies and reacted for TNFalpha, IL-1beta, and IL-8 release in a complex manner. Within the frequencies studied (0-125 Hz), we found stimulation as well as depression of the release. In a second run the cells were activated by pretreatment with 10 microg/ml lipopolysaccharide (LPS) and exposed in the same way as the nonactivated cells. Again the modulation frequency influenced, in a complex way, the induction of TNFalpha, IL-1beta, and IL-8, resulting in a pattern of stimulation and depression of release different from that found in nonactivated cells. For IL-6 production no significant changes were detected in activated or non-activated cells.     

THE PROCESS OF DIFFERENTIATION OF EMBRYOID BODIES IN THE LUNG OF SYNGENEIC MICE

The process of differentiation of embryoid bodies of mouse teratocarcinoma OTT6050 transplanted into the lung of syngeneic mice (129/Sv) is described. Embryoid bodies took more than 2 weeks to differentiate, and several kinds of differentiated tissues appeared often in the colonies derived from a single embryoid body. All the colonies with differentiated tissues were larger than 100μm in diameter.
Three steps on the differentiation of embryoid bodies can be distinguished by microscopic observations on histological preparations of tumors at different periods after injection. The first step is the deformation of the embryoid bodies and the disappearance of the outer endodermal cells, which occurs within a few days after injection. In the second step, which begins 5–7 days after injection, clusters of embryonal carcinoma cells in the colony are identified by the PAS reaction. The third step starts about 10 days after injection, and is characterized by the formation of tubular structures in some clusters.     

定向诱导小鼠ES细胞向心肌细胞的分化

为了提高体外诱导ES细胞向心肌细胞分化的效率 ,对以往的诱导方法加以改进 ,采用直接悬浮培养和 0 8%DMSO诱导 ,建立了简便、高效的定向诱导ES细胞向心肌细胞分化的体系 .诱导第 9d起可见自发性、有节律跳动的类胚体出现 ,第 14d达到高峰 ,约有 70 %的拟胚体产生跳动 .用RT PCR的方法在跳动的拟胚体中检测到心肌细胞特异性标志物的表达 ,采用免疫荧光染色的方法在蛋白水平检测到心肌特异的α辅肌动蛋白 (α actinin)的表达 ,并可见清晰肌小节 ,表明在改进的体外诱导条件下ES细胞可分化为成熟的心肌细胞 .     

Comparison of coupling of humans to electric and magnetic fields with frequencies between 100 Hz and 100 kHz

Recent laboratory and epidemiological results have stimulated interest in the hypothesis that human beings may exhibit biological responses to magnetic and/or electric field transients with frequencies in the range between 100 Hz and 100 kHz. Much can be learned about the response of a system to a transient stimulation by understanding its response to sinusoidal disturbances over the entire frequency range of interest. Thus, the main effort of this paper was to compare the strengths of the electric fields induced in homogeneous ellipsoidal models by uniform 100 Hz through 100 kHz electric and magnetic fields. Over this frequency range, external electric fields of about 25–2000 V/m (depending primarily on the orientation of the body relative to the field) are required to induce electric fields inside models of adults and children that are similar in strength to those induced by an external 1 μT magnetic field. Additional analysis indicates that electric fields induced by uniform external electric and magnetic fields and by the nonuniform electric and magnetic fields produced by idealized point sources will not differ by more than a factor of two until the sources are brought close to the body. Published data on electric and magnetic field transients in residential environments indicate that, for most field orientations, the magnetic component will induce stronger electric fields inside adults and children than the electric component. This conclusion is also true for the currents induced in humans by typical levels of 60 Hz electric and magnetic fields in U.S. residences. Bioelectromagnetics 18:67–76, 1997. © 1997 Wiley-Liss, Inc.     

抑制NHE1活性对干细胞向心肌分化的影响

Na /H 交换蛋白1(NHE1)在心肌细胞发育过程中发挥重要的调节功能.为深入探索NHEl活性对干细胞向心肌分化过程中产生的影响,采用二甲基亚砜(DMSO)诱导P19干细胞向心肌细胞分化,同时在培养液中添加NHE1抑制荆EMD87580,对诱导后形成的类胚体进行检测.通过细胞形态观察、免疫组织化学染色及检测心肌特异表达基因等方法证明,经诱导形成的类胚体贴壁生长后,会向心肌细胞分化并出现跳动细胞团.而经过抑制剂处理的P19干细胞尽管能够形成类胚体且贴壁培养后细胞仍具有增殖活力,细胞团周边也较整齐,但未出现向心肌细胞分化的现象.这一结果表明,抑制NHE1的活性,能够影响P19干细胞向心肌细胞的分化作用.     

Cyclosporin A Induces Cardiac Differentiation but Inhibits Hemato-Endothelial Differentiation of P19 Cells

Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA) on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB) formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO) during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.