Embryonic Teratocarcinoma Cells Research Articles

Microscopic Analysis of Heterochromatin, Euchromatin and Cohesin in Cancer Cell Models and under Anti-Cancer Treatment.

The spatial organization of euchromatin (EC) and heterochromatin (HC) appears as a cell-type specific network, which seems to have an impact on gene regulation and cell fate. The spatial organization of cohesin should thus also be characteristic for a cell type since it is involved in a TAD (topologically associating domain) formation, and thus in gene regulation or DNA repair processes. Based on the previous hypotheses and results on the general importance of heterochromatin organization on genome functions in particular, the configurations of these organizational units (EC represented by H3K4me3-positive regions, HC represented by H3K9me3-positive regions, cohesins) are investigated in the cell nuclei of different cancer and non-cancerous cell types and under different anti-cancer treatments. Confocal microscopic images of the model cell systems were used and analyzed using analytical processes of quantification created in Fiji, an imaging tool box well established in different fields of science. Human fibroblasts, breast cancer and glioblastoma cells as well as murine embryonal terato-carcinoma cells were used as these cell models and compared according to the different parameters of spatial arrangements. In addition, proliferating, quiescent and from the quiescent state reactivated fibroblasts were analyzed. In some selected cases, the cells were treated with X-rays or azacitidine. Heterogeneous results were obtained by the analyses of the configurations of the three different organizational units: granulation and a loss of H3K4me3-positive regions (EC) occurred after irradiation with 4 Gy or azacitidine treatment. While fibroblasts responded to irradiation with an increase in cohesin and granulation, in breast cancer cells, it resulted in decreases in cohesin and changes in granulation. H3K9me3-positive regions (HC) in fibroblasts experienced increased granulation, whereas in breast cancer cells, the amount of such regions increased. After azacitidine treatment, murine stem cells showed losses of cohesin and granulation and an increase in the granulation of H3K9me3-positive regions. Fibroblasts that were irradiated with 2 Gy only showed irregularities in structural amounts and granulation. Quiescent fibroblasts contained less euchromatin-related H3K4me3-positive signals and cohesin levels as well as higher heterochromatin-related H3K9me3-positive signals than non-quiescent ones. In general, fibroblasts responded more intensely to X-ray irradiation than breast cancer cells. The results indicate the usefulness of model cell systems and show that, in general, characteristic differences initially existing in chromatin and cohesin organizations result in specific responses to anti-cancer treatment.

Bis-Bibenzyls from the Liverwort Pellia endiviifolia and Their Biological Activity.

Based on previous investigations where bis-bibenzyls isolated from liverworts showed various biological activities (cytotoxic, antimicrobial, and antiviral), we investigated their cytotoxic activity in several human cancer cell lines. From the methylene-chloride/methanol extract of the liverwort Pellia endiviifolia, three bis-bibenzyls of the perrottetin type were isolated, namely perrottetin E, 10′-hydroxyperrottetin E, and 10,10′-dihydroxyperrottetin E. The last two were found for the first time in this species. Their structures were resolved using 1D and 2D NMR, as well as by comparison with data in the literature. Cytotoxic activity of the isolated compounds was tested on three human leukemia cell lines, HL-60 (acute promyelocytic leukemia cells), U-937 (acute monocytic leukemia cells), and K-562 (human chronic myelogenous leukemia cells), as well as on human embryonal teratocarcinoma cell line (NT2/D1) and human glioblastoma cell lines A-172 and U-251, and compared to the previously isolated bis-bibenzyls (perrottetins) of similar structure. The isolated compounds exhibited modest activity against leukemia cells and significant activity against NT2/D1 and A-172. Overall, the most active cytotoxic compounds in this investigation were perrottetin E (1), isolated in this work from Pellia endiviifolia, and perrottetin F phenanthrene derivative (7), previously isolated from Lunularia cruciata and added for a comparison of their cytotoxic activity.

Retinoic acid and CTCF play key roles in inducing the collinear expression of the Hoxa cluster.

During the development of an embryo, the initiation of the collinear expression of Hox genes is essential for the proper formation of the anteroposterior body axis. Retinoic acid (RA), a natural derivative of vitamin A, plays a role in vertebrate development by regulating Hox gene expression. CCCTC-binding factor (CTCF), an insulator protein that controls gene transcription, also regulates the expression of Hox genes by binding to the CTCF-binding sites (CBSs). It has been reported that upon RA signaling, retinoic acid response elements (RAREs) located in the Hox clusters become occupied. Interestingly, RAREs exist in close proximity with CBSs, and therefore when RA is bound, CTCF cannot bind. Without CTCF and its insulator activities, the repressive domain in the chromatin becomes open for gene transcription. Here, we examine the relationship between RA and CTCF during the RA-induced expression of the Hoxa cluster genes, using F9 murine embryonic teratocarcinoma cells as a model system. We treated F9 cells with RA for different time, confirmed the collinear expression of Hoxa genes, and validated CTCF-binding in F9 cells as well as in CTCF-overexpressing F9 cells, in the presence of RA. The present study suggests that RA and CTCF pose antagonistic effects on each other during vertebrate development to attain Hox gene collinearity.

Novel evidence that pituitary sex hormones regulate migration, adhesion, and proliferation of embryonic stem cells and teratocarcinoma cells.

The pituitary sex hormones (SexHs): follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) regulate several functions crucial for reproduction, including oogenesis, spermatogenesis, and lactation. An important source of prolactin-like hormones, known as lactogens, is the placenta, and lactogens bind to the PRL receptor (PRLR) with high affinity and thereby mimic the actions of PRL. Recently, it has been demonstrated that pituitary SexHs were involved in metastatic lung cancer, certain sarcomas, and leukemia. In the present study we aimed to investigate whether FSH, LH, and PRL were able to stimulate stem cells involved in early development. To address this issue we employed a murine embryonic stem cell line (ES-D3) as well as two teratocarcinoma cell lines, P19 (murine) and NTera2 (human). We determined that all these cells expressed SexH receptors at the mRNA and protein levels and that stimulation of these receptors induced phosphorylation of p42/44 MAPK, p38 MAPK, and AKT. Moreover, ES-D3, P19, and NTera2 cells responded with increased migration and adhesion to physiological concentrations of pituitary SexHs. In view of these findings we proposed that maternal-derived pituitary SexHs regulate the biology of stem cells involved in early development.

Tumorigenic and Differentiation Potentials of Embryonic Stem Cells Depend on TGFβ Family Signaling: Lessons from Teratocarcinoma Cells Stimulated to Differentiate with Retinoic Acid.

A significant challenge for the development of safe pluripotent stem cell-based therapies is the incomplete in vitro differentiation of the pluripotent stem cells and the presence of residual undifferentiated cells initiating teratoma development after transplantation in recipients. To understand the mechanisms of incomplete differentiation, a comparative study of retinoic acid-induced differentiation of mouse embryonic stem (ES) and teratocarcinoma (EC) cells was conducted. The present study identified differences in proliferative activity, differentiation, and tumorigenic potentials between ES and EC cells. Higher expression of Nanog and Mvh, as well as Activin A and BMP4, was found in undifferentiated ES cells than in EC cells. However, the expression levels of Activin A and BMP4 increased more sharply in the EC cells during retinoic acid-induced differentiation. Stimulation of the Activin/Nodal and BMP signaling cascades and inhibition of the MEK/ERK and PI3K/Act signaling pathways resulted in a significant decrease in the number of Oct4-expressing ES cells and a loss of tumorigenicity, similar to retinoic acid-stimulated EC cells. Thus, this study demonstrates that a differentiation strategy that modulates prodifferentiation and antiproliferative signaling in ES cells may be effective for eliminating tumorigenic cells and may represent a valuable tool for the development of safe stem cell therapeutics.

Overexpression of Cathepsin E Interferes with Neuronal Differentiation of P19 Embryonal Teratocarcinoma Cells by Degradation of N-cadherin.

Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.

F9 EC 세포에서 레티노산에 의해 유도되는 Hoxc 유전자의 발현에 히스톤 메틸화가 미치는 영향

Department of Anatomy, Embryology Laboratory, Brain Korea 21 Pl us project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, KoreaReceived December 1, 2014/Revised March 11, 2015/Accepted March 12, 2015Hox genes encode a highly conserved family of homeodomain-containing transcription factors control-ling vertebrate pattern formation along the anteroposterior body axis during embryogenesis. Retinoic acid (RA) is a key morphogen in embryogenesis and a critical regulator of both adult and embryonic cellular activity. Specifically, RA regulates Hox gene expression in mouse- or human-derived embry-onic carcinoma (EC) cells. Histone modification has been reported to play a pivotal role in the process of RA-induced gene expression and cell differentiation. As histone modification is thought to play an essential role in RA-induced Hox gene expression, we examined RA-induced initiation of collinear ex-pression of Hox genes and the corresponding histone modifications in F9 murine embryonic ter-atocarcinoma (EC) cells. Hox expression patterns and histone modifications were analyzed by semi- quantitative RT-PCR, RNA-sequencing, and chromatin immuno-precipitation (ChIP)-PCR analyses. The Hoxc4 gene (D0) was initiated earlier than the Hoxc5 to –c10 genes (D3) upon RA treatment (day 0 [D0], day 1 [D1], and day 3 [D3]). The Hox nonexpressing D0 sample had a strong repressive marker, H3K27me3, than the D1 and D3 samples. In the D1 and D3 samples, reduced enrichment of the H3K27me3 marker was observed in the whole cluster. The active H3K4me3 marker was closely asso-ciated with the collinear expression of Hoxc genes. Thus, the Hoxc4 gene (D1) and all Hoxc genes (D3) expressed H3K4me3 upon transcription activation. In conclusion, these data indicated that removing H3K27me3 and acquiring H3K4me3 regulated RA-induced Hoxc gene collinearity in F9 cells. Keywords :

PI3K/Akt pathway regulates retinoic acid-induced Hox gene expression in F9 cells.

Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA-induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA-induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA-induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time-course gene expression profiles for all 39 Hox genes located in four different clusters-Hoxa, Hoxb, Hoxc, and Hoxd-were analyzed. Collinear expression of Hoxa and -b cluster genes was initiated earlier than that of the -c and -d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA-induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.

Low expression of activin a in mouse and human embryonic teratocarcinoma cells

TGFP3 family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFbeta family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGF@[beta] family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty 1, TGFbeta1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFbeta family (ACTIVINA, NODAL, LEFTY 1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells: Thus, in mouse and human nullipotent teratocarcinoma cells, theexpression of ActivinA is significantly reduced com- pared ivith embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.

Mir-302 cluster exhibits tumor suppressor properties on human unrestricted somatic stem cells

Many studies have reported that miR-302-367 cluster acts in different ways in various cell types. For instance, this cluster is shown to have a potential role in stemness regulation in embryonic stem cells (ESCs). On the other hand, this cluster inhibits the tumorigenicity of human pluripotent stem cells by coordinated suppression of CDK2 and CDK4/6 cell cycle pathways. Indeed, this cluster has a significant posttranscriptional impact on cell cycle progression. Previous reports have shown the participation of miR-302-367 cluster in cell cycle regulation of hESCs, MCF7, HepG2, and Teta-2 embryonal teratocarcinoma cells, but its effect on unrestricted somatic stem cells (USSCs) as a new source of human somatic stem cells from the umbilical cord blood remains to be elucidated. Therefore, in this study, we aimed to investigate the effect of miR-302-367 cluster on cell proliferation by MTT assay, cell cycle analysis, and colony formation assay. In addition, the expression of candidate cell cycle regulatory performance and tumor suppressor genes was determined. In this study, for the first time, we found that miR-302-367 cluster not only did not reprogram human USSCs into a pluripotent ESC-like state, but also inhibited the proliferation of human USSCs. Moreover, analyzing the cell cycle curve revealed a significant apoptotic phase upon viral introduction of miR-302-367. Our gene expression study revealed the overexpression of candidate genes after transduction of USSCs with miR-302-367 cluster. In conclusion, the controversial role of miR-302-367 in different cell types may provide better understanding for its role in stemness level and its antitumorigenicity potential in different contexts.

Development of Experimental Tumors Formed by Mouse and Human Embryonic Stem and Teratocarcinoma Cells after Subcutaneous and Intraperitoneal Transplantations into Immunodeficient and Immunocompetent Mice

Pluripotent stem cells represent an attractive cell source for regenerative medicine. However, the risk of teratoma formation after transplantation restricts their clinical application. Therefore, to adequately evaluate the potential risk of tumorigenicity after cell transplantation into human tissues, effective animal transplantation assays need to be developed. We performed a multiparameter (cell number, transplantation site, cell type, host) comparative analysis of the efficiency of tumor development after transplantation of mouse and human embryonic stem (ES) cells and their malignant counterparts, teratocarcinoma (EC) cells, into animal recipients and revealed several key correlations. We found that the efficiency of tumor growth was higher after intraperitoneal than after subcutaneous transplantations of all cell lines studied. The minimal cell numbers sufficient for tumor growth in immunodeficient nude mice were 100-fold lower for intraperitoneal than for subcutaneous transplantations of mouse and human ES cells (10(3) vs. 10(5) and 10(4) vs. 10(6), respectively). Moreover, mouse ES and EC cells formed tumors in immunodeficient and immunocompetent mice more effectively than human ES and EC cells. After intraperitoneal transplantation of 10(3), 10(4), and 10(5) mouse ES cells, teratomas developed in 83%, 100%, and 100% of nude mice, whereas after human ES cell transplantation, teratomas developed in 0%, 17%, and 60%, respectively. In addition, malignant mouse and human EC cells initiated tumor growth after intraperitoneal transplantation significantly faster and more effectively than ES cells. Mouse and human ES cells formed different types of teratomas containing derivatives of three germ layers but different numbers of undifferentiated cells. ES cell-like sublines with differentiation potential similar to the parental cell line were recloned only from mouse, but not from human, ES cell teratomas. These findings provide new information about the possibility and efficiency of tumor growth after transplantation of pluripotent stem cells. This information allows one to predict and possibly prevent the possible risks of tumorigenicity that could arise from stem cell therapeutics.

Role of Signaling Crosstalk in the Regulation of Neonatal Gonocytes and Embryonal Teratocarcinoma Cell Differentiation.

Role of Signaling Crosstalk in the Regulation of Neonatal Gonocytes and Embryonal Teratocarcinoma Cell Differentiation.

Antiproliferative and cytotoxic effects of different type cytostatics on mouse pluripotent stem and teratocarcinoma cells

Pluripotent stem cells are able to proliferate indefinitely and differentiate in vitro into various cell types. However, in most cases in vitro differentiation of the pluripotent stem cells is asynchronous and incomplete, and the residual undifferentiated cells can initiate teratoma development after transplantation into recipients. These features of the pluripotent stem cells are the major issue for development of safe cell therapy technologies based on pluripotent stem cells. Considering significant resemblance of growth rates of pluripotent stem and cancer cells we investigated antiproliferative and cytotoxic effects of different type cytostatics (mitomycin C, etoposide, vinblastine and cycloheximide) on the undifferentiated and differentiating mouse embryonic stem cells, embryonic germ cells, blastocyst and on mouse embryonal teratocarcinoma cells and mouse embryonic fibroblasts. The findings showed that all cytostatics used induced both antiproliferative effects and acute toxic processes in undifferentiated pluripotent stem cells and embryonal teratocarcinoma cells whereas these effects were less in differentiating embryonic stem cells and embryonic fibroblast. Moreover, the trophoblast cells of mouse blastocysts were less sensitive to damaging effects of cytostatics than inner cell mass cells. The examination of deferred effects ofcytostatics revealed that the effects ofmitomycin C, etoposide and vinblastine, but not cycloheximide, were irreversible because survived cells were not able to proliferate. Nevertheless, the numbers of embryonic fibroblasts exposed to etoposide or vinblastine remained unchanged while vast majority of undifferentiated pluripotent cells treated underwent apoptosis. Thus, diverse effects of etoposide and vinblastine on the undifferentiated pluripotent stem cells and differentiated embryonic cells allow us to consider these cytostatics and their analogs as drug-candidates for selective elimination of the residual undifferentiated pluripotent stem cells from population of differentiating cells. These findings demonstrate for the first time the possibility of selective elimination of undifferentiated pluripotent stem cells using cytostatic drugs approved for clinic practice. However, to improve effectiveness and safety of this approach and to prevent mutagenic, carcinogenic and teratogenic effects on undifferentiated pluripotent stem cells and their differentiated cell derivatives large-scale studies ofcytostatic effects using different experimental design and active doses must be performed.

Expression patterns of germ line specific genes in mouse and human pluripotent stem cells are associated with regulation of ground and primed state of pluripotency

One of the main criteria of pluripotency is ability of cell lines to differentiate into the germ line. Pluripotent stem cell lines in ground state of pluripotency differ from the lines in primed state by their ability to give rise to the mature gametes. To understand molecular mechanisms involved in regulation of different states of pluripotency we investigated the expression patterns of germ line specific genes in different type pluripotent stem cells and mouse and human embryonic teratocarcinoma cells. We found that pluripotent stem cells in vitro, in blastocyst and gonocytes at stage E13.5 had similar expression patterns in contrast to the epiblast cells at stage E6.5. Quantitative real time PCR analysis showed that Vasa/Ddx4 expression in mouse and human embryonic stem cells was significantly lower than in blastocyst and gonocytes. Moreover, Vasa/Ddx4 and E_ras expression was significantly higher in mouse embryonic stem cells than in human embryonic stem cells. Our analysis of germ line specific gene expression in differentiating mouse embryonic stem and embryonic germ as well as in mouse embryonic teratocarcinoma cells maintained under conditions promoting cell reprogramming from primed to ground state of pluripotency (2i + LIF) revealed that only pluripotent stem cells are able to regulate the expression level of Oct4 and Vasa/Ddx4 and restore initial ground state, while in embryonic teratocarcinoma cells the expression level of these genes remained unchanged. We suggest that expression patterns of germ lines specific genes, in particular of Vasa/Ddx4, can underlie the regulation of ground and primed states of pluripotency. [corrected].

Histone H1 Variants Are Differentially Expressed and Incorporated into Chromatin during Differentiation and Reprogramming to Pluripotency

There are seven linker histone variants in human somatic cells (H1.0 to H1.5 and H1X), and their prevalence varies as a function of cell type and differentiation stage, suggesting that the different variants may have distinct roles. We have revisited this notion by using new methodologies to study pluripotency and differentiation, including the in vitro differentiation of human embryonic stem (ES) and teratocarcinoma cells and the reprogramming of keratinocytes to induced pluripotent stem cells. Our results show that pluripotent cells (PCs) have decreased levels of H1.0 and increased levels of H1.1, H1.3, and H1.5 compared with differentiated cells. PCs have a more diverse repertoire of H1 variants, whereas in differentiated cells, H1.0 expression represents ∼80% of the H1 transcripts. In agreement with their prevalent expression in ES cells, the regulatory regions of H1.3 and H1.5 genes were found to be occupied by pluripotency factors. Moreover, the H1.0 gene promoter contains bivalent domains (H3K4me2 and H3K27me3) in PCs, suggesting that this variant is likely to have an important role during differentiation. Indeed, the knockdown of H1.0 in human ES did not affect self-renewal but impaired differentiation. Accordingly, H1.0 was recruited to the regulatory regions of differentiation and pluripotency genes during differentiation, confirming that this histone variant plays a critical role in the regulation of these genes. Thus, histone H1 variant expression is controlled by a variety of mechanisms that produce distinct but consistent H1 repertoires in pluripotent and differentiated cells that appear critical to maintain the functionality of such cells.

Identification of electrophysiological endpoints in stem cell-based systems for developmental neurotoxicity testing.

Event Abstract Back to Event Identification of electrophysiological endpoints in stem cell-based systems for developmental neurotoxicity testing. Grzegorz Podrygajlo1, 2*, Samora Okujeni1, 2, 3, Patrick Dini1, 2, 3, Matthew Goddard1, 2 and Ulrich Egert1, 2 1 University of Freiburg, Department of Microsystems Engineering – IMTEK, Germany 2 University of Freiburg, Bernstein Center Freiburg, Germany 3 University of Freiburg, Faculty of Biology, Germany The toxicity of chemicals on brain development is of major concern. A predictive in vitro test for potential developmental neurotoxicity (DNT) needs to be an inexpensive, quick, standardized and predictive alternative to present in vivo methods. Here, we combine endpoints based on attachment, proliferation, differentiation and electrophysiological analyses, comparing four neuronal systems (murine embryonic stem cells, human neural progenitor cells and human teratocarcinoma cells) grown on microelectrode arrays (MEAs). Electrophysiological recordings show a spontaneous electrical activity of the networks derived from murine embryonic stem cells and human teratocarcinoma cells neuronal networks only. Comparison of their properties at different developmental stages shows activity increasing from 1st to 3rd week. Supplementary to the MEAs data we provided measurements of Ca+2 transient curves, which support existence of active neuronal networks in case of murine embryonic stem cells and human teratocarcinoma cells. Moreover, we optimized the differentiation of human teratocarcinoma cells, which now yields up to 2 times more neurons in a 3 to 5 days shorter time. In summary, we analyzed functional endpoints for electrical activity and network properties of 3 cell lines during the period of neuronal maturation. Acknowledgements NT2 cells were obtained from G. Bicker (Tiho, Hannover, Germany) mESC cells were obtained from A. Seiler (BfR, Berlin, Germany) hNPC cells were obtained from E. Fritsche (IUF, Duesseldorf, Germany) Supported by the German BMBF FKZ 0315522B Keywords: developmental toxicity DNT, microelectrode arrays MEAs, networks, Neurons, Stem Cells Conference: BC11 : Computational Neuroscience & Neurotechnology Bernstein Conference & Neurex Annual Meeting 2011, Freiburg, Germany, 4 Oct - 6 Oct, 2011. Presentation Type: Poster Topic: neurons, networks and dynamical systems (please use "neurons, networks and dynamical systems" as keywords) Citation: Podrygajlo G, Okujeni S, Dini P, Goddard M and Egert U (2011). Identification of electrophysiological endpoints in stem cell-based systems for developmental neurotoxicity testing.. Front. Comput. Neurosci. Conference Abstract: BC11 : Computational Neuroscience & Neurotechnology Bernstein Conference & Neurex Annual Meeting 2011. doi: 10.3389/conf.fncom.2011.53.00057 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Aug 2011; Published Online: 04 Oct 2011. * Correspondence: Dr. Grzegorz Podrygajlo, University of Freiburg, Department of Microsystems Engineering – IMTEK, Freiburg, Germany, grzegorz.podrygajlo@imtek.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Grzegorz Podrygajlo Samora Okujeni Patrick Dini Matthew Goddard Ulrich Egert Google Grzegorz Podrygajlo Samora Okujeni Patrick Dini Matthew Goddard Ulrich Egert Google Scholar Grzegorz Podrygajlo Samora Okujeni Patrick Dini Matthew Goddard Ulrich Egert PubMed Grzegorz Podrygajlo Samora Okujeni Patrick Dini Matthew Goddard Ulrich Egert Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Dishevelled-3 C-terminal His single amino acid repeats are obligate for Wnt5a activation of non-canonical signaling

BackgroundThe Wnt non-canonical pathway (Wnt5a > Frizzled-2 > cyclic GMP phosphodiesterase/Ca2+-mobilization pathway regulates the activation of NF-AT) is mediated by three mammalian Dishevelleds (Dvl1, Dvl2, and Dvl3) and the role of the C-terminal region unique to Dvl3 was interrogated.ResultsDvl1, Dvl2, and Dvl3 are expressed at varying levels in mouse totipotent F9 embryonal teratocarcinoma cells. The expression of each endogenous Dvl isoform, as defined by knock-down with siRNA, was obligate for Wnt5a to activate NF-AT-sensitive transcription. Elements upstream of effectors, e.g., cGMP phosphodiesterase and Ca2+-mobilization, were blocked by knock-down of any one of the Dvls; thus, with respect to Wnt5a activation of NF-AT Dvls are not redundant. Among the three Dvl isoforms, the C-terminal sequence of Dvl3 is the most divergent. Deletion of region of Dvl3 abolishes Wnt5a-stimulated signaling. Alanine (Ala)-substitution of histidine (His) single amino acid repeats at 637,638 and/or 647,648 in Dvl3, like C-terminal deletion, abolishes Wnt 5a signal propagation. Phenylalanine (Phe)-substitution of the same His-repeats in Dvl3 mimics Wnt5a stimulated NF-AT-sensitive transcription.ConclusionsThe C-terminal third of Dvl3 and His single amino acid repeats 637,638 and 647,648 (which are unique to and conserved in Dvl3) are essential for Wnt5a activation of the non-canonical pathway, but not the Wnt3a activation of the canonical pathway.

Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells

Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.

Chromatin organization and transcriptional activation ofHoxgenes

Spatially and temporally programmed expression of the Hox genes along the antero-posterior (A-P) axis is essential for correct pattern formation during embryonic development. An accumulating body of evidence indicates the pivotal role of spatial chromatin organization for the coordination of gene regulation. Recently, chromosome conformation capture (3C) technique has been developed and opened a new way to study chromosomal interactions in the nucleus. In this study, we describe 3C method we applied in F9 embryonic teratocarcinoma cells and demonstrate that the chromosomal interactions at Hox loci are successfully detected. Interestingly, at Hoxc loci, the abundance of intrachromosomal interactions with neighboring fragments was drastically decreased when the genes are expressed. These results indicate the possibility of the dynamic pattern of chromosomal interaction in association with the transcriptional regulation of Hox genes.

Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells and teratocarcinoma cells by TGFβ family signaling factors

The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.