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Sample records for embryonic cortical cultures

  1. Duration of culture and sonic hedgehog signaling differentially specify PV versus SST cortical interneuron fates from embryonic stem cells.

    PubMed

    Tyson, Jennifer A; Goldberg, Ethan M; Maroof, Asif M; Xu, Qing; Petros, Timothy J; Anderson, Stewart A

    2015-04-01

    Medial ganglionic eminence (MGE)-derived GABAergic cortical interneurons (cINs) consist of multiple subtypes that are involved in many cortical functions. They also have a remarkable capacity to migrate, survive and integrate into cortical circuitry after transplantation into postnatal cortex. These features have engendered considerable interest in generating distinct subgroups of interneurons from pluripotent stem cells (PSCs) for the study of interneuron fate and function, and for the development of cell-based therapies. Although advances have been made, the capacity to generate highly enriched pools of subgroup fate-committed interneuron progenitors from PSCs has remained elusive. Previous studies have suggested that the two main MGE-derived interneuron subgroups--those expressing somatostatin (SST) and those expressing parvalbumin (PV)--are specified in the MGE from Nkx2.1-expressing progenitors at higher or lower levels of sonic hedgehog (Shh) signaling, respectively. To further explore the role of Shh and other factors in cIN fate determination, we generated a reporter line such that Nkx2.1-expressing progenitors express mCherry and postmitotic Lhx6-expressing MGE-derived interneurons express GFP. Manipulations of Shh exposure and time in culture influenced the subgroup fates of ESC-derived interneurons. Exposure to higher Shh levels, and collecting GFP-expressing precursors at 12 days in culture, resulted in the strongest enrichment for SST interneurons over those expressing PV, whereas the strongest enrichment for PV interneurons was produced by lower Shh and by collecting mCherry-expressing cells after 17 days in culture. These findings confirm that fate determination of cIN subgroups is crucially influenced by Shh signaling, and provide a system for the further study of interneuron fate and function.

  2. Mouse Embryonic Retina Delivers Information Controlling Cortical Neurogenesis

    PubMed Central

    Bonetti, Ciro; Surace, Enrico Maria

    2010-01-01

    The relative contribution of extrinsic and intrinsic mechanisms to cortical development is an intensely debated issue and an outstanding question in neurobiology. Currently, the emerging view is that interplay between intrinsic genetic mechanisms and extrinsic information shape different stages of cortical development [1]. Yet, whereas the intrinsic program of early neocortical developmental events has been at least in part decoded [2], the exact nature and impact of extrinsic signaling are still elusive and controversial. We found that in the mouse developing visual system, acute pharmacological inhibition of spontaneous retinal activity (retinal waves-RWs) during embryonic stages increase the rate of corticogenesis (cell cycle withdrawal). Furthermore, early perturbation of retinal spontaneous activity leads to changes of cortical layer structure at a later time point. These data suggest that mouse embryonic retina delivers long-distance information capable of modulating cell genesis in the developing visual cortex and that spontaneous activity is the candidate long-distance acting extrinsic cue mediating this process. In addition, these data may support spontaneous activity to be a general signal coordinating neurogenesis in other developing sensory pathways or areas of the central nervous system. PMID:21170332

  3. Culture and Manipulation of Embryonic Cells

    PubMed Central

    Edgar, Lois G.; Goldstein, Bob

    2012-01-01

    The direct manipulation of embryonic cells is an important tool for addressing key questions in cell and developmental biology. C. elegans is relatively unique among genetic model systems in being amenable to manipulation of embryonic cells. Embryonic cell manipulation has allowed the identification of cell interactions by direct means, and it has been an important technique for dissecting mechanisms by which cell fates are specified, cell divisions are oriented, and morphogenesis is accomplished. Here, we present detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans. PMID:22226523

  4. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  5. Embryonic Stem Cells: Isolation, Characterization and Culture

    NASA Astrophysics Data System (ADS)

    Amit, Michal; Itskovitz-Eldor, Joseph

    Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

  6. FoxP2 regulates neurogenesis during embryonic cortical development.

    PubMed

    Tsui, David; Vessey, John P; Tomita, Hideaki; Kaplan, David R; Miller, Freda D

    2013-01-02

    The transcription factor FoxP2 has been associated with the development of human speech but the underlying cellular function of FoxP2 is still unclear. Here we provide evidence that FoxP2 regulates genesis of some intermediate progenitors and neurons in the mammalian cortex, one of the key centers for human speech. Specifically, knockdown of FoxP2 in embryonic cortical precursors inhibits neurogenesis, at least in part by inhibiting the transition from radial glial precursors to neurogenic intermediate progenitors. Moreover, overexpression of human, but not mouse, FoxP2 enhances the genesis of intermediate progenitors and neurons. In contrast, expression of a human FoxP2 mutant that causes vocalization deficits decreases neurogenesis, suggesting that in the murine system human FoxP2 acts as a gain-of-function protein, while a human FoxP2 mutant acts as a dominant-inhibitory protein. These results support the idea that FoxP2 regulates the transition from neural precursors to transit-amplifying progenitors and ultimately neurons, and shed light upon the molecular changes that might contribute to evolution of the mammalian cortex.

  7. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells.

    PubMed

    Maroof, Asif M; Keros, Sotirios; Tyson, Jennifer A; Ying, Shui-Wang; Ganat, Yosif M; Merkle, Florian T; Liu, Becky; Goulburn, Adam; Stanley, Edouard G; Elefanty, Andrew G; Widmer, Hans Ruedi; Eggan, Kevin; Goldstein, Peter A; Anderson, Stewart A; Studer, Lorenz

    2013-05-02

    Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.

  8. Organotypic slice culture of embryonic brain tissue.

    PubMed

    Daza, Ray A M; Englund, Chris; Hevner, Robert F

    2007-12-01

    INTRODUCTIONThis protocol describes how to dissect, assemble, and cultivate mouse embryonic (E) brain tissue from age E11.5 to E18.5 (days) for organotypic slice culture. These preparations can be used for a variety of assays and studies including coculture of different brain regions, cell migration assays, axon guidance assays, and DNA electroporation experiments. During electroporation, an electric current is applied to the surface of a specific target area of the brain slice in order to open holes in the plasma membrane and introduce a plasmid of coding DNA. The floating slice-on-membrane construct helps to preserve the structural integrity of the brain slices, while maintaining easy experimental access and optimal viability. Experiments can be monitored in living slices (e.g., with confocal imaging), and further studies can be completed using slices that have been fixed and cryosectioned at the end of the experiment. Any region of embryonic brain or spinal tissue can be used in this protocol.

  9. Development and Maturation of Embryonic Cortical Neurons Grafted into the Damaged Adult Motor Cortex

    PubMed Central

    Ballout, Nissrine; Frappé, Isabelle; Péron, Sophie; Jaber, Mohamed; Zibara, Kazem; Gaillard, Afsaneh

    2016-01-01

    Injury to the human central nervous system can lead to devastating consequences due to its poor ability to self-repair. Neural transplantation aimed at replacing lost neurons and restore functional circuitry has proven to be a promising therapeutical avenue. We previously reported in adult rodent animal models with cortical lesions that grafted fetal cortical neurons could effectively re-establish specific patterns of projections and synapses. The current study was designed to provide a detailed characterization of the spatio-temporal in vivo development of fetal cortical transplanted cells within the lesioned adult motor cortex and their corresponding axonal projections. We show here that as early as 2 weeks after grafting, cortical neuroblasts transplanted into damaged adult motor cortex developed appropriate projections to cortical and subcortical targets. Grafted cells initially exhibited characteristics of immature neurons, which then differentiated into mature neurons with appropriate cortical phenotypes where most were glutamatergic and few were GABAergic. All cortical subtypes identified with the specific markers CTIP2, Cux1, FOXP2, and Tbr1 were generated after grafting as evidenced with BrdU co-labeling. The set of data provided here is of interest as it sets biological standards for future studies aimed at replacing fetal cells with embryonic stem cells as a source of cortical neurons. PMID:27536221

  10. FGF SIGNALING EXPANDS EMBRYONIC CORTICAL SURFACE AREA BY REGULATING NOTCH-DEPENDENT NEUROGENESIS

    PubMed Central

    Rash, Brian G.; Lim, H. David; Breunig, Joshua J.; Vaccarino, Flora M.

    2011-01-01

    The processes regulating cortical surface area expansion during development and evolution are unknown. We show that loss of function of all Fibroblast Growth Factor Receptors (FgfR) expressed at the earliest stages of cortical development causes severe deficits in surface area growth by embryonic day (E) 12.5 in the mouse. In FgfR mutants, accelerated production of neurons led to severe loss of radial progenitors and premature termination of neurogenesis. Nevertheless, these mutants showed remarkably little change in cortical layer structure. Birthdating experiments indicated that a greater proportion of layer fates was generated during early neurogenic stages, revealing that FgfR activity normally slows the temporal progression of cortical layer fates. Electroporation of a dominant negative FgfR at E11.5 increased cortical neurogenesis in normal mice—an effect that was blocked by simultaneous activation of the Notch pathway. Together with changes in the expression of Notch pathway genes in FgfR mutant embryos, these findings indicate that Notch lies downstream of FgfR signaling in the same pathway regulating cortical neurogenesis and begin to establish a mechanism for regulating cortical surface expansion. PMID:22031906

  11. Culturing murine embryonic organs: Pros, cons, tips and tricks.

    PubMed

    McClelland, Kathryn S; Bowles, Josephine

    2016-01-01

    There are three established techniques described for ex vivo culture of the early embryonic organs: filter culture, agar block culture and hanging drop culture. Each of these protocols has advantages and disadvantages; here we assess the merits of each approach. Agar block culture has a long history and has been well described. This method results in good embryonic organ morphology. Filter culture has been used to culture a number of different embryonic organs and there are a variety of filter choices available. The key disadvantage of agar-block and filter based culture is that the large amount of media required can make the approach expensive, especially if biologicals such as growth factors are necessary; in addition, using these methods it can be difficult to track particular samples. Hanging drop culture is most commonly used to enable the aggregation of embryonic stem cells into embryoid bodies but it has also been employed for ex vivo organ culture. This method requires only 40μL of media per drop and isolates every organ to a trackable unit. We describe each of these methods and the use of different medias and provide the user with a matrix to help determine the optimal culture method for their needs. Glass-based culture methods required for live imaging are not discussed here.

  12. Density gradient electrophoresis of cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

    Plank, L. D.; Kunze, M. E.; Giranda, V.; Todd, P. W.

    1985-01-01

    Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

  13. Development of Scalable Culture Systems for Human Embryonic Stem Cells

    PubMed Central

    Azarin, Samira M.; Palecek, Sean P.

    2009-01-01

    The use of human pluripotent stem cells, including embryonic and induced pluripotent stem cells, in therapeutic applications will require the development of robust, scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs), but challenges specific to hESCs will also have to be addressed, including development of defined, humanized culture media and substrates, monitoring spontaneous differentiation and heterogeneity in the cultures, and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. PMID:20161686

  14. GABA(B) receptors mediate motility signals for migrating embryonic cortical cells.

    PubMed

    Behar, T N; Smith, S V; Kennedy, R T; McKenzie, J M; Maric, I; Barker, J L

    2001-08-01

    During development, postmitotic neurons migrate from germinal regions into the cortical plate (cp), where lamination occurs. In rats, GABA is transiently expressed in the cp, near target destinations for migrating neurons. In vitro GABA stimulates neuronal motility, suggesting cp cells release GABA, which acts as a chemoattractant during corticogenesis. Pharmacological studies indicate GABA stimulates migration via GABA(B)-receptor (GABA(B)-R) activation. Using immunohistochemistry, RT-PCR and Western blotting, we examined embryonic cortical cell expression of GABA(B)-Rs in vivo. At E17, GABA(B)-R1(+) cells were identified in the ventricular zone (vz) and cp. RT-PCR and Western blotting demonstrated the presence of GABA(B)-R1a and GABA(B)-R1b mRNA and proteins. Using immuno- cytochemistry, GABA(B)-R expression was examined in vz and cp cell dissociates before and after migration to GABA in an in vitro chemotaxis assay. GABA-induced migration resulted in an increase of GABA(B)-R(+) cells in the migrated population. While <20% of each starting dissociate was GABA(B)-R(+), >70% of migrated cells were immunopositive. We used a microchemotaxis assay to analyze cp cell release of diffusible chemotropic factor(s). In vitro, cp dissociates induced vz cell migration in a cell density-dependent manner that was blocked by micromolar saclofen (a GABA(B)-R antagonist). HPLC demonstrated cp cells release micromolar levels of GABA and taurine in several hours. Micromolar levels of both molecules stimulated cell migration that was blocked by micromolar saclofen. Thus, migratory cortical cells express GABA(B)-Rs, cp cells release GABA and taurine, and both molecules stimulate cortical cell movement. Together these findings suggest GABA and/or taurine act as chemoattractants for neurons during rat cortical histogenesis via mechanisms involving GABA(B)-Rs.

  15. Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity.

    PubMed

    Xu, Jin-Chong; Fan, Jing; Wang, Xueqing; Eacker, Stephen M; Kam, Tae-In; Chen, Li; Yin, Xiling; Zhu, Juehua; Chi, Zhikai; Jiang, Haisong; Chen, Rong; Dawson, Ted M; Dawson, Valina L

    2016-04-06

    Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells or human inducible pluripotent stem cells that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid to FOXG1(+) neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuronal cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and poly(ADP-ribose) polymerase (PARP) (a cell death pathway called parthanatos that is distinct from apoptosis, necroptosis, and other forms of cell death). Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons.

  16. Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

    PubMed Central

    Meucci, Olimpia

    2011-01-01

    This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1. At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do

  17. Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells

    PubMed Central

    Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

    2016-01-01

    Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy. PMID:27904699

  18. Rat embryonic palatal shelves respond to TCDD in organ culture

    SciTech Connect

    Abbott, B.D.; Birnbaum, L.S. )

    1990-05-01

    TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental contaminant, is teratogenic in mice, inducing cleft palate (CP) and hydronephrosis at doses which are not overtly maternally or embryo toxic. Palatal shelves of embryonic mice respond to TCDD, both in vivo and in organ culture, with altered differentiation of medial epithelial cells. By contrast, in the rat TCDD produces substantial maternal, embryonic, and fetal toxicity, including fetal lethality, with few malformations. In this study the possible effects of maternal toxicity on induction of cleft palate were eliminated by exposure of embryonic rat palatal shelves in organ culture. The shelves were examined for specific TCDD-induced alterations in differentiation of the medial cells. On Gestation Day (GD) 14 or 15 palatal shelves from embryonic F344 rats were placed in organ culture for 2 to 3 days (IMEM:F12 medium, 5% FBS, 0.1% DMSO) containing 0, 1 x 10(-8), 1 x 10(-9), 1 x 10(-10), or 5 x 10(-11) M TCDD. The medial epithelial peridermal cells degenerated on shelves exposed to control media or 5 x 10(-11) M TCDD. Exposure to 10(-10), 10(-9), and 10(-8) M TCDD inhibited this degeneration in 20, 36, and 60% of the shelves, respectively, and was statistically significant at the two highest doses. A normally occurring decrease in (3H)TdR incorporation was inhibited in some GD 15 shelves cultured with 10(-10) and 10(-9) M TCDD. The medial cells of TCDD-exposed shelves continued to express high levels of immunohistochemically detected EGF receptors. The altered differentiation of rat medial epithelium is similar to that reported for TCDD-exposed mouse medial cells in vivo and in vitro. However, in order to obtain these responses, the cultured rat shelves require much higher concentrations of TCDD than the mouse shelves.

  19. Reduced Synaptic Vesicle Recycling during Hypoxia in Cultured Cortical Neurons

    PubMed Central

    Fedorovich, Sergei; Hofmeijer, Jeannette; van Putten, Michel J. A. M.; le Feber, Joost

    2017-01-01

    Improvement of neuronal recovery in the ischemic penumbra, an area around the core of a brain infarct with some remaining perfusion, has a large potential for the development of therapy against acute ischemic stroke. However, mechanisms that lead to either recovery or secondary damage in the penumbra largely remain unclear. Recent studies in cultured networks of cortical neurons showed that failure of synaptic transmission (referred to as synaptic failure) is a critical factor in the penumbral area, but the mechanisms that lead to synaptic failure are still under investigation. Here we used a Styryl dye, FM1-43, to quantify endocytosis and exocytosis in cultures of rat cortical neurons under normoxic and hypoxic conditions. Hypoxia in cultured cortical networks rapidly depressed endocytosis and, to a lesser extent, exocytosis. These findings support electrophysiological findings that synaptic failure occurs quickly after the induction of hypoxia, and confirms that the failing processes are at least in part presynaptic. PMID:28261063

  20. Cortical plasticity induced by transplantation of embryonic somatostatin or parvalbumin interneurons.

    PubMed

    Tang, Yunshuo; Stryker, Michael P; Alvarez-Buylla, Arturo; Espinosa, Juan Sebastian

    2014-12-23

    GABAergic inhibition has been shown to play an important role in the opening of critical periods of brain plasticity. We recently have shown that transplantation of GABAergic precursors from the embryonic medial ganglionic eminence (MGE), the source of neocortical parvalbumin- (PV(+)) and somatostatin-expressing (SST(+)) interneurons, can induce a new period of ocular dominance plasticity (ODP) after the endogenous period has closed. Among the diverse subtypes of GABAergic interneurons PV(+) cells have been thought to play the crucial role in ODP. Here we have used MGE transplantation carrying a conditional allele of diphtheria toxin alpha subunit and cell-specific expression of Cre recombinase to deplete PV(+) or SST(+) interneurons selectively and to investigate the contributions of each of these types of interneurons to ODP. As expected, robust plasticity was observed in transplants containing PV(+) cells but in which the majority of SST(+) interneurons were depleted. Surprisingly, transplants in which the majority of PV(+) cells were depleted induced plasticity as effectively as those containing PV(+) cells. In contrast, depleting both cell types blocked induction of plasticity. These findings reveal that PV(+) cells do not play an exclusive role in ODP; SST(+) interneurons also can drive cortical plasticity and contribute to the reshaping of neural networks. The ability of both PV(+) and SST(+) interneurons to induce de novo cortical plasticity could help develop new therapeutic approaches for brain repair.

  1. Dixdc1 is a critical regulator of DISC1 and embryonic cortical development

    PubMed Central

    Singh, Karun K.; Ge, Xuecai; Mao, Yingwei; Drane, Laurel; Meletis, Konstantinos; Samuels, Benjamin A.; Tsai, Li-Huei

    2010-01-01

    Summary The psychiatric illness risk gene Disrupted in Schizophrenia-1 (DISC1) plays an important role in brain development, however, it is unclear how DISC1 is regulated during cortical development. Here, we report that DISC1 is regulated during embryonic neural progenitor proliferation and neuronal migration through an interaction with DIX domain containing-1 (Dixdc1), the third mammalian gene discovered to contain a Disheveled-Axin (DIX) domain. We determined that Dixdc1 functionally interacts with DISC1 to regulate neural progenitor proliferation by co-modulating Wnt-GSK3β/β-catenin signaling. However, DISC1 and Dixdc1 do not regulate migration via this pathway. During neuronal migration, we discovered that phosphorylation of Dixdc1 by cyclin-dependent kinase 5 (Cdk5) facilitates its interaction with the DISC1-binding partner Ndel1. Furthermore, Dixdc1 phosphorylation and its interaction with DISC1/Ndel1 in vivo is required for neuronal migration. Together, these data reveal that Dixdc1 integrates DISC1 into Wnt-GSK3β/β-catenin-dependent and -independent signaling pathways during cortical development, and further delineate how DISC1 contributes to neuropsychiatric disorders. PMID:20624590

  2. Olesoxime protects embryonic cortical neurons from camptothecin intoxication by a mechanism distinct from BDNF

    PubMed Central

    Gouarné, Caroline; Giraudon-Paoli, Marc; Seimandi, Mathieu; Biscarrat, Clotilde; Tardif, Gwenaëlle; Pruss, Rebecca M; Bordet, Thierry

    2013-01-01

    Background and Purpose Olesoxime is a small cholesterol–oxime promoting rat embryonic motor neurons survival in the absence of trophic factors. Because olesoxime can substitute for neurotrophic factors in many situations, and to gain further understanding of its mechanism of action, we wondered if it could prevent neuronal death induced by camptothecin (CPT) and compared its effects with those of brain-derived neurotrophic factor (BDNF). Experimental Approach E17 rat embryonic cortical neurons were treated with olesoxime, BDNF or vehicle and intoxicated with CPT. Caspase-dependent and caspase-independent death pathways along with pro-survival pathways activation were explored. Key Results As previously reported for BDNF, olesoxime dose-dependently delayed CPT-induced cell death. Both compounds acted downstream of p53 activation preventing cytochrome c release and caspases activation. When caspase activation was blocked, both olesoxime and BDNF provided additional neuroprotective effect, potentially through the prevention of apoptosis-inducing factor release from mitochondria. While BDNF activates both the PI3K/Akt and the ERK pathway, olesoxime induced only a late activation of the ERK pathways, which did not seem to play a major role in its neuroprotection against CPT. Rather, our results favour preserved mitochondrial membrane integrity by olesoxime. Conclusions and Implications Albeit different, olesoxime and BDNF mechanisms for neuroprotection converge to preserve mitochondrial function. These findings emphasize the importance of targeting the mitochondria in the process of neurodegeneration. Importantly olesoxime, by mimicking neurotrophin pro-survival activities without impacting PI3K/Akt and ERK signalling, may have greater therapeutic potential in many diseases where neurotrophins were considered as a therapeutic solution. PMID:23278424

  3. Embryonic mouse pre-metatarsal development in organ culture

    NASA Technical Reports Server (NTRS)

    Klement, B. J.; Spooner, B. S.

    1993-01-01

    Embryonic mouse pre-metatarsals were removed from embryos at 13 days of gestation and cultured in a defined, serum-free medium for up to 15 days. By histological analysis, we observe that the cultured pre-metatarsal tissue undergoes a similar developmental profile as pre-metatarsals growing normally in vivo. The initial mesenchyme condensation regions undergo differentiation and morphogenesis to form distinct rods made up of cartilage tissue. A marker of this differentiation step is the synthesis of type II collagen. Metabolic labelling, pepsin digestion, SDS-PAGE, and autoradiography were used to demonstrate this protein when cartilage tissue is present in the cultures. After additional culture time, terminal chondrocyte differentiation and morphogenesis take place in specific regions of the cartilage rods to form bands of hypertrophied chondrocytes. One marker of this differentiation step is the synthesis of the enzyme alkaline phosphatase. We have measured the activity of this enzyme throughout the culture period and see a substantial increase at the time of terminal chondrocyte differentiation. Another feature of hypertrophied chondrocytes is that the matrix around the cells becomes calcified. Calcified matrix in our cultured pre-metatarsals was visualized by staining with alizarin red. By supplementing the defined culture medium with ITS, we observed that terminal chondrocyte differentiation took place in a shorter culture time. Supplementation of the medium with serum results in a similar acceleration of terminal differentiation, and, with additional culture time, an osteoid-like matrix forms around the central region of the rods.

  4. Urokinase production by electrophoretically separated cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

    Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

    1985-01-01

    Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

  5. Cortical Inhibition Modified by Embryonic Neural Precursors Grafted into the Postnatal Brain

    PubMed Central

    Alvarez-Dolado, Manuel; Calcagnotto, Maria Elisa; Karkar, Kameel M.; Southwell, Derek G.; Jones-Davis, Dorothy M.; Estrada, Rosanne C.; Rubenstein, John L. R.; Alvarez-Buylla, Arturo; Baraban, Scott C.

    2006-01-01

    Embryonic medial ganglionic eminence (MGE) cells transplanted into the adult brain can disperse, migrate, and differentiate to neurons expressing GABA, the primary inhibitory neurotransmitter. It has been hypothesized that grafted MGE precursors could have important therapeutic applications increasing local inhibition, but there is no evidence that MGE cells can modify neural circuits when grafted into the postnatal brain. Here we demonstrate that MGE cells grafted into one location of the neonatal rodent brain migrate widely into cortex. Grafted MGE-derived cells differentiate into mature cortical interneurons; the majority of these new interneurons express GABA. Based on their morphology and expression of somatostatin, neuropeptide Y, parvalbumin, or calretinin, we infer that graft-derived cells integrate into local circuits and function as GABA-producing inhibitory cells. Whole-cell current-clamp recordings obtained from MGE-derived cells indicate firing properties typical of mature interneurons. Moreover, patch-clamp recordings of IPSCs on pyramidal neurons in the host brain, 30 and 60 d after transplantation, indicated a significant increase in GABA-mediated synaptic inhibition in regions containing transplanted MGE cells. In contrast, synaptic excitation is not altered in the host brain. Grafted MGE cells, therefore, can be used to modify neural circuits and selectively increase local inhibition. These findings could have important implications for reparative cell therapies for brain disorders. PMID:16837585

  6. Mineralization and growth of cultured embryonic skeletal tissue in microgravity

    NASA Technical Reports Server (NTRS)

    Klement, B. J.; Spooner, B. S.

    1999-01-01

    Microgravity provides a unique environment in which to study normal and pathological phenomenon. Very few studies have been done to examine the effects of microgravity on developing skeletal tissue such as growth plate formation and maintenance, elongation of bone primordia, or the mineralization of growth plate cartilage. Embryonic mouse premetatarsal triads were cultured on three space shuttle flights to study cartilage growth, differentiation, and mineralization, in a microgravity environment. The premetatarsal triads that were cultured in microgravity all formed cartilage rods and grew in length. However, the premetatarsal cartilage rods cultured in microgravity grew less in length than the ground control cartilage rods. Terminal chondrocyte differentiation also occurred during culture in microgravity, as well as in the ground controls, and the matrix around the hypertrophied chondrocytes was capable of mineralizing in both groups. The same percentage of premetatarsals mineralized in the microgravity cultures as mineralized in the ground control cultures. In addition, the sizes of the mineralized areas between the two groups were very similar. However, the amount of 45Ca incorporated into the mineralized areas was significantly lower in the microgravity cultures, suggesting that the composition or density of the mineralized regions was compromised in microgravity. There was no significant difference in the amount of 45Ca liberated from prelabeled explants in microgravity or in the ground controls.

  7. Cortical Morphogenesis during Embryonic Development Is Regulated by miR-34c and miR-204

    PubMed Central

    Venø, Morten T.; Venø, Susanne T.; Rehberg, Kati; van Asperen, Jessy V.; Clausen, Bettina H.; Holm, Ida E.; Pasterkamp, R. Jeroen; Finsen, Bente; Kjems, Jørgen

    2017-01-01

    The porcine brain closely resembles the human brain in aspects such as development and morphology. Temporal miRNA profiling in the developing embryonic porcine cortex revealed a distinct set of miRNAs, including miR-34c and miR-204, which exhibited a highly specific expression profile across the time of cortical folding. These miRNAs were found to target Doublecortin (DCX), known to be involved in neuron migration during cortical folding of gyrencephalic brains. In vivo modulation of miRNA expression in mouse embryos confirmed that miR-34c and miR-204 can control neuronal migration and cortical morphogenesis, presumably by posttranscriptional regulation of DCX. PMID:28232790

  8. Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

    PubMed Central

    Mahler, Gretchen; Gould, Russell; Butcher, Johnathan

    2010-01-01

    Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis. PMID:21085095

  9. BMP4 acts as a dorsal telencephalic morphogen in a mouse embryonic stem cell culture system.

    PubMed

    Watanabe, Momoko; Fung, Ernest S; Chan, Felicia B; Wong, Jessica S; Coutts, Margaret; Monuki, Edwin S

    2016-12-15

    The concept of a morphogen - a molecule that specifies two or more cell fates in a concentration-dependent manner - is paradigmatic in developmental biology. Much remains unknown, however, about the existence of morphogens in the developing vertebrate central nervous system (CNS), including the mouse dorsal telencephalic midline (DTM). Bone morphogenetic proteins (BMPs) are candidate DTM morphogens, and our previous work demonstrated BMP4 sufficiency to induce one DTM cell fate - that of choroid plexus epithelial cells (CPECs) - in a mouse embryonic stem cell (mESC) culture system. Here we used BMP4 in a modified mESC culture system to derive a second DTM fate, the cortical hem (CH). CH and CPEC markers were induced by BMP4 in a concentration-dependent manner consistent with in vivo development. BMP4 concentrations that led to CH fate also promoted markers for Cajal-Retzius neurons, which are known CH derivatives. Interestingly, single BMP4 administrations also sufficed for appropriate temporal regulation of CH, CPEC, and cortical genes, with initially broad and overlapping dose-response profiles that sharpened over time. BMP4 concentrations that yielded CH- or CPEC-enriched populations also had different steady-state levels of phospho-SMAD1/5/8, suggesting that differences in BMP signaling intensity underlie DTM fate choice. Surprisingly, inactivation of the cortical selector gene Lhx2 did not affect DTM expression levels, dose-response profiles, or timing in response to BMP4, although neural progenitor genes were downregulated. These data indicate that BMP4 can act as a classic morphogen to orchestrate both spatial and temporal aspects of DTM fate acquisition, and can do so in the absence of Lhx2.

  10. BMP4 acts as a dorsal telencephalic morphogen in a mouse embryonic stem cell culture system

    PubMed Central

    Watanabe, Momoko; Fung, Ernest S.; Chan, Felicia B.; Wong, Jessica S.; Coutts, Margaret

    2016-01-01

    ABSTRACT The concept of a morphogen – a molecule that specifies two or more cell fates in a concentration-dependent manner – is paradigmatic in developmental biology. Much remains unknown, however, about the existence of morphogens in the developing vertebrate central nervous system (CNS), including the mouse dorsal telencephalic midline (DTM). Bone morphogenetic proteins (BMPs) are candidate DTM morphogens, and our previous work demonstrated BMP4 sufficiency to induce one DTM cell fate – that of choroid plexus epithelial cells (CPECs) – in a mouse embryonic stem cell (mESC) culture system. Here we used BMP4 in a modified mESC culture system to derive a second DTM fate, the cortical hem (CH). CH and CPEC markers were induced by BMP4 in a concentration-dependent manner consistent with in vivo development. BMP4 concentrations that led to CH fate also promoted markers for Cajal–Retzius neurons, which are known CH derivatives. Interestingly, single BMP4 administrations also sufficed for appropriate temporal regulation of CH, CPEC, and cortical genes, with initially broad and overlapping dose-response profiles that sharpened over time. BMP4 concentrations that yielded CH- or CPEC-enriched populations also had different steady-state levels of phospho-SMAD1/5/8, suggesting that differences in BMP signaling intensity underlie DTM fate choice. Surprisingly, inactivation of the cortical selector gene Lhx2 did not affect DTM expression levels, dose-response profiles, or timing in response to BMP4, although neural progenitor genes were downregulated. These data indicate that BMP4 can act as a classic morphogen to orchestrate both spatial and temporal aspects of DTM fate acquisition, and can do so in the absence of Lhx2. PMID:27815243

  11. [Effects of different culture system of isolating and passage of sheep embryonic stem-like cells].

    PubMed

    Bai, Changming; Liu, Chousheng; Wang, Zhigang; Wang, Xinzhuang

    2008-07-01

    In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal.

  12. Potentiated necrosis of cultured cortical neurons by neurotrophins.

    PubMed

    Koh, J Y; Gwag, B J; Lobner, D; Choi, D W

    1995-04-28

    The effects of neurotrophins on several forms of neuronal degeneration in murine cortical cell cultures were examined. Consistent with other studies, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 all attenuated the apoptotic death induced by serum deprivation or exposure to the calcium channel antagonist nimodipine. Unexpectedly, however, 24-hour pretreatment with these same neurotrophins markedly potentiated the necrotic death induced by exposure to oxygen-glucose deprivation or N-methyl-D-aspartate. Thus, certain neurotrophins may have opposing effects on different types of death in the same neurons.

  13. The effects of simulated microgravity on cultured chicken embryonic chondrocytes

    NASA Astrophysics Data System (ADS)

    Zhang, X.; Li, X. B.; Yang, S. Z.; Li, S. G.; Jiang, P. D.; Lin, Z. H.

    2003-10-01

    Using the cultured chicken embryonic chondrocytes as a model, the effects of simulated microgravity on the microtubular system of the cellular skeleton, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration and mitochondrial ATP synthase activity with its oligomycin inhibition rate were studied with a clinostat. The microtubular content was measured by a flow cytometer. The decrease of microtubular content showed the impairment of the cellular skeleton system. Observation on the extracellualr matrix by the scanning electron microscopy showed that it decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly than that of the control group. It can be concluded that the simulated microgravity can affect the secreting and assembly of the extracellular matrix. In contrast to the control, there was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. These results indicate that simulated microgravity can suppress matrix calcification of cultured chondrocytes, and intracellular free calcium may be involved in the regulation of matrix calcification as the second signal transmitter. No significant changes happened in the mitochondrial ATP synthase activity and its oligomycin inhibition rate. Perhaps the energy metabolism wasn't affected by the simulated microgravity. The possible mechanisms about them were discussed.

  14. In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

    PubMed Central

    Rice, Heather; Suth, Seiyam; Cavanaugh, William; Bai, Jilin; Young-Pearse, Tracy L.

    2010-01-01

    In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells. The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth. PMID:20972409

  15. Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

    PubMed

    Sleeboom-Faulkner, Margaret

    2010-01-01

    This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

  16. Human embryonic stem cells: Derivation, culture, and differentiation: A review

    PubMed Central

    Vazin, Tandis; Freed, William J.

    2010-01-01

    The greatest therapeutic promise of human embryonic stem cells (hESC) is to generate specialized cells to replace damaged tissue in patients suffering from various degenerative diseases. However, the signaling mechanisms involved in lineage restriction of ESC to adopt various cellular phenotypes are still under investigation. Furthermore, for progression of hESC-based therapies towards clinical applications, appropriate culture conditions must be developed to generate genetically stable homogenous populations of cells, to hinder possible adverse effects following transplantation. Other critical challenges that must be addressed for successful cell implantation include problems related to survival and functional efficacy of the grafted cells. This review initially describes the derivation of hESC and focuses on recent advances in generation, characterization, and maintenance of these cells. We also give an overview of original and emerging differentiation strategies used to convert hESC to different cell types. Finally, we will discuss transplantation studies of hESC-derived cells with respect to safety and functional recovery. PMID:20714081

  17. Signal transfer within a cultured asymmetric cortical neuron circuit

    NASA Astrophysics Data System (ADS)

    Isomura, Takuya; Shimba, Kenta; Takayama, Yuzo; Takeuchi, Akimasa; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2015-12-01

    Objective. Simplified neuronal circuits are required for investigating information representation in nervous systems and for validating theoretical neural network models. Here, we developed patterned neuronal circuits using micro fabricated devices, comprising a micro-well array bonded to a microelectrode-array substrate. Approach. The micro-well array consisted of micrometre-scale wells connected by tunnels, all contained within a silicone slab called a micro-chamber. The design of the micro-chamber confined somata to the wells and allowed axons to grow through the tunnels bidirectionally but with a designed, unidirectional bias. We guided axons into the point of the arrow structure where one of the two tunnel entrances is located, making that the preferred direction. Main results. When rat cortical neurons were cultured in the wells, their axons grew through the tunnels and connected to neurons in adjoining wells. Unidirectional burst transfers and other asymmetric signal-propagation phenomena were observed via the substrate-embedded electrodes. Seventy-nine percent of burst transfers were in the forward direction. We also observed rapid propagation of activity from sites of local electrical stimulation, and significant effects of inhibitory synapse blockade on bursting activity. Significance. These results suggest that this simple, substrate-controlled neuronal circuit can be applied to develop in vitro models of the function of cortical microcircuits or deep neural networks, better to elucidate the laws governing the dynamics of neuronal networks.

  18. Pharmacological Characterization of the Native Store-Operated Calcium Channels of Cortical Neurons from Embryonic Mouse Brain

    PubMed Central

    Chauvet, Sylvain; Jarvis, Louis; Chevallet, Mireille; Shrestha, Niroj; Groschner, Klaus; Bouron, Alexandre

    2016-01-01

    In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of the transient receptor potential channel of C type 3 (TRPC3 channel). However, Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to clarify the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2 and Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction. E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, and GSK-7975A) were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6 and GSK-7975A, as well as to EVP4593, zinc, copper, and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ∼10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex. PMID:28018223

  19. Spaceflight effects on cultured embryonic chick bone cells

    NASA Technical Reports Server (NTRS)

    Landis, W. J.; Hodgens, K. J.; Block, D.; Toma, C. D.; Gerstenfeld, L. C.

    2000-01-01

    A model calcifying system of primary osteoblast cell cultures derived from normal embryonic chicken calvaria has been flown aboard the shuttle, Endeavour, during the National Aeronautics and Space Administration (NASA) mission STS-59 (April 9-20, 1994) to characterize unloading and other spaceflight effects on the bone cells. Aliquots of cells (approximately 7 x 10(6)) grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were mixed with microcarrier beads, inoculated into cartridge culture units of artificial hollow fiber capillaries, and carried on the shuttle. To promote cell differentiation, cartridge media were supplemented with 12.5 microg/ml ascorbate and 10 mM beta-glycerophosphate for varying time periods before and during flight. Four cartridges contained cells from 17-day-old embryos grown for 5 days in the presence of ascorbate prior to launch (defined as flight cells committed to the osteoblastic lineage) and four cartridges supported cells from 14-day-old embryos grown for 10 days with ascorbate before launch (uncommitted flight cells). Eight cartridges prepared in the same manner were maintained under normal gravity throughout the flight (control cells) and four additional identical cartridges under normal gravity were terminated on the day of launch (basal cells). From shuttle launch to landing, all cartridges were contained in closed hardware units maintaining 5% CO2, 37 degrees C, and media delivery at a rate of approximately 1.5 ml/6 h. During day 3 and day 5 of flight, duplicate aliquots of conditioned media and accumulated cell products were collected in both the flight and the control hardware units. At the mission end, comparisons among flight, basal, and control samples were made in cell metabolism, gene expression for type I collagen and osteocalcin, and ultrastructure. Both committed and uncommitted flight cells were metabolically active, as measured by glucose uptake and lactate production, at approximately the

  20. [Derivation of germ cells from mouse embryonic stem cells in culture].

    PubMed

    Fuhrmann, G

    2005-10-01

    Mouse embryonic stem cells derive from the inner cell mass of the blastocyst and give rise to the three primitive embryonic layers, which later will form all the different tissue types of an adult. Embryonic stem cells are thus defined as totipotent cells. In vitro, these cells can give rise to all the somatic cells. Different laboratories have now shown that cultured embryonic stem cells can also differentiate into germline cells. By using the transcription factor Oct-4 as a tool for the visualization of germ cells, it has been shown the derivation of oocytes from mouse embryonic stem cells. These works should contribute to various areas, including therapeutic cloning which associates nuclear transfer and selective production of a specific cell type.

  1. Iron Accumulation and Neurotoxicity in Cortical Cultures Treated with Holotransferrin

    PubMed Central

    Chen-Roetling, Jing; Liu, Wenpei; Regan, Raymond F.

    2012-01-01

    Nonheme iron accumulates in CNS tissue after ischemic and hemorrhagic insults, and may contribute to cell loss. The source of this iron has not been precisely defined. After blood-brain barrier disruption, CNS cells may be exposed to plasma concentrations of transferrin-bound iron (TBI), which exceed that in CSF by over 50-fold. In this study, the hypothesis that these concentrations of TBI produce cell iron accumulation and neurotoxicity was tested in primary cortical cultures. Treatment with 0.5-3 mg/ml holotransferrin for 24 hours resulted in loss of 20-40% of neurons, associated with increases in malondialdehyde, ferritin, heme oxygenase-1 and iron; transferrin receptor-1 expression was reduced by about 50%. Deferoxamine, 2,2′-bipyridyl, Trolox, and ascorbate prevented all injury, but apotransferrin was ineffective. Cell TBI accumulation was significantly reduced by deferoxamine, 2,2′-bipyridyl, and apotransferrin, but not by ascorbate or Trolox. After treatment with 55Fe-transferrin, approximately 40% of cell iron was exported within 16 hours. Net export was increased by deferoxamine and 2,2′-bipyridyl, but not by apotransferrin. These results suggest that downregulation of transferrin receptor-1 expression is insufficient to prevent iron-mediated death when neurons are exposed to plasma concentrations of TBI. Chelator therapy may be beneficial for acute CNS injuries associated with loss of blood-brain barrier integrity. PMID:21939754

  2. AN EMBRYONIC CHICK PANCREAS ORGAN CULTURE MODEL: CHARACTERIZATION AND NEURAL CONTROL OF EXOCRINE RELEASE

    EPA Science Inventory

    An embryonic chick (Gallus domesticus) whole-organ pancreas culture system was developed for use as an in vitro model to study cholinergic regulation of exocrine pancreatic function. The culture system was examined for characteristic exocrine function and viability by measuring e...

  3. Active cortical innervation protects striatal neurons from slow degeneration in culture.

    PubMed

    Fishbein, Ianai; Segal, Menahem

    2011-03-01

    Spiny striatal GABAergic neurons receive most of their excitatory input from the neocortex. In culture, striatal neurons form inhibitory connections, but the lack of intrinsic excitatory afferents prevents the development of spontaneous network activity. Addition of cortical neurons to the striatal culture provides the necessary excitatory input to the striatal neurons, and in the presence of these neurons, striatal cultures do express spontaneous network activity. We have confirmed that cortical neurons provide excitatory drive to striatal neurons in culture using paired recording from cortical and striatal neurons. In the presence of tetrodotoxin (TTX), which blocks action potential discharges, the connections between cortical and striatal neurons are still formed, and in fact synaptic currents generated between them when TTX is removed are far larger than in control, undrugged cultures. Interestingly, the continuous presence of TTX in the co-culture caused striatal cell death. These observations indicate that the mere presence of cortical neurons is not sufficient to preserve striatal neurons in culture, but their synchronous activity, triggered by cortical excitatory synapses, is critical for the maintenance of viability of striatal neurons. These results have important implications for understanding the role of activity in neurodegenerative diseases of the striatum.

  4. Human embryonic stem cells: culture, differentiation, and genetic modification for regenerative medicine applications.

    PubMed

    Lebkowski, J S; Gold, J; Xu, C; Funk, W; Chiu, C P; Carpenter, M K

    2001-01-01

    Human embryonic stem (hES) cells can proliferate extensively in culture and can differentiate into representatives of all three embryonic germ layers in vitro and in vivo. The undifferentiated hES cells have now been cultured for more than 50 passages in vitro, yet maintain a normal karyotype. The hES cells express a series of specific surface antigens, as well as OCT-4 and human telomerase, proteins associated with a pluripotent and immortal phenotype. On differentiation, OCT-4 and human telomerase expression decreases with the emergence of a maturing population of cells. During hES cell differentiation, modulation of the expression of many genes has been evaluated using microarray analysis. To improve the ease, reproducibility, and scalability of hES culture, methods have been developed to propagate the cells in the absence of mouse embryonic cell feeders. hES cells maintained in culture using extracellular matrix factors together with mouse embryonic cell conditioned medium proliferate indefinitely while maintaining a normal karyotype, proliferation rate, and complement of undifferentiated cell markers. hES cells cultured without feeder layers retain their capacity to differentiate into cells of all three germ layers in vitro and in teratomas. The hES cells can also be genetically modified transiently or stably using both plasmid and viral gene transfer agents. These analyses and technological developments will aid in the realization of the full potential of hES cells for both research and therapeutic applications.

  5. The culture of human embryonic stem cells in microchannel perfusion bioreactors

    NASA Astrophysics Data System (ADS)

    Korin, Natanel; Bransky, Avishay; Dinnar, Uri; Levenberg, Shulamit

    2007-12-01

    The culture of human Embryonic Stem (ES) cells in microchannel bioreactors can be highly beneficial for ES cell biology studies and ES tissue engineering applications. In the present study we examine the use of Human Foreskin Fibroblasts (HFF) cells as feeder cells for human ES culture in a microchannel perfusion bioreactor. PDMS microchannels (depth:130 micron) were fabricated using conventional soft-lithography techniques. The channels were sterilized, coated with a human fibronectin solution and seeded with cells. Following a period of static incubation, culture medium was perfused through the channels at various flow rates and cell growth was monitored throughout the culture process. Mass transport and fluid mechanics models were used to evaluate the culture conditions (shear stress, oxygen levels within the micro-bioreactor as a function of the medium flow rate. The conditions for successful long-term culture (>7 days) of HFF under flow were established. Experiments with human embryonic stem cells cultured in microchannels show that the conditions essential to co-culture human ES cell on HFF cells under perfusion differ from the conditions necessary for HFF cell culture. Human ES cells were found to be highly sensitive to flow and culture conditions and did not grow under flow rates which were suitable for HFF long-term culture. Successful culture of undifferentiated human ES cell colonies in a perfusion micro-bioreactor is a basic step towards utilizing microfluidic techniques to explore stem cell biology.

  6. Totipotent embryonic stem cells arise in ground-state culture conditions.

    PubMed

    Morgani, Sophie M; Canham, Maurice A; Nichols, Jennifer; Sharov, Alexei A; Migueles, Rosa Portero; Ko, Minoru S H; Brickman, Joshua M

    2013-06-27

    Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.

  7. Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    PubMed Central

    Morgani, Sophie M.; Canham, Maurice A.; Nichols, Jennifer; Sharov, Alexei A.; Migueles, Rosa Portero; Ko, Minoru S.H.; Brickman, Joshua M.

    2013-01-01

    Summary Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants. PMID:23746443

  8. Effects of ethanol on embryonic and neonatal rat testes in organ cultures.

    PubMed

    Li, Hui; Kim, Kwan Hee

    2003-01-01

    Ethanol exposure in adult animals and humans has shown to elicit significant inhibitory effects on the function of male reproduction, but consequences of ethanol exposure on the embryonic and early postnatal testis development are not known. The current study investigated the effect of ethanol on embryonic and neonatal testis development using an organ culture technique. In embryonic day 13 (E13) testis organ cultures, ethanol had no effect on the testicular cord formation, the expression of Müllerian-inhibiting substance (MIS) in Sertoli cells or the number of gonocytes. Similarly, in the ethanol-treated embryonic day 18 (E18) testes, both the number of gonocytes and the expression of GATA-4 and MIS were similar to those from the control testes. In contrast, in postnatal day 3 (P3) testes, ethanol at concentrations of 150 and 200 mM significantly decreased the number of gonocytes without affecting the expression of GATA-4 and MIS in Sertoli cells. This effect was shown to be resulting from the enhanced apoptosis of gonocytes. In addition, ethanol abnormally activated retinoic acid receptor alpha (RARalpha), as indicated by increased nuclear localization of RARalpha with increasing doses of ethanol treatment. These observations suggest that the effect of ethanol on testis varies at different stages during embryonic and neonatal testis development. Furthermore, germ cells may be the main target for the action of ethanol on the early postnatal testis.

  9. Maintenance of human embryonic stem cells in animal serum- and feeder layer-free culture conditions.

    PubMed

    Amit, Michal; Itskovitz-Eldor, Joseph

    2006-01-01

    The availability of human embryonic stem cells (hESCs) reflects their outstanding potential for research areas such as human developmental biology, teratology, and cell-based therapies. To allow their continuous growth as undifferentiated cells, isolation and culturing were traditionally conducted on mouse embryonic fibroblast feeder layers, using medium supplemented with fetal bovine serum. However, these conditions allow possible exposure of the cells to animal pathogens. Because both research and future clinical application require an animal-free and well-defined culture system for hESCs, these conventional conditions would prevent the use of hESCs in human therapy. This chapter describes optional culture conditions based on either animal-free or feeder-free culture methods for hESCs.

  10. Effect of passage number on electrophoretic mobility distributions of cultured human embryonic kidney cells

    NASA Technical Reports Server (NTRS)

    Kunze, M. E.

    1985-01-01

    A systematic investigation was undertaken to characterize population shifts that occur in cultured human embryonic kidney cells as a function of passage number in vitro after original explantation. This approach to cell population shift analysis follows the suggestion of Mehreshi, Klein and Revesz that perturbed cell populations can be characterized by electrophoretic mobility distributions if they contain subpopulations with different electrophoretic mobilities. It was shown that this is the case with early passage cultured human embryo cells.

  11. Proteome analysis of chicken embryonic gonads: identification of major proteins from cultured gonadal primordial germ cells.

    PubMed

    Han, Beom Ku; Kim, Jin Nam; Shin, Ji Hye; Kim, Jin-Kyoo; Jo, Do Hyun; Kim, Heebal; Han, Jae Yong

    2005-12-01

    The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGCs) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 days of incubation, and the gPGCs were cultured in vitro until colony formed. After 7-10 days in culture, gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of this type will serve as an important reference for germ cell biology and transgenic research.

  12. Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture.

    PubMed

    Cossu, G; Eusebi, F; Grassi, F; Wanke, E

    1987-09-01

    The expression and the physiological properties of acetylcholine receptors (AChRs) of mononucleated myogenic cells, isolated from either embryonic or adult muscle of the mouse, have been investigated using the gigaohm seal patch-clamp technique in combination with immunocytochemistry (with an anti-myosin antibody) and alpha-bungarotoxin binding techniques. Undifferentiated (myosin-negative) embryonic myoblasts, grown either in mass culture or under clonal conditions, were found to be unresponsive to ACh and did not bind alpha-bungarotoxin. On the contrary, undifferentiated satellite cells (from adult muscle) exhibited channels activated by ACh and alpha-bungarotoxin binding sites similar to those observed in differentiated (myosin-positive) embryonic myoblasts and myotubes. Two classes of ACh-activated channels with different opening frequencies were identified. The major class of channels had a conductance of about 42 pS and mean open time of 3.1-8.2 msec. The minor class of channels had smaller conductance (about 17 pS) and similar open time. During differentiation, the conductance of the two channels did not change significantly, while channel lifetime became shorter in myotubes derived from satellite cells but not in myotubes derived from embryonic myoblasts. The relative proportion of small over large channels was significantly larger in embryonic than in adult myogenic cells.

  13. Ghrelin accelerates synapse formation and activity development in cultured cortical networks

    PubMed Central

    2014-01-01

    Background While ghrelin was initially related to appetite stimulation and growth hormone secretion, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of those processes is related to synaptic efficacy and plasticity. Previous studies have shown that ghrelin not only stimulates synapse formation in cultured cortical neurons and hippocampal slices, but also alters some of the electrophysiological properties of neurons in the hypothalamus, amygdala and other subcortical areas. However, direct evidence for ghrelin’s ability to modulate the activity in cortical neurons is not available yet. In this study, we investigated the effect of acylated ghrelin on the development of the activity level and activity patterns in cortical neurons, in relation to its effect on synaptogenesis. Additionally, we quantitatively evaluated the expression of the receptor for acylated ghrelin – growth hormone secretagogue receptor-1a (GHSR-1a) during development. Results We performed electrophysiology and immunohistochemistry on dissociated cortical cultures from neonates, treated chronically with acylated ghrelin. On average 76 ± 4.6% of the cortical neurons expressed GHSR-1a. Synapse density was found to be much higher in ghrelin treated cultures than in controls across all age groups (1, 2 or 3 weeks). In all cultures (control and ghrelin treated), network activity gradually increased until it reached a maximum after approximately 3 weeks, followed by a slight decrease towards a plateau. During early developmental stages (1–2 weeks), the activity was much higher in ghrelin treated cultures and consequently, they reached the plateau value almost a week earlier than controls. Conclusions Acylated ghrelin leads to earlier network formation and activation in cultured cortical neuronal networks, the latter being a possibly consequence of accelerated synaptogenesis. PMID:24742241

  14. Comparison of three embryo culture methods for derivation of human embryonic stem cells from discarded embryos.

    PubMed

    Liu, Ying; Li, Yang; Hwang, Andrew; Wang, Shu-yu; Jia, Chan-wei; Yu, Lan; Li, Jian

    2011-06-01

    Human embryonic stem cells (hESC) are self-renewing and pluripotent cells that hold great promise. Our objective was to compare the effect of three different embryo culture methods for derivation of human embryonic stem cells from discarded embryos. A prospective and randomized trial was conducted using 381 discarded human embryos at days 2-3 postfertilization in Beijing Obstetrics and Gynecology Hospital IVF center. After removal of the zona pellucida, discarded human embryos were cultured by three different methods as multiple embryo aggregates, single embryo, and blastomeres. Outgrowth of embryos and hESC derivation were observed. The outgrowth rate of embryos cultured as multiple embryo aggregates was higher than that of those cultured as single embryos or blastomeres (p < 0.05). Three propagating hESC lines were derived from poor quality day 2-3 postfertilization nonblastocyst embryos cultured as multiple embryo aggregates. Derived hESC lines expressed hESC-specific markers of pluripotency and had normal diploid karyotype. The cells were able to form derivatives of all three germ layers in vivo as teratomas. Our results demonstrate that culturing these discarded embryos as multiple embryo aggregates was more profitable for outgrowth and derivation of ESC line than culturing these as single embryo or blastomeres.

  15. Mouse embryonic stem cell-derived cardiac myocytes in a cell culture dish.

    PubMed

    Glass, Carley; Singla, Reetu; Arora, Anshu; Singla, Dinender K

    2015-01-01

    Embryonic stem (ES) cells are pluripotent stem cells capable of self-renewal and have broad differentiation potential yielding cell types from all three germ layers. In the absence of differentiation inhibitory factors, when cultured in suspension, ES cells spontaneously differentiate and form three-dimensional cell aggregates termed embryoid bodies (EBs). Although various methods exist for the generation of EBs, the hanging drop method offers reproducibility and homogeneity from a predetermined number of ES cells. Herein, we describe the in vitro differentiation of mouse embryonic stem cells into cardiac myocytes using the hanging drop method and immunocytochemistry to identify cardiomyogenic differentiation. In brief, ES cells, placed in droplets on the lid of culture dishes following a 2-day incubation, yield embryoid bodies, which are resuspended and plated. 1-2 weeks following plating of the EBs, spontaneous beating areas can be observed and staining for specific cardiac markers can be achieved.

  16. The role of preparation technique, culture media and incubation time for embryonation of Heterakis gallinarum eggs.

    PubMed

    Püllen, U; Cheat, Sophal; Moors, E; Gauly, M

    2008-01-01

    The importance of preparation technique, culture media and incubation time in the embryonation of the infective egg stages of the intestinal nematode parasite Heterakis gallinarum was studied. Mature H. gallinarum worms were isolated from the caeca of infected chickens and separated by sex. In a first experiment intact female worms were kept for the development of their eggs in four different media (0.5% formalin, 2% formalin, 0.1 N sulphuric acid, 0.1% potassium dichromate) and incubated under constant temperature (20-22 degrees C) for 2, 4, 6 or 8 weeks. Afterwards the body of the worms were ruptured and the numbers of unembryonated and embryonated eggs were determined using a McMaster egg counting chamber, and the percentage of embryonated eggs was calculated. After 8 weeks of incubation in 0.5% formalin, 0.1 N sulphuric acid or 0.1% potassium dichromate 27.6%, 26.7% and 29.4% of the eggs, respectively, embryonated into third stage larvae (p > 0.05). In contrast, incubation in 2% formalin resulted in an embryonation of 18.6% only (p < 0.05). In a second experiment H. gallinarum eggs were directly harvested from worm uteri and cultivated afterwards in different media (2% formalin, 0.1 N sulphuric acid, 0.1% potassium dichromate) at 20 to 22 degrees C for 6 weeks. An incubation of isolated eggs in 2.0% formalin or 0.1% potassium dichromate during 6 weeks resulted in a significantly higher percentage of embryonation in comparison to the incubation of intact worms (first experiment). The results suggest that preparation technique, media and time of incubation has an essential influence on the development rate of H. gallinarum eggs.

  17. Electrophoretic mobilities of cultured human embryonic kidney cells in various buffers

    NASA Technical Reports Server (NTRS)

    1985-01-01

    Data on the electrophoretic mobility distributions of cells in the new D-1 buffer and the interlaboratory standardization of urokinase assay methods are presented. A table of cell strains and recent data on cell dispersal methods are also included. It was decided that glycerol in A-1 electrophoretic mobility data on cultured human embryonic kidney cells subjected to electrophoresis in this buffer. The buffer composition is presented.

  18. A novel approach for studying the temporal modulation of embryonic skeletal development using organotypic bone cultures and microcomputed tomography.

    PubMed

    Kanczler, Janos M; Smith, Emma L; Roberts, Carol A; Oreffo, Richard O C

    2012-10-01

    Understanding the structural development of embryonic bone in a three dimensional framework is fundamental to developing new strategies for the recapitulation of bone tissue in latter life. We present an innovative combined approach of an organotypic embryonic femur culture model, microcomputed tomography (μCT) and immunohistochemistry to examine the development and modulation of the three dimensional structures of the developing embryonic femur. Isolated embryonic chick femurs were organotypic (air/liquid interface) cultured for 10 days in either basal, chondrogenic, or osteogenic supplemented culture conditions. The growth development and modulating effects of basal, chondrogenic, or osteogenic culture media of the embryonic chick femurs was investigated using μCT, immunohistochemistry, and histology. The growth and development of noncultured embryonic chick femur stages E10, E11, E12, E13, E15, and E17 were very closely correlated with increased morphometric indices of bone formation as determined by μCT. After 10 days in the organotpyic culture set up, the early aged femurs (E10 and E11) demonstrated a dramatic response to the chondrogenic or osteogenic culture conditions compared to the basal cultured femurs as determined by a change in μCT morphometric indices and modified expression of chondrogenic and osteogenic markers. Although the later aged femurs (E12 and E13) increased in size and structure after 10 days organotpypic culture, the effects of the osteogenic and chondrogenic organotypic cultures on these femurs were not significantly altered compared to basal conditions. We have demonstrated that the embryonic chick femur organotpyic culture model combined with the μCT and immunohistochemical analysis can provide an integral methodology for investigating the modulation of bone development in an ex vivo culture setting. Hence, these interdisciplinary techniques of μCT and whole organ bone cultures will enable us to delineate some of the temporal

  19. Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system

    PubMed Central

    Abraham, Sheena; Sheridan, Steven D.; Laurent, Louise C.; Albert, Kelsey; Stubban, Christopher; Ulitsky, Igor; Miller, Bradley; Loring, Jeanne F.; Rao, Raj R.

    2010-01-01

    We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols. PMID:20117095

  20. Robust, microfabricated culture devices with improved control over the soluble microenvironment for the culture of embryonic stem cells.

    PubMed

    Macown, Rhys J; Veraitch, Farlan S; Szita, Nicolas

    2014-06-01

    The commercial use of stem cells continues to be constrained by the difficulty and high cost of developing efficient and reliable production protocols. The use of microfabricated systems combines good control over the cellular microenvironment with reduced use of resources in process optimization. Our previously reported microfabricated culture device was shown to be suitable for the culture of embryonic stem cells but required improvements to robustness, ease of use, and dissolved gas control. In this report, we describe a number of improvements to the design of the microfabricated system to significantly improve the control over shear stress and soluble factors, particularly dissolved oxygen. These control improvements are investigated by finite element modeling. Design improvements also make the system easier to use and improve the robustness. The culture device could be applied to the optimization of pluripotent stem cell growth and differentiation, as well as the development of monitoring and control strategies and improved culture systems at various scales.

  1. Robust, microfabricated culture devices with improved control over the soluble microenvironment for the culture of embryonic stem cells

    PubMed Central

    Macown, Rhys J; Veraitch, Farlan S; Szita, Nicolas

    2014-01-01

    The commercial use of stem cells continues to be constrained by the difficulty and high cost of developing efficient and reliable production protocols. The use of microfabricated systems combines good control over the cellular microenvironment with reduced use of resources in process optimization. Our previously reported microfabricated culture device was shown to be suitable for the culture of embryonic stem cells but required improvements to robustness, ease of use, and dissolved gas control. In this report, we describe a number of improvements to the design of the microfabricated system to significantly improve the control over shear stress and soluble factors, particularly dissolved oxygen. These control improvements are investigated by finite element modeling. Design improvements also make the system easier to use and improve the robustness. The culture device could be applied to the optimization of pluripotent stem cell growth and differentiation, as well as the development of monitoring and control strategies and improved culture systems at various scales. PMID:24677785

  2. Sulfite triggers sustained calcium overload in cultured cortical neurons via a redox-dependent mechanism.

    PubMed

    Wang, Xiao; Cao, Hui; Guan, Xin-Lei; Long, Li-Hong; Hu, Zhuang-Li; Ni, Lan; Wang, Fang; Chen, Jian-Guo; Wu, Peng-Fei

    2016-09-06

    Sulfite is a compound commonly used as preservative in foods and pharmaceuticals. Many studies have examined the neurotoxicity of sulfite, but its effect on neuronal calcium homeostasis has not yet been reported. Here, we observed the effect of sulfite on the cytosolic free calcium concentration ([Ca(2+)]i) in cultured cortical neurons using Fura-2/AM based calcium imaging technique. Sulfite (250-1000μM) caused a sustained increase in [Ca(2+)]i in the neurons via a dose-dependent manner. In Ca(2+)-free solution, sulfite failed to increase [Ca(2+)]i. After the depletion of the intracellular calcium store, the effect of sulfite on the [Ca(2+)]i was largely abolished. Pharmacological inhibition of phospholipase C (PLC)-inositol 1,4,5-triphosphate (IP3) signaling pathway blocked sulfite-induced increase of [Ca(2+)]i. Interestingly, antioxidants such as trolox and dithiothreitol, abolished the increase of [Ca(2+)]i induced by sulfite. Exposure to sulfite triggered generation of sulfur- and oxygen-centered free radicals in neurons and increased oxidative stress both in the cultured cortical neurons and the prefrontal cortex of rats. Furthemore, sulfite decreased cell viability in cultured cortical neurons via a calcium-dependent manner. Thus, our current study suggests that the redox-dependent calcium overload triggered by sulfite in cortical neuronsmay be involved in its neurotoxicity.

  3. Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation.

    PubMed

    Gokhale, Paul J; Au-Young, Janice K; Dadi, SriVidya; Keys, David N; Harrison, Neil J; Jones, Mark; Soneji, Shamit; Enver, Tariq; Sherlock, Jon K; Andrews, Peter W

    2015-01-01

    We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.

  4. Scalable Culture and Cryopreservation of Human Embryonic Stem Cells on Microcarriers

    PubMed Central

    Nie, Ying; Bergendahl, Veit; Hei, Derek J.; Jones, Jeffrey M.; Palecek, Sean P.

    2009-01-01

    As a result of their pluripotency and potential for unlimited self-renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large-scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor-intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel-coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF-microcarriers was less than that on MEF-plates, the doubling time of hESCs on Matrigel-microcarriers was indistinguishable from that of hESCs expanded on Matrigel-coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier-based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. PMID:19197994

  5. Secreted factors from ventral telencephalon induce the differentiation of GABAergic neurons in cortical cultures.

    PubMed

    Trinh, H-h; Reid, J; Shin, E; Liapi, A; Parnavelas, J G; Nadarajah, B

    2006-12-01

    It is widely believed that the pyramidal cells and interneurons of the cerebral cortex are distinct in their origin, lineage and genetic make up. In view of these findings, the current thesis is that the phenotype determination of cortical neurons is primarily directed by genetic mechanisms. Using in vitro assays, the present study demonstrates that secreted factors from ganglionic eminence (GE) of the ventral telencephalon have the potency to induce the differentiation of a subset of cortical neurons towards gamma-aminobutyric acid (GABA)ergic lineage. Characterization of cortical cultures that were exposed to medium derived from GE illustrated a significant increase in the number of GABA-, calretinin- and calbindin-positive neurons. Calcium imaging together with pharmacological studies showed that the application of exogenous medium significantly elevated the intracellular calcium transients in cortical neurons through the activation of ionotropic glutamate receptors. The increase in GABA+ neurons appeared to be associated with the elevated calcium activity; treatment with blockers specific for glutamate receptors abolished both the synchronized transients and reduced the differentiation of GABAergic neurons. Such studies demonstrate that although intrinsic mechanisms determine the fate of cortical interneurons, extrinsic factors have the potency to influence their neurochemical differentiation and contribute towards their molecular diversity.

  6. Morphology of human embryonic kidney cells in culture after space flight

    NASA Technical Reports Server (NTRS)

    Todd, P.; Kunze, M. E.; Williams, K.; Morrison, D. R.; Lewis, M. L.; Barlow, G. H.

    1985-01-01

    The ability of human embyronic kidney cells to differentiate into small epithelioid, large epithelioid, domed, and fenestrated morphological cell types following space flight is examined. Kidney cells exposed to 1 day at 1 g, then 1 day in orbit, and a 12 minute passage through the electrophoretic separator are compared with control cultures. The data reveal that 70 percent of small epithelioid, 16 percent of large epithelioid, 9 percent of dome-forming, and 5 percent of fenestrated cells formed in the space exposed cells; the distributions correlate well with control data. The formation of domed cells from cells cultured from low electrophoretic mobility fractions and small epithelioid cells from high mobility fractions is unaffected by space flight conditions. It is concluded that storage under microgravity conditions does not influence the morphological differentiation of human embryonic kidney cells in low-passage culture.

  7. Purothionin from wheat endosperm reversibly blocks myogenic differentiation of chick embryonic muscle cells in culture

    SciTech Connect

    Kyu Bong Kwak; Young Sup Lee; Se Won Suh; Doo Bong Ha; Chin Ha Chung ); Chin Sang Chung )

    1989-08-01

    Purothionin from wheat endosperm is a cysteine-rich, basic polypeptide of about 5,000 Da, which modifies membrane permeability of cultured mammalian cells. This peptide was found to block fusion of chick embryonic muscle cells in culture but allows proliferation and alignment. A purothionin concentration of 6 {mu}m/ml was necessary for the complete prevention of myotube formation. Under similar conditions, incorporation of ({sup 35}S) methionine occurred normally but the synthesis of muscle-specific proteins including creatin kinase and acetylcholine receptor was strongly inhibited. In addition, purothionin blocked the uptake of {sup 86}Rb{sup +}, immediately after its addition to the cultured myoblasts. These results suggest that purothionin exerts its regulatory effect on the transition from proliferative to differentiative myoblasts by interfering with membrane permeability or intercellular contact and recognition, which are necessary for the initiation of muscle differentiation.

  8. TOPICAL REVIEW: Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

    NASA Astrophysics Data System (ADS)

    Amranul Haque, Md; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

    2010-02-01

    Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

  9. Controlled respiratory gas delivery to embryonic renal epithelial explants in perfusion culture.

    PubMed

    Strehl, R; Schumacher, K; Minuth, Will W

    2004-01-01

    During generation of artificial tissues high levels of oxygen are usually available whereas after implantation into a recipient's body the implant is not vascularized immediately, which leads to low oxygen partial pressures within the implanted tissue. Under these conditions cells will experience an oxygen shortage, contrasting with the abundance of oxygen during culture. It is uncertain whether tissues can be trained to tolerate such an acute hypoxic situation so that nonphysiological stress reactions and tissue necrosis can be avoided. To investigate the effects of varying oxygen levels on embryonic renal tissue in vitro we have been developing a model system combining continuous medium renewal with the ability to control levels of oxygen and carbon dioxide by gas equilibration through gas-permeable tubing. Renal embryonic tissue from neonatal rabbit was cultured in serum-free Iscove's modified Dulbecco's medium at 45, 90, 115, and 160 mmHg oxygen partial pressure for 14 days under continuous medium exchange in such a setup. After a 14-day culture period tissue sections were analyzed by cell biological methods and compared with fresh tissue histology. Surprisingly, embryonic renal explants survive and maintain good morphology for 14 days under all O(2) conditions tested. Expression of cytokeratin 19 within the established epithelium remains unchanged, indicating a structurally intact tissue. However, Na/K-ATPase is clearly downregulated under low O(2) conditions, whereas COX-2 expression increases drastically. An antiparallel effect of decreased O(2) concentrations on glycoprotein expression can be demonstrated with the lectin Dolichos biflorus agglutinin. Scanning electron microscopy reveals oxygen-dependent changes in cellular surface differentiation of developed collecting duct epithelium.

  10. Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems.

    PubMed

    Desai, Nina; Rambhia, Pooja; Gishto, Arsela

    2015-02-22

    Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers (ectoderm, endoderm and mesoderm). Harnessing the power of these pluripotent stem cells could potentially offer new therapeutic treatment options for a variety of medical conditions. Since the initial derivation of hESC lines in 1998, tremendous headway has been made in better understanding stem cell biology and culture requirements for maintenance of pluripotency. The approval of the first clinical trials of hESC cells for treatment of spinal cord injury and macular degeneration in 2010 marked the beginning of a new era in regenerative medicine. Yet it was clearly recognized that the clinical utility of hESC transplantation was still limited by several challenges. One of the most immediate issues has been the exposure of stem cells to animal pathogens, during hESC derivation and during in vitro propagation. Initial culture protocols used co-culture with inactivated mouse fibroblast feeder (MEF) or human feeder layers with fetal bovine serum or alternatively serum replacement proteins to support stem cell proliferation. Most hESC lines currently in use have been exposed to animal products, thus carrying the risk of xeno-transmitted infections and immune reaction. This mini review provides a historic perspective on human embryonic stem cell culture and the evolution of new culture models. We highlight the challenges and advances being made towards the development of xeno-free culture systems suitable for therapeutic applications.

  11. Concentration-dependent gene expression responses to flusilazole in embryonic stem cell differentiation cultures

    SciTech Connect

    Dartel, Dorien A.M. van; Pennings, Jeroen L.A.; Fonteyne, Liset J.J. de la; Brauers, Karen J.J.; Claessen, Sandra; Delft, Joost H. van; Kleinjans, Jos C.S.; Piersma, Aldert H.

    2011-03-01

    The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 {mu}M) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.

  12. The Effects of Simulated Micro-gravity on Cultured Chicken Embryonic Chondrocytes

    NASA Astrophysics Data System (ADS)

    Li, X.; Zhang, X.; Yang, S.; Li, S.; Peidong, J.; Lin, Z.

    T he effects of simulated microgravity on the microtubular system, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration, mitochondrial ATP synthase activity and oligomycin inhibition rate of cultured chicken embryonic chondrocytes were studied with a clinostat. The microtubular content decreased. The extracellualr matrix decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly. There was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. No significant changes happened in the mitochondrial ATP synthase activity and oligomycin inhibition rate. The possible mechanisms about them were discussed.

  13. Patenting, morality and human embryonic stem cell science: bioethics and cultural politics in Europe.

    PubMed

    Salter, Brian

    2007-05-01

    As the recent experience of the European Patent Office graphically demonstrates, there is an inherent political tension between the individual ownership rights necessary for the operation of an international market in human embryonic stem cell science and the communal values of the many cultures in which such markets operate. This report examines the basis of the conflict between patenting and morality at national and international levels, the manifestation of those tensions in European patenting policy, and the contribution of bioethics to the attempt by European institutions to develop a governance response.

  14. Regulation of embryonic size in early mouse development in vitro culture system.

    PubMed

    Hisaki, Tomoka; Kawai, Ikuma; Sugiura, Koji; Naito, Kunihiko; Kano, Kiyoshi

    2014-08-01

    Mammals self-regulate their body size throughout development. In the uterus, embryos are properly regulated to be a specific size at birth. Previously, size and cell number in aggregated embryos, which were made from two or more morulae, and half embryos, which were halved at the 2-cell stage, have been analysed in vivo in preimplantation and post-implantation development in mice. Here, we examined whether or not the mouse embryo has the capacity to self-regulate growth using an in vitro culture system. To elucidate embryonic histology, cells were counted in aggregated or half embryos in comparison with control embryos. Both double- and triple-aggregated embryos contained more cells than did control embryos during all culture periods, and the relative growth ratios showed no growth inhibition in an in vitro culture system. Meanwhile, half embryos contained fewer cells than control embryos, but the number grew throughout the culture period. Our data suggest that the growth of aggregated embryos is not affected and continues in an in vitro culture system. On the other hand, the growth of half embryos accelerates and continues in an in vitro culture system. This situation, in turn, implied that post-implantation mouse embryos might have some potential to regulate their own growth and size as seen by using an in vitro culture system without uterus factors. In conclusion, our results indicated that embryos have some ways in which to regulate their own size in mouse early development.

  15. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    PubMed

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  16. COMMUNICATION: Folate and S-adenosylmethionine modulate synaptic activity in cultured cortical neurons: acute differential impact on normal and apolipoprotein-deficient mice

    NASA Astrophysics Data System (ADS)

    Serra, Michael; Chan, Amy; Dubey, Maya; Gilman, Vladimir; Shea, Thomas B.

    2008-12-01

    Folate deficiency is accompanied by a decline in the cognitive neurotransmitter acetylcholine and a decline in cognitive performance in mice lacking apolipoprotein E (ApoE-/- mice), a low-density lipoprotein that regulates aspects of lipid metabolism. One direct consequence of folate deficiency is a decline in S-adenosylmethionine (SAM). Since dietary SAM supplementation maintains acetylcholine levels and cognitive performance in the absence of folate, we examined herein the impact of folate and SAM on neuronal synaptic activity. Embryonic cortical neurons from mice expressing or lacking ApoE (ApoE+/+ or -/-, respectively) were cultured for 1 month on multi-electrode arrays, and signaling was recorded. ApoE+/+ cultures displayed significantly more frequent spontaneous signals than ApoE-/- cultures. Supplementation with 166 µm SAM (not normally present in culture medium) increased signal frequency and decreased signal amplitude in ApoE+/+ cultures. SAM also increased the frequency of tightly clustered signal bursts. Folate deprivation reversibly reduced signal frequency in ApoE+/+ cultures; SAM supplementation maintained signal frequency despite folate deprivation. These findings support the importance of dietary supplementation with folate and SAM on neuronal health. Supplementation with 166 µm SAM did not alter signaling in ApoE-/- cultures, which may be a reflection of the reduced SAM levels in ApoE-/- mice. The differential impact of SAM on ApoE+/+ and -/- neurons underscores the combined impact of nutritional and genetic deficiencies on neuronal homeostasis.

  17. Locust bean gum as an alternative polymeric coating for embryonic stem cell culture.

    PubMed

    Perestrelo, Ana Rubina; Grenha, Ana; Rosa da Costa, Ana M; Belo, José António

    2014-07-01

    Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro

  18. Differentiation of cartilaginous anlage in entire embryonic mouse limbs cultured in a rotating bioreactor.

    NASA Astrophysics Data System (ADS)

    Duke, P.; Oakley, C.; Montufar-Solis, D.

    The embryonic mammalian limb is sensitive both in vivo and in vitro to changes in gravitational force. Hypergravity of centrifugation and microgravity of space decreased size of elements due to precocious or delayed chondrogenesis respectively. In recapitulating spaceflight experiments, premetatarsals were cultured in suspension in a low stress, low sheer rotating bioreactor, and found to be shorter than those cultured in standard culture dishes, and cartilage development was delayed. This study only measured length of the metatarsals, and did not account for possible changes in width and/or in form of the skeletal elements. Shorter cartilage elements in limbbuds cultured in the bioreactor may be due to the ability of the system to reproduce a more in vivo 3D shape than traditional organ cultures. Tissues subjected to traditional organ cultures become flattened by their own weight, attachment to the filter, and restrictions imposed by nutrient diffusion. The purpose of the current experiment was to determine if entire limb buds could be successfully cultured in the bioreactor, and to compare the effects on 3D shape with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were placed either in the bioreactor, in Trowell culture, or fixed as controls. Limbbuds were cultured for six days, fixed, and processed either as whole mounts or embedded for histology. Qualitative analysis revealed that the Trowell culture specimens were flattened, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections of limbbuds from both types of cultures had excellent cartilage differentiation, with apparently more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Morphometric quantitation of the cartilaginous elements for comparisons of the two culture systems was complicated due to some limb buds fusing together during culture. This problem was especially noticeable in the younger limbs, and

  19. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    PubMed

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  20. Cyclooxygenase-2 contributes to VX-induced cell death in cultured cortical neurons.

    PubMed

    Tenn, Catherine C; Weiss, M Tracy; Beaup, Claire; Peinnequin, Andre; Wang, Yushan; Dorandeu, Frederic

    2012-04-05

    The link between cell death and increased cyclooxygenases-2 (COX-2) activity has not been clearly established. In this study, we examined whether COX-2 activation contributed to the mechanism of neurotoxicity produced by an organophosphorous nerve agent in cultured rat cortical neurons. Exposure of neuronal cells to the nerve agent, VX resulted in an increase in COX enzyme activity in the culture media. A concentration dependent increase in the activity levels of COX-2 enzyme was observed while there was little to no effect on COX-1. In addition, COX-2 mRNA and protein levels increased several hours post-VX exposure. Pre-treatment of the cortical cells with the COX-2 selective inhibitor, NS 398 resulted in a decrease in both the enzyme activity and prostaglandin (PGE(2) and PGF(2α)) release, as well as in a reduction in cell death. These findings indicate that the increase in COX-2 activity may contribute to the mechanism of VX-induced neurotoxicity in cultured rat cortical neuron.

  1. Enhancement of synaptic transmission induced by BDNF in cultured cortical neurons

    NASA Astrophysics Data System (ADS)

    He, Jun; Gong, Hui; Zeng, Shaoqun; Li, Yanling; Luo, Qingming

    2005-03-01

    Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, BDNF has been reported to exert an acute potentiation of synaptic activity and are critically involved in long-term potentiation (LTP). We found that BDNF rapidly induced potentiation of synaptic activity and an increase in the intracellular Ca2+ concentration in cultured cortical neurons. Within minutes of BDNF application to cultured cortical neurons, spontaneous firing rate was dramatically increased as were the frequency and amplitude of excitatory spontaneous postsynaptic currents (EPSCs). Fura-2 recordings showed that BDNF acutely elicited an increase in intracellular calcium concentration ([Ca2+]c). This effect was partially dependent on [Ca2+]o; The BDNF-induced increase in [Ca2+]c can not be completely blocked by Ca2+-free solution. It was completely blocked by K252a and partially blocked by Cd2+ and TTX. The results demonstrate that BDNF can enhances synaptic transmission and that this effect is accompanied by a rise in [Ca2+]c that requires two route: the release of Ca2+ from intracellular calcium stores and influx of extracellular Ca2+ through voltage-dependent Ca2+ channels in cultured cortical neurons.

  2. The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system.

    PubMed

    Ahir, Bhavesh K; Pratten, Margaret K

    2016-07-01

    Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell-to-cell communication pathways, resulting in an inability to co-ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 μm (P < 0.05), while not affecting cell viability and total protein, in the embryonic chick cardiomyocyte micromass culture system. The effects of caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in-cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non-cytotoxic concentrations (≥2000 μm), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 μm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd.

  3. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    NASA Technical Reports Server (NTRS)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

  4. Controlling Bursting in Cortical Cultures with Closed-Loop Multi-Electrode Stimulation

    PubMed Central

    Wagenaar, Daniel A.; Madhavan, Radhika; Pine, Jerome; Potter, Steve M.

    2009-01-01

    One of the major modes of activity of high-density cultures of dissociated neurons is globally synchronized bursting. Unlike in vivo, neuronal ensembles in culture maintain activity patterns dominated by global bursts for the lifetime of the culture (up to 2 years). We hypothesize that persistence of bursting is caused by a lack of input from other brain areas. To study this hypothesis, we grew small but dense monolayer cultures of cortical neurons and glia from rat embryos on multi-electrode arrays and used electrical stimulation to substitute for afferents. We quantified the burstiness of the firing of the cultures in spontaneous activity and during several stimulation protocols. Although slow stimulation through individual electrodes increased burstiness as a result of burst entrainment, rapid stimulation reduced burstiness. Distributing stimuli across several electrodes, as well as continuously fine-tuning stimulus strength with closed-loop feedback, greatly enhanced burst control. We conclude that externally applied electrical stimulation can substitute for natural inputs to cortical neuronal ensembles in transforming burst-dominated activity to dispersed spiking, more reminiscent of the awake cortex in vivo. This nonpharmacological method of controlling bursts will be a critical tool for exploring the information processing capacities of neuronal ensembles in vitro and has potential applications for the treatment of epilepsy. PMID:15659605

  5. Development of Ascorbate Transporters in Brain Cortical Capillary Endothelial Cells in Culture

    PubMed Central

    Qiao, Huan; May, James M.

    2008-01-01

    Ascorbic acid in its reduced form is not transported across the capillary endothelial cell blood-brain barrier. This is thought to be due to absence of the SVCT2, a specific transporter for ascorbate. To assess this directly we prepared primary cultures of mouse cortical microvascular endothelial cells. When still in the capillaries, these cells did not express the SVCT2 protein as assessed by immunocytochemistry and by immunoblotting. However, during several days in culture, they developed SVCT2 expression and showed ascorbate transport rates comparable to those in immortalized endothelial cell lines. SVCT2 expression was inversely proportional to cell density, was enhanced by culture at low physiologic plasma ascorbate concentrations, was inhibited by ascorbate concentrations expected in the brain interstitium, and was stimulated by cobalt ions. Expression of the SVCT2 was associated with ascorbate-dependent maturation and release of type IV collagen by the cells in culture. Although the SVCT2 is induced by culture of cortical capillary endothelial cells, its absence in vivo remains perplexing, given the need for intracellular ascorbate to facilitate type IV collagen maturation and release by endothelial cells. PMID:18394593

  6. Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.

    PubMed

    Odintsova, Nelly A; Ageenko, Natalya V; Kipryushina, Yulia O; Maiorova, Mariia A; Boroda, Andrey V

    2015-08-01

    This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%).

  7. Formation of electrical coupling between embryonic Xenopus muscle cells in culture.

    PubMed Central

    Chow, I; Poo, M M

    1984-01-01

    Electrical coupling between embryonic Xenopus muscle cells in 1-5 day old cultures was studied after isolated cells were manipulated into contact for various periods. The coupling was examined by measuring the electrotonic spread of acetylcholine (ACh)-induced membrane depolarizations or of potential changes induced by intracellular current injection. In 1 day old culture, cells developed coupling rapidly after contact. Strong coupling was observed within 20 min after contact was made. The rate of coupling formation was age dependent. The percentage of cell pairs that established detectable coupling within 30 min of contact decreased from 66% in 1 day culture to 0% in 5 day culture. Older cells, when put into contact for prolonged periods, developed substantial coupling, suggesting that the age of the culture affects the rate of coupling formation rather than the final extent of coupling. Pre-treatment of older cells with colchicine, metabolic inhibitors, Ca2+ and Mg2+-free saline, or trypsin significantly increased the rate of coupling formation to a level close to that of younger cells. This suggests that the reduced rate of coupling was not due to a lack of membrane precursors for the intercellular channels, but was probably due to the appearance of extramembranous constraints for the channel assembly. PMID:6699773

  8. Germ-cell culture conditions facilitate the production of mouse embryonic stem cells.

    PubMed

    Ramos-Ibeas, Priscila; Pericuesta, Eva; Fernández-González, Raúl; Gutiérrez-Adán, Alfonso; Ramírez, Miguel Ángel

    2014-09-01

    The derivation of embryonic stem-cell (ESC) lines from blastocysts is a very inefficient process. Murine ESCs are thought to arise from epiblast cells that are already predisposed to a primordial-germ-cell fate. During the process of ESC derivation from B6D2 F1 hybrid mice, if we first culture the embryo from the two-cell stage in medium supplemented with LIF, we improve the quality of the blastocyst. When the blastocyst is then cultured in a germ-line stem-cell culture medium (GSCm), we are able to more efficiently (28.3%) obtain quality ESC lines that have a normal karyotype, proper degree of chimerism, and exhibit germ-line transmission when microinjected into blastocysts. Although germ-cell-specific genes were expressed in all culture medium conditions, GSCm did not shift the transcriptome towards germ-cell specification. A correlation was further observed between ESC derivation efficiency and the expression of some imprinted genes and retrotransposable elements. In conclusion, the combination of LIF supplementation followed by culture in GSCm establishes a higher efficiency method for ESC derivation.

  9. Benzodiazepine receptor turnover in embryonic chick brain and spinal cord cell cultures

    SciTech Connect

    Borden, L.A.

    1985-01-01

    The turnover (synthesis and degradation) of the benzodiazepine receptor (BZD-R) in embryonic chick brain and spinal cord cell cultures was monitored using flunitrazepam (GNZM) as a photoaffinity label. To measure BZD-R appearance, intact cell cultures were incubated with 100 nM RNZM and irradiated with ultraviolet light; this process, referred to as photoinactivation, resulted in a 75% decrease in the subsequent reversible binding of 5 nM (/sup 3/H)FNZM. Following photoinactivation, (/sup 3/H)FNZM binding sites reappeared at a rate of 6 +/- 1.5%/hour (n = 7) in brain cultures and at 8%/hour (n = 2) in spinal cord cultures. Reappearance reflects de novo receptors synthesis. To examine the degradation of existing receptors, cultures were photolabeled with 5 nM (/sup 3/H)FNZM, washed, and then the decrease in cell-associated radioactivity, or the efflux of radioactivity into the medium, was monitored. The released radioactivity did not comigrate with authentic FNZM on thin-layer-chromatographs, indicating that release did not represent dissociation of ligand from the photolabeled receptor. The BZD-R appears to be degraded by an energy-dependent, non-lysosomal pathway. These experiments represent the first direct examination of the turnover of a neurotransmitter receptor localized to the central nervous system; this information will be valuable in elucidating the mechanisms by which receptor levels are altered following chronic drug treatment.

  10. Ghrelin stimulates synaptic formation in cultured cortical networks in a dose-dependent manner.

    PubMed

    Stoyanova, Irina I; le Feber, Joost; Rutten, Wim L C

    2013-09-10

    Ghrelin was initially related to appetite stimulation and growth hormone secretion. However, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of these processes is related to synaptic efficacy and plasticity. Previous studies indicated that ghrelin has an excitatory effect on neuronal activity, and stimulates synaptic plasticity in vivo. Plasticity in the adult brain occurs in many different ways, including changes in synapse morphology and number. Therefore, we used in vitro neuronal cultures to investigate how ghrelin affects synaptogenesis. We used dissociated cortical cultures of newborn rats, chronically treated with different doses of ghrelin (0.5, 1, 1.5 and 2μM). After one-, two-, three- or four weeks cultures were immunostained for the presynaptic marker synaptophysin. In parallel, additional groups of non-treated cultures were immunostained for detection of ghrelin receptor (GHSR1). During development, GHSR1was increasingly expressed in all type of neurons, as well as the synaptophysin. Synaptic density depended on ghrelin concentration, and was much higher than in controls in all age groups. In conclusion, ghrelin leads to earlier network formation in dissociated cortical networks and an increase in number of synapses. The effect is probably mediated by GHSR1. These findings suggest that ghrelin may provide a novel therapeutic strategy for the treatment of disorders related to synaptic impairment.

  11. Network Bursting Dynamics in Excitatory Cortical Neuron Cultures Results from the Combination of Different Adaptive Mechanism

    PubMed Central

    Masquelier, Timothée; Deco, Gustavo

    2013-01-01

    In the brain, synchronization among cells of an assembly is a common phenomenon, and thought to be functionally relevant. Here we used an in vitro experimental model of cell assemblies, cortical cultures, combined with numerical simulations of a spiking neural network (SNN) to investigate how and why spontaneous synchronization occurs. In order to deal with excitation only, we pharmacologically blocked GABAAergic transmission using bicuculline. Synchronous events in cortical cultures tend to involve almost every cell and to display relatively constant durations. We have thus named these “network spikes” (NS). The inter-NS-intervals (INSIs) proved to be a more interesting phenomenon. In most cortical cultures NSs typically come in series or bursts (“bursts of NSs”, BNS), with short (∼1 s) INSIs and separated by long silent intervals (tens of s), which leads to bimodal INSI distributions. This suggests that a facilitating mechanism is at work, presumably short-term synaptic facilitation, as well as two fatigue mechanisms: one with a short timescale, presumably short-term synaptic depression, and another one with a longer timescale, presumably cellular adaptation. We thus incorporated these three mechanisms into the SNN, which, indeed, produced realistic BNSs. Next, we systematically varied the recurrent excitation for various adaptation timescales. Strong excitability led to frequent, quasi-periodic BNSs (CV∼0), and weak excitability led to rare BNSs, approaching a Poisson process (CV∼1). Experimental cultures appear to operate within an intermediate weakly-synchronized regime (CV∼0.5), with an adaptation timescale in the 2–8 s range, and well described by a Poisson-with-refractory-period model. Taken together, our results demonstrate that the INSI statistics are indeed informative: they allowed us to infer the mechanisms at work, and many parameters that we cannot access experimentally. PMID:24146781

  12. Network bursting dynamics in excitatory cortical neuron cultures results from the combination of different adaptive mechanisms.

    PubMed

    Masquelier, Timothée; Deco, Gustavo

    2013-01-01

    In the brain, synchronization among cells of an assembly is a common phenomenon, and thought to be functionally relevant. Here we used an in vitro experimental model of cell assemblies, cortical cultures, combined with numerical simulations of a spiking neural network (SNN) to investigate how and why spontaneous synchronization occurs. In order to deal with excitation only, we pharmacologically blocked GABAAergic transmission using bicuculline. Synchronous events in cortical cultures tend to involve almost every cell and to display relatively constant durations. We have thus named these "network spikes" (NS). The inter-NS-intervals (INSIs) proved to be a more interesting phenomenon. In most cortical cultures NSs typically come in series or bursts ("bursts of NSs", BNS), with short (~1 s) INSIs and separated by long silent intervals (tens of s), which leads to bimodal INSI distributions. This suggests that a facilitating mechanism is at work, presumably short-term synaptic facilitation, as well as two fatigue mechanisms: one with a short timescale, presumably short-term synaptic depression, and another one with a longer timescale, presumably cellular adaptation. We thus incorporated these three mechanisms into the SNN, which, indeed, produced realistic BNSs. Next, we systematically varied the recurrent excitation for various adaptation timescales. Strong excitability led to frequent, quasi-periodic BNSs (CV~0), and weak excitability led to rare BNSs, approaching a Poisson process (CV~1). Experimental cultures appear to operate within an intermediate weakly-synchronized regime (CV~0.5), with an adaptation timescale in the 2-8 s range, and well described by a Poisson-with-refractory-period model. Taken together, our results demonstrate that the INSI statistics are indeed informative: they allowed us to infer the mechanisms at work, and many parameters that we cannot access experimentally.

  13. Ethanol-induced oxidative stress precedes mitochondrially mediated apoptotic death of cultured fetal cortical neurons.

    PubMed

    Ramachandran, Vinitha; Watts, Lora Talley; Maffi, Shivani Kaushal; Chen, Juanjuan; Schenker, Steven; Henderson, George

    2003-11-15

    In utero ethanol exposure elicits apoptotic cell death in the fetal brain, and this may be mediated by oxidative stress. Our studies utilize cultured fetal rat cortical neurons and illustrate that ethanol elicits a rapid onset of oxidative stress, which culminates in mitochondrially mediated apoptotic cell death. Cells exposed to ethanol (2.5 mg/ml) remained attached to their polylysine matrix during a 24-hr exposure, but they exhibited distinct signs of oxidative stress, decreased viability, and apoptosis. Confocal microscopy of live cortical neurons pretreated with dichlorodihydrofluorescein diacetate demonstrated an increase in reactive oxygen species (ROS) within 5 min of ethanol exposure. The levels of ROS further increased by 58% within 1 hr (P <.05) and by 82% within 2 hr (P <.05), accompanied by increases of mitochondrial 4-hydroxynonenal (HNE). These early events were followed by decreased trypan blue exclusion of 10% to 32% (P <.05) at the 6- to 24-hr time points, respectively. This culminates in apoptotic death, with increases of Annexin V binding of 43%, 89%, 123%, and 238%, at 2, 6, 12, and 24 hr of ethanol treatment, respectively, as well as DNA fragmentation increases of 50% and 65% by 12 and 24 hr, respectively. Release of cytochrome c by mitochondria increased by 53% at 6 hr of exposure (P <.05), concomitant with activation of caspase 3 (52% at 12 hr, P <.05). Pretreatment with N-acetylcysteine increased cellular glutathione and prevented apoptosis. These studies provide a time line illustrating that oxidative stress and formation of a proapoptotic lipid peroxidation product, HNE, precede a cascade of mitochondrially mediated events in cultured fetal cortical neurons, culminating in apoptotic death. The prevention of apoptosis by augmentation of glutathione stores also strongly supports a role for oxidative stress in ethanol-mediated apoptotic death of fetal cortical neurons.

  14. Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

    PubMed Central

    MacRae, Vicky E.; Farquharson, Colin

    2016-01-01

    The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth. PMID:28060328

  15. Emergence of flat cells from glia in stationary cultures of embryonic chick neural retina.

    PubMed

    Moyer, M; Bullrich, F; Sheffield, J B

    1990-11-01

    When embryonic retina is dissociated into a single cell suspension and maintained in stationary culture, a population of flat cells is found on the culture dish. We have carried out a morphologic and immunologic study of the emergence of this population in vitro. Ten- and fourteen-day-old chick embryo retinas were dissociated with trypsin, seeded on glass cover slips for various times, and prepared for scanning electron microscopy (SEM) and immunofluorescence (IF) for Vimentin, an intermediate filament protein. SEM indicates that the characteristic flat cell morphology is initiated in some cells in as little as 30 min after the start of the culture. Not all of the cells that attach flatten. As incubation proceeds, small clusters of cells that had formed in suspension attach to the substrate, and flat cells emerge from them. The flattened cells are positive for Vimentin by IF within 10 min of attachment. The percent of fluorescent cells found on the substrate is constant during the time in culture. This suggests that flat cells do not attach first, followed by neural cells, but that the neural cells and flat cells attach to the dish at the same rate. When aggregates that had formed in suspension attach to the substrate, they are anchored by flat cells that migrate out of the aggregate. Since Vimentin appears in the cultured cells within 10 min, it is unlikely that it has been newly synthesized. Thus, the same cells that contained Vimentin in the retina now express it as flat cells. This supports the hypothesis that flat cells derive from the same cells in the retina that give rise to Müller cells. We have also observed the emergence of a population of cells with short (0.5 micron) microvilli that appear within 8 h of culture. They seem to be a distinct subpopulation of the cells on the upper portion of attached clusters.

  16. Cardiac stem cell niche, MMP9, and culture and differentiation of embryonic stem cells.

    PubMed

    Mishra, Paras Kumar; Kuypers, Nicholas John; Singh, Shree Ram; Leiberh, Noel Diaz; Chavali, Vishalakshi; Tyagi, Suresh C

    2013-01-01

    Embryonic stem cells (ESC) are totipotent, self-renewing, and clonogenic, having potential to differentiate into a wide variety of cell types. Due to regenerative capability, it has tremendous potential for treating myocardial infarction (death of myocardial tissue) and type 1 diabetes (death of pancreatic beta cells). Understanding the components regulating ESC differentiation is the key to unlock the regenerative potential of ESC-based therapies. Both the stiffness of extracellular matrix (ECM) and surrounding niche/microenvironment play pivotal roles in ESC differentiation. Matrix metalloproteinase-9 (MMP9) induces fibrosis that causes stiffness of the ECM and impairs differentiation of cardiac stem cells into cardiomyocytes. Here, we describe the method of ESC culture and differentiation, and the expression of MMP9 and its inhibitor, tissue inhibitor of metalloproteinase-4 (TIMP4) in differentiating ESC.

  17. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    PubMed

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  18. Minimum neuron density for synchronized bursts in a rat cortical culture on multi-electrode arrays.

    PubMed

    Ito, D; Tamate, H; Nagayama, M; Uchida, T; Kudoh, S N; Gohara, K

    2010-11-24

    To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat cortical neurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbecco's Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

  19. Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors.

    PubMed

    Cormier, Jaymi T; zur Nieden, Nicole I; Rancourt, Derrick E; Kallos, Michael S

    2006-11-01

    Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors.

  20. Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability

    PubMed Central

    Jacobs, Kurt; Zambelli, Filippo; Mertzanidou, Afroditi; Smolders, Ilse; Geens, Mieke; Nguyen, Ha Thi; Barbé, Lise; Sermon, Karen; Spits, Claudia

    2016-01-01

    Summary Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem. PMID:26923824

  1. A Novel Core-Shell Microcapsule for Encapsulation and 3D Culture of Embryonic Stem Cells

    PubMed Central

    Zhang, Wujie; Zhao, Shuting; Rao, Wei; Snyder, Jedidiah; Choi, Jung K.; Wang, Jifu; Khan, Iftheker A.; Saleh, Navid B.; Mohler, Peter J.; Yu, Jianhua; Hund, Thomas J.; Tang, Chuanbing; He, Xiaoming

    2013-01-01

    In this study, we report the preparation of a novel microcapsule of ~ 100 μm with a liquid (as compared to solid-like alginate hydrogel) core and an alginate-chitosan-alginate (ACA) shell for encapsulation and culture of embryonic stem (ES) cells in the miniaturized 3D space of the liquid core. Murine R1 ES cells cultured in the microcapsules were found to survive (> 90%) well and proliferate to form either a single aggregate of pluripotent cells or embryoid body (EB) of more differentiated cells in each microcapsule within 7 days, dependent on the culture medium used. This novel microcapsule technology allows massive production of the cell aggregates or EBs of uniform size and controllable pluripotency, which is important for the practical application of stem cell based therapy. Moreover, the semipermeable ACA shell was found to significantly reduce immunoglobulin G (IgG) binding to the encapsulated cells by up to 8.2 times, compared to non-encapsulated cardiac fibroblasts, mesenchymal stem cells, and ES cells. This reduction should minimize inflammatory and immune responses induced damage to the cells implanted in vivo becasue IgG binding is an important first step of the undesired host responses. Therefore, the ACA microcapsule with selective shell permeability should be of importance to advance the emerging cell-based medicine. PMID:23505611

  2. Short exposure to collagenase and coculture with mouse embryonic pancreas improve human dermal fibroblast culture.

    PubMed

    Pandamooz, Sareh; Hadipour, Abbas; Akhavan-Niaki, Haleh; Pourghasem, Mohsen; Abedian, Zeinab; Ardekani, Ali Motevallizadeh; Golpour, Monireh; Hassan, Zuhair Mohammad; Mostafazadeh, Amrollah

    2012-01-01

    The dermal fibroblast as a major component of connective tissue has attracted much attention in the past few years, and application of these very fast growing cells in several fields has been intensively studied. Isolating dermal fibroblasts is an appropriate way to expand these fast growing cells in vitro. Although using a dissociated fibroblast culture method is more convenient than skin explant culture, its enzymatic digestion is critical because a large number of cells can be lost over prolonged exposure to collagenase. This study was performed to increase the number of viable cells after digestion of fresh human foreskin of donors aged from 1 to 3 months with collagenase and also by to design a coculture system for resuscitation of the injured fibroblast. Our results demonstrate that we can maximize cell yield while maintaining cell viability by cutting the specimens into very small pieces (1-2 mm³) after removing the epidermal layer with dispase II and also by collecting released cells every 20 Min subsequent to digesting the dermal layer with collagenase. Moreover, our data strongly indicate that coculturing of isolated fibroblasts with embryonic pancreas explants can enhance the rate of proliferation in cultured fibroblasts.

  3. Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid

    PubMed Central

    Makoolati, Zohreh; Movahedin, Mansoureh; Forouzandeh-Moghadam, Mehdi

    2016-01-01

    An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0–5  μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh),  α6 integrin,  β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3  μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs. PMID:27834666

  4. Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens.

    PubMed

    Naito, Mitsuru; Harumi, Takashi; Kuwana, Takashi

    2015-02-01

    Production of germline chimaeric chickens by the transfer of cultured primordial germ cells (PGC) is a useful system for germline manipulation. A novel culture system was developed for chicken PGC isolated from embryonic blood. The isolated PGC were cultured on feeder cells derived from chicken embryonic fibroblast. The cultured PGC formed colonies and they proliferated about 300-times during the first 30 days. The cultured PGC retained the ability to migrate to recipient gonads and were also chicken VASA homologue (CVH)-positive. Female PGC were present in the mixed-sex PGC populations cultured for more than 90 days and gave rise to viable offspring efficiently via germline chimaeric chickens. Male cultured PGC were transferred to recipient embryos and produced putative chimaeric chickens. The DNA derived from the cultured PGC was detected in the sperm samples of male putative chimaeric chickens, but no donor derived offspring were obtained. Donor-derived offspring were also obtained from germline chimaeric chickens by the transfer of frozen-thawed cultured PGC. The culture method for PGC developed in the present study is useful for manipulation of the germline in chickens, such as preservation of genetic resources and gene transfer.

  5. [The features of postsynaptic currents in primary culture of rat cortical neurons].

    PubMed

    Sibarov, D A; Antonov, S M

    2013-06-01

    The generation features of postsynaptic currents were studied in primary culture of cortical neurons at 7-20 days in vitro (DIV). The use of specific blockers of postsynaptic ion channels after 10 DIV revealed all types of electrical activity found in adult cortex including miniature inhibitory (mIPSCs), excitatory (mEPSCs) and spontaneous giant excitatory currents and spikes. The frequency of mEPSCs increased exponentially from 7 to 20 DIV doubling every 2.2 days in parallel with changes in action potentials generation. The mEPSCs generated by NMDA and AMPA or by only AMPA receptor activation were found. The inhibition of NMDA receptors by magnesium ions or AP5 were shown to modulate the frequency and amplitude of mEPSCs, which differ primary culture from brain slices possibly because of the lack of glial control of synaptic transmission.

  6. Inhibition of microRNA 128 promotes excitability of cultured cortical neuronal networks

    PubMed Central

    McSweeney, K. Melodi; Gussow, Ayal B.; Bradrick, Shelton S.; Dugger, Sarah A.; Gelfman, Sahar; Wang, Quanli; Petrovski, Slavé; Frankel, Wayne N.; Boland, Michael J.; Goldstein, David B.

    2016-01-01

    Cultured neuronal networks monitored with microelectrode arrays (MEAs) have been used widely to evaluate pharmaceutical compounds for potential neurotoxic effects. A newer application of MEAs has been in the development of in vitro models of neurological disease. Here, we directly evaluated the utility of MEAs to recapitulate in vivo phenotypes of mature microRNA-128 (miR-128) deficiency, which causes fatal seizures in mice. We show that inhibition of miR-128 results in significantly increased neuronal activity in cultured neuronal networks derived from primary mouse cortical neurons. These results support the utility of MEAs in developing in vitro models of neuroexcitability disorders, such as epilepsy, and further suggest that MEAs provide an effective tool for the rapid identification of microRNAs that promote seizures when dysregulated. PMID:27516621

  7. Inhibition of microRNA 128 promotes excitability of cultured cortical neuronal networks.

    PubMed

    McSweeney, K Melodi; Gussow, Ayal B; Bradrick, Shelton S; Dugger, Sarah A; Gelfman, Sahar; Wang, Quanli; Petrovski, Slavé; Frankel, Wayne N; Boland, Michael J; Goldstein, David B

    2016-10-01

    Cultured neuronal networks monitored with microelectrode arrays (MEAs) have been used widely to evaluate pharmaceutical compounds for potential neurotoxic effects. A newer application of MEAs has been in the development of in vitro models of neurological disease. Here, we directly evaluated the utility of MEAs to recapitulate in vivo phenotypes of mature microRNA-128 (miR-128) deficiency, which causes fatal seizures in mice. We show that inhibition of miR-128 results in significantly increased neuronal activity in cultured neuronal networks derived from primary mouse cortical neurons. These results support the utility of MEAs in developing in vitro models of neuroexcitability disorders, such as epilepsy, and further suggest that MEAs provide an effective tool for the rapid identification of microRNAs that promote seizures when dysregulated.

  8. Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

    SciTech Connect

    Zeng, Xiang Jun; Yu, Shan Ping; Zhang, Like; Wei, Ling

    2010-07-01

    The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca{sup 2+} accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.

  9. Cerium oxide nanoparticles prevent apoptosis in primary cortical culture by stabilizing mitochondrial membrane potential.

    PubMed

    Arya, A; Sethy, N K; Das, M; Singh, S K; Das, A; Ujjain, S K; Sharma, R K; Sharma, M; Bhargava, K

    2014-07-01

    Cerium oxide nanoparticles (CNPs) of spherical shape have unique antioxidant capacity primarily due to alternating + 3 and + 4 oxidation states and crystal defects. Several studies revealed the protective efficacies of CNPs in cells and tissues against the oxidative damage. However, its effect on mitochondrial functioning, downstream effectors of radical burst and apoptosis remains unknown. In this study, we investigated whether CNPs treatment could protect the primary cortical cells from loss of mitochondrial membrane potential (Δψm) and Δψm-dependent cell death. CNPs with spherical morphology and size range 7-10 nm were synthesized and utilized at a concentration of 25 nM on primary neuronal culture challenged with 50 μM of hydrogen peroxide (H2O2). We showed that optimal dose of CNPs minimized ROS content of the cells and also curbed related surge in cellular calcium flux. Importantly, CNPs treatment prevented apoptotic loss of cell viability. Reduction in the apoptosis could be successfully attributed to the maintenance of Δψm and restoration of major redox equivalents NADH/NAD(+) ratio and cellular ATP. These findings, therefore, suggest possible route of CNPs protective efficacies in primary cortical culture.

  10. Morphogenesis of human embryonic stem cells into mature neurons under in vitro culture conditions

    PubMed Central

    Shroff, Geeta

    2016-01-01

    AIM To describe the morphogenesis of different neuronal cells from the human embryonic stem cell (hESC) line, SCT-N, under in vitro culture conditions. METHODS The directed neuronal cell line was produced from a single, spare, pre-implantation stage fertilized ovum that was obtained during a natural in vitro fertilization process. The hESCs were cultured and maintained as per our proprietary in-house technology in a Good Manufacturing Practice, Good Laboratory Practice and Good Tissue Practice compliant laboratory. The cell line was derived and incubated in aerobic conditions. The cells were examined daily under a phase contrast microscope for their growth and differentiation. RESULTS Different neural progenitor cells (NPCs) and differentiating neurons were observed under the culture conditions. Multipotent NPCs differentiated into all three types of nervous system cells, i.e., neurons, oligodendrocytes and astrocytes. Small projections resembling neurites or dendrites, and protrusion coming out of the cells, were observed. Differentiating cells were observed at day 18 to 20. The differentiating neurons, neuronal bodies, axons, and neuronal tissue were observed on day 21 and day 30 of the culture. On day 25 and day 30, prominent neurons, axons and neuronal tissue were observed under phase contrast microscopy. 4’, 6-diamidino-2-phenylindole staining also indicated the pattern of differentiating neurons, axonal structure and neuronal tissue. CONCLUSION This study describes the generation of different neuronal cells from an hESC line derived from biopsy of blastomeres at the two-cell cleavage stage from a discarded embryo. PMID:27909687

  11. Long-term culture of mouse embryonic stem cell-derived adherent neurospheres and functional neurons.

    PubMed

    Hayashi, Mirian A F; Guerreiro, Juliano R; Cassola, Antonio C; Lizier, Nelson F; Kerkis, Alexandre; Camargo, Antonio C M; Kerkis, Irina

    2010-12-01

    Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of

  12. Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

    PubMed

    Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz

    2009-03-01

    Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

  13. Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors.

    PubMed

    Stachelscheid, H; Wulf-Goldenberg, A; Eckert, K; Jensen, J; Edsbagge, J; Björquist, P; Rivero, M; Strehl, R; Jozefczuk, J; Prigione, A; Adjaye, J; Urbaniak, T; Bussmann, P; Zeilinger, K; Gerlach, J C

    2013-09-01

    Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models.

  14. Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

    NASA Technical Reports Server (NTRS)

    Chen, Silvia S.; Revoltella, Roberto P.; Papini, Sandra; Michelini, Monica; Fitzgerald, Wendy; Zimmerberg, Joshua; Margolis, Leonid

    2003-01-01

    In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

  15. The presence of cortical neurons in striatal-cortical co-cultures alters the effects of dopamine and BDNF on medium spiny neuron dendritic development

    PubMed Central

    Penrod, Rachel D.; Campagna, Justin; Panneck, Travis; Preese, Laura; Lanier, Lorene M.

    2015-01-01

    Medium spiny neurons (MSNs) are the major striatal neuron and receive synaptic input from both glutamatergic and dopaminergic afferents. These synapses are made on MSN dendritic spines, which undergo density and morphology changes in association with numerous disease and experience-dependent states. Despite wide interest in the structure and function of mature MSNs, relatively little is known about MSN development. Furthermore, most in vitro studies of MSN development have been done in simple striatal cultures that lack any type of non-autologous synaptic input, leaving open the question of how MSN development is affected by a complex environment that includes other types of neurons, glia, and accompanying secreted and cell-associated cues. Here we characterize the development of MSNs in striatal-cortical co-culture, including quantitative morphological analysis of dendritic arborization and spine development, describing progressive changes in density and morphology of developing spines. Overall, MSN growth is much more robust in the striatal-cortical co-culture compared to striatal mono-culture. Inclusion of dopamine (DA) in the co-culture further enhances MSN dendritic arborization and spine density, but the effects of DA on dendritic branching are only significant at later times in development. In contrast, exogenous Brain Derived Neurotrophic Factor (BDNF) has only a minimal effect on MSN development in the co-culture, but significantly enhances MSN dendritic arborization in striatal mono-culture. Importantly, inhibition of NMDA receptors in the co-culture significantly enhances the effect of exogenous BDNF, suggesting that the efficacy of BDNF depends on the cellular environment. Combined, these studies identify specific periods of MSN development that may be particularly sensitive to perturbation by external factors and demonstrate the importance of studying MSN development in a complex signaling environment. PMID:26257605

  16. Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

    PubMed

    Ziv, Omer; Zaritsky, Assaf; Yaffe, Yakey; Mutukula, Naresh; Edri, Reuven; Elkabetz, Yechiel

    2015-10-01

    Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

  17. Involvement of transglutaminase in myofibril assembly of chick embryonic myoblasts in culture

    PubMed Central

    1995-01-01

    Involvement of transglutaminase in myofibrillogenesis of chick embryonic myoblasts has been investigated in vitro. Both the activity and protein level of transglutaminase initially decreased to a minimal level at the time of burst of myoblast fusion but gradually increased thereafter. The localization of transglutaminase underwent a dramatic change from the whole cytoplasm in a diffuse pattern to the cross- striated sarcomeric A band, being strictly colocalized with the myosin thick filaments. For a brief period prior to the appearance of cross- striation, transglutaminase was localized in nonstriated filamental structures that coincided with the stress fiber-like structures. When 12-o-tetradecanoyl phorbol acetate was added to muscle cell cultures to induce the sequential disassembly of thin and thick filaments, transglutaminase was strictly colocalized with the myosin thick filaments even in the myosacs, of which most of the thin filaments were disrupted. Moreover, monodansylcadaverine, a competitive inhibitor of transglutaminase, reversibly inhibited the myofibril maturation. In addition, myosin heavy chain behaved as one of the potential intracellular substrates for transglutaminase. The cross-linked myosin complex constituted approximately 5% of the total Triton X-100- insoluble pool of myosin molecules in developing muscle cells, and its level was reduced to below 1% upon treatment with monodansylcadaverine. These results suggest that transglutaminase plays a crucial role in myofibrillogenesis of developing chick skeletal muscle. PMID:7657697

  18. Neuronal differentiation of PC12 and embryonic stem cells in two- and three-dimensional in vitro culture.

    PubMed

    Sadri, Soheil; Khazaei, Mozafar; Ghanbari, Ali; Khazaei, Mohammad Rasool; Shah, Palak

    2014-04-01

    The quality of neuronal differentiation and reduction in apoptosis that occurred in two-dimensional (2D) and three-dimensional (3D) culture conditions is compared. PC12 and embryonic stem cells are two commonly utilized cell lines for the study of neuronal regeneration. These cells were induced to neuronally differentiate by adding NGF and retinoic acid respectively. Total neurite length and expression of neuronal markers (MAP-2 and beta-tubulin) was assessed by morphometry and immunocytochemistry. Also, TUNEL assay was used to detect apoptosis. Upon exposure to a differentiation media in the 3D fibrin gel, PC12 and embryonic stem cells stopped dividing, had increased adhesion to the substratum, extended neurite processes and expressed neuronal markers. The same results, however, were not observedwith the 2D culture. Also, the apoptosis index performed by TUNEL a ss ay demonstrated a reduction in th e degree of apoptosis in the 3D culture compared to 2D culture. Fibrin matrix supports growth and n euronal differentiation of PC12 andembryonic stem cells. In addition, the 3D culture enhanced cellular resistance to apoptosis when compared to the 2D culture. It appears as if a 3D culture system may offer a better technique for future neuronal tissue engineering investigations.

  19. Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions

    PubMed Central

    Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

    2016-01-01

    Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies. PMID:27294211

  20. Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture.

    PubMed

    López, E; Arce, C; Oset-Gasque, M J; Cañadas, S; González, M P

    2006-03-15

    Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in cortical neurons in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a) depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action. This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular glutathione levels and induced catalase and SOD activation in these cells.

  1. Activity Changes Induced by Spatio-Temporally Correlated Stimuli in Cultured Cortical Networks

    NASA Astrophysics Data System (ADS)

    Takayama, Yuzo; Moriguchi, Hiroyuki; Jimbo, Yasuhiko

    Activity-dependent plasticity probably plays a key role in learning and memory in biological information processing systems. Though long-term potentiation and depression have been extensively studied in the filed of neuroscience, little is known on the mechanisms for integrating these modifications on network-wide activity changes. In this report, we studied effects of spatio-temporally correlated stimuli on the neuronal network activity. Rat cortical neurons were cultured on substrates with 64 embedded micro-electrodes and the evoked responses were extracellularly recorded and analyzed. We compared spatio-temporal patterns of the responses between before and after repetitive application of correlated stimuli. After the correlated stimuli, the networks showed significantly different responses from those in the initial states. The modified activity reflected structures of the repeatedly applied correlated stimuli. The results suggested that spatiotemporally correlated inputs systematically induced modification of synaptic strengths in neuronal networks, which could serve as an underlying mechanism of associative memory.

  2. Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

    PubMed Central

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-01-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells. PMID:24938227

  3. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    PubMed

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  4. Identification of prothymosin-α1, the necrosis–apoptosis switch molecule in cortical neuronal cultures

    PubMed Central

    Ueda, Hiroshi; Fujita, Ryousuke; Yoshida, Akira; Matsunaga, Hayato; Ueda, Mutsumi

    2007-01-01

    We initially identified a nuclear protein, prothymosin-α1 (ProTα), as a key protein inhibiting necrosis by subjecting conditioned media from serum-free cultures of cortical neurons to a few chromatography steps. ProTα inhibited necrosis of cultured neurons by preventing rapid loss of cellular adenosine triphosphate levels by reversing the decreased membrane localization of glucose transporters but caused apoptosis through up-regulation of proapoptotic Bcl2-family proteins. The apoptosis caused by ProTα was further inhibited by growth factors, including brain-derived neurotrophic factor. The ProTα-induced cell death mode switch from necrosis to apoptosis was also reproduced in experimental ischemia-reperfusion culture experiments, although the apoptosis level was markedly reduced, possibly because of the presence of growth factors in the reperfused serum. Knock down of PKCβII expression prevented this cell death mode switch. Collectively, these results suggest that ProTα is an extracellular signal protein that acts as a cell death mode switch and could be a promising candidate for preventing brain strokes with the help of known apoptosis inhibitors. PMID:17353361

  5. The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures.

    PubMed

    Chen, Yongmei; Swanson, Raymond A

    2003-03-01

    Cysteine availability is normally the rate-limiting factor in glutathione synthesis. How neurons obtain cysteine from extracellular space is not well established. Here we used mouse cortical neuron cultures to examine the role of the excitatory amino acid transporters (EAATs) in neuronal cysteine uptake. The cultured neurons expressed both EAAT2 and EAAT3. Cysteine uptake was predominantly (> 85%) Na+-dependent, with an apparent Km of 37 microm. Cysteine uptake was reduced by the EAAT substrates l-glutamate and l-aspartate and by synthetic EAAT inhibitors. The non-selective EAAT inhibitor threo-beta-hydroxyaspartate had a significantly greater maximal inhibitory effect than did the EAAT2-selective inhibitor, dihydrokainate, indicating uptake by both EAAT2 and EAAT3. Serine, a substrate of ASC uptake system, had negligible effects on cysteine uptake at 10-fold excess concentrations. To assess the functional importance of EAAT-mediated cysteine uptake in neuronal glutathione synthesis, cultures were treated with diethylmaleate to deplete glutathione, then incubated with cysteine in the presence or absence of EAAT inhibitors. Threo-beta-benzyloxyaspartate and the non-transportable inhibitor threo-beta-hydroxyaspartate both inhibited the cysteine-dependent glutathione synthesis. The findings suggest that neuronal EAAT activity can be a rate-limiting step for neuronal glutathione synthesis and that the primary function of EAATs expressed by neurons in vivo may be to transport cysteine.

  6. Effects of inorganic lead on the differentiation and growth of cortical neurons in culture.

    PubMed

    Kern, M; Audesirk, T; Audesirk, G

    1993-01-01

    Lead exposure has devastating effects on the developing nervous system, producing morphological, cognitive, and behavioral deficits. To elucidate some of the mechanisms of lead neurotoxicity, we have examined its effects on the differentiation of several types of cultured neurons. Previously, we reported the effects of inorganic lead on several parameters of growth and differentiation of E18 rat hippocampal neurons and two types of neuroblastoma cells cultured in medium with 2% fetal calf serum (FCS) (Audesirk et al., 1991). In the present study, we report the effects of concentrations of lead ranging from 1nM to 1 mM on the differentiation of hippocampal neurons cultured in medium containing 10% FCS. In addition, we investigated lead effects on neurons isolated from the motor cortex region of the E18 rat embryo. Cortical neurons were exposed to lead in concentrations ranging from 0.1 nM to 1 mM in medium with either 10% FCS or 2% FCS for 48 hr. The effects of lead tended to be multimodal. Neurite initiation, which is highly sensitive to neurotoxic compounds, was inhibited by lead at both high and low concentrations, with no effects at intermediate levels. Medium with 10% FCS enhanced certain growth parameters and tended to reduce the effects of lead. There was an overall consistency in the effects of lead on motor cortex and hippocampal neurons.

  7. Sphingosine-1-Phosphate Enhancement of Cortical Actomyosin Organization in Cultured Human Schlemm's Canal Endothelial Cell Monolayers

    PubMed Central

    Sumida, Grant M.

    2010-01-01

    Purpose. Perfusion of sphingosine-1-phosphate (S1P) in whole eye organ culture models decreases outflow facility, whereas S1P promotes stress fiber formation and contractility in cultured trabecular meshwork (TM) cells. Because of S1P's known effect of increasing barrier function in endothelial cells, the authors hypothesized that Schlemm's canal (SC) cells in culture respond to S1P by increasing actomyosin organization at the cell cortex. Methods. Using primary cultures of human SC cells, the authors determined S1P activation of the GTP-binding proteins, RhoA and Rac (1,2,3). Time- and dose-dependent myosin light chain (MLC) phosphorylation in response to S1P and total expression of MLC were determined. Immunocytochemistry after S1P treatment was used to monitor filamentous actin (F-actin) and phospho-MLC organization and the localization of β-catenin, a component of adherens junctions. TM and human umbilical vein endothelial cell monolayers were used as controls. Results. S1P (1 μM) activated RhoA and Rac after 5- and 30-minute treatments. S1P increased MLC phosphorylation with a similar time- and dose-dependent response in SC (EC50 = 0.83 μM) compared with TM (EC50 = 1.33 μM), though MLC expression was significantly greater in TM. In response to 1 μM S1P treatment, phospho-MLC concentrated in the SC cell periphery, coincident with cortical actin assembly and recruitment of β-catenin to the cell periphery. Conclusions. Results obtained in this study support the hypothesis that S1P increases actomyosin organization at the SC cell cortex and promotes intercellular junctions at the level of the inner wall of SC to increase transendothelial resistance and in part explains the S1P-induced decrease of outflow facility in organ culture. PMID:20592229

  8. Embryonic tongue morphogenesis in an organ culture model of mouse mandibular arches: blocking Sonic hedgehog signaling leads to microglossia.

    PubMed

    Torii, Daisuke; Soeno, Yuuichi; Fujita, Kazuya; Sato, Kaori; Aoba, Takaaki; Taya, Yuji

    2016-01-01

    Mouse tongue development is initiated with the formation of lateral lingual swellings just before fusion between the mediodorsal surfaces of the mandibular arches at around embryonic day 11.0. Here, we investigated the role of Sonic hedgehog (Shh) signaling in embryonic mouse tongue morphogenesis. For this, we used an organ culture model of the mandibular arches from mouse embryos at embryonic day 10.5. When the Shh signaling inhibitor jervine was added to the culture medium for 24-96 h, the formation of lateral lingual swellings and subsequent epithelial invagination into the mesenchyme were impaired markedly, leading to a hypoplastic tongue with an incomplete oral sulcus. Notably, jervine treatment reduced the proliferation of non-myogenic mesenchymal cells at the onset of forming the lateral lingual swellings, whereas it did not affect the proliferation and differentiation of a myogenic cell lineage, which created a cell community at the central circumferential region of the lateral lingual swellings as seen in vivo and in control cultures lacking the inhibitor. Thus, epithelium-derived Shh signaling stimulates the proliferation of non-myogenic mesenchymal cells essential for forming lateral lingual swellings and contributes to epithelial invagination into the mesenchyme during early tongue development.

  9. Toxicity of organotin compounds in primary cultures of rat cortical astrocytes.

    PubMed

    Röhl, C; Gülden, M; Seibert, H

    2001-01-01

    The neurotoxic organotin compounds trimethyl (TMT) and triethyltin (TET) are known to induce astrogliosis in vivo, which is indicated by an increased synthesis of glial fibrillary acidic protein (GFAP) in astrocytes. In contrast, tributyltin (TBT) does not induce astrogliosis. The aim of this study was to investigate whether trialkyltin derivatives can induce an increased GFAP synthesis in astrocyte cultures in the absence of neurons and whether differences between the action of TMT, TET, and TBT can be detected. Primary cultures of rat cortical astrocytes from 2-day-old rats were grown in 96-well plates until confluency and then exposed to various concentrations of TMT, TET, and TBT for 40 h. Effects on basal cell functions were measured by colorimetric determination of cell protein contents and by assessment of viability by means of the MTT assay. An indirect sandwich ELISA for 96-well plates was used for quantitative measurements of the GFAP content of the cells. All three compounds induced a concentration-dependent cytotoxicity indicated by parallel decreases of protein contents and MTT reduction. Half-maximum cytotoxic concentrations were 3 micromol/L (TBT), 30 micromol/L (TET), and 800 micromol/L (TMT). Cellular GFAP contents were reduced in parallel to cytotoxic action but no increase in GFAP expression at subcytotoxic concentrations could be observed. Thus, the astrocytes were not able to respond to TMT or TET exposure by an increased synthesis of GFAP in the absence of neuronal signals.

  10. Cultured Cortical Neurons Can Perform Blind Source Separation According to the Free-Energy Principle

    PubMed Central

    Isomura, Takuya; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2015-01-01

    Blind source separation is the computation underlying the cocktail party effect––a partygoer can distinguish a particular talker’s voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes’ principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico) demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle. PMID:26690814

  11. Panaxydol and panaxynol protect cultured cortical neurons against Abeta25-35-induced toxicity.

    PubMed

    Nie, Bao-Ming; Jiang, Xiao-Yan; Cai, Jin-Xian; Fu, Sai-Li; Yang, Li-Min; Lin, Lin; Hang, Qin; Lu, Pei-Lua; Lu, Yang

    2008-04-01

    Amyloid beta protein (Abeta), the central constituent of senile plaques in Alzheimer's disease (AD), is known to exert toxic effects on cultured neurons. In the present study, the protective effect of panaxydol (PND) and panaxynol (PNN) on Abeta25-35-induced neuronal apoptosis and potential mechanisms were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN prior to 10 microM Abeta25-35 exposure resulted significantly in elevation of cell survival determined by MTT assay, TUNEL/Hoechst staining and western blot. Furthermore, a marked increase in calcium influx and intracellular free radical generation was found after Abeta25-35 exposure, which could be almost completely reversed by pretreatment of PND or PNN. PND and PNN could also alleviate Abeta25-35-induced early-stage neuronal degeneration. These results indicated that inhibition of calcium influx and free radical generation is a mechanism of the anti-apoptotic action of PND and PNN. Since Abeta plays critical roles in the pathogenesis of AD, these findings raise the possibility that PND and PNN reduce neurodegeneration in AD.

  12. The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking.

    PubMed

    Englund, Mikael C O; Caisander, Gunilla; Noaksson, Karin; Emanuelsson, Katarina; Lundin, Kersti; Bergh, Christina; Hansson, Charles; Semb, Henrik; Strehl, Raimund; Hyllner, Johan

    2010-04-01

    This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.

  13. A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture.

    PubMed

    Meng, Guoliang; Liu, Shiying; Krawetz, Roman; Chan, Michael; Chernos, Judy; Rancourt, Derrick E

    2008-06-01

    Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.

  14. Isolation and culture of neural crest cells from embryonic murine neural tube.

    PubMed

    Pfaltzgraff, Elise R; Mundell, Nathan A; Labosky, Patricia A

    2012-06-02

    The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from

  15. Rosuvastatin induces delayed preconditioning against oxygen-glucose deprivation in cultured cortical neurons.

    PubMed

    Domoki, Ferenc; Kis, Béla; Gáspár, Tamás; Snipes, James A; Parks, John S; Bari, Ferenc; Busija, David W

    2009-01-01

    We tested whether rosuvastatin (RST) protected against oxygen-glucose deprivation (OGD)-induced cell death in primary rat cortical neuronal cultures. OGD reduced neuronal viability (%naive controls, mean +/- SE, n = 24-96, P < 0.05) to 44 +/- 1%, but 3-day pretreatment with RST (5 microM) increased survival to 82 +/- 2% (P < 0.05). One-day RST treatment was not protective. RST-induced neuroprotection was abolished by mevalonate or geranylgeranyl pyrophosphate (GGPP), but not by cholesterol coapplication. Furthermore, RST-induced decreases in neuronal cholesterol levels were abolished by mevalonate but not by GGPP. Reactive oxygen species (ROS) levels were reduced in RST-preconditioned neurons after OGD, and this effect was also reversed by both mevalonate and GGPP. These data suggested that GGPP, but not cholesterol depletion, were responsible for the induction of neuroprotection. Therefore, we tested whether 3-day treatments with perillic acid, a nonspecific inhibitor of both geranylgeranyl transferase (GGT) GGT 1 and Rab GGT, and the GGT 1-specific inhibitor GGTI-286 would reproduce the effects of RST. Perillic acid, but not GGTI-286, elicited robust neuronal preconditioning against OGD. RST, GGTI-286, and perillic acid all decreased mitochondrial membrane potential and lactate dehydrogenase activity in the cultured neurons, but only RST and perillic acid reduced neuronal ATP and membrane Rab3a protein levels. In conclusion, RST preconditions cultured neurons against OGD via depletion of GGPP, leading to decreased geranylgeranylation of proteins that are probably not isoprenylated by GGT 1. Reduced neuronal ATP levels and ROS production after OGD may be directly involved in the mechanism of neuroprotection.

  16. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

    PubMed

    Miller-Pinsler, Lutfiya; Wells, Peter G

    2015-09-15

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p<0.001). Maternal pretreatment of C57BL/6 WT dams with 50kU/kg PEG-catalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p<0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p<0.01), and trends for reduced anterior neuropore closure, turning and crown-rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p<0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies.

  17. Astrocytes regulate developmental changes in the chloride ion gradient of embryonic rat ventral spinal cord neurons in culture

    PubMed Central

    Li, Yong-Xin; Schaffner, Anne E; Walton, Marc K; Barker, Jeffery L

    1998-01-01

    Embryonic rat ventral spinal cord neurons were dissociated at day 15 and grown on: (i) poly-D-lysine (PDL); (ii) a confluent monolayer of type I astrocytes; or (iii) PDL in astrocyte-conditioned medium (ACM) to examine the influence of astroglia on the regulation of GABAA receptor/Cl− channel properties. Potentiometric oxonol dye recordings of intact cells indicated that embryonic neurons were uniformly depolarized by muscimol. The depolarizing effects disappeared in cells dissociated during the early postnatal period and recovered in culture for 24 h. Similar recordings using the calcium-imaging dye fura-2 AM revealed that GABA or muscimol triggered a sustained rise in cytosolic Ca2+ (Cac2+) in embryonic neurons that was dependent on extracellular Ca2+, blocked by bicuculline and nifedipine and sensitive to changes in extracellular chloride. The incidence and amplitude of the Ca2+ response decreased with time in vitro and was accelerated in neurons cultured on astrocytes compared with those on PDL. Perforated patch-clamp recordings revealed that GABA depolarized neurons in a Cl−-dependent and bicuculline-sensitive manner. Both the resting membrane potential and the GABA equilibrium potential became more hyperpolarized with time in vitro. Astrocytes and ACM accelerated the transformation of GABAergic potential responses from depolarizing to hyperpolarizing. The change occurred over the first 4 days in co-culture or in ACM but took more than 2 weeks in neurons cultured on PDL alone. The intrinsic, elementary properties of GABAA receptor/Cl− channels including open time and unitary conductance changed independently of the presence of astrocytes or ACM. Mean open time of the dominant kinetic component decreased and conductance increased with time in vitro. In sum, astrocytes accelerate the developmental change in the Cl− ion gradient extrinsic to GABAA receptor/Cl− channels, which is critical for triggering Ca2+ entry, without influencing parallel changes in

  18. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

    SciTech Connect

    Miller-Pinsler, Lutfiya; Wells, Peter G.

    2015-09-15

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat{sup b}/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • Et

  19. Interaction of electrically evoked activity with intrinsic dynamics of cultured cortical networks with and without functional fast GABAergic synaptic transmission

    PubMed Central

    Baltz, Thomas; Voigt, Thomas

    2015-01-01

    The modulation of neuronal activity by means of electrical stimulation is a successful therapeutic approach for patients suffering from a variety of central nervous system disorders. Prototypic networks formed by cultured cortical neurons represent an important model system to gain general insights in the input–output relationships of neuronal tissue. These networks undergo a multitude of developmental changes during their maturation, such as the excitatory–inhibitory shift of the neurotransmitter GABA. Very few studies have addressed how the output properties to a given stimulus change with ongoing development. Here, we investigate input–output relationships of cultured cortical networks by probing cultures with and without functional GABAAergic synaptic transmission with a set of stimulation paradigms at various stages of maturation. On the cellular level, low stimulation rates (<15 Hz) led to reliable neuronal responses; higher rates were increasingly ineffective. Similarly, on the network level, lowest stimulation rates (<0.1 Hz) lead to maximal output rates at all ages, indicating a network wide refractory period after each stimulus. In cultures aged 3 weeks and older, a gradual recovery of the network excitability within tens of milliseconds was in contrast to an abrupt recovery after about 5 s in cultures with absent GABAAergic synaptic transmission. In these GABA deficient cultures evoked responses were prolonged and had multiple discharges. Furthermore, the network excitability changed periodically, with a very slow spontaneous change of the overall network activity in the minute range, which was not observed in cultures with absent GABAAergic synaptic transmission. The electrically evoked activity of cultured cortical networks, therefore, is governed by at least two potentially interacting mechanisms: A refractory period in the order of a few seconds and a very slow GABA dependent oscillation of the network excitability. PMID:26236196

  20. Latency-Related Development of Functional Connections in Cultured Cortical Networks

    PubMed Central

    le Feber, J.; van Pelt, J.; Rutten, W.L.C.

    2009-01-01

    Abstract To study plasticity, we cultured cortical networks on multielectrode arrays, enabling simultaneous recording from multiple neurons. We used conditional firing probabilities to describe functional network connections by their strength and latency. These are abstract representations of neuronal pathways and may arise from direct pathways between two neurons or from a common input. Functional connections based on direct pathways should reflect synaptic properties. Therefore, we searched for long-term potentiation (this mechanism occurs in vivo when presynaptic action potentials precede postsynaptic ones with interspike intervals up to ∼20 ms) in vitro. To investigate if the strength of functional connections showed a similar latency-related development, we selected periods of monotonously increasing or decreasing strength. We observed increased incidence of short latencies (5–30 ms) during strengthening, whereas these rarely occurred during weakening. Furthermore, we saw an increased incidence of 40–65 ms latencies during weakening. Conversely, functional connections tended to strengthen in periods with short latency, whereas strengthening was significantly less than average during long latency. Our data suggest that functional connections contain information about synaptic connections, that conditional firing probability analysis is sensitive enough to detect it and that a substantial fraction of all functional connections is based on direct pathways. PMID:19383487

  1. Anesthetic actions of thiopental remain largely unaffected during cholinergic overstimulation in cultured cortical networks.

    PubMed

    Weimer, Isabel; Worek, Franz; Seeger, Thomas; Thiermann, Horst; Grasshoff, Christian; Antkowiak, Bernd; Balk, Monika

    2016-02-26

    In case of military or terrorist use of organophosphorus (OP) compounds victims are likely to suffer from not only intoxication but physical trauma as well. Appropriate emergency care may therefore include general anesthesia to allow life-saving surgical intervention. Since there is evidence that drug potency and efficacy of several anesthetics are attenuated by high concentrations of acetylcholine in the CNS, this study was designed to evaluate the anesthetic actions of thiopental during cholinergic overstimulation. Making use of organotypic slice cultures derived from the mouse neocortex, drug effects were assessed by extracellular voltage recordings of network activity at basal cholinergic tone and during simulated cholinergic crisis (high cholinergic tone). The latter was achieved by inhibition of acetylcholinesterases via soman and an ambient acetylcholine concentration of 10μM. The induction of cholinergic crisis in vitro increased the network activity of cortical neurons significantly. Surprisingly, differences in network activity between basal and high cholinergic tone became less pronounced with rising concentrations of thiopental and drug potency and efficacy were almost equivalent. These results clearly distinguish thiopental from previously tested general anesthetics and make it a promising candidate for in vivo studies to identify suitable anesthetics for victims of OP intoxication.

  2. Lycopene Prevents Amyloid [Beta]-Induced Mitochondrial Oxidative Stress and Dysfunctions in Cultured Rat Cortical Neurons.

    PubMed

    Qu, Mingyue; Jiang, Zheng; Liao, Yuanxiang; Song, Zhenyao; Nan, Xinzhong

    2016-06-01

    Brains affected by Alzheimer's disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between β-amyloid (Aβ) and mitochondrial dysfunction has been established in cellular models of AD. We observed previously that lycopene, a member of the carotenoid family of phytochemicals, could counteract neuronal apoptosis and cell damage induced by Aβ and other neurotoxic substances, and that this neuroprotective action somehow involved the mitochondria. The present study aims to investigate the effects of lycopene on mitochondria in cultured rat cortical neurons exposed to Aβ. It was found that lycopene attenuated Aβ-induced oxidative stress, as evidenced by the decreased intracellular reactive oxygen species generation and mitochondria-derived superoxide production. Additionally, lycopene ameliorated Aβ-induced mitochondrial morphological alteration, opening of the mitochondrial permeability transition pores and the consequent cytochrome c release. Lycopene also improved mitochondrial complex activities and restored ATP levels in Aβ-treated neuron. Furthermore, lycopene prevented mitochondrial DNA damages and improved the protein level of mitochondrial transcription factor A in mitochondria. Those results indicate that lycopene protects mitochondria against Aβ-induced damages, at least in part by inhibiting mitochondrial oxidative stress and improving mitochondrial function. These beneficial effects of lycopene may account for its protection against Aβ-induced neurotoxicity.

  3. Concentration-Dependent Dual Role of Thrombin In Protection of Cultured Rat Cortical Neurons

    PubMed Central

    García, Paul S.; Ciavatta, Vincent T.; Fidler, Jonathan A.; Woodbury, Anna; Levy, Jerrold H.; Tyor, William R.

    2015-01-01

    Background Thrombin’s role in the nervous system is not well understood. Under conditions of blood-brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. Methods We investigated the effects of physiologically relevant concentrations of thrombin on cortical neurons using two culture-based assays. We examined thrombin’s effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin’s effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. Results We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor - 1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. Conclusions An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin’s receptor

  4. Serum-based culture conditions provoke gene expression variability in mouse embryonic stem cells as revealed by single cell analysis

    PubMed Central

    Guo, Guoji; Pinello, Luca; Han, Xiaoping; Lai, Shujing; Shen, Li; Lin, Ta-Wei; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

    2015-01-01

    Summary Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. We define highly variable gene clusters with distinct chromatin states; and show that bivalent genes are prone to expression variation. At the same time, we identify an ESC priming pathway that initiates the exit from the naïve ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum free culture reduces cellular heterogeneity and transcriptome variation in ESCs. PMID:26804902

  5. An endothelial cultured condition medium embedded porous PLGA scaffold for the enhancement of mouse embryonic stem cell differentiation.

    PubMed

    Li, Ching-Wen; Pan, Wei-Ting; Ju, Jyh-Cherng; Wang, Gou-Jen

    2016-04-12

    In this study, we have developed a microporous poly(lactic-co-glycolic acid) (PLGA) scaffold that combines a continuous release property and a three-dimensional (3D) scaffolding technique for the precise and efficient formation of endothelial cell lineage from embryonic stem cells (ESCs). Eight PLGA scaffolds (14.29%, 16.67%, 20% and 25% concentrations of PLGA solutions) mixed with two crystal sizes of sodium chloride (NaCl) were fabricated by leaching. Then, vascular endothelial cell conditioned medium (ECCM) mixed with gelatin was embedded into the scaffold for culturing of mouse embryonic stem cells (mESCs). The 14.29% PLGA scaffolds fabricated using non-ground NaCl particles (NG-PLGA) and the 25% PLGA containing scaffolds fabricated using ground NaCl particles (G-PLGA) possessed minimum and maximum moisture content and bovine serum albumin (BSA) content properties, respectively. These two groups of scaffolds were used for future experiments in this study. Cell culture results demonstrated that the proposed porous scaffolds without growth factors were sufficient to induce mouse ESCs to differentiate into endothelial-like cells in the early culture stages, and combined with embedded ECCM could provide a long-term inducing system for ESC differentiation.

  6. Chondrogenesis of embryonic limb bud cells in micromass culture progresses rapidly to hypertrophy and is modulated by hydrostatic pressure.

    PubMed

    Saha, Anurati; Rolfe, Rebecca; Carroll, Simon; Kelly, Daniel J; Murphy, Paula

    2017-04-01

    Chondrogenesis in vivo is precisely controlled in time and space. The entire limb skeleton forms from cells at the core of the early limb bud that condense and undergo chondrogenic differentiation. Whether they form stable cartilage at the articular surface of the joint or transient cartilage that progresses to hypertrophy as endochondral bone, replacing the cartilage template of the skeletal rudiment, is spatially controlled over several days in the embryo. Here, we follow the differentiation of cells taken from the early limb bud (embryonic day 11.5), grown in high-density micromass culture and show that a self-organising pattern of evenly spaced cartilage nodules occurs spontaneously in growth medium. Although chondrogenesis is enhanced by addition of BMP6 to the medium, the spatial pattern of nodule formation is disrupted. We show rapid progression of the entire nodule to hypertrophy in culture and therefore loss of the local signals required to direct formation of stable cartilage. Dynamic hydrostatic pressure, which we have previously predicted to be a feature of the forming embryonic joint region, had a stabilising effect on chondrogenesis, reducing expression of hypertrophic marker genes. This demonstrates the use of micromass culture as a relatively simple assay to compare the effect of both biophysical and molecular signals on spatial and temporal control of chondrogenesis that could be used to examine the response of different types of progenitor cell, both adult- and embryo-derived.

  7. Isolation and Culture of Embryonic Stem Cells, Mesenchymal Stem Cells, and Dendritic Cells from Humans and Mice.

    PubMed

    Kar, Srabani; Mitra, Shinjini; Banerjee, Ena Ray

    2016-01-01

    Stem cells are cells capable of proliferation, self-renewal, and differentiation into specific phenotypes. They are an essential part of tissue engineering, which is used in regenerative medicine in case of degenerative diseases. In this chapter, we describe the methods of isolating and culturing various types of stem cells, like human embryonic stem cells (hESCs), human umbilical cord derived mesenchymal stem cells (hUC-MSCs), murine bone marrow derived mesenchymal stem cells (mBM-MSCs), murine adipose tissue derived mesenchymal stem cells (mAD-MSCs), and murine bone marrow derived dendritic cells (mBMDCs). All these cell types can be used in tissue engineering techniques.

  8. Neuronal avalanches are diverse and precise activity patterns that are stable for many hours in cortical slice cultures.

    PubMed

    Beggs, John M; Plenz, Dietmar

    2004-06-02

    A major goal of neuroscience is to elucidate mechanisms of cortical information processing and storage. Previous work from our laboratory (Beggs and Plenz, 2003) revealed that propagation of local field potentials (LFPs) in cortical circuits could be described by the same equations that govern avalanches. Whereas modeling studies suggested that these "neuronal avalanches" were optimal for information transmission, it was not clear what role they could play in information storage. Work from numerous other laboratories has shown that cortical structures can generate reproducible spatiotemporal patterns of activity that could be used as a substrate for memory. Here, we show that although neuronal avalanches lasted only a few milliseconds, their spatiotemporal patterns were also stable and significantly repeatable even many hours later. To investigate these issues, we cultured coronal slices of rat cortex for 4 weeks on 60-channel microelectrode arrays and recorded spontaneous extracellular LFPs continuously for 10 hr. Using correlation-based clustering and a global contrast function, we found that each cortical culture spontaneously produced 4736 +/- 2769 (mean +/- SD) neuronal avalanches per hour that clustered into 30 +/- 14 statistically significant families of spatiotemporal patterns. In 10 hr of recording, over 98% of the mutual information shared by these avalanche patterns were retained. Additionally, jittering analysis revealed that the correlations between avalanches were temporally precise to within +/-4 msec. The long-term stability, diversity, and temporal precision of these avalanches indicate that they fulfill many of the requirements expected of a substrate for memory and suggest that they play a central role in both information transmission and storage within cortical networks.

  9. Periodic "flow-stop" perfusion microchannel bioreactors for mammalian and human embryonic stem cell long-term culture.

    PubMed

    Korin, Natanel; Bransky, Avishay; Dinnar, Uri; Levenberg, Shulamit

    2009-02-01

    The present study examines the use of automated periodic "flow-stop" perfusion systems for long-term culture of mammalian cells in a microchannel bioreactor. The method is used to culture Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) for long periods of time (>7 d) in a microchannel (height 100 mum). Design parameters, mass transport and shear stress issues are theoretically examined via numerical simulations. Cell growth and morphology are experimentally monitored and an enhanced growth rate was measured compared to constant perfusion micro-reactors and to traditional culture in Petri dishes. Moreover, we demonstrate the use of the method to co-culture undifferentiated colonies of human Embryonic Stem Cells (hESC) on HFF feeder cells in microchannels. The successful hESC-HFF co-culture in the microbioreactor is achieved due to two vital characteristics of the developed method-short temporal exposure to flow followed by long static incubation periods. The short pulsed exposure to shear enables shear sensitive cells (e.g., hESC) to withstand the medium renewal flow. The long static incubation period may enable secreted factors (e.g., feeder cells secreted factors) to accumulate locally. Thus the developed method may be suitable for long-term culture of sensitive multi-cellular complexes in microsystems.

  10. Promotion of hair follicle development and trichogenesis by Wnt-10b in cultured embryonic skin and in reconstituted skin

    SciTech Connect

    Ouji, Yukiteru . E-mail: oujix@naramed-u.ac.jp; Yoshikawa, Masahide; Shiroi, Akira; Ishizaka, Shigeaki

    2006-06-30

    We previously showed that Wnt-10b promoted the differentiation of primary skin epithelial cells (MPSEC) toward hair shaft and inner root sheath of the hair follicle (IRS) cells in vitro. In the present study, we found that Wnt-10b promotes the development of hair follicles using a culture of mouse embryonic skin tissue and trichogenesis using a reconstitution experiment with nude mice. Hair follicle development was observed in skin taken from mouse embryos on embryonic day 10.5 following a 2-day culture with recombinant Wnt-10b (rWnt-10b), however, not without rWnt-10b. Brown hair growth was observed at the site of reconstituted skin in Balb/c nude mice where dermal fibroblasts and keratinocytes, derived from C3H/HeN new born mice, were transplanted with Wnt-10b-producing COS cells (Wnt-COS). Without the co-transplantation of Wnt-COS, no hair growth was observed. Our results suggest an important role of Wnt-10b in the initiation of hair follicle development and following trichogenesis.

  11. Ultrastructural identification of Ricinus communis agglutinin-1 positive cells in primary dissociated cell cultures of human embryonic brain.

    PubMed

    Bobryshev, Y; Ashwell, K

    1994-12-01

    While Ricinus communis agglutinin 1 (RCA-1) can be used as a specific marker to study the development and differentiation of microglial cells in human embryogenesis, little is known about the structural heterogeneity and nature of RCA-1+ cells. To analyse the structural peculiarities of RCA-1+ cells, we have used primary dissociated cultures of human embryonic brain. These have been used as models for investigating many of the aspects of central nervous system (CNS) HIV infection. We have shown that primary dissociated cultures from human embryos as young as 10 weeks gestation contain RCA-1+ cells. The RCA-1+ cells exist in two forms, those without (type I) and those with (type II) processes. The former have a poorly developed ultrastructure, while the latter have well developed ultrastructural features, such as rough endoplasmic reticulum with short cisternae, abundant ribosomes, mitochondria, lysosomes and vacuoles. Furthermore, some of these cells with processes have well developed cytoskeletal features. In this paper, the classification of RCA-1+ cells of embryonic human brain is considered and their morphology compared to microglia identified in rodent CNS.

  12. [Study on the effect of alcohol on embryonic development by using in vitro post-implantation rat whole embryo culture].

    PubMed

    Qu, W; Zhang, B; Wu, D; Wu, W

    2000-01-30

    In order to explore the effects of drinking alcohol during pregnancy on embryonic development and its mechanisms, a post-implantation whole embryo culture(WEC) technique was used. The 9.5 day rat embryos were explanted in rat serum medium(immediately centrifugal serum, ICS) with alcohol(0.0.4.1.0, 2.00 and 4.00 g/L), and cultured for 48 hours. The index of embryo development and morphological scores induced by alcohol were observed. The result showed that alcohol had obviously effects on the development and growth of embryos with a dose-response relationship. Embryonic development of 0.4 g/L group was not significantly different from the control group, whereas 1.0 g/L group could interfere with the development score of mid-brain, forebrain, neurotube, and visceral yolk sac(VYS) circle obviously. All scores of the 2.00 g/L group were significantly lower than that of control group (P < 0.05). Moreover, the rate of embryo lethality and teratogenecity were obvious increased. It is concluded that alcohol has developmental toxicity and teratogenicity. The target organ affected by alcohol is brain. The effects of alcohol on the developmental differentiation of visceral yolk sac and DNA synthesis are probably related to its developmental abnormalities.

  13. Generation of viable embryos and embryonic stem cell-like cells from cultured primary follicles in mice.

    PubMed

    Choi, Jun Hee; Kim, Gil Ah; Park, Jong Heum; Song, Gwon Hwa; Park, Jun Won; Kim, Dae Yong; Lim, Jeong Mook

    2011-10-01

    Primary follicles retrieved from B6CBAF1 prepubertal mice were cultured in a stepwise manner in an alpha-minimum essential medium-based medium to generate viable embryos and embryonic stem cell (ESC)-like cells. A significant increase in follicle growth and oocyte maturation accompanied by increased secretion of 17beta-estradiol and progesterone was achieved by exposing primary follicles to 100 or 200 mIU of follicle-stimulating hormone (FSH) during culture. More oocytes developed into blastocysts following in vitro fertilization (IVF) or parthenogenetic activation after culture with 200 mIU of FSH during the entire culture period than with 100 mIU. Eleven ESC-like cell lines, consisting of four heterozygotic and seven homozygotic phenotypes, were established from 25 trials of primary follicle culture combined with IVF or parthenogenetic activation. In conclusion, primary follicles can potentially yield developmentally competent oocytes, which produce viable embryos and ESC-like cell lines following in vitro manipulation. We suggest a method to utilize immature follicles, which are most abundant in ovaries, to improve reproductive efficiency and for use in regenerative medicine.

  14. Green Tea Polyphenols Attenuated Glutamate Excitotoxicity via Antioxidative and Antiapoptotic Pathway in the Primary Cultured Cortical Neurons.

    PubMed

    Cong, Lin; Cao, Chang; Cheng, Yong; Qin, Xiao-Yan

    2016-01-01

    Green tea polyphenols are a natural product which has antioxidative and antiapoptotic effects. It has been shown that glutamate excitotoxicity induced oxidative stress is linked to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In this study we explored the neuroprotective effect of green teen polyphenols against glutamate excitotoxicity in the primary cultured cortical neurons. We found that green tea polyphenols protected against glutamate induced neurotoxicity in the cortical neurons as measured by MTT and TUNEL assays. Green tea polyphenols were then showed to inhibit the glutamate induced ROS release and SOD activity reduction in the neurons. Furthermore, our results demonstrated that green tea polyphenols restored the dysfunction of mitochondrial pro- or antiapoptotic proteins Bax, Bcl-2, and caspase-3 caused by glutamate. Interestingly, the neuroprotective effect of green tea polyphenols was abrogated when the neurons were incubated with siBcl-2. Taken together, these results demonstrated that green tea polyphenols protected against glutamate excitotoxicity through antioxidative and antiapoptotic pathways.

  15. Embryonic Cell Grafts in a Culture Model of Spinal Cord Lesion: Neuronal Relay Formation Is Essential for Functional Regeneration

    PubMed Central

    Tscherter, Anne; Heidemann, Martina; Kleinlogel, Sonja; Streit, Jürg

    2016-01-01

    Presently there exists no cure for spinal cord injury (SCI). However, transplantation of embryonic tissue into spinal cord (SC) lesions resulted in axon outgrowth across the lesion site and some functional recovery, fostering hope for future stem cell therapies. Although in vivo evidence for functional recovery is given, the exact cellular mechanism of the graft support remains elusive: either the grafted cells provide a permissive environment for the host tissue to regenerate itself or the grafts actually integrate functionally into the host neuronal network reconnecting the separated SC circuits. We tested the two hypotheses in an in vitro SC lesion model that is based on propagation of activity between two rat organotypic SC slices in culture. Transplantation of dissociated cells from E14 rat SC or forebrain (FB) re-established the relay of activity over the lesion site and thus, provoked functional regeneration. Combining patch-clamp recordings from transplanted cells with network activity measurements from the host tissue on multi-electrode arrays (MEAs) we here show that neurons differentiate from the grafted cells and integrate into the host circuits. Optogenetic silencing of neurons developed from transplanted embryonic mouse FB cells provides clear evidence that they replace the lost neuronal connections to relay and synchronize activity between the separated SC circuits. In contrast, transplantation of neurospheres (NS) induced neither the differentiation of mature neurons from the grafts nor an improvement of functional regeneration. Together these findings suggest, that the formation of neuronal relays from grafted embryonic cells is essential to re-connect segregated SC circuits. PMID:27708562

  16. Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions

    PubMed Central

    Garitaonandia, Ibon; Amir, Hadar; Boscolo, Francesca Sesillo; Wambua, Gerald K.; Schultheisz, Heather L.; Sabatini, Karen; Morey, Robert; Waltz, Shannon; Wang, Yu-Chieh; Tran, Ha; Leonardo, Trevor R.; Nazor, Kristopher; Slavin, Ileana; Lynch, Candace; Li, Yingchun; Coleman, Ronald; Gallego Romero, Irene; Altun, Gulsah; Reynolds, David; Dalton, Stephen; Parast, Mana; Loring, Jeanne F.; Laurent, Louise C.

    2015-01-01

    The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies. PMID:25714340

  17. Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture.

    PubMed

    Hongisto, Heidi; Vuoristo, Sanna; Mikhailova, Alexandra; Suuronen, Riitta; Virtanen, Ismo; Otonkoski, Timo; Skottman, Heli

    2012-01-01

    Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.

  18. A low ethanol dose affects all types of cells in mixed long-term embryonic cultures of the cerebellum.

    PubMed

    Pickering, Chris; Wicher, Grzegorz; Rosendahl, Sofi; Schiöth, Helgi B; Fex-Svenningsen, Asa

    2010-06-01

    The beneficial effect of the '1-drink-a-day' lifestyle is suggested by studies of cardiovascular health, and this recommendation is increasingly followed in many countries. The main objective of this study was to determine whether this pattern of ethanol use would be detrimental to a pregnant woman. We exposed a primary culture of rat cerebellum from embryonic day 17 (corresponding to second trimester in humans) to ethanol at a concentration of 17.6 mM which is roughly equivalent to one glass of wine. Acutely, there was no change in cell viability after 5 or 8 days of exposure relative to control. By 11 days, a reduction in the number of viable cells was observed without an accompanying change in caspase-3 activity (marker of apoptotic cell death), suggesting changes in cell proliferation. As the proportion of nestin-positive cells was higher in the ethanol-treated cultures after 5 days, we hypothesized that an increase in differentiation to neurons would compensate for the ongoing neuronal death. However, there were limits to this compensatory ability as the relative proportion of nestin-positive cells was decreased after 11 days. To further illustrate the negative long-term effects of this ethanol dose, cultures were exposed for 30 days. After this period, virtually no neurons or myelinating oligodendrocytes were present in the ethanol-treated cultures. In conclusion, chronic exposure to ethanol, even at small doses, dramatically and persistently affects normal development.

  19. Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

    PubMed Central

    Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh

    2016-01-01

    Abstract Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell−1 s−1, and 5 and 35 amol cell−1 s−1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies. PMID:27214658

  20. Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

    PubMed

    Super, Alexandre; Jaccard, Nicolas; Cardoso Marques, Marco Paulo; Macown, Rhys Jarred; Griffin, Lewis Donald; Veraitch, Farlan Singh; Szita, Nicolas

    2016-09-01

    Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies.

  1. The effects of BmNPV on biochemical changes in primary cultures of Bombyx mori embryonic tissue.

    PubMed

    Matindoost, Leila; Sendi, Jalal Jalali; Soleimanjahi, Hoorieh; Etebari, Kayvan; Rahbarizade, Fateme

    2008-01-01

    The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phosphatase (ALP) activity decreased in infected cells.

  2. Activation of antioxidant response element in mouse primary cortical cultures with sesquiterpene lactones isolated from Tanacetum parthenium

    PubMed Central

    Fischedick, Justin T; Standiford, Miranda; Johnson, Delinda A.; De Vos, Ric C.H.; Todorović, Slađana; Banjanac, Tijana; Verpoorte, Rob; Johnson, Jeffrey A.

    2012-01-01

    Tanacetum parthenium (Asteraceae) produces biologically active sesquiterpene lactones (SL). Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate a series of genes termed the antioxidant response element (ARE). Activation of the Nrf2/ARE may be useful for the treatment of neurodegenerative disease. In this study we isolated 11 sesquiterpene lactones from T. parthenium with centrifugal partition chromatography and semi-preparative HPLC. Compounds were screened in-vitro for their ability to activate the ARE on primary mouse cortical cultures as well as for their toxicity towards the cultures. All sesquiterpene lactones containing the α-methylene-γ-lactone moiety were able to activate the ARE although a number of compounds displayed significant cellular toxicity towards the cultures. The structure activity relationship of the sesquiterpene lactones indicate that the guaianolides isolated were more active and less toxic then the germacranolides. PMID:22923197

  3. Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

    PubMed Central

    Sharma, Ruchi; Greenhough, Sebastian; Medine, Claire N.; Hay, David C.

    2010-01-01

    The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays. PMID:20169088

  4. Measurement of saturation processes in glutamatergic and GABAergic synapse densities during long-term development of cultured rat cortical networks.

    PubMed

    Ito, Daisuke; Komatsu, Takumi; Gohara, Kazutoshi

    2013-10-09

    The aim of this study was to clarify the saturation processes of excitatory and inhibitory synapse densities during the long-term development of cultured neuronal networks. For this purpose, we performed a long-term culture of rat cortical cells for 35 days in vitro (DIV). During this culture period, we labeled glutamatergic and GABAergic synapses separately using antibodies against vesicular glutamate transporter 1 (VGluT1) and vesicular transporter of γ-aminobutyric acid (VGAT). The densities and distributions of both types of synaptic terminals were measured simultaneously. Observations and subsequent measurements of immunofluorescence demonstrated that the densities of both types of antibody-labeled terminals increased gradually from 7 to 21-28 DIV. The densities did not show a further increase at 35 DIV and tended to become saturated. Triple staining with VGluT1, VGAT, and microtubule-associated protein 2 (MAP2) enabled analysis of the distribution of both types of synapses, and revealed that the densities of the two types of synaptic terminals on somata were not significantly different, but that glutamatergic synapses predominated on the dendrites during long-term culture. However, some neurons did not fall within this distribution, suggesting differences in synapse distribution on target neurons. The electrical activity also showed an initial increase and subsequent saturation of the firing rate and synchronized burst rate during long-term culture, and the number of days of culture to saturation from the initial increase followed the same pattern under this culture condition.

  5. Functional down-regulation of volume-regulated anion channels in AQP4 knockdown cultured rat cortical astrocytes.

    PubMed

    Benfenati, Valentina; Nicchia, Grazia Paola; Svelto, Maria; Rapisarda, Carmela; Frigeri, Antonio; Ferroni, Stefano

    2007-01-01

    In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin-4 (AQP4), and the swelling-activated Cl(-) current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume-regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process-bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling-activated Cl(-) current provided evidence that in AQP4 KD astrocytes, there was a down-regulation of chloride channel-2 (CIC-2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.

  6. Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

    PubMed Central

    THONGKITTIDILOK, Chommanart; THARASANIT, Theerawat; SONGSASEN, Nucharin; SANANMUANG, Thanida; BUARPUNG, Sirirak; TECHAKUMPHU, Mongkol

    2015-01-01

    This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages. PMID:25985792

  7. Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos.

    PubMed

    Thongkittidilok, Chommanart; Tharasanit, Theerawat; Songsasen, Nucharin; Sananmuang, Thanida; Buarpung, Sirirak; Techakumphu, Mongkol

    2015-01-01

    This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2-4-cell embryos, 8-16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.

  8. Cryopreservation of Quercus suber and Quercus ilex embryonic axes: in vitro culture, desiccation and cooling factors.

    PubMed

    Gonzalez-Benito, M Elena; Prieto, Roberto-Moreno; Herradon, Esther; Martin, Carmen

    2002-01-01

    This study examines different factors included in the cryopreservation protocols for Quercus ilex and Q. suber embryonic axes. In vitro incubation temperature played an important role in the appropriate development of Q. ilex axes, as 15 degrees C was superior to 25 degrees C. Q. suber axes proved to be more sensitive to desiccation and cooling. Poor survival (35%) was observed when axes were included into cryovials and then in liquid nitrogen, and none when immersed in sub-cooled liquid nitrogen (-210 degrees C). Q. ilex axes showed poorly organised development in vitro (c. 50% of non-cooled axes showed shoot development). However, c. 80% survival was observed after cryopreservation (either in liquid nitrogen or sub-cooled liquid nitrogen at 0.34 g water / g dry weight), of which c. 15% showed shoot development.

  9. A simple slice culture system for the imaging of nerve development in embryonic mouse.

    PubMed

    Brachmann, Isabel; Jakubick, Vera Catherine; Shakèd, Maya; Unsicker, Klaus; Tucker, Kerry Lee

    2007-12-01

    Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest.

  10. Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds.

    PubMed

    Kang, Xihai; Xie, Yubing; Powell, Heather M; James Lee, L; Belury, Martha A; Lannutti, John J; Kniss, Douglas A

    2007-01-01

    A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.

  11. The Three-Dimensional Culture of Epithelial Organoids Derived from Embryonic Chicken Intestine.

    PubMed

    Pierzchalska, Malgorzata; Panek, Malgorzata; Czyrnek, Malgorzata; Grabacka, Maja

    2016-10-28

    The intestinal epithelium isolated from chicken embryos in last 3 days of development can be used to establish the 3D culture of intestinal organoids. When fragments of epithelial tissue released by incubation with EGTA (2.5 mM, 2 h) are embedded in Matrigel matrix on cell culture inserts the formation of empty spheres covered by epithelial cells is observed in first 24 h of culture. The growth and survival of organoids are supported by the addition of R-spondin 1, Noggin, and prostaglandin E2 to the culture medium. The organoids are accompanied by myofibroblasts which become visible in the next 2 days of culture. The intestinal enteroids (free of myofibroblasts) can be obtained from adult chicken intestine.

  12. Ethanol neuronotoxicity in the embryonic chick brain in ovo and in culture: interaction of the neural cell adhesion molecule (NCAM).

    PubMed

    Kentroti, S; Rahman, H; Grove, J; Vernadakis, A

    1995-12-01

    The present study was undertaken to investigate the involvement of NCAM in the neuroteratogenic effects of ethanol demonstrated by us and others. In the first experiment we examined the effect of in-ovo ethanol exposure on expression of NCAM in various regions of the embryonic CNS throughout development. Chick embryos received ethanol (10 mg/50 microliters/day) or saline (control) at days 1-3 of development (E1-E3), were sacrificed at various embryonic ages and whole brain (WB), cerebral hemispheres (CH) and cerebellum (CE) processed for SDS-polyacrylamide gel electrophoresis. The normal developmental profile of NCAM in the chick brain exhibited the same dynamics as previously reported by others. When compared to age-matched control brains, an increase was observed in expression of high molecular weight forms of NCAM in cerebral hemispheres between E8 and E10. These bands represented highly sialated (> 180 kDa) forms of NCAM. In fact, the NCAM hand from ethanol-treated embryos at E8 migrated at a higher molecular weight than did its control counterpart, indicating an increase in sialic acid content. In contrast, no clear change was observed in NCAM expression in cerebellum from E10 through E20 as a result of ethanol exposure. In the second experiment, we examined the involvement of NCAM in the alterations in neuronal growth patterns observed in ethanol-exposed cultures. Neuroblast-enriched cultures derived from three-day-old whole chick embryos (E3WE) were maintained on poly-L-lysine pre-coated Petri dishes in DMEM+5% fetal bovine serum with or without 50 mM ethanol. Cultures were fixed at 3, 6 or 9 DIV and co-stained for NCAM and neurofilament (160 kDa). E3WE cultures exhibited intense NCAM immunoreactivity at 3 and 6 DIV decreasing by 9 DIV.NCAM positive structures included all neuronal perikarya, neuritic processes and growth cones. Addition of 50 mM ethanol to the medium resulted in profound alterations in growth patterns of developing neurons which continued

  13. Isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains.

    PubMed

    Weinert, Maria; Selvakumar, Tharakeswari; Tierney, Travis S; Alavian, Kambiz N

    2015-02-19

    Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson's diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

  14. Mechanisms of Reduced Astrocyte Surface Coverage in Cortical Neuron-Glia Co-cultures on Nanoporous Gold Surfaces.

    PubMed

    Chapman, Christopher A R; Chen, Hao; Stamou, Marianna; Lein, Pamela J; Seker, Erkin

    2016-09-01

    Nanoporous gold (np-Au) is a promising multifunctional material for neural electrodes. We have previously shown that np-Au nanotopography reduces astrocyte surface coverage (linked to undesirable gliosis) while maintaining high neuronal coverage in a cortical primary neuron-glia co-culture model as long as two weeks in vitro. Here, we investigate the potential influence of secreted soluble factors from cells grown on np-Au on the cell type-specific surface coverage of cells grown on conventional tissue culture plastic and test the hypothesis that secretion of factors is responsible for inhibiting astrocyte coverage on np-Au. In order to assess whether factors secreted from cells grown on np-Au surfaces reduced surface coverage by astrocytes, we seeded fresh primary rat neuron-glia co-cultures on conventional polystyrene culture dishes, but maintained the cells in conditioned media from co-cultures grown on np-Au surfaces. After one week in vitro, a preferential reduction in astrocyte surface coverage was not observed, suggesting that soluble factors are not playing a role. In contrast, four hours after cell seeding there were a significant number of non-adhered, yet still viable, cells for the cultures on np-Au surfaces. We hypothesize that the non-adherent cells are mainly astrocytes, because: (i) there was no difference in neuronal cell coverage between np-Au and pl-Au for long culture durations and (ii) neurons are post-mitotic and not expected to increase in number upon attaching to the surface. Overall, the results suggest that the np-Au topography leads to preferential neuronal attachment shortly after cell seeding and limits astrocyte-specific np-Au surface coverage at longer culture durations.

  15. Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery.

    PubMed

    Kiprilov, Enko N; Awan, Aashir; Desprat, Romain; Velho, Michelle; Clement, Christian A; Byskov, Anne Grete; Andersen, Claus Y; Satir, Peter; Bouhassira, Eric E; Christensen, Søren T; Hirsch, Rhoda Elison

    2008-03-10

    Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.

  16. Accumulation of pyrethroid compounds in primary cultures of rat cortical neurons

    EPA Science Inventory

    Recent studies have demonstrated that lipophilic compounds (e.g. methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neur...

  17. Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells

    PubMed Central

    Scheerlinck, Ellen; Van Steendam, Katleen; Daled, Simon; Govaert, Elisabeth; Vossaert, Liesbeth; Meert, Paulien; Van Nieuwerburgh, Filip; Van Soom, Ann; Peelman, Luc; De Sutter, Petra; Heindryckx, Björn; Dhaenens, Maarten

    2016-01-01

    We present a fully defined culture system (adapted Essential8TM [E8TM] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8TM medium was modified by adding (1) l‐proline, (2) l‐ornithine, (3) Nω‐hydroxy‐nor‐l‐arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l‐ornithine, followed by 3.5 mM l‐proline and by lowering the arginine concentration in the medium to 99.5 μM. No major changes in pluripotency and cell amount could be observed for the adapted E8TM media with ornithine and proline. However, our subsequent ion mobility assisted data‐independent acquisition (high‐definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion. PMID:27392809

  18. Efficient embryonic culture method for the Japanese striped snake, Elaphe quadrivirgata, and its early developmental stages.

    PubMed

    Matsubara, Yoshiyuki; Sakai, Atsushi; Kuroiwa, Atsushi; Suzuki, Takayuki

    2014-10-01

    The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named "sausage-style (SS) culture", allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9 min, between 60/70 and 80/91 somite stages 42.4 min, and between 113-115 and 126/127 somite stages 71 min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E. quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full-color images with high-resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo.

  19. Accumulation of neurons differentiated from mouse embryonic stem cells in particular areas of culture plate surface.

    PubMed

    Kitazawa, Ayako; Naka, Yukie; Yamaguchi, Hiroko; Shimizu, Norio

    2010-08-01

    Nanoscale magnetic beads coated with nerve growth factor (NGF) allow us to accumulate neurons differentiated from mouse ES cells in a selected area of the culture plate surface using a magnet. Neurons with neurite outgrowths within a particular area expressed TrkA and incorporated beads in the soma.

  20. Hepatic Differentiation and Maturation of Human Embryonic Stem Cells Cultured in a Perfused Three-Dimensional Bioreactor

    PubMed Central

    Synnergren, Jane; Jensen, Janne; Björquist, Petter; Ingelman-Sundberg, Magnus

    2013-01-01

    Drug-induced liver injury is a serious and frequently occurring adverse drug reaction in the clinics and is hard to predict during preclinical studies. Today, primary hepatocytes are the most frequently used cell model for drug discovery and prediction of toxicity. However, their use is marred by high donor variability regarding drug metabolism and toxicity, and instable expression levels of liver-specific genes such as cytochromes P450. An in vitro model system based on human embryonic stem cells (hESC), with their unique properties of pluripotency and self-renewal, has potential to provide a stable and unlimited supply of human hepatocytes. Much effort has been made to direct hESC toward the hepatic lineage, mostly using 2-dimensional (2D) cultures. Although the results are encouraging, these cells lack important functionality. Here, we investigate if hepatic differentiation of hESC can be improved by using a 3-dimensional (3D) bioreactor system. Human ESCs were differentiated toward the hepatic lineage using the same cells in either the 3D or 2D system. A global transcriptional analysis identified important differences between the 2 differentiation regimes, and we identified 10 pathways, highly related to liver functions, which were significantly upregulated in cells differentiated in the bioreactor compared to 2D control cultures. The enhanced hepatic differentiation observed in the bioreactor system was also supported by immunocytochemistry. Taken together, our results suggest that hepatic differentiation of hESC is improved when using this 3D bioreactor technology as compared to 2D culture systems. PMID:22970843

  1. Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells.

    PubMed

    Hara, Jared; Tottori, Jordan; Anders, Megan; Dadhwal, Smritee; Asuri, Prashanth; Mobed-Miremadi, Maryam

    2017-05-01

    Post cryopreservation viability of human embryonic kidney (HEK) cells under two-dimensional (2D) and three-dimensional (3D) culture conditions was studied using trehalose as the sole cryoprotective agent. An L9 (3(4)) Taguchi design was used to optimize the cryoprotection cocktail seeding process prior to slow-freezing with the specific aim of maximizing cell viability measured 7 days post thaw, using the combinatorial cell viability and in-vitro cytotoxicity WST assay. At low (200 mM) and medium (800 mM) levels of trehalose concentration, encapsulation in alginate offered a greater protection to cryopreservation. However, at the highest trehalose concentration (1200 mM) and in the absence of the pre-incubation step, there was no statistical difference at the 95% CI (p = 0.0212) between the viability of the HEK cells under 2D and 3D culture conditions estimated to be 17.9 ± 4.6% and 14.0 ± 3.6%, respectively. A parallel comparison between cryoprotective agents conducted at the optimal levels of the L9 study, using trehalose, dimethylsulfoxide and glycerol in alginate microcapsules yielded a viability of 36.0 ± 7.4% for trehalose, in average 75% higher than the results associated with the other two cell membrane-permeating compounds. In summary, the effectiveness of trehalose has been demonstrated by the fact that 3D cell cultures can readily be equilibrated with trehalose before cryopreservation, thus mitigating the cytotoxic effects of glycerol and dimethylsulfoxide.

  2. Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

    NASA Technical Reports Server (NTRS)

    Spooner, B. S.; Bassett, K. E.; Spooner, B. S. Jr

    1993-01-01

    The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.

  3. Differentiation of human embryonic stem cells to cardiomyocytes on microcarrier cultures.

    PubMed

    Ting, Sherwin; Lecina, Marti; Reuveny, Shaul; Oh, Steve

    2012-05-01

    We have developed an improved cardiomyocyte differentiation protocol where we stabilized embryoid bodies (EB) in serum- and insulin-free medium (bSFS) supplemented with p38 MAP kinase inhibitor (SB203580) by addition of 10 µm laminin-coated positively charged (protamine sulfate derivatized TSKgel Tresyl-5PW) microcarriers. This protocol achieved a maximum 3-fold cell expansion, differentiation efficiency of 20%, and an overall cardiomyocyte yield of 3 × 10⁵ CM/ml in static conditions. In comparison, EB cultures achieved 1.5-fold cell expansion, differentiation efficiency of 15%, and an overall cardiomyocyte yield of 1.1 × 10⁵ CM/ml. The scalability of this platform was demonstrated in suspended spinner cultures, producing a maximum of 2.14 × 10⁵ CM/ml in 50-ml cultures. This yield is two-fold higher than the control static EB-based platform (1.1 × 10⁵ CM/ml), and seven-fold higher than yields reported in literature, 3.1-9 × 10⁴ CM/ml. The robustness of this protocol was tested with HES-3 and H1 cell lines.

  4. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    PubMed

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes.

  5. Dibenzocyclooctadiene lignans from Schisandra chinensis protect primary cultures of rat cortical cells from glutamate-induced toxicity.

    PubMed

    Kim, So Ra; Lee, Mi Kyeong; Koo, Kyung Ah; Kim, Seung Hyun; Sung, Sang Hyun; Lee, Na Gyong; Markelonis, George J; Oh, Tae H; Yang, Jae Ho; Kim, Young Choong

    2004-05-01

    A methanolic extract of dried Schisandra fruit (Schisandra chinensis Baill.; Schisandraceae) significantly attenuated the neurotoxicity induced by L-glutamate in primary cultures of rat cortical cells. Five dibenzocyclooctadiene lignans (deoxyschisandrin, gomisin N, gomisin A, schisandrin, and wuweizisu C) were isolated from the methanolic extract; their protective effects against glutamate-induced neurotoxicity were then evaluated. Among the five lignans, deoxyschisandrin, gomisin N, and wuweizisu C significantly attenuated glutamate-induced neurotoxicity as measured by 1). an inhibition in the increase of intracellular [Ca(2+)]; 2). an improvement in the glutathione defense system, the level of glutathione, and the activity of glutathione peroxidase; and 3). an inhibition in the formation of cellular peroxide. These results suggest that dibenzocyclooctadiene lignans from Schisandra chinensis may possess therapeutic potential against oxidative neuronal damage induced by excitotoxin.

  6. Neuroprotection of Ilex latifolia and caffeoylquinic acid derivatives against excitotoxic and hypoxic damage of cultured rat cortical neurons.

    PubMed

    Kim, Joo Youn; Lee, Hong Kyu; Hwang, Bang Yeon; Kim, SeungHwan; Yoo, Jae Kuk; Seong, Yeon Hee

    2012-06-01

    Ilex latifolia (Aquifoliaceae), one of the primary components of "Ku-ding-cha", has been used in Chinese folk medicine to treat headaches and various inflammatory diseases. A previous study demonstrated that the ethanol extract of I. latifolia could protect against ischemic apoptotic brain damage in rats. The present study investigated the protective activity of I. latifolia against glutamate-induced neurotoxicity using cultured rat cortical neurons in order to explain a possible mechanism related to its inhibitory effect on ischemic brain damage and identified potentially active compounds from it. Exposure of cultured cortical neurons to 500 μM glutamate for 12 h triggered neuronal cell death. I. latifolia (10-100 μg/mL) inhibited glutamate-induced neuronal death, elevation of intracellular calcium ([Ca(2+)](i)), generation of reactive oxygen species (ROS), the increase of a pro-apoptotic protein, BAX, and the decrease of an anti-apoptotic protein, BcL-2. Hypoxia-induced neuronal cell death was also inhibited by I. latifolia. 3,4-Dicaffeoylquinic acid (diCQA), 3,5-diCQA, and 3,5-diCQA methyl ester isolated from I. latifolia also inhibited the glutamate-induced increase in [Ca(2+)](i), generation of ROS, the change of apoptosis-related proteins, and neuronal cell death; and hypoxia-induced neuronal cell death. These results suggest that I. latifolia and its active compounds prevented glutamate-induced neuronal cell damage by inhibiting increase of [Ca(2+)](i), generation of ROS, and resultantly apoptotic pathway. In addition, the neuroprotective effects of I. latifolia on ischemia-induced brain damage might be associated with the anti-excitatory and anti-oxidative actions and could be attributable to these active compounds, CQAs.

  7. Embryonic body formation using the tapered soft stencil for cluster culture device.

    PubMed

    Yukawa, Hiroshi; Ikeuchi, Masashi; Noguchi, Hirofumi; Miyamoto, Yoshitaka; Ikuta, Koji; Hayashi, Shuji

    2011-05-01

    Induced pluripotent stem (iPS) cells are expected to provide a source of tissue, a renewable cell source for tissue engineering, and a method for in vitro drug screening for patient-specific or disease-specific treatment. A simple technology by which iPS cells can be differentiated effectively and in large quantities is strongly desired. In this paper, a new device (Tapered Soft Stencil for Cluster Culture: TASCL) is proposed for the easy and efficient formation of EBs which can be used in regenerative medicine. This device was compared with the two major methods currently being evaluated, namely the HD method and the Terasaki® plate (MWC substitution), in terms of the efficiency, morphology and acquired number of EB formation. Using the TASCL device, the shape of the EBs formed was almost a perfect sphere, and the formation was also faster than for the two other methods. There was little variability in the number of cells. Moreover, EBs formed using the TASCL device had the ability to differentiate into all three germ layers, and differentiation of EBs from the TASCL culture into hepatic cells was confirmed. In conclusion, it appears that the TASCL device can be utilized for EB formation to generate cells for regenerative medicine applications.

  8. Analytical characterization of spontaneous firing in networks of developing rat cultured cortical neurons

    NASA Astrophysics Data System (ADS)

    Tateno, Takashi; Kawana, Akio; Jimbo, Yasuhiko

    2002-05-01

    We have used a multiunit electrode array in extracellular recording to investigate changes in the firing patterns in networks of developing rat cortical neurons. The spontaneous activity of continual asynchronous firing or the alternation of asynchronous spikes and synchronous bursts changed over time so that activity in the later stages consisted exclusively of synchronized bursts. The spontaneous coordinated activity in bursts produced a variability in interburst interval (IBI) sequences that is referred to as ``form.'' The stochastic and nonlinear dynamical analysis of IBI sequences revealed that these sequences reflected a largely random process and that the form for relatively immature neurons was largely oscillatory while the form for the more mature neurons was Poisson-like. The observed IBI sequences thus showed changes in form associated with both the intrinsic properties of the developing cells and the neural response to correlated synaptic inputs due to interaction between the developing neural circuits.

  9. ANEPIII, a new recombinant neurotoxic polypeptide derived from scorpion peptide, inhibits delayed rectifier, but not A-type potassium currents in rat primary cultured hippocampal and cortical neurons.

    PubMed

    Li, Chun-Li; Zhang, Jing-Hai; Yang, Bao-Feng; Jiao, Jun-Dong; Wang, Ling; Wu, Chun-Fu

    2006-01-15

    A new recombinant neurotoxic polypeptide ANEPIII (BmK ANEPIII) derived from Scorpion peptide, which was demonstrated with antineuroexcitation properties in animal models, was examined for its action on K+ currents in primary cultured rat hippocampal and cortical neurons using the patch clamp technique in the whole-cell configuration. The delayed rectifier K+ current (I(k)) was inhibited by externally applied recombinant BmK ANEPIII, while the transient A-current (I(A)) remained virtually unaffected. BmK ANEPIII 3 microM, reduced the delayed rectifier current by 28.2% and 23.6% in cultured rat hippocampal and cortical neurons, respectively. The concentration of half-maximal block was 155.1 nM for hippocampal neurons and 227.2 nM for cortical neurons, respectively. These results suggest that BmK ANEPIII affect K+ currents, which may lead to a reduction in neuronal excitability.

  10. Pyrethroid insecticide accumulation in primary cultures of cortical neurons in vitro

    EPA Science Inventory

    Primary cultures of neurons have been widely utilized to study the actions of pyrethroids and other neurotoxicants, with the presumption that the media concentration accurately reflects the dose received by the cells. However, recent studies have demonstrated that lipophilic comp...

  11. Assessment of ‘one-step’ versus ‘sequential’ embryo culture conditions through embryonic genome methylation and hydroxymethylation changes

    PubMed Central

    Salvaing, J.; Peynot, N.; Bedhane, M. N.; Veniel, S.; Pellier, E.; Boulesteix, C.; Beaujean, N.; Daniel, N.; Duranthon, V.

    2016-01-01

    STUDY QUESTION In comparison to in vivo development, how do different conditions of in vitro culture (‘one step’ versus ‘sequential medium’) impact DNA methylation and hydroxymethylation in preimplantation embryos? SUMMARY ANSWER Using rabbit as a model, we show that DNA methylation and hydroxymethylation are both affected by in vitro culture of preimplantation embryos and the effect observed depends on the culture medium used. WHAT IS KNOWN ALREADY Correct regulation of DNA methylation is essential for embryonic development and DNA hydroxymethylation appears more and more to be a key player. Modifications of the environment of early embryos are known to have long term effects on adult phenotypes and health; these probably rely on epigenetic alterations. STUDY DESIGN SIZE, DURATION The study design we used is both cross sectional (control versus treatment) and longitudinal (time-course). Each individual in vivo experiment used embryos flushed from the donor at the 2-, 4-, 8-, 16- or morula stage. Each stage was analyzed in at least two independent experiments. Each individual in vitro experiment used embryos flushed from donors at the 1-cell stage (19 h post-coïtum) which were then cultured in parallel in the two tested media until the 2-, 4-, 8- 16-cell or morula stages. Each stage was analyzed in at least three independent experiments. In both the in vivo and in vitro experiments, 4-cell stage embryos were always included as an internal reference. PARTICIPANTS/MATERIALS, SETTING, METHODS Immunofluorescence with antibodies specific for 5-methylcytosine (5meC) and 5-hydroxymethylcytosine (5hmeC) was used to quantify DNA methylation and hydroxymethylation levels in preimplantation embryos. We assessed the expression of DNA methyltransferases (DNMT), of ten eleven translocation (TET) dioxigenases and of two endogenous retroviral sequences (ERV) using RT-qPCR, since the expression of endogenous retroviral sequences is known to be regulated by DNA methylation

  12. Molecular mechanisms and pathways involved in bovine embryonic genome activation and their regulation by alternative in vivo and in vitro culture conditions.

    PubMed

    Gad, Ahmed; Hoelker, Michael; Besenfelder, Urban; Havlicek, Vitezslav; Cinar, Ulas; Rings, Franca; Held, Eva; Dufort, Isabelle; Sirard, Marc-André; Schellander, Karl; Tesfaye, Dawit

    2012-10-01

    Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro- and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE's bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

  13. Ion permeation properties of the glutamate receptor channel in cultured embryonic Drosophila myotubes.

    PubMed Central

    Chang, H; Ciani, S; Kidokoro, Y

    1994-01-01

    Ion permeation properties of the glutamate receptor channel in cultured myotubes of Drosophila embryos were studied using the inside-out configuration of the patch-clamp technique. Lowering the NaCl concentration in the bath (intracellular solution), while maintaining that of the external solution constant, caused a shift of the reversal potential in the positive direction, thus indicating a higher permeability of the channel to Na+ than to Cl- (PCl/PNa < 0.04), and suggesting that the channel is cation selective. With 145 mM Na+ on both sides of the membrane, the single-channel current-voltage relation was almost linear in the voltage range between -80 and +80 mV, the conductance showing some variability in the range between 140 and 170 pS. All monovalent alkali cations tested, as well as NH4+, permeated the channel effectively. Using the Goldman-Hodgkin-Katz equation for the reversal potential, the permeability ratios with respect to Na+ were estimated to be: 1.32 for K+, 1.18 for NH4+, 1.15 for Rb+, 1.09 for Cs+, and 0.57 for Li+. Divalent cations, i.e. Mg2+ and Ca2+, in the external solution depressed not only the inward but also the outward Na+ currents, although reversal potential measurements indicated that both ions have considerably higher permeabilities than Na+ (PMg/PNa = 2.31; PCa/PNa = 9.55). The conductance-activity relation for Na+ was described by a hyperbolic curve. The maximal conductance was about 195 pS and the half-saturating activity 45 mM. This result suggests that Na+ ions bind to sites in the channel. All data were fitted by a model based on the Eyring's reaction rate theory, in which the receptor channel is a one-ion pore with three energy barriers and two internal sites. PMID:7519261

  14. Repeated Stimulation of Cultured Networks of Rat Cortical Neurons Induces Parallel Memory Traces

    ERIC Educational Resources Information Center

    le Feber, Joost; Witteveen, Tim; van Veenendaal, Tamar M.; Dijkstra, Jelle

    2015-01-01

    During systems consolidation, memories are spontaneously replayed favoring information transfer from hippocampus to neocortex. However, at present no empirically supported mechanism to accomplish a transfer of memory from hippocampal to extra-hippocampal sites has been offered. We used cultured neuronal networks on multielectrode arrays and…

  15. Self-renewal and pluripotency is maintained in human embryonic stem cells by co-culture with human fetal liver stromal cells expressing hypoxia inducible factor 1alpha.

    PubMed

    Ji, Lei; Liu, Yu-xiao; Yang, Chao; Yue, Wen; Shi, Shuang-shuang; Bai, Ci-xian; Xi, Jia-fei; Nan, Xue; Pei, Xue-Tao

    2009-10-01

    Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Recent evidence suggests that stem cells are localized in the microenvironment of low oxygen. We hypothesized that hypoxia could maintain the undifferentiated phenotype of embryonic stem cells. We have co-cultured a human embryonic cell line with human fetal liver stromal cells (hFLSCs) feeder cells stably expressing hypoxia-inducible factor-1 alpha (HIF-1alpha), which is known as the key transcription factor in hypoxia. The results suggested HIF-1alpha was critical for preventing differentiation of hES cells in culture. Consistent with this observation, hypoxia upregulated the expression of Nanog and Oct-4, the key factors expressed in undifferentiated stem cells. We further demonstrated that HIF-1alpha could upregulate the expression of some soluble factors including bFGF and SDF-1alpha, which are released into the microenvironment to maintain the undifferentiated status of hES cells. This suggests that the targets of HIF-1alpha are secreted soluble factors rather than a cell-cell contact mechanism, and defines an important mechanism for the inhibition of hESCs differentiation by hypoxia. Our findings developed a transgene feeder co-culture system and will provide a more reliable alternative for future therapeutic applications of hES cells.

  16. Neurotrophic effects of GnRH on neurite outgrowth and neurofilament protein expression in cultured cerebral cortical neurons of rat embryos.

    PubMed

    Quintanar, J Luis; Salinas, Eva

    2008-06-01

    The presence of GnRH receptor in cerebral cortical neurons of rat embryos and adult rats has been described. In this work, we studied the effects of GnRH on outgrowth and length of neurites and cytoskeletal neurofilament proteins expression (NF-68 and NF-200 kDa) by immunoblot of cultured cerebral cortical neurons of rat embryos. Our results show that GnRH increases both outgrowth and length of neurites accompanied by an increase in neurofilaments expression. It is conceivable that GnRH plays a role in neuronal plasticity parallel to its gonadal function.

  17. Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells.

    PubMed

    Heng, Boon Chin; Cao, Tong; Liu, Hua; Rufaihah, Abdul Jalil

    2005-01-01

    This study attempted to investigate whether different levels of mitotic activity exist within different physical regions of a human embryonic stem (hES) cell colony. Incorporation of 5-bromo-2-deoxyuridine (BrdU) within newly-synthesized DNA, followed by immunocytochemical staining was used as a means of detecting mitotically-active cells within hES colonies. The results showed rather surprisingly that the highest levels of mitotic activity are primarily concentrated within the central regions of hES colonies, whereas the peripheral regions exhibited reduced levels of cellular proliferation. Two hypothetical mechanisms are therefore proposed for hES colony growth and expansion. Firstly, it is envisaged that the less mitotically-active hES cells at the periphery of the colony are continually migrating outwards, thereby providing space for newly-divided daughter cells within the more mitotically-active central region of the hES colony. Secondly, it is proposed that the newly-divided hES cells within the central region of the colony somehow migrate to the outer periphery. This could possibly explain why the periphery of hES colonies are less mitotically-active, since there would obviously be an extended time-lag before newly-divided daughter cells are ready again for the next cell division. Further investigations need to be carried out to characterize the atypical mechanisms by which hES colonies grow and expand in size.

  18. Differential effects of ciguatoxin and maitotoxin in primary cultures of cortical neurons.

    PubMed

    Martin, Victor; Vale, Carmen; Antelo, Alvaro; Hirama, Masahiro; Yamashita, Shuji; Vieytes, Mercedes R; Botana, Luis M

    2014-08-18

    Ciguatoxins (CTXs) and maitotoxins (MTXs) are polyether ladder shaped toxins derived from the dinoflagellate Gambierdiscus toxicus. Despite the fact that MTXs are 3 times larger than CTXs, part of the structure of MTXs resembles that of CTXs. To date, the synthetic ciguatoxin, CTX 3C has been reported to activate voltage-gated sodium channels, whereas the main effect of MTX is inducing calcium influx into the cell leading to cell death. However, there is a lack of information regarding the effects of these toxins in a common cellular model. Here, in order to have an overview of the main effects of these toxins in mice cortical neurons, we examined the effects of MTX and the synthetic ciguatoxin CTX 3C on the main voltage dependent ion channels in neurons, sodium, potassium, and calcium channels as well as on membrane potential, cytosolic calcium concentration ([Ca(2+)]c), intracellular pH (pHi), and neuronal viability. Regarding voltage-gated ion channels, neither CTX 3C nor MTX affected voltage-gated calcium or potassium channels, but while CTX 3C had a large effect on voltage-gated sodium channels (VGSC) by shifting the activation and inactivation curves to more hyperpolarized potentials and decreasing peak sodium channel amplitude, MTX, at 5 nM, had no effect on VGSC activation and inactivation but decreased peak sodium current amplitude. Other major differences between both toxins were the massive calcium influx and intracellular acidification produced by MTX but not by CTX 3C. Indeed, the novel finding that MTX produces acidosis supports a pathway recently described in which MTX produces calcium influx via the sodium-hydrogen exchanger (NHX). For the first time, we found that VGSC blockers partially blocked the MTX-induced calcium influx, intracellular acidification, and protected against the short-term MTX-induced cytotoxicity. The results presented here provide the first report that shows the comparative effects of two prototypical ciguatera toxins, CTX 3C

  19. A Common Stem Cell for Murine Cortical and Medullary Thymic Epithelial Cells?

    PubMed Central

    Van Soest, Peter; Platenburg, Peter Paul; Van Ewijk, Willem

    1995-01-01

    We have addressed the question whether the epithelial stroma in the thymus is derived from a common stem cell or whether cortical and medullary epithelial cells are derived from different embryonic stem cells emerging, for example, from endoderm and ectoderm. By the use of rapidly expanding cultures of thymic epithelial cells (TEC) from 14 to 16 day-old murine fetuses and by specific antibodies against cortical and medullary epithelium, respectively, we were able to demonstrate a small subpopulation of double-labeled TEC in the cultures. These cells were not present in TEC cultures initiated from thymuses of neonatal mice. Double-labeled TEC were also found in tissue sections from fetal thymuses. These findings may indicate that TEC populations of the cortex and the medulla are derived from a common stem cell, with potential for differentiation toward both cortical and medullary TEC. PMID:9700364

  20. Block of Na+,K+-ATPase and induction of hybrid death by 4-aminopyridine in cultured cortical neurons.

    PubMed

    Wang, Xue Qing; Xiao, Ai Ying; Yang, Aizhen; LaRose, Lori; Wei, Ling; Yu, Shan Ping

    2003-05-01

    K(+) channel blockers such as 4-aminopyridine (4-AP) can be toxic to neurons; the cellular mechanism underlying the toxicity, however, is obscure. In cultured mouse cortical neurons, we tested the hypothesis that the toxic effect of 4-AP might result from inhibiting the Na(+),K(+)-ATPase (Na(+),K(+)-pump) and thereafter induction of a hybrid death of concomitant apoptosis and necrosis. The Na(+),K(+)-pump activity, monitored as whole-cell membrane currents, was markedly blocked by 4-AP in concentration- and voltage-dependent manners in low millimolar ranges. At similar concentrations, 4-AP induced a neuronal death sensitive to attenuation by the caspase inhibitor Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethyl ketone) or Ca(2+) chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Electron microscopy confirmed hybrid ultrastructural features of coexisting apoptotic and necrotic components in same cells. We suggest that 4-AP is a potent antagonist of the Na(+),K(+)-ATPase and an inducer of the hybrid death of central neurons.

  1. [Neuroprotective effects of the effective components group of xiaoshuantongluo against oxygen-glucose deprivation in primary cultured rat cortical neurons].

    PubMed

    Xie, Xin-Mei; Pang, Xiao-Bin; Zhao, Yan; Wang, Bao-Quan; Chen, Ruo-Yun; Du, Guan-Hua

    2014-08-01

    This study is to investigate the effect of the effective components group of Xiaoshuantongluo (XECG) on neuronal injury induced by oxygen-glucose deprivation (OGD) in primary cortical cultures isolated from SD rat cortex at day 3 and the possible mechanism. Cells were divided into control group, OGD model group and XECG group (1, 3 and 10 mg x L(-1)). The cell viability was assessed with MTT assay and the LDH release rate was measured by enzyme label kit. The cell apoptosis was analyzed using Hoechst staining. RT-PCR was applied to detect the mRNA levels of JAK2 and STAT3. Western blotting was used to detect the expressions of Bcl-2, Bax, p-JAK2 and p-STAT3 proteins. Results showed that XECG resulted in an obvious resistance to oxygen-glucose deprivation-induced cell apoptosis and decrement of cell viability, decrease the cell LDH release rate. XECG could adjust the expression of Bcl-2 and Bax proteins and increase Bcl-2/Bax ratio, up-regulate the expression of p-JAK2 and p-STAT3. In conclusion, XECG could protect against the neuronal injury cells exposed to OGD, which may be relevant to the promotion of JAK2/STAT3 signaling pathway, and impact the expression of Bax and Bcl-2.

  2. Enhanced neuroprotective effects of resveratrol delivered by nanoparticles on hydrogen peroxide-induced oxidative stress in rat cortical cell culture.

    PubMed

    Lu, Xiaowei; Xu, Huae; Sun, Bo; Zhu, Zhenshu; Zheng, Donghui; Li, Xiaolin

    2013-05-06

    Resveratrol (RES) has recently been reported as a potential antioxidant in treatment of ischemia/reperfusion injury through attenuating oxidative stress and apoptosis. However, application of RES is limited for its insolubility and short half-time. Latest evidence raises the possibility of developing nanoparticle-based delivery systems with improved solubility, stability and cytotoxicity of lipophilic drug. Here, we reported first a simple way to produce RES-loaded nanoparticles (RES-NPs) based on poly(N-vinylpyrrolidone)-b-poly(ε-caprolactone) polymer and further evaluated the protective effect of RES-NPs on hydrogen peroxide-induced oxidative stress and apoptosis in rat cortical cell culture. The controlled release pattern of RES-loaded nanoparticles was characterized by in vitro release experiments. Cytotoxicity tests proved cytocompatibility of these nanoparticles with neurons. Shown by coumarin-6 loaded nanoparticles, the uptake of nanoparticles by neurons was considered through endocytosis, which could lead to higher uptake efficiency at lower concentration. Thereby, the hypothesis is raised that RES-NPs could demonstrate enhanced neuroprotection compared to an equivalent dose of free RES at lower concentration, especially. It was further supported by enhanced reduction of LDH release, elimination of ROS and MDA, and attenuation of apoptosis signal (ratio of Bax/Bcl-2, activation of caspase-3). RES-NPs could be a potential treatment needing intensive research for ischemia/reperfusion related disorder including stroke.

  3. AMPK Mediates Glucocorticoids Stress-Induced Downregulation of the Glucocorticoid Receptor in Cultured Rat Prefrontal Cortical Astrocytes

    PubMed Central

    Lu, Wei; Zhou, Hai-Yun; Long, Li-Hong; Hu, Zhuang-Li; Ni, Lan; Wang, Yi; Chen, Jian-Guo; Wang, Fang

    2016-01-01

    Chronic stress induces altered energy metabolism and plays important roles in the etiology of depression, in which the glucocorticoid negative feedback is disrupted due to imbalanced glucocorticoid receptor (GR) functions. The mechanism underlying the dysregulation of GR by chronic stress remains elusive. In this study, we investigated the role of AMP-activated protein kinase (AMPK), the key enzyme regulating cellular energy metabolism, and related signaling pathways in chronic stress-induced GR dysregulation. In cultured rat cortical astrocytes, glucocorticoid treatment decreased the level, which was accompanied by the decreased expression of liver kinase B1 (LKB1) and reduced phosphorylation of AMPK. Glucocorticoid-induced effects were attenuated by glucocorticoid-inducible kinase 1 (SGK1) inhibitor GSK650394, which also inhibited glucocorticoid induced phosphorylation of Forkhead box O3a (FOXO3a). Furthermore, glucocorticoid-induced down-regulation of GR was mimicked by the inhibition of AMPK and abolished by the AMPK activators or the histone deacetylase 5 (HDAC5) inhibitors. In line with the role of AMPK in GR expression, AMPK activator metformin reversed glucocorticoid-induced reduction of AMPK phosphorylation and GR expression as well as behavioral alteration of rats. Taken together, these results suggest that chronic stress activates SGK1 and suppresses the expression of LKB1 via inhibitory phosphorylation of FOXO3a. Downregulated LKB1 contributes to reduced activation of AMPK, leading to the dephosphorylation of HDAC5 and the suppression of transcription of GR. PMID:27513844

  4. A modified culture medium increases blastocyst formation and the efficiency of human embryonic stem cell derivation from poor-quality embryos.

    PubMed

    FAN, Yong; LUO, Yumei; CHEN, Xinjie; SUN, Xiaofang

    2010-10-01

    Human embryonic stem cells (HESCs) are defined as self-renewing cells that retain their ability to differentiate into all cell types of the body. They have enormous potential in medical applications and as a model for early human development. There is a need for derivation of new HESC lines to meet emerging requirements for their use in cell replacement therapies, disease modeling, and basic research. Here, we describe a modified culture medium containing human recombinant leukemia inhibitory factor and human basic fibroblast growth factor that significantly increases the number of human blastocysts formed and their quality, as well as the efficiency of HESC derivation from poor-quality embryos. Culturing poor-quality embryos in modified medium resulted in a two-fold increase in the blastocyst formation rate and a seven-fold increase over the derivation efficiency in conventional medium. We derived 15 HESC lines from poor-quality embryos cultured in modified culture medium and two HESC lines from quality embryos cultured in conventional culture medium. All cell lines shared typical human pluripotent stem cell features including similar morphology, normal karyotypes, expression of alkaline phosphatase, pluripotency genes, such as Oct4, and cell surface markers (SSEA-4, TRA-1-60, TRA-1-81), the ability to form teratomas in SCID mice, and the ability to differentiate into cells of three embryonic germ layers in vitro. Our data suggest that poor-quality embryos that have reached the blastocyst stage in our modified culture medium are a robust source for normal HESC line derivation.

  5. Simultaneous calcium fluorescence imaging and MR of ex vivo organotypic cortical cultures: a new test bed for functional MRI.

    PubMed

    Bai, Ruiliang; Klaus, Andreas; Bellay, Tim; Stewart, Craig; Pajevic, Sinisa; Nevo, Uri; Merkle, Hellmut; Plenz, Dietmar; Basser, Peter J

    2015-12-01

    Recently, several new functional (f)MRI contrast mechanisms including diffusion, phase imaging, proton density, etc. have been proposed to measure neuronal activity more directly and accurately than blood-oxygen-level dependent (BOLD) fMRI. However, these approaches have proved difficult to reproduce, mainly because of the dearth of reliable and robust test systems to vet and validate them. Here we describe the development and testing of such a test bed for non-BOLD fMRI. Organotypic cortical cultures were used as a stable and reproducible biological model of neuronal activity that shows spontaneous activity similar to that of in vivo brain cortex without any hemodynamic confounds. An open-access, single-sided magnetic resonance (MR) "profiler" consisting of four permanent magnets with magnetic field of 0.32 T was used in this study to perform MR acquisition. A fluorescence microscope with long working distance objective was mounted on the top of a custom-designed chamber that keeps the organotypic culture vital, and the MR system was mounted on the bottom of the chamber to achieve real-time simultaneous calcium fluorescence optical imaging and MR acquisition on the same specimen. In this study, the reliability and performance of the proposed test bed were demonstrated by a conventional CPMG MR sequence acquired simultaneously with calcium imaging, which is a well-characterized measurement of neuronal activity. This experimental design will make it possible to correlate directly the other candidate functional MR signals to the optical indicia of neuronal activity in the future.

  6. In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice.

    PubMed

    Amps, K J; Jones, M; Baker, D; Moore, H D

    2010-06-01

    The development of efficient and robust methods for the cryopreservation of human embryonic stem cells (hESCs) is important for the production of master and working cell banks for future clinical applications. Such methods must meet requirements of good manufacturing practice (GMP) and maintain genetic stability of the cell line. We investigated the culture of four Shef hESC lines in gas permeable 'culture cassettes' which met GMP compliance. hESCs adhered rapidly to the membrane and colonies displayed good proliferation and expansion. After 5-7 days of culture, hESCs were cryopreserved in situ using 10% dimethyl sulphoxide in foetal calf serum at approximately 1 degrees C/min. This method was compared with a control of standard flask culture and cryopreservation in vials. Post-thaw cassette culture displayed relative proliferation ratios (fold increase above flask/cryovial culture) of 114 (Shef 4), 8.2 (Shef 5), 195 (shef 6) and 17.5 (Shef 7). The proportion of cells expressing pluripotency markers after cryopreservation was consistently greater in cassette culture than for the control with the markers SSEA3 and SSEA4 exhibiting a significant increase (P> or =0.05). The efficiency of cell line culture in cassette was associated with the overall passage number of the cell line. The procedure enables cryopreservation of relatively large quantities of hESCs in situ, whilst returning high yields of viable, undifferentiated stem cells, thereby increasing capacity to scale up with greater efficacy.

  7. Action potential propagation through embryonic dorsal root ganglion cells in culture. II. Decrease of conduction reliability during repetitive stimulation.

    PubMed

    Lüscher, C; Streit, J; Lipp, P; Lüscher, H R

    1994-08-01

    1. The reliability of the propagation of action potentials (AP) through dorsal root ganglion (DRG) cells in embryonic slice cultures was investigated during repetitive stimulation at 1-20 Hz. Membrane potentials of DRG cells were recorded intracellularly while the axons were stimulated by an extracellular electrode. 2. In analogy to the double-pulse experiments reported previously, either one or two types of propagation failures were recorded during repetitive stimulation, depending on the cell morphology. In contrast to the double-pulse experiments, the failures appeared at longer interpulse intervals and usually only after several tens of stimuli with reliable propagation. 3. In the period with reliable propagation before the failures, a decrease in the conduction velocity and in the amplitude of the afterhyperpolarization (AHP), an increase in the total membrane conductance, and the disappearance of the action potential "shoulder" were observed. 4. The reliability of conduction during repetitive stimulation was improved by lowering the extracellular calcium concentration or by replacing the extracellular calcium by strontium. The reliability of conduction decreased by the application of cadmium, a calcium channel blocker, 4-amino pyridine, a fast potassium channel blocker, or apamin or muscarine, the blockers of calcium-dependent potassium channels. The reliability of conduction was not effected by blocking the sodium potassium pump with ouabain or by replacing extracellular sodium with lithium. 5. In the period with reliable propagation cadmium, apamin, and muscarine reduced the amplitude of the AHP. The shoulder of the action potential was more pronounced and not sensitive to repetitive stimulation when extracellular calcium was replaced by strontium. It disappeared when cadmium was applied. 6. In DRG somata changes of the intracellular Ca2+ concentration were monitored by measuring the fluorescence of the Ca2+ indicator Fluo-3 with a laser-scanning confocal

  8. Repeated stimulation of cultured networks of rat cortical neurons induces parallel memory traces

    PubMed Central

    Witteveen, Tim; van Veenendaal, Tamar M.; Dijkstra, Jelle

    2015-01-01

    During systems consolidation, memories are spontaneously replayed favoring information transfer from hippocampus to neocortex. However, at present no empirically supported mechanism to accomplish a transfer of memory from hippocampal to extra-hippocampal sites has been offered. We used cultured neuronal networks on multielectrode arrays and small-scale computational models to study the effect of memory replay on the formation of memory traces. We show that input-deprived networks develop an activity⇔connectivity balance where dominant activity patterns support current connectivity. Electrical stimulation at one electrode disturbs this balance and induces connectivity changes. Intrinsic forces in recurrent networks lead to a new equilibrium with activity patterns that include the stimulus response. The new connectivity is no longer disrupted by this stimulus, indicating that networks memorize it. A different stimulus again induces connectivity changes upon first application but not subsequently, demonstrating the formation of a second memory trace. Returning to the first stimulus does not affect connectivity, indicating parallel storage of both traces. A computer model robustly reproduced experimental results, suggesting that spike-timing-dependent plasticity and short time depression suffice to store parallel memory traces, even in networks without particular circuitry constraints. PMID:26572650

  9. Serum-Based Culture Conditions Provoke Gene Expression Variability in Mouse Embryonic Stem Cells as Revealed by Single-Cell Analysis.

    PubMed

    Guo, Guoji; Pinello, Luca; Han, Xiaoping; Lai, Shujing; Shen, Li; Lin, Ta-Wei; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H

    2016-02-02

    Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single-cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. We define highly variable gene clusters with distinct chromatin states and show that bivalent genes are prone to expression variation. At the same time, we identify an ESC-priming pathway that initiates the exit from the naive ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum-free culture reduces cellular heterogeneity and transcriptome variation in ESCs.

  10. Electrophoretic separation and analysis of living cells from solid tissues by several methods - Human embryonic kidney cell cultures as a model

    NASA Technical Reports Server (NTRS)

    Todd, Paul; Plank, Lindsay D.; Kunze, M. Elaine; Lewis, Marian L.; Morrison, Dennis R.

    1986-01-01

    The use of free-fluid electrophoresis methods to separate tissue cells having a specific function is discussed. It is shown that cells suspended by trypsinization from cultures of human embryonic kidney are electrophoretically heterogeneous and tolerate a wide range of electrophoresis buffers and conditions without significant attenuation of function. Moreover, these cells do not separate electrophoretically on the basis of size or cell position alone and can be separated according to their ability to give rise to progeny that produce specific plasminogen activators.

  11. Neuroprotective effects of genistein and folic acid on apoptosis of rat cultured cortical neurons induced by beta-amyloid 31-35.

    PubMed

    Yu, Huan-Ling; Li, Li; Zhang, Xiao-Hong; Xiang, Li; Zhang, Jie; Feng, Jin-Fang; Xiao, Rong

    2009-09-01

    Genistein and folic acid have been reported respectively to protect against the development of cognitive dysfunction; however, the underlying mechanism(s) for this protection remain unknown. In this report, the mechanism(s) contributing to the neuroprotective effects of genistein and folic acid were explored using rat cortical neuron cultures. We found that genistein and folic acid, both separately and collaboratively, increased cell viability and mitochondrial membrane potential in beta-amyloid (Abeta) 31-35-treated neurons. Furthermore, reduced percentage of comet cells and shortened tail length were observed in the neurons treated with genistein or folic acid. A more significant reduction in tail length of the comet neurons was observed in the co-administered neurons. RT-PCR analysis of the cultured cortical neurons showed down-regulated expression of p53, bax and caspase-3, but up-regulated expression of bcl-2 in the three neuroprotective treatment groups compared with neurons from the Abeta31-35 solo-treated group. In a nuclear dyeing experiment using Hoechst 33342, we found that both genistein and folic acid prevent neuronal apoptosis. Collectively, these findings suggest that the mechanism underlying the neuroprotection of genistein and folic acid singly or in combination observed in cultured cortical neuron studies might be related to their anti-apoptotic properties.

  12. Trophic and proliferative effects of Shh on motor neurons in embryonic spinal cord culture from wildtype and G93A SOD1 mice

    PubMed Central

    2013-01-01

    Background The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the support of terminally-differentiated motor neurons, of potential relevance to motor neuron disease. In addition, it may support the proliferation and differentiation of endogenous stem cells along motor neuronal lineages. As such, we have examined the trophic and proliferative effects of Shh supplementation or Shh antagonism in embryonic spinal cord cell cultures derived from wildtype or G93A SOD1 mice, a mouse model of amyotrophic lateral sclerosis. Results Shh supported survival, and stimulated growth of motor neurons, neurite outgrowth, and neurosphere formation in primary culture derived from both G93A SOD1 and WT mice. Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture. Shh-treated cultures showed increased neuronal proliferation compared to controls and especially cyclopamine treated cultures, from G93A SOD1 and WT mice. Moreover, Shh enhanced cell survival and differentiation of motor neuron precursors in WT culture. Conclusions Shh is neurotrophic to motor neurons and has mitogenic effects in WT and mSOD1 G93A culture in vitro. PMID:24119209

  13. Multidisciplinary Inquiry-Based Investigation Learning Using an Ex Ovo Chicken Culture Platform: Role of Vitamin A on Embryonic Morphogenesis

    ERIC Educational Resources Information Center

    Buskohl, Philip R.; Gould, Russell A.; Curran, Susan; Archer, Shivaun D.; Butcher, Jonathan T.

    2012-01-01

    Embryonic development offers a unique perspective on the function of many biological processes because of embryos' heightened sensitivity to environmental factors. This hands-on lesson investigates the effects of elevated vitamin A on the morphogenesis of chicken embryos. The active form of vitamin A (retinoic acid) is applied to shell-less (ex…

  14. Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion.

    EPA Science Inventory

    Morphogenetic tissue fusion is a critical and complex event in embryonic development and failure of this event leads to birth defects, such as cleft palate. Palatal fusion requires adhesion and subsequent dissolution of the medial epithelial layer of the mesenchymal palatal shelv...

  15. Effects of the analgesic acetaminophen (Paracetamol) and its para-aminophenol metabolite on viability of mouse-cultured cortical neurons.

    PubMed

    Schultz, Stephen; DeSilva, Mauris; Gu, Ting Ting; Qiang, Mei; Whang, Kyumin

    2012-02-01

    Acetaminophen has been used as an analgesic for more than a hundred years, but its mechanism of action has remained elusive. Recently, it has been shown that acetaminophen produces analgesia by the activation of the brain endocannabinoid receptor CB1 through its para-aminophenol (p-aminophenol) metabolite. The objective of this study was to determine whether p-aminophenol could be toxic for in vitro developing mouse cortical neurons as a first step in establishing a link between acetaminophen use and neuronal apoptosis. We exposed developing mouse cortical neurons to various concentrations of drugs for 24 hr in vitro. Acetaminophen itself was not toxic to developing mouse cortical neurons at therapeutic concentrations of 10-250 μg/ml. However, concentrations of p-aminophenol from 1 to 100 μg/ml produced significant (p < 0.05) loss of mouse cortical neuron viability at 24 hr compared to the controls. The naturally occurring endocannabinoid anandamide also caused similar 24-hr loss of cell viability in developing mouse cortical neurons at concentrations from 1 to 100 μg/ml, which indicates the mechanism of cell death could be through the cannabinoid receptors. The results of our experiments have shown a detrimental effect of the acetaminophen metabolite p-aminophenol on in vitro developing cortical neuron viability which could act through CB1 receptors of the endocannabinoid system. These results could be especially important in recommending an analgesic for children or individuals with traumatic brain injury who have developing cortical neurons.

  16. Estrogen and Tamoxifen Protect against Mn-Induced Toxicity in Rat Cortical Primary Cultures of Neurons and Astrocytes

    PubMed Central

    Lee, Eun-Sook Y.; Yin, Zhaobao; Milatovic, Dejan; Jiang, Haiyan; Aschner, Michael

    2009-01-01

    Chronic exposure to manganese (Mn) leads to a neurological disorder, manganism, which shares multiple common features with idiopathic Parkinson disease (IPD). 17β-Estradiol (E2) and some selective estrogen receptor modulators, including tamoxifen (TX), afford neuroprotection in various experimental models of neurodegeneration. However, the neuroprotective effects and mechanisms of E2/TX in Mn-induced toxicity have yet to be documented. Herein, we studied the ability of E2/TX to protect rat cortical primary neuronal and astroglial cultures from Mn-induced toxicity. Cell viability, Western blot, and reactive oxygen species (ROS) generation were assessed. Results established that both E2 (10nM) and TX (1μM) attenuated Mn-induced toxicity. The protective effects of E2/TX were more pronounced in astrocytes versus neurons. The E2-mediated attenuation of Mn-induced ROS generation in astrocytes at 6-h treatment (where no cell death was detected) was mediated by a classical estrogen receptor (ER) pathway and the TX-mediated effect on Mn-induced ROS generation was not mediated via classical ER-dependent mechanisms and likely by its antioxidant properties. The phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway was involved in both E2- and TX-induced attenuation of Mn-induced ROS formation (6 h) in astrocytes. Treatments with Mn for a longer duration (24 h) led to significant cell death, and the protective effects of E2 and TX were (1) not mediated by a classical ER pathway and (2) associated with activation of both mitogen-activated protein kinase/extracellular signal-regulated kinase and PI3K/Akt signaling pathways. Taken together, the results suggest that both E2 and TX offer effective therapeutic means for neuroprotection against Mn-induced toxicity. PMID:19383943

  17. Estrogen and tamoxifen protect against Mn-induced toxicity in rat cortical primary cultures of neurons and astrocytes.

    PubMed

    Lee, Eun-Sook Y; Yin, Zhaobao; Milatovic, Dejan; Jiang, Haiyan; Aschner, Michael

    2009-07-01

    Chronic exposure to manganese (Mn) leads to a neurological disorder, manganism, which shares multiple common features with idiopathic Parkinson disease (IPD). 17beta-Estradiol (E2) and some selective estrogen receptor modulators, including tamoxifen (TX), afford neuroprotection in various experimental models of neurodegeneration. However, the neuroprotective effects and mechanisms of E2/TX in Mn-induced toxicity have yet to be documented. Herein, we studied the ability of E2/TX to protect rat cortical primary neuronal and astroglial cultures from Mn-induced toxicity. Cell viability, Western blot, and reactive oxygen species (ROS) generation were assessed. Results established that both E2 (10nM) and TX (1 microM) attenuated Mn-induced toxicity. The protective effects of E2/TX were more pronounced in astrocytes versus neurons. The E2-mediated attenuation of Mn-induced ROS generation in astrocytes at 6-h treatment (where no cell death was detected) was mediated by a classical estrogen receptor (ER) pathway and the TX-mediated effect on Mn-induced ROS generation was not mediated via classical ER-dependent mechanisms and likely by its antioxidant properties. The phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway was involved in both E2- and TX-induced attenuation of Mn-induced ROS formation (6 h) in astrocytes. Treatments with Mn for a longer duration (24 h) led to significant cell death, and the protective effects of E2 and TX were (1) not mediated by a classical ER pathway and (2) associated with activation of both mitogen-activated protein kinase/extracellular signal-regulated kinase and PI3K/Akt signaling pathways. Taken together, the results suggest that both E2 and TX offer effective therapeutic means for neuroprotection against Mn-induced toxicity.

  18. Chronic intermittent ethanol treatment selectively alters N-methyl-D-aspartate receptor subunit surface expression in cultured cortical neurons.

    PubMed

    Qiang, Mei; Denny, Ashley D; Ticku, Maharaj K

    2007-07-01

    A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression. Cultured cortical neurons were exposed to 75 mM ethanol for 14 h followed by 10 h of withdrawal, repeated this cycle five times, and followed by 2 or 5 days of withdrawal. Surface-expressed NMDA receptor subunits and their endocytosis were measured by biotinylation and Western blots. CIE significantly increased NMDA receptor (NR) 1 and NR2B but not NR2A subunit surface expression after 5 days of treatment. However, CIE treatment did not reduce the NMDA receptor endocytosis. Quantification of immunocytochemistry confirmed CIE-induced increase in both the total number of NR1 and NR2B subunit clusters and their targeting to synaptic sites. It is noteworthy that this effect persisted even after ethanol withdrawal with a peak expression occurring between 0 and 2 days after withdrawal, and the expression on the plasma membrane was still at high levels after 5 days of withdrawal. In addition, this was accompanied by significant increases in postsynaptic density protein 95 clusters. Protein kinase A inhibitor completely reversed CIE-induced increase in NR1 and partially in NR2B surface level and a long-lasting effect. These changes may contribute to the development of ethanol-induced neurotoxicity and ethanol dependence.

  19. [Developing of a new feeder-free system and characterization of human embryonic stem cell sublines derived in this system under autogenic and allogenic culturing].

    PubMed

    Kol'tsova, A M; Voronkina, I V; Gordeeva, O F; Zenin, V V; Lifantseva, N V; Musorina, A S; Smagina, L V; Iakovleva, T K; Polianskaia, G G

    2012-01-01

    A new feeder-free culture system for human embryonic stem cells (hESC) was developed. It consist of extracellular matrix proteins synthesized by feeder cells--mesenchymal stem cell line SC5-MSC, which was derived from initial hESC line SC5. The major ECM proteins--fibronectin and laminin--that maintain hESC growth in feeder-free system were identified. An essential component of this system is a SC5-MSC-conditioned medium. Two hESC sublines were derived. The subline SC5-FF was cultured in autogenic and subline SC7-FF in allogenic system. Sublines SC5-FF and SC7-FF passed through more than 300 and 115 cell population doublings, retained normal diploid karyotype and an ability of in vitro differentiation into derivates of three germ layers. These sublines express markers of undifferentiated hESC: alkaline phosphatase, Oct-4, SSEA-4, TRA-1-81 and multidrug resistance transporter--ABCG2. The RT-PCR analysis revealed that undifferentiated cells SC5-FF subline, like cells of initial feeder-maintained hESC line SC5, expressed genes OCT4 and NANOG, and germ line specific genes such as DPPA3/STELLA and DAZL. An expression of OCT4, NANOG, DPPA3/STELLA ans DAZL was down-regulated during embryonic bodies differentiation, whereas expression of somatic lineages specific genes like GATA4 and AFP (extra embryonic and embryonic endoderm), PAX6 (neuroectoderm) and BRY (mesoderm) was up-regulated. The comparative analysis of some typical features (karyotype structure, the average population doubling time and the number of undifferentiated cells in populations) did not reveal essential differences between initial SC5 and SC7 lines and their sublines SC5-FF and SC7-FF. This shows that feeder-free culture systems, which are much more stable than any feeder systems, do not break main hESC features during long cultivation and can be recommended for fundamental, biomedicine and pharmacological investigations, using hESCs.

  20. EFFECT OF AROCLOR 1254 ON THE TRANSCRIPTION FACTOR CREB AND CELL VIABILITY IN A PRIMARY CULTURE OF IMMATURE CORTICAL CELLS.

    EPA Science Inventory

    Considerable work indicates that elevations in Ca2+ levels and kinase activity are sensitive responses to polychlorinated biphenyls (PCBs), which are developmental neurotoxicants. In cortical cells in vitro the PCB mixture Aroclor 1254 (A1254) induces temporally and mechanistica...

  1. Selective removal of undifferentiated embryonic stem cells from differentiation cultures through HSV1 thymidine kinase and ganciclovir treatment.

    PubMed

    Naujok, Ortwin; Kaldrack, Joanna; Taivankhuu, Terbish; Jörns, Anne; Lenzen, Sigurd

    2010-09-01

    Pluripotent cell lines such as embryonic stem cells are an attractive source for a potential cell replacement therapy. However, transplantation of differentiated cells harbors the risk of teratoma formation, presenting a serious health risk. To overcome this obstacle, a negative selection system was established that permits selective removal of undifferentiated cells during in vitro differentiation. Use of the HSV1 thymidine kinase and eGFP under the control of the Oct4 promoter allowed the destruction of undifferentiated ES cells by ganciclovir treatment; differentiated cells were unharmed. Clonal ES cells remained pluripotent and showed positive staining for a wide range of embryonic markers. Thus, treatment with ganciclovir during in vitro differentiation effectively removed the population of undifferentiated cells and provided a pure population of completely differentiated cells. This approach may pave the way for a safe application of ES cells in regenerative medicine in the future.

  2. Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro.

    PubMed

    Yuan, Chao; He, Qiang; Li, Jun-ming; Azzam, Mahmoud Mostafa; Lu, Jian-jun; Zou, Xiao-ting

    2015-06-01

    The present study evaluates the effects of embryonic age and proteolytic enzymes on the isolation and primary culture of chicken enterocyte and to establish an effective technique for chicken intestinal epithelial cell (IEC) cultivation. Fourteen-day-old, 16-day-old and 18-day-old embryos (average weight: 52.23 ± 0.76 g, 50.86 ± 0.99 g, 48.98 ± 1.03 g) were the source for preparation of enterocyte culture, and trypsin-ethylene diamine tetraacetic acid, collagenase, thermolysin and combination of collagenase and thermolysin were used for digestion medium. Optimal culture protocols were determined by qualitative assays of proliferation. Cells isolated by using 14-day-old embryo and collagenase obtain the best attachment and growth in culture, and the production of continuously growing IEC cultures. Thus, we conclude that the use of collagenase as a dissociating enzyme and 14-day-old embryo as a source can be advantageously applied to the isolation of chicken IEC and this method may be useful for various applications and basic studies of the intestinal tract concerning such objects as physiology, immunology and toxicology.

  3. Stimulation of Cultured H9 Human Embryonic Stem Cells with Thyroid Stimulating Hormone Does Not Lead to Formation of Thyroid-Like Cells

    PubMed Central

    Onyshchenko, Mykola I.; Panyutin, Igor G.; Panyutin, Irina V.; Neumann, Ronald D.

    2012-01-01

    The sodium-iodine symporter (NIS) is expressed on the cell membrane of many thyroid cancer cells, and is responsible for the radioactive iodine accumulation. However, treatment of anaplastic thyroid cancer is ineffective due to the low expression of NIS on cell membranes of these tumor cells. Human embryonic stem cells (ESCs) provide a potential vehicle to study the mechanisms of NIS expression regulation during differentiation. Human ESCs were maintained on feeder-independent culture conditions. RT-qPCR and immunocytochemistry were used to study differentiation marker expression, 125I uptake to study NIS function. We designed a two-step protocol for human ESC differentiation into thyroid-like cells, as was previously done for mouse embryonic stem cells. First, we obtained definitive endoderm from human ESCs. Second, we directed differentiation of definitive endoderm cells into thyroid-like cells using various factors, with thyroid stimulating hormone (TSH) as the main differentiating factor. Expression of pluripotency, endoderm and thyroid markers and 125I uptake were monitored throughout the differentiation steps. These approaches did not result in efficient induction of thyroid-like cells. We conclude that differentiation of human ESCs into thyroid cells cannot be induced by TSH media supplementation alone and most likely involves complicated developmental patterns that are yet to be understood. PMID:22619683

  4. Concentration dependent survival and neural differentiation of murine embryonic stem cells cultured on polyethylene glycol dimethacrylate hydrogels possessing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle.

    PubMed

    Lim, Hyun Ju; Mosley, Matthew C; Kurosu, Yuki; Smith Callahan, Laura A

    2016-12-01

    N-cadherin cell-cell signaling plays a key role in the structure and function of the nervous system. However, few studies have incorporated bioactive signaling from n-cadherin into tissue engineering matrices. The present study uses a continuous gradient approach in polyethylene glycol dimethacrylate hydrogels to identify concentration dependent effects of n-cadherin peptide, His-Ala-Val-Asp-Lle (HAVDI), on murine embryonic stem cell survival and neural differentiation. The n-cadherin peptide was found to affect the expression of pluripotency marker, alkaline phosphatase, in murine embryonic stem cells cultured on n-cadherin peptide containing hydrogels in a concentration dependent manner. Increasing n-cadherin peptide concentrations in the hydrogels elicited a biphasic response in neurite extension length and mRNA expression of neural differentiation marker, neuron-specific class III β-tubulin, in murine embryonic stem cells cultured on the hydrogels. High concentrations of n-cadherin peptide in the hydrogels were found to increase the expression of apoptotic marker, caspase 3/7, in murine embryonic stem cells compared to that of murine embryonic stem cell cultures on hydrogels containing lower concentrations of n-cadherin peptide. Increasing the n-cadherin peptide concentration in the hydrogels facilitated greater survival of murine embryonic stem cells exposed to increasing oxidative stress caused by hydrogen peroxide exposure. The combinatorial approach presented in this work demonstrates concentration dependent effects of n-cadherin signaling on mouse embryonic stem cell behavior, underscoring the need for the greater use of systematic approaches in tissue engineering matrix design in order to understand and optimize bioactive signaling in the matrix for tissue formation.

  5. Conditional ablation of p63 indicates that it is essential for embryonic development of the central nervous system.

    PubMed

    Cancino, Gonzalo I; Fatt, Michael P; Miller, Freda D; Kaplan, David R

    2015-01-01

    p63 is a member of the p53 family that regulates the survival of neural precursors in the adult brain. However, the relative importance of p63 in the developing brain is still unclear, since embryonic p63(-/-) mice display no apparent deficits in neural development. Here, we have used a more definitive conditional knockout mouse approach to address this issue, crossing p63(fl/fl) mice to mice carrying a nestin-CreERT2 transgene that drives inducible recombination in neural precursors following tamoxifen treatment. Inducible ablation of p63 following tamoxifen treatment of mice on embryonic day 12 resulted in highly perturbed forebrain morphology including a thinner cortex and enlarged lateral ventricles 3 d later. While the normal cortical layers were still present following acute p63 ablation, cortical precursors and neurons were both reduced in number due to widespread cellular apoptosis. This apoptosis was cell-autonomous, since it also occurred when p63 was inducibly ablated in primary cultured cortical precursors. Finally, we demonstrate increased expression of the mRNA encoding another p53 family member, ΔNp73, in cortical precursors of p63(-/-) but not tamoxifen-treated p63(fl/fl);R26YFP(fl/fl);nestin-CreERT2(+/Ø) embryos. Since ΔNp73 promotes cell survival, then this compensatory increase likely explains the lack of an embryonic brain phenotype in p63(-/-) mice. Thus, p63 plays a key prosurvival role in the developing mammalian brain.

  6. Induction of sestrin2 as an endogenous protective mechanism against amyloid beta-peptide neurotoxicity in primary cortical culture.

    PubMed

    Chen, Yueh-Sheng; Chen, Shang-Der; Wu, Chia-Lin; Huang, Shiang-Suo; Yang, Ding-I

    2014-03-01

    Accumulation of amyloid β-peptide (Aβ) in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neurodegeneration. Recent studies suggested sestrin2 as a crucial mediator for reactive oxygen species (ROS) scavenging and autophagy regulation that both play a pivotal role in age-dependent neurodegenerative diseases. However, the potential link between sestrin2 and Aβ neurotoxicity has never been explored. The present study was therefore undertaken to test whether sestrin2 may be induced by Aβ and its possible role in modulating Aβ neurotoxicity. We showed that sestrin2 expression was elevated in primary rat cortical neurons upon Aβ exposure; a heightened extent of sestrin2 expression was also detected in the cortices of 12-month-old APPswe/PSEN1dE9 transgenic mice. Exposure of cortical neurons to Aβ led to formation of LC3B-II, an autophagic marker; an increased LC3B-II level was also observed in the cortices of 12-month-old AD transgenic mice. More importantly, downregulation of sestrin2 by siRNA abolished LC3B-II formation caused by Aβ that was accompanied by more severe neuronal death. Inhibition of autophagy by bafilomycin A1 also enhanced Aβ neurotoxicity. Together, these results indicate that sestrin2 induced by Aβ plays a protective role against Aβ neurotoxicity through, at least in part, regulation of autophagy.

  7. Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.

    PubMed

    Amer, Luke D; Holtzinger, Audrey; Keller, Gordon; Mahoney, Melissa J; Bryant, Stephanie J

    2015-08-01

    This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15)μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced under both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (∼ 2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1(+)/Nkx6.1(+) cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates.

  8. Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures — a new donor for cell therapy

    PubMed Central

    Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

    2016-01-01

    Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases. PMID:27009841

  9. Changes in NMDA receptor-induced cyclic nucleotide synthesis regulate the age-dependent increase in PDE4A expression in primary cortical cultures

    PubMed Central

    Hajjhussein, Hassan; Suvarna, Neesha U.; Gremillion, Carmen; Judson Chandler, L.; O’Donnell, James M.

    2007-01-01

    NMDA receptor-induced cAMP and cGMP are selectively hydrolyzed by PDE4 and PDE2, respectively, in rat primary cerebral cortical and hippocampal cultures. Because cAMP levels regulate the expression of PDE4 in rat primary cortical cultures, we examined the manner in which NMDA receptor activity regulates the age-dependent increase in the expression of PDE4A observed in vivo and in vitro. Inhibiting the activity of NR2B subunit with ifenprodil blocked NMDA receptor-induced cGMP synthesis and increased NMDA receptor-induced cAMP levels in a manner that reduced PDE4 activity. Therefore, NR1/NR2B receptor-induced cGMP signaling is involved in an acute cross-talk regulation of NR1/NR2A receptor-induced cAMP levels, mediated by PDE4. Chronic inhibition of NMDA receptor activity with MK-801 reduced PDE4A1 and PDE4A5 expression and activity in a time-dependent manner; this effect was reversed by adding the PKA activator dbr-cAMP. Inhibiting GABA receptors with bicuculline increased NMDA receptor-induced cAMP synthesis and PDE4A expression in cultures treated between DIV 16 and DIV 21 but not in cultures treated between DIV 8 and DIV 13. This effect was due to a high tone of NMDA receptor-induced cGMP in younger cultures, which negatively regulated the expression of PDE4A by a PKG-mediated process. The present results are consistent with behavioral data showing that both PDE4 and PDE2 are involved in NMDA receptor-mediated memory processes. PMID:17407767

  10. [Expression of TGFbeta family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems].

    PubMed

    Lifantseva, N V; Kol'tsova, A M; Polianskaia, G G; Gordeeva, O F

    2013-01-01

    Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases ofpluripotency, we examined the expression of TGFbeta family factors (ActivinA, Nodal, Leftyl, TGFbeta1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFbeta1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFbeta1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3BMP/Smad1/5/8 endogenous branches of TGFbeta signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFbeta family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smadl/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primary states of pluripotency demonstrate diverse involvement of this

  11. Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

    PubMed Central

    Singh, Ratnesh K.; Mallela, Ramya K.; Cornuet, Pamela K.; Reifler, Aaron N.; Chervenak, Andrew P.; West, Michael D.; Wong, Kwoon Y.; Nasonkin, Igor O.

    2015-01-01

    Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for

  12. 2,3,7,8-Tetrachlorodibenzo-p-dioxin specifically reduces mRNA for the mineralization-related dentin sialophosphoprotein in cultured mouse embryonic molar teeth

    SciTech Connect

    Kiukkonen, Anu . E-mail: Anu.Kiukkonen@helsinki.fi; Sahlberg, Carin; Lukinmaa, Pirjo-Liisa; Alaluusua, Satu; Peltonen, Eija; Partanen, Anna-Maija

    2006-11-01

    Previous studies show that the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), interferes with mineralization of the dental matrices in developing mouse and rat teeth. Culture of mouse embryonic molar teeth with TCDD leads to the failure of enamel to be deposited and dentin to undergo mineralization. Lactationally exposed rats show defectively matured enamel and retardation of dentin mineralization. To see if the impaired mineralization is associated with changes in the expression of dentin sialophosphoprotein (Dspp), Bono1 and/or matrix metalloproteinase-20 (MMP-20), thought to be involved in mineralization of the dental hard tissues, we cultured mouse (NMRI) E18 mandibular molars for 3, 5 or 7 days and exposed them to 1 {mu}M TCDD after 2 days of culture. As detected by in situ hybridization of tissue sections, localization and intensity of Bono1 and MMP-20 expression showed no definite difference between the control and exposed tooth explants, suggesting that TCDD does not affect their expression. On the contrary, TCDD reduced or prevented the expression of Dspp in secretory odontoblasts and decreased it in presecretory ameloblasts. The results suggest that the retardation of dentin mineralization by TCDD in mouse molar teeth involves specific interference with Dspp expression.

  13. Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

    PubMed Central

    Tsuji, Yukiiko; Yoshimura, Naoko; Aoki, Hitomi; Sharov, Alexei A.; Ko, Minoru S.H.; Motohashi, Tsutomu; Kunisada, Takahiro

    2008-01-01

    The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products. PMID:18624284

  14. Distinct effects of Abelson kinase mutations on myocytes and neurons in dissociated Drosophila embryonic cultures: mimicking of high temperature.

    PubMed

    Liu, Lijuan; Wu, Chun-Fang

    2014-01-01

    Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl(1) and Abl(4) ) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development

  15. Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

    PubMed

    Pennington, Britney O; Clegg, Dennis O; Melkoumian, Zara K; Hikita, Sherry T

    2015-02-01

    Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies.

  16. Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

    PubMed

    Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

    2013-01-01

    We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

  17. Live-Cell, Label-Free Identification of GABAergic and Non-GABAergic Neurons in Primary Cortical Cultures Using Micropatterned Surface

    PubMed Central

    Kono, Sho; Kushida, Takatoshi; Hirano-Iwata, Ayumi; Niwano, Michio; Tanii, Takashi

    2016-01-01

    Excitatory and inhibitory neurons have distinct roles in cortical dynamics. Here we present a novel method for identifying inhibitory GABAergic neurons from non-GABAergic neurons, which are mostly excitatory glutamatergic neurons, in primary cortical cultures. This was achieved using an asymmetrically designed micropattern that directs an axonal process to the longest pathway. In the current work, we first modified the micropattern geometry to improve cell viability and then studied the axon length from 2 to 7 days in vitro (DIV). The cell types of neurons were evaluated retrospectively based on immunoreactivity against GAD67, a marker for inhibitory GABAergic neurons. We found that axons of non-GABAergic neurons grow significantly longer than those of GABAergic neurons in the early stages of development. The optimal threshold for identifying GABAergic and non-GABAergic neurons was evaluated to be 110 μm at 6 DIV. The method does not require any fluorescence labelling and can be carried out on live cells. The accuracy of identification was 98.2%. We confirmed that the high accuracy was due to the use of a micropattern, which standardized the development of cultured neurons. The method promises to be beneficial both for engineering neuronal networks in vitro and for basic cellular neuroscience research. PMID:27513933

  18. Protective effects of N-methyl-D-aspartate receptor antagonism on VX-induced neuronal cell death in cultured rat cortical neurons.

    PubMed

    Wang, Yushan; Weiss, M Tracy; Yin, Junfei; Tenn, Catherine C; Nelson, Peggy D; Mikler, John R

    2008-01-01

    Exposure of the central nervous system to organophosphorus (OP) nerve agents induces seizures and neuronal cell death. Here we report that the OP nerve agent, VX, induces apoptotic-like cell death in cultured rat cortical neurons. The VX effects on neurons were concentration-dependent, with an IC(50) of approximately 30 microM. Blockade of N-methyl-D-aspartate receptors (NMDAR) with 50 microM. D-2-amino-5-phosphonovalerate (APV) diminished 30 microM VX-induced total cell death, as assessed by alamarBlue assay and Hoechst staining. In contrast, neither antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) nor metabotropic glutamate receptors (mGluRs) had any effect on VX-induced neurotoxicity. VX-induced neuronal cell death could not be solely attributed to acetylcholinesterase (AChE) inhibition, since neither the reversible pharmacological cholinesterase inhibitor, physostigmine, nor the muscarinic receptor antagonist, atropine, affected VX-induced cell death. Importantly, APV was found to be therapeutically effective against VX-induced cell death up to 2 h post VX exposure. These results suggest that NMDARs, but not AMPARs or mGluRs, play important roles in VX-induced cell death in cultured rat cortical neurons. Based on their therapeutic effects, NMDAR antagonists may be beneficial in the treatment of VX-induced neurotoxicities.

  19. Active components from Siberian ginseng (Eleutherococcus senticosus) for protection of amyloid β(25-35)-induced neuritic atrophy in cultured rat cortical neurons.

    PubMed

    Bai, Yanjing; Tohda, Chihiro; Zhu, Shu; Hattori, Masao; Komatsu, Katsuko

    2011-07-01

    Not only neuronal death but also neuritic atrophy and synaptic loss underlie the pathogenesis of Alzheimer's disease as direct causes of the memory deficit. Extracts of Siberian ginseng (the rhizome of Eleutherococcus senticosus) were shown to have protective effects on the regeneration of neurites and the reconstruction of synapses in rat cultured cortical neurons damaged by amyloid β (Aβ)(25-35), and eleutheroside B was one of the active constituents. In this study, a comprehensive evaluation of constituents was conducted to explore active components from Siberian ginseng which can protect against neuritic atrophy induced by Aβ(25-35) in cultured rat cortical neurons. The ethyl acetate, n-butanol and water fractions from the methanol extract of Siberian ginseng showed protective effects against Aβ-induced neuritic atrophy. Twelve compounds were isolated from the active fractions and identified. Among them, eleutheroside B, eleutheroside E and isofraxidin showed obvious protective effects against Aβ(25-35)-induced atrophies of axons and dendrites at 1 and 10 μM.

  20. [Cortical blindness].

    PubMed

    Chokron, S

    2014-02-01

    Cortical blindness refers to a visual loss induced by a bilateral occipital lesion. The very strong cooperation between psychophysics, cognitive psychology, neurophysiology and neuropsychology these latter twenty years as well as recent progress in cerebral imagery have led to a better understanding of neurovisual deficits, such as cortical blindness. It thus becomes possible now to propose an earlier diagnosis of cortical blindness as well as new perspectives for rehabilitation in children as well as in adults. On the other hand, studying complex neurovisual deficits, such as cortical blindness is a way to infer normal functioning of the visual system.

  1. Evaluation of a hybrid artificial liver module based on a spheroid culture system of embryonic stem cell-derived hepatic cells.

    PubMed

    Mizumoto, Hiroshi; Hayashi, Shunsuke; Matsumoto, Kinya; Ikeda, Kaoru; Kusumi, Tomoaki; Inamori, Masakazu; Nakazawa, Kohji; Ijima, Hiroyuki; Funatsu, Kazumori; Kajiwara, Toshihisa

    2012-01-01

    Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a hybrid artificial liver is obtaining an adequate cell source. In this study, we attempt to differentiate embryonic stem (ES) cells by hepatic lineage using a polyurethane foam (PUF)/spheroid culture in which the cultured cells spontaneously form spherical multicellular aggregates (spheroids) in the pores of the PUF. We also demonstrate the feasibility of the PUF-HAL system by comparing ES cells to primary hepatocytes in in vitro and ex vivo experiments. Mouse ES cells formed multicellular spheroids in the pores of PUF. ES cells expressed liver-specific functions (ammonia removal and albumin secretion) after treatment with the differentiation-promoting agent, sodium butyrate (SB). We designed a PUF-HAL module comprised of a cylindrical PUF block with many medium-flow capillaries for hepatic differentiation of ES cells. The PUF-HAL module cells expressed ammonia removal and albumin secretion functions after 2 weeks of SB culture. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of albumin secretion function per unit volume of module was comparable to that seen in primary mouse hepatocyte culture. In the animal experiments with rats, the PUF-HAL differentiating ES cells appeared to partially contribute to recovery from liver failure. This outcome indicates that the PUF module containing differentiating ES cells may be a useful biocomponent of a hybrid artificial liver support system.

  2. Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina.

    PubMed

    Gonzalez-Cordero, Anai; West, Emma L; Pearson, Rachael A; Duran, Yanai; Carvalho, Livia S; Chu, Colin J; Naeem, Arifa; Blackford, Samuel J I; Georgiadis, Anastasios; Lakowski, Jorn; Hubank, Mike; Smith, Alexander J; Bainbridge, James W B; Sowden, Jane C; Ali, Robin R

    2013-08-01

    Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.

  3. Analysis of blastocyst culture of discarded embryos and its significance for establishing human embryonic stem cell lines.

    PubMed

    Wang, Fang; Kong, Hui-Juan; Kan, Quan-Cheng; Liang, Ju-Yan; Zhao, Fang; Bai, Ai-Hong; Li, Peng-Fen; Sun, Ying-Pu

    2012-12-01

    In recent years, applications of stem cells have already involved in all domains of life science and biomedicine. People try to establish human embryonic stem cell lines (hESCs) in order to carry out hESC-related studies. In this study, we explored what embryos are conducive to the establishment of hESCs. The discarded embryos from in vitro fertilization-embryo transfer (IVF-ET) cycles were sequentially incubated into blastocysts, and then the inner cell mass (ICM) was isolated and incubated in the mixed feeder layer. The cell lines which underwent serial passage were identified. After a total of 1,725 discarded embryos from 754 patients were incubated, 448 blastocysts were formed with 123 high-quality blastocysts. The blastulation rate was significantly higher in the discarded embryos with non-pronucleus (0PN) or 1PN than in the discarded embryos with 2PN or ≥3PN. The blastulation rate of the D3 embryos with 7-9 blastomeres was higher. Among the originally incubated 389 ICMs, 22 hESCs with normal karyotype were established, and identified to be ESCs. Therefore, in establishing hESCs with discarded embryos, D(3) 0PN or 1PN embryos with 7-9 blastomeres should be first selected, because they can improve high-quality blastulation rate which can increase the efficiency of hESC establishment.

  4. Exogenous Fibroblast Growth Factor-10 Induces Cystic Lung Development with Altered Target Gene Expression in the Presence of Heparin in Cultures of Embryonic Rat Lung

    PubMed Central

    Hashimoto, Shuichi; Nakano, Hiroshi; Suguta, Yuko; Irie, Seiko; Jianhua, Luo; Katyal, Sikardar L.

    2012-01-01

    Objectives Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs. Methods Embryonic day 14 rat lungs were cultured with FGF-10 (0–250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry. Results Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh(sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively. Conclusions Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC

  5. Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method

    PubMed Central

    Kim, So-Jung; Jung, Ji-Won; Ha, Hye-Yeong; Koo, Soo Kyung; Kim, Eung-Gook

    2017-01-01

    Background Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. Methods Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. Results Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34+CD43+ hematopoietic progenitor cells (HPCs) and CD34+CD45+ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro. Conclusion In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

  6. Lack of changes in cytosolic ionized calcium in primary cultures of rat kidney cortical cells exposed to cytotoxic concentrations of gentamicin.

    PubMed

    Swann, J D; Ulrich, R; Acosta, D

    1990-10-01

    Gentamicin nephrotoxicity in vivo has a delayed onset. Our assessment of gentamicin-induced cell death in vitro, by measuring the release of cytosolic lactate dehydrogenase (LDH), indicated a prolonged onset as well. A recent study, which showed that gentamicin caused an abrupt increase in the concentration of cytosolic free calcium ([Ca2+]i) in a trypsin-harvested kidney cell line, suggested that immediate changes in calcium homeostasis may initiate the pathogenesis of gentamicin nephrotoxicity. To study the immediate effect of gentamicin on [Ca2+]i, gentamicin was perfused for 1 hr over primary monolayer cultures of renal cortical epithelial cells, and suspensions of trypsin-harvested renal cells (from primary cultures and a cell line) were treated with gentamicin for 30 min. [Ca2+]i was determined using the fluorescent probe fura-2. Positive controls (ionomycin and mercury) reliably increased [Ca2+]i in each experimental model, but no increase in [Ca2+]i was observed with gentamicin. Because enzyme release data indicated that significant cytotoxicity did not occur until 48 hr of exposure to 2 mM gentamicin, primary cultures were exposed to gentamicin (1-2 mM) for 24-48 hr and [Ca2+]i was measured. No gentamicin-induced increase in [Ca2+]i was observed in these longer exposures, whether or not significant LDH release occurred. These results do not support a role for elevated [Ca2+]i in the cytotoxicity of gentamicin in cultured kidney cells, either immediately after exposure or following prolonged exposures.

  7. Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water.

    PubMed

    Silva, José R V; van den Hurk, Robert; Costa, Sonia H F; Andrade, Evelyn R; Nunes, Ana P A; Ferreira, Francisco V A; Lôbo, Raimundo N B; Figueiredo, José R

    2004-04-01

    The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In

  8. Embryonic stem cells: testing the germ-cell theory.

    PubMed

    Hochedlinger, Konrad

    2011-10-25

    The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state.

  9. Trace levels of mitomycin C disrupt genomic integrity and lead to DNA damage response defect in long-term-cultured human embryonic stem cells.

    PubMed

    Zhou, Di; Lin, Ge; Zeng, Si-Cong; Xiong, Bo; Xie, Ping-Yuan; Cheng, De-Hua; Zheng, Qing; Ouyang, Qi; Zhou, Xiao-Ying; Tang, Wei-Ling; Sun, Yi; Lu, Guang-Ying; Lu, Guang-Xiu

    2015-01-01

    How to maintain the genetic integrity of cultured human embryonic stem (hES) cells is raising crucial concerns for future clinical use in regenerative medicine. Mitomycin C(MMC), a DNA damage agent, is widely used for preparation of feeder cells in many laboratories. However, to what extent MMC affects the karyotypic stability of hES cells is not clear. Here, we measured residual MMC using High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry following each step of feeder preparation and found that 2.26 ± 0.77 and 3.50 ± 0.92 ng/ml remained in mouse feeder cells and human feeder cells, respectively. In addition, different amounts of MMC caused different chromosomal aberrations in hES cells. In particular, one abnormality, dup(1)(p32p36), was the same identical to one we previously reported in another hES cell line. Using Affymetrix SNP 6.0 arrays, the copy number variation changes of the hES cells maintained on MMC-inactivated feeders (MMC-feeder) were significantly more than those cultured on γ-inactivated feeder (IR-feeder) cells. Furthermore, DNA damage response (DDR) genes were down-regulated during long-term culture in the MMC-containing system, leading to DDR defect and shortened telomeres of hES cells, a sign of genomic instability. Therefore, MMC-feeder and MMC-induced genomic variation present an important safety problem that would limit such hES from being applied for future clinic use and drug screening.

  10. Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells.

    PubMed

    Mamidi, Murali Krishna; Pal, Rajarshi; Mori, Nor Azah Binti; Arumugam, Greetha; Thrichelvam, Saratha Thevi; Noor, Puteri J; Abdullah, Hj Mohamad Farouk; Gupta, Pawan Kumar; Das, Anjan Kumar; Zakaria, Zubaidah; Bhonde, Ramesh

    2011-05-01

    Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.

  11. Transfer and Detection of Freshly Isolated or Cultured Chicken (Gallus gallus) and Exotic Species’ Embryonic Gonadal Germ Stem Cells in Host Embryos

    PubMed Central

    Imus, Nastassja; Roe, Mandi; Charter, Suellen; Durrant, Barbara; Jensen, Thomas

    2015-01-01

    The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ova sexing of embryos to adjust population sex ratios. Currently, however, no attention has been given to the loss of genetic diversity following sex-selective incubation, even with respect to individuals from critically endangered species. This project evaluated the possibility of using xenotransfer of embryonic gonadal germline stem cells (GGCs) for future reintroduction of their germplasm into the gene pool. We examined and compared the host gonad colonization of freshly isolated and 3 day (3d) cultured donor GGCs from chicken and 13 species of exotic embryos. Following 3d-culture of GGCs, there was a significant increase in the percentage of stem cell marker (SSEA-1, -3, -4) positive cells. However, the percentage of positive host gonads with chicken donor-derived cells decreased from 68% (fresh) to 22% (3d), while the percentage of exotic species donor-cells positive host gonads decreased from 61% (fresh) to 49% (3d-cultured). Donor GGCs from both chicken and exotic species were localized within the caudal endoderm, including the region encompassing the gonadal ridge by 16 hours post-injection. Furthermore, donor-derived cells isolated from stage 36 host embryos were antigenic for anti SSEA-1, VASA/DDX4 and EMA-1 antibodies, presumably indicating maintenance of stem cell identity. This study demonstrates that GGCs from multiple species can migrate to the gonadal region and maintain presumed stemness following xenotransfer into a chicken host embryo, suggesting that germline stem cell migration is highly conserved in birds. PMID:24882096

  12. Three-dimensional culture of single embryonic stem-derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling.

    PubMed

    Bento, Ana R; Quelhas, Pedro; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2016-12-29

    In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES-NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES-NSPCs retained high cell viability and proliferated within small-sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII-tubulin and NF200 over time. At day 14, cell-matrix constructs mainly comprised NSPCs and neurons (46.5% βIII-tubulin(+) cells). Gamma-aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES-NSPCs expressed matriptase and secreted MMP-2/9, with MMP-2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES-NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three-dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES-NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin-based hydrogels for ES-NSPC delivery into the injured central nervous system. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Transfer and detection of freshly isolated or cultured chicken (Gallus gallus) and exotic species' embryonic gonadal germ stem cells in host embryos.

    PubMed

    Imus, Nastassja; Roe, Mandi; Charter, Suellen; Durrant, Barbara; Jensen, Thomas

    2014-06-01

    The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ovo sexing of embryos to adjust population sex ratios. Currently, however, no attention has been given to the loss of genetic diversity following sex-selective incubation, even with respect to individuals from critically endangered species. This project evaluated the possibility of using xenotransfer of embryonic gonadal germline stem cells (GGCs) for future reintroduction of their germplasm into the gene pool. We examined and compared the host gonad colonization of freshly isolated and 3 day (3d) cultured donor GGCs from chicken and 13 species of exotic embryos. Following 3d-culture of GGCs, there was a significant increase in the percentage of stem cell marker (SSEA-1, -3, -4) positive cells. However, the percentage of positive host gonads with chicken donor-derived cells decreased from 68% (fresh) to 22% (3d), while the percentage of exotic species donor-cells positive host gonads decreased from 61% (fresh) to 49% (3d-cultured). Donor GGCs from both chicken and exotic species were localized within the caudal endoderm, including the region encompassing the gonadal ridge by 16 hours post-injection. Furthermore, donor-derived cells isolated from stage 36 host embryos were antigenic for anti SSEA-1, VASA/DDX4 and EMA-1 antibodies, presumably indicating maintenance of stem cell identity. This study demonstrates that GGCs from multiple species can migrate to the gonadal region and maintain presumed stemness following xenotransfer into a chicken host embryo, suggesting that germline stem cell migration is highly conserved in birds.

  14. Selective beta-cell differentiation of dissociated embryonic pancreatic precursor cells cultured in synthetic polyethylene glycol hydrogels.

    PubMed

    Mason, Mariah N; Mahoney, Melissa J

    2009-06-01

    Continuing advances in islet cell transplantation have been promising; however, several limitations, including severe shortage of transplantable islets, hinder the widespread use of this therapy. Pancreatic precursor cells are one alternative to cadaveric donor islets. These cells found in the developing pancreatic buds are capable of self-renewal and also have the innate ability to become insulin-producing beta-cells. For this work, bioinert polyethylene glycol (PEG) hydrogels were chosen as the supportive three-dimensional matrix for encapsulation of dissociated pancreatic precursor cells obtained from the dorsal pancreatic bud of day-15 rat embryos. This culture system was selected in order to eliminate cell-extracellular matrix and cell-cell signal heterogeneity present when intact pancreatic buds are embedded in protein-based gels, the typical in vitro culture conditions used to study this cell population. In this study it was found that (1) dissociated precursor cells maintain a robust viability for 7 days in PEG hydrogel culture, (2) encapsulated cells selectively differentiate into insulin-expressing beta-cells, and (3) differentiated beta-cells have releasable insulin stores, but are not achieving a mature, glucose responsive phenotype. These findings suggest that encapsulating dissociated pancreatic precursor cells in an environment designed to minimize the heterogeneous signaling cues present during development or in standard culture conditions generates a population highly enriched in pancreatic beta-cells; however, future efforts must focus on achieving glucose responsiveness in this cell population. Further, these results indicate that differentiation down a beta-cell lineage may be the default pathway in pancreatic development.

  15. Embryonic Stem Cell-Derived Neurons are a Novel, Highly Sensitive Tissue Culture Platform for Botulinum Research

    DTIC Science & Technology

    2011-01-01

    drug discovery while dramatically decreasing animal use. Published by Elsevier Inc. 1. Introduction The Clostridium botulinum neurotoxins (BoNTs) are...Quinn, Clostridium botulinum neurotoxins act with a wide range of potencies on SH-SY5Y human neuroblastoma cells, Neurotoxicology 22 (2001) 447–453...tissue culture platform for botulinum research 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) McNutt, P, Celver, J, Hamilton, T, Mesngon

  16. ESP-102, a combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, protects against glutamate-induced toxicity in primary cultures of rat cortical cells.

    PubMed

    Ma, Choong Je; Kim, Seung Hyun; Lee, Ki Yong; Oh, Taehwan; Kim, Sun Yeou; Sung, Sang Hyun; Kim, Young Choong

    2009-11-01

    It was reported previously that ESP-102, a combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, significantly improved scopolamine-induced memory impairment in mice and protected primary cultured rat cortical cells against glutamate-induced toxicity. To corroborate this effect, the action patterns of ESP-102 were elucidated using the same in vitro system. ESP-102 decreased the cellular calcium concentration increased by glutamate, and inhibited the subsequent overproduction of cellular nitric oxide and reactive oxygen species to the level of control cells. It also preserved cellular activities of antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and glutathione reductase reduced in the glutamate-injured neuronal cells. While a loss of mitochondrial membrane potential was observed in glutamate treated cells, the mitochondrial membrane potential was maintained by ESP-102. These results support that the actual mechanism of neuroprotective activity of ESP-102 against glutamate-induced oxidative stress might be its antioxidative activity.

  17. Neuroprotective effects of a sesquiterpene lactone and flavanones from Paulownia tomentosa Steud. against glutamate-induced neurotoxicity in primary cultured rat cortical cells.

    PubMed

    Kim, Soo-Ki; Cho, Sang-Buem; Moon, Hyung-In

    2010-12-01

    The neuroprotective effects of Paulownia tomentosa against glutamate-induced neurotoxicity were studied in primary cultured rat cortical cells. It was found that the aqueous extract of this medicinal plant significantly attenuated glutamate-induced toxicity. In order to clarify the mechanism(s) underlying this neuroprotective effect, the active fractions and components were isolated and identified. Five compounds were isolated as the methanol extracts from air-dried flowers of P. tomentosa. Isoatriplicolide tiglate exhibited significant neuroprotective activity against glutamate-induced toxicity at concentrations ranging from 1 μM to 10 μM, and exhibited cell viability of approximately 43-78%. Therefore, the neuroprotective effect of P. tomentosa might be due to the inhibition of glutamate-induced toxicity by the sesquiterpene lactone derivative it contains.

  18. BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons.

    PubMed

    Haubensak, W; Narz, F; Heumann, R; Lessmann, V

    1998-06-01

    The protein family of mammalian neurotrophins, comprising nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 and -4/5 (NT-3, NT-4/5), supports the survival and the phenotype of neurons from the central as well as the peripheral nervous system (CNS, PNS). In addition, exogenous application of neurotrophins has recently been found to modulate synaptic transmission in the rodent CNS. However, to provide evidence for a role of neurotophins as endogenous fast acting modulators of synaptic transmission, the synaptic localization and secretion of neurotrophins needs to be shown. We have now constructed a fusion protein consisting of N-terminal BDNF (the most abundant neurotrophin in the rodent hippocampus and neocortex) and C-terminal green fluorescent protein (GFP) to elucidate the cellular localization of BDNF in cortical neurons. Transient expression of BDNF-GFP in COS-7 cells revealed that the cellular localization in the trans-Golgi network (TGN), the processing of precursor proteins and the secretion of mature BDNF-GFP is indistinguishable from the properties of untagged BDNF. Upon transient transfection of primary rat cortical neurons, BDNF-GFP was found in secretory granules of the regulated pathway of secretion, as indicated by colocalization with the secretory granule marker secretogranin II. BDNF-GFP vesicles were found in the neurites of transfected neurons with a pattern reminiscent of the localization of endogenous BDNF in untransfected cortical neurons. BDNF-GFP vesicles were found predominantly in the somatodendritic compartment of the neurons, whereas additional axonal localization was found less frequently. Immunocytochemical staining of synaptic terminals with synapsin I antibodies revealed that the density of BDNF-GFP vesicles is elevated in the vicinity of synaptic junctions, indicating that BDNF is localized appropriately to function as an acute modulator of synaptic transmission. These data suggest that BDNF-GFP will

  19. Short Term Culture of Vitrified Human Ovarian Cortical Tissue to Assess the Cryopreservation Outcome: Molecular and Morphological Analysis

    PubMed Central

    Ramezani, Mehdi; Salehnia, Mojdeh; Jafarabadi, Mina

    2017-01-01

    Background: The aim of the present study was to evaluate the effectiveness of human ovarian vitrification protocol followed with in vitro culture at the morphological and molecular levels. Methods: Ovarian tissues were obtained from 10 normal transsexual women and cut into small pieces and were divided into non-vitrified and vitrified groups and some of the tissues fragments in both groups were randomly cultured for two weeks. The morphological study using hematoxylin and eosin and Masson’s trichrome staining was done. The analysis of mean follicular density, 17-β estradiol (E2) and anti mullerian hormone (AMH), and real-time RT-PCR was down for the evaluation of expression of genes related to folliculogenesis. Data were compared by paired-samples and independent-samples T test. Values of p<0.05 were considered statistically significant. Results: The proportion of normal follicles did not show significant difference between vitrified and non-vitrified groups before and after culture but these rates and the mean follicle density significantly decreased in both cultured tissues (p<0.05). The expression of genes was similar in vitrified and non-vitrified groups but in cultured tissues the expression of GDF9 and FSHR genes increased and the expression of FIGLA and KIT-L genes decreased (p<0.05). An increase in E2 and AMH concentration was observed after 14 days of culture in both groups. Conclusion: In conclusion, the present study indicated that the follicular development and gene expression in vitrified ovarian tissue was not altered before and after in vitro culture, thus this method could be useful for fertility preservation; however, additional studies are needed to improve the culture condition. PMID:28377895

  20. Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: an in vitro and in vivo analysis.

    PubMed

    Furmanski, Orion; Gajavelli, Shyam; Lee, Jeung Woon; Collado, Maria E; Jergova, Stanislava; Sagen, Jacqueline

    2009-07-01

    Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of gamma-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaete-scute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and beta-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased beta-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full

  1. A chimeric vitronectin: IGF-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells.

    PubMed

    Manton, Kerry J; Richards, Sean; Van Lonkhuyzen, Derek; Cormack, Luke; Leavesley, David; Upton, Zee

    2010-09-01

    The therapeutic use of human embryonic stem (hES) cells is severely limited by safety concerns regarding their culture in media containing animal-derived or nondefined factors and on animal-derived feeder cells. Thus, there is a pressing need to develop culture techniques that are xeno-free, fully defined, and synthetic. Our laboratory has discovered that insulin-like growth factor (IGF) and vitronectin (VN) bind to each other resulting in synergistic short-term functional effects in several cell types, including keratinocytes and breast epithelial cells. We have further refined this complex into a single chimeric VN:IGF-I protein that functionally mimics the effects obtained upon binding of IGF-I to VN. The aim of the current study was to determine whether hES cells can be serially propagated in feeder-cell-free and serum-free conditions using medium containing our novel chimeric VN:IGF-I protein. Here we demonstrate that hES cells can be serially propagated and retain their undifferentiated state in vitro for up to 35 passages in our feeder-cell-free, serum-free, chemically defined media. We have utilized real-time polymerase chain reaction (PCR), immunofluorescence, and fluorescence-activated cell sorter (FACS) analysis to show that the hES cells have maintained an undifferentiated phenotype. In vitro differentiation assays demonstrated that the hES cells retain their pluripotent potential and the karyotype of the hES cells remains unchanged. This study demonstrates that the novel, fully defined, synthetic VN:IGF-I chimera-containing medium described herein is a viable alternative to media containing serum, and that in conjunction with laminin-coated plates facilitates feeder-cell-free and serum-free growth of hES.

  2. Regulation of the distribution and function of [(125)I]epibatidine binding sites by chronic nicotine in mouse embryonic neuronal cultures.

    PubMed

    Zambrano, Cristian A; Salamander, Rakel M; Collins, Allan C; Grady, Sharon R; Marks, Michael J

    2012-08-01

    Chronic nicotine produces up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) (* denotes that an additional subunit may be part of the receptor). However, the extent of up-regulation to persistent ligand exposure varies across brain regions. The aim of this work was to study the cellular distribution and function of nAChRs after chronic nicotine treatment in primary cultures of mouse brain neurons. Initially, high-affinity [(125)I]epibatidine binding to cell membrane homogenates from primary neuronal cultures obtained from diencephalon and hippocampus of C57BL/6J mouse embryos (embryonic days 16-18) was measured. An increase in α4β2*-nAChR binding sites was observed in hippocampus, but not in diencephalon, after 24 h of treatment with 1 μM nicotine. However, a nicotine dose-dependent up-regulation of approximately 3.5- and 0.4-fold in hippocampus and diencephalon, respectively, was found after 96 h of nicotine treatment. A significant fraction of total [(125)I]epibatidine binding sites in both hippocampus (45%) and diencephalon (65%) was located on the cell surface. Chronic nicotine (96 h) up-regulated both intracellular and surface binding in both brain regions without changing the proportion of those binding sites compared with control neurons. The increase in surface binding was not accompanied by an increase in nicotine-stimulated Ca(2+) influx, suggesting persistent desensitization or inactivation of receptors at the plasma membrane occurred. Given the differences observed between hippocampus and diencephalon neurons exposed to nicotine, multiple mechanisms may play a role in the regulation of nAChR expression and function.

  3. Cadmium-Induced Apoptosis in Primary Rat Cerebral Cortical Neurons Culture Is Mediated by a Calcium Signaling Pathway

    PubMed Central

    Xu, Hui; Sun, Ya; Hu, Fei-fei; Bian, Jian-chun; Liu, Xue-zhong; Gu, Jian-hong; Liu, Zong-ping

    2013-01-01

    Cadmium (Cd) is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca2+]i) homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca2+]i and inhibition of Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities. This Cd-induced elevation of [Ca2+]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca2+ is involved. In addition, we observed elevation of reactive oxygen species (ROS) levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III), depletion of mitochondrial membrane potential (ΔΨm), and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca2+ chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca2+ homeostasis, by compromising ATPases activities and ER-regulated Ca2+, and this elevation in Ca2+ triggers the activation of the Ca2+-mitochondria apoptotic signaling pathway. This

  4. Effects of frutalin on early follicle morphology, ultrastructure and gene expression in cultured goat ovarian cortical tissue.

    PubMed

    Soares, Maria A A; Costa, José J N; Vasconcelos, Gisvani L; Ribeiro, Regislane P; Souza, José C; Silva, André L C; Van den Hurk, Robert; Silva, José R V

    2017-02-15

    Frutalin is a galactose-binding lectin that has an irreversible cytotoxic effect on HeLa cervical cancer cells, by inducing apoptosis and inhibiting cell proliferation. It was previously shown that after in vitro incubation, frutalin is internalized into HeLa cells nucleus, which indicates that frutalin apoptosis-inducing activity might be linked with its nuclear localization. Considering that drugs commonly used for cancer treatment have a deleterious effect on germ cells, the aim of this study was to evaluate the effect of frutalin on the activation, survival, ultrastructure and gene expression in follicles cultured within ovarian tissue. Goat ovarian fragments were cultured for 6 days in α-MEM+ alone or supplemented with frutalin (1, 10, 50, 100 or 200 µg/ml). Non-culturad and cultured tissues were processed for histological and ultrastructural analysis and they were also stored to evaluate the expression of anti- and pro-apoptotic genes by quantitative polymerase chain reaction (qPCR). The results showed that the frutalin, at all concentrations tested, reduced follicular survival when compared with control medium. Higher concentrations of frutalin (50, 100 or 200 µg/ml) also reduced follicular survival when compared with those tissues cultured with 1 or 10 µg/ml of frutalin. The ultrastructural analysis showed that atretic cultured follicles had retracted oocytes and a large number of vacuoles spread throughout the cytoplasm. In addition, signs of damage of mitochondrial membranes and cristae were observed. Moreover, although a dose-response effect on gene expression has not been observed, when compared with tissues culture in control medium, the presence of frutalin increased in mRNA expression pro-apoptotic genes. In conclusion, frutalin reduces follicular survival at all concentrations tested, its effects being more pronounced when high concentrations of this lectin (50, 100 and 200 µg/ml) are used. Gene expression profile and ultrastrutural features of

  5. Short-Term Culture of Ovarian Cortical Strips From Capuchin Monkeys (Sapajus apella): A Morphological, Viability, and Molecular Study of Preantral Follicular Development In Vitro

    PubMed Central

    Brito, A. B.; van den Hurk, R.; Lima, J. S.; Miranda, M. S.; Ohashi, O. M.; Domingues, S. F. S.

    2013-01-01

    The aim of this study was to evaluate whether an in vitro culture (IVC) medium containing either or not β-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) could be able to promote the development of capuchin monkeys’ preantral follicles enclosed in ovarian cortical strips. Follicular viability after IVC was similar to control (89.32%). Primordial follicle recruitment to primary stage was not reached with IVC, but the rate of secondary follicle formation was increased in the medium supplemented with BME, BMP4, and PMSG (44.86%) when compared to IVC control (9.20%). In the medium supplemented with BME, BMP4, and PMSG, contrary to other media, anti-müllerian hormone-messenger RNA (mRNA) expression in ovarian tissue was upregulated (3.4-fold), while that of growth differentiation factor-9 was maintained. The BMP4-mRNA expression, however, appeared downregulated in all cultured tissues. Our findings show a favorable effect of BME, BMP4, and PMSG on the in vitro development of secondary follicles from capuchin monkeys. PMID:23314959

  6. Short-term culture of ovarian cortical strips from capuchin monkeys (Sapajus apella): a morphological, viability, and molecular study of preantral follicular development in vitro.

    PubMed

    Brito, A B; Santos, R R; van den Hurk, R; Lima, J S; Miranda, M S; Ohashi, O M; Domingues, S F S

    2013-08-01

    The aim of this study was to evaluate whether an in vitro culture (IVC) medium containing either or not β-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) could be able to promote the development of capuchin monkeys' preantral follicles enclosed in ovarian cortical strips. Follicular viability after IVC was similar to control (89.32%). Primordial follicle recruitment to primary stage was not reached with IVC, but the rate of secondary follicle formation was increased in the medium supplemented with BME, BMP4, and PMSG (44.86%) when compared to IVC control (9.20%). In the medium supplemented with BME, BMP4, and PMSG, contrary to other media, anti-müllerian hormone-messenger RNA (mRNA) expression in ovarian tissue was upregulated (3.4-fold), while that of growth differentiation factor-9 was maintained. The BMP4-mRNA expression, however, appeared downregulated in all cultured tissues. Our findings show a favorable effect of BME, BMP4, and PMSG on the in vitro development of secondary follicles from capuchin monkeys.

  7. Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution

    PubMed Central

    Nomura, Tadashi; Yamashita, Wataru; Gotoh, Hitoshi; Ono, Katsuhiko

    2015-01-01

    The mammalian neocortex is a remarkable structure that is characterized by tangential surface expansion and six-layered lamination. However, how the mammalian neocortex emerged during evolution remains elusive. Because all modern reptiles have a homolog of the neocortex at the dorsal pallium, developmental analyses of the reptilian cortex are valuable to explore the origin of the neocortex. However, reptilian cortical development and the underlying molecular mechanisms remain unclear, mainly due to technical difficulties with sample collection and embryonic manipulation. Here, we introduce a method of embryonic manipulations for the Madagascar ground gecko and Chinese softshell turtle. We established in ovo electroporation and an ex ovo culture system to address neural stem cell dynamics, neuronal differentiation and migration. Applications of these techniques illuminate the developmental mechanisms underlying reptilian corticogenesis, which provides significant insight into the evolutionary steps of different types of cortex and the origin of the mammalian neocortex. PMID:25759636

  8. Impairments in brain-derived neurotrophic factor-induced glutamate release in cultured cortical neurons derived from rats with intrauterine growth retardation: possible involvement of suppression of TrkB/phospholipase C-γ activation.

    PubMed

    Numakawa, Tadahiro; Matsumoto, Tomoya; Ooshima, Yoshiko; Chiba, Shuichi; Furuta, Miyako; Izumi, Aiko; Ninomiya-Baba, Midori; Odaka, Haruki; Hashido, Kazuo; Adachi, Naoki; Kunugi, Hiroshi

    2014-04-01

    Low birth weight due to intrauterine growth retardation (IUGR) is suggested to be a risk factor for various psychiatric disorders such as schizophrenia. It has been reported that developmental cortical dysfunction and neurocognitive deficits are observed in individuals with IUGR, however, the underlying molecular mechanisms have yet to be elucidated. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are associated with schizophrenia and play a role in cortical development. We previously demonstrated that BDNF induced glutamate release through activation of the TrkB/phospholipase C-γ (PLC-γ) pathway in developing cultured cortical neurons, and that, using a rat model for IUGR caused by maternal administration of thromboxane A2, cortical levels of TrkB were significantly reduced in IUGR rats at birth. These studies prompted us to hypothesize that TrkB reduction in IUGR cortex led to impairment of BDNF-dependent glutamatergic neurotransmission. In the present study, we found that BDNF-induced glutamate release was strongly impaired in cultured IUGR cortical neurons where TrkB reduction was maintained. Impairment of BDNF-induced glutamate release in IUGR neurons was ameliorated by transfection of human TrkB (hTrkB). Although BDNF-stimulated phosphorylation of TrkB and of PLC-γ was decreased in IUGR neurons, the hTrkB transfection recovered the deficits in their phosphorylation. These results suggest that TrkB reduction causes impairment of BDNF-stimulated glutamatergic function via suppression of TrkB/PLC-γ activation in IUGR cortical neurons. Our findings provide molecular insights into how IUGR links to downregulation of BDNF function in the cortex, which might be involved in the development of IUGR-related diseases such as schizophrenia.

  9. Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures.

    PubMed

    Dawson, V L; Brahmbhatt, H P; Mong, J A; Dawson, T M

    1994-11-01

    Nitric oxide (NO) is a potent biological messenger molecule in the central nervous system (CNS). There are several potential sources of NO production in the CNS, including neurons and endothelial cells which express NO synthase (NOS) constitutively. Astrocytes and microglia can be induced by cytokines to express a NOS isoform similar to macrophage NOS (mNOS). Primary mixed glial cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and gamma-interferon (INF-gamma) produce nitrite, a breakdown product of NO formation, in a dose-dependent manner. Nitrite production is detectable at 12 hr, peaks at 48 hr and is sustained for at least 96 hr. The NOS inhibitor, nitro-L-arginine (NArg), inhibits nitrite formation, but the immunosuppressant agent, FK506, does not. In mixed glial-neuronal cultures exposed to 50 ng LPS or 5 ng LPS and 1 microgram INF-gamma, neurons begin to die at 48 hr, approx. 24-36 hr after detectable nitrite production. Neurotoxicity is attenuated by 100 microM NArg. These data indicate that expression of inducible mNOS causes delayed neurotoxicity.

  10. Role of neuronal glutamate transporter in the cysteine uptake and intracellular glutathione levels in cultured cortical neurons.

    PubMed

    Himi, T; Ikeda, M; Yasuhara, T; Nishida, M; Morita, I

    2003-12-01

    Cysteine uptake is the rate-limiting process in glutathione synthesis. Previously we have shown that the inhibitors of excitatory amino acid transporters (EAATs) significantly enhance glutamate toxicity via depletion of intracellular glutathione. In this study we show evidence that the neuronal glutamate transporter EAAT3 is directly enrolled in cysteine uptake in cultured neurons. Neuronal cysteine uptake was dependent on the extracellular sodium, and was suppressed by EAAT inhibitors. Cysteine uptake was suppressed by extracellular glutamate and aspartate, substrates of EAATs, and not by substrates of cysteine transporters. Intracellular glutathione levels were reduced by EAAT inhibitors, and not by inhibitors of cysteine transporters. Knock down of EAAT3 expression using antisense oligonucleotide significantly reduced cysteine uptake, intracellular glutathione level, and neuronal viability against oxidative stress. These facts indicate that EAAT3 functions as a cysteine transporter, and this function seems to be unique and distinct from cysteine transporters that have been reported.

  11. Induction of ER stress in response to oxygen-glucose deprivation of cortical cultures involves the activation of the PERK and IRE-1 pathways and of caspase-12

    PubMed Central

    Badiola, N; Penas, C; Miñano-Molina, A; Barneda-Zahonero, B; Fadó, R; Sánchez-Opazo, G; Comella, J X; Sabriá, J; Zhu, C; Blomgren, K; Casas, C; Rodríguez-Alvarez, J

    2011-01-01

    Disturbance of calcium homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) are considered contributory components of cell death after ischemia. However, the signal-transducing events that are activated by ER stress after cerebral ischemia are incompletely understood. In this study, we show that caspase-12 and the PERK and IRE pathways are activated following oxygen-glucose deprivation (OGD) of mixed cortical cultures or neonatal hypoxia–ischemia (HI). Activation of PERK led to a transient phosphorylation of eIF2α, an increase in ATF4 levels and the induction of gadd34 (a subunit of an eIF2α-directed phosphatase). Interestingly, the upregulation of ATF4 did not lead to an increase in the levels of CHOP. Additionally, IRE1 activation was mediated by the increase in the processed form of xbp1, which would be responsible for the observed expression of edem2 and the increased levels of the chaperones GRP78 and GRP94. We were also able to detect caspase-12 proteolysis after HI or OGD. Processing of procaspase-12 was mediated by NMDA receptor and calpain activation. Moreover, our data suggest that caspase-12 activation is independent of the unfolded protein response activated by ER stress. PMID:21525936

  12. Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

    PubMed

    Numakawa, Yumiko; Numakawa, Tadahiro; Matsumoto, Tomoya; Yagasaki, Yuki; Kumamaru, Emi; Kunugi, Hiroshi; Taguchi, Takahisa; Niki, Etsuo

    2006-05-01

    The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre-treatment with vitamin E analogs including alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up-regulation of Bcl-2 (survival promoting molecule) was induced by alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-up-regulated Bcl-2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.

  13. The trans-species core SELF: the emergence of active cultural and neuro-ecological agents through self-related processing within subcortical-cortical midline networks.

    PubMed

    Panksepp, Jaak; Northoff, Georg

    2009-03-01

    The nature of "the self" has been one of the central problems in philosophy and more recently in neuroscience. This raises various questions: (i) Can we attribute a self to animals? (ii) Do animals and humans share certain aspects of their core selves, yielding a trans-species concept of self? (iii) What are the neural processes that underlie a possible trans-species concept of self? (iv) What are the developmental aspects and do they result in various levels of self-representation? Drawing on recent literature from both human and animal research, we suggest a trans-species concept of self that is based upon what has been called a "core-self" which can be described by self-related processing (SRP) as a specific mode of interaction between organism and environment. When we refer to specific neural networks, we will here refer to the underlying system as the "core-SELF." The core-SELF provides primordial neural coordinates that represent organisms as living creatures-at the lowest level this elaborates interoceptive states along with raw emotional feelings (i.e., the intentions in action of a primordial core-SELF) while higher medial cortical levels facilitate affective-cognitive integration (yielding a fully-developed nomothetic core-self). Developmentally, SRP allows stimuli from the environment to be related and linked to organismic needs, signaled and processed within core-self structures within subcorical-cortical midline structures (SCMS) that provide the foundation for epigenetic emergence of ecologically framed, higher idiographic forms of selfhood across different individuals within a species. These functions ultimately operate as a coordinated network. We postulate that core SRP operates automatically, is deeply affective, and is developmentally and epigenetically connected to sensory-motor and higher cognitive abilities. This core-self is mediated by SCMS, embedded in visceral and instinctual representations of the body that are well integrated with basic

  14. Drugs for stroke: action of nitrone (Z)-N-(2-bromo-5-hydroxy-4-methoxybenzylidene)-2-methylpropan-2-amine oxide on rat cortical neurons in culture subjected to oxygen-glucose-deprivation.

    PubMed

    Arce, Carmen; Diaz-Castroverde, Sabela; Canales, María J; Marco-Contelles, José; Samadi, Abdelouahid; Oset-Gasque, María J; González, María P

    2012-09-01

    The action of (Z)-N-(2-bromo-5-hydroxy-4-methoxybenzylidene)-2-methylpropan-2-amine oxide (RP6) on rat cortical neurons in culture, under oxygen-glucose-deprivation conditions, is reported. Cortical neurons in culture were treated during 1 h with OGD. After, they were placed under normal conditions during 24 h (reperfusion) in absence and presence of RP6. Different parameters were measured under each condition (control, 1 h OGD and 1 h OGD + reperfusion in absence and presence of RP6). RP6 protects neurons against ROS generation, lipid peroxidation levels, LDH release and mitochondrial membrane potential alteration, when administered during reperfusion after the OGD damage. Consequently, these results show that nitrone RP6 protects cells against ischemia injury produced during the reoxygenation, and could be a potential drug for the ictus therapy.

  15. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    SciTech Connect

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R.

    2011-11-15

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  16. Pyruvate and cilostazol protect cultured rat cortical pericytes against tissue plasminogen activator (tPA)-induced cell death.

    PubMed

    Kim, Ha Na; Kim, Tae-Youn; Yoon, Young Hee; Koh, Jae-Young

    2015-12-02

    Since even a brief ischemia can cause permanent brain damage, rapid restoration of blood flow is critical to limiting damage. Although intravenous tPA during the acute stage is the treatment of choice for achieving reperfusion, this treatment is sometimes associated with brain hemorrhage. Agents that reduce tPA-related bleeding risk may help expand its therapeutic window. This study assessed whether zinc dyshomeostasis underlies the toxic effect of tPA on brain vascular pericytes; whether pyruvate, an inhibitor of zinc toxicity, protects pericytes against tPA-induced cell death; and whether cilostazol, which protects pericytes against tPA-induced cell death, affects zinc dyshomeostasis associated with tPA toxicity. Cultured pericytes from newborn rat brains were treated with 10-200 μg/ml tPA for 24 h, inducing cell death in a concentration-dependent manner. tPA-induced cell death was preceded by increases in intracellular free zinc levels, and was substantially attenuated by plasminogen activator inhibitor-1 (PAI-1) or TPEN. Pyruvate completely blocked direct zinc toxicity and tPA-induced pericyte cell death. Both cAMP and cilostazol, a PDE3 inhibitor that attenuates tPA-induced pericyte cell death in vitro and tPA-induced brain hemorrhage in vivo, reduced zinc- and tPA-induced pericyte cell death, suggesting that zinc dyshomeostasis may be targeted by cilostazol in tPA toxicity. These findings show that tPA-induced pericyte cell death may involve zinc dyshomeostasis, and that pyruvate and cilostazol attenuate tPA-induced cell death by reducing the toxic cascade triggered by zinc dyshomeostasis. Since pyruvate is an endogenous metabolite and cilostazol is an FDA-approved drug, in vivo testing of both as protectors against tPA-induced brain hemorrhage may be warranted. This article is part of a Special Issue entitled SI: Neuroprotection.

  17. Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

    SciTech Connect

    Gu, Angel; Tsark, Walter; Holmes, Kathryn V.; Shively, John E.

    2009-06-10

    CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (- 8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as - 5 to - 3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased

  18. Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device.

    PubMed

    van der Meer, Andries D; Orlova, Valeria V; ten Dijke, Peter; van den Berg, Albert; Mummery, Christine L

    2013-09-21

    Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here, we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells, human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h, the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell-cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels, inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity, highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary, we have

  19. Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning: fungiform papillae double in number and form in novel locations in dorsal lingual epithelium.

    PubMed

    Mistretta, Charlotte M; Liu, Hong-Xiang; Gaffield, William; MacCallum, Donald K

    2003-02-01

    From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline

  20. Amyloid beta protein inhibits cellular MTT reduction not by suppression of mitochondrial succinate dehydrogenase but by acceleration of MTT formazan exocytosis in cultured rat cortical astrocytes.

    PubMed

    Abe, K; Saito, H

    1998-08-01

    Alzheimer's disease amyloid beta protein (Abeta) inhibits cellular reduction of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Kaneko et al. have previously hypothesized that Abeta works by suppressing mitochondrial succinate dehydrogenase (SDH), but Liu and Schubert have recently demonstrated that Abeta decreases cellular MTT reduction by accelerating the exocytosis of MTT formazan in neuronal cells. To ask which is the case in astrocytes, we compared the effects of Abeta and 3-nitropropionic acid (3-NP), a specific SDH inhibitor, on MTT reduction in cultured rat cortical astrocytes. Treatment with 3-NP (10 mM) decreased cellular activity of MTT reduction, regardless of the time of incubation with MTT. On the other hand. Abeta-induced inhibition of cellular MTT reduction was dependent on the time of incubation with MTT. The cells treated with Abeta (0.1-1000 nM) exhibited normal capacity for MTT reduction at an early stage of incubation ( < 30 min), but ceased to reduce MTT at the late stage (> 1 h). Microscopic examination revealed that Abeta treatment accelerated the appearance of needle-like MTT formazan crystals at the cell surface. These observations support that Abeta accelerates the exocytosis of MTT formazan in astrocytes. In addition to inhibition of MTT reduction, Abeta is known to induce morphological changes in astrocytes. Following addition of Abeta (20 microM), polygonal astrocytes changed into process-bearing stellate cells. To explore a possible linkage between these two effects of Abeta, we tested if astrocyte stellation is induced by agents that mimic the effect of Abeta on MTT reduction. Cholesterol (5 5000 nM) and lysophosphatidic acid (0.2-20 microg/ml) were found to accelerate the exocytosis of MTT formazan in a similar manner to Abeta, but failed to induce astrocyte stellation. Therefore, Abeta-induced inhibition of MTT reduction is unlikely to be directly linked to its effect on astrocyte morphology.

  1. Explant culture of mouse embryonic whole lung, isolated epithelium, or mesenchyme under chemically defined conditions as a system to evaluate the molecular mechanism of branching morphogenesis and cellular differentiation.

    PubMed

    Del Moral, Pierre-Marie; Warburton, David

    2010-01-01

    Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages, requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceed under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors, and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown. Additionally, to further study epithelial-mesenchymal interactions, the relative roles of epithelium versus mesenchyme signaling can also be determined using tissue recombination (e.g., epithelial and mesenchymal separation) and microbead studies.

  2. Cortical Visual Impairment

    MedlinePlus

    ... Frequently Asked Questions Español Condiciones Chinese Conditions Cortical Visual Impairment En Español Read in Chinese What is cortical visual impairment? Cortical visual impairment (CVI) is a decreased ...

  3. Primary cultures of rat cortical microglia treated with nicotine increases in the expression of excitatory amino acid transporter 1 (GLAST) via the activation of the α7 nicotinic acetylcholine receptor.

    PubMed

    Morioka, N; Tokuhara, M; Nakamura, Y; Idenoshita, Y; Harano, S; Zhang, F F; Hisaoka-Nakashima, K; Nakata, Y

    2014-01-31

    Although the clearance of glutamate from the synapse under physiological conditions is performed by astrocytic glutamate transporters, their expression might be diminished under pathological conditions. Microglia glutamate transporters, however, might serve as a back-up system when astrocytic glutamate uptake is impaired, and could have a prominent neuroprotective function under pathological conditions. In the current study, the effect of nicotine, well known as a neuroprotective molecule, on the function of glutamate transporters in cultured rat cortical microglia was examined. Reverse transcription polymerase chain reaction and pharmacological approaches demonstrated that, glutamate/aspartate transporter (GLAST), not glutamate transporter 1 (GLT-1), is the major functional glutamate transporter in cultured cortical microglia. Furthermore, the α7 subunit was demonstrated to be the key subunit comprising nicotinic acetylcholine (nACh) receptors in these cells. Treatment of cortical microglia with nicotine led to a significant increase of GLAST mRNA expression and (14)C-glutamate uptake in a concentration- and time-dependent manner, which were markedly inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The nicotine-induced expression of GLAST mRNA and protein is mediated through an inositol trisphosphate (IP3) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) depend intracellular pathway, since pretreatment with either xestospongin C, an IP3 receptor antagonist, or KN-93, a CaMKII inhibitor, blocked GLAST expression. Together, these findings indicate that activation of nACh receptors, specifically those expressing the α7 subunit, on cortical microglia could be a key mechanism of the neuroprotective effect of nACh receptor ligands such as nicotine.

  4. Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-{beta}- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures

    SciTech Connect

    Kamanga-Sollo, E.; Pampusch, M.S.; White, M.E.; Hathaway, M.R.; Dayton, W.R. . E-mail: wdayton@umn.edu

    2005-11-15

    We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-{beta} superfamily members myostatin and TGF-{beta}{sub 1} have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-{beta}{sub 1} or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-{beta}{sub 1} and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-{beta}{sub 1} or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-{beta} and myostatin to suppress proliferation of PEMC.

  5. Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat.

    PubMed

    Fischer, Wolfgang; Nörenberg, Wolfgang; Franke, Heike; Schaefer, Michael; Illes, Peter

    2009-10-10

    The expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura-2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X(2,4,6) and P2Y(1,2) subtypes. P2X(1) and P2X(7) receptor immunoreactivity (IR) was found on thin axon-like processes and presynaptic structures, respectively. Application of ATP caused a small concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in most investigated neurons, whereas only about the half of these cells responded to 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP), ADPbetaS, 2MeSADP, or 2MeSATP and even fewer cells to UTP. In contrast, alpha,beta-meATP, UDP, and UDP-glucose failed to produce any [Ca2+]i signaling. The response to ATP itself was inhibited by pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Reactive Blue 2, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179), and suramin (300 microM) as well as by a cyclopiazonic acid-induced depletion of intracellular Ca2+ stores. A Ca2+-free external medium tended to decrease the ATP-induced [Ca2+]i transients, although this action did not reach statistical significance. Various blockers of voltage-sensitive Ca2+ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(-)-2-amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) decreased it. Cross-desensitization experiments between ADPbetaS or UTP and ATP suggested that ATP acts on the one hand via P2Y(1,2) receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca2

  6. Isolation, characterization, and extra-embryonic secretion of the Xenopus laevis embryonic epidermal lectin, XEEL.

    PubMed

    Nagata, Saburo

    2005-03-01

    The Xenopus laevis embryonic epidermal lectin (XEEL) is a novel member of a group of lectins including mammalian intelectins, frog oocyte cortical granule lectins, and plasma lectins in lower vertebrates and ascidians. We isolated the XEEL protein from the extract of tailbud embryos by affinity chromatography on a galactose-Sepharose column. The XEEL protein is a homohexamer of 43-kDa N-glycosylated peptide subunits linked by disulfide bonds. It requires Ca(2+) for saccharide binding and shows a higher affinity to pentoses than hexoses and disaccharides. HEK-293T cells transfected with an expression vector containing the XEEL cDNA secrete into the culture medium the recombinant XEEL (rXEEL) that is similar to the purified XEEL in its molecular nature and saccharide-binding properties. Substitution of Asn-192 to Gln removed the N-linked carbohydrate and inhibited secretion of rXEEL but did not abolish the activity to bind to galactose-Sepharose. The embryo's XEEL content, as estimated by western blot analyses, increases during neurula/tailbud stages and declines after 1 week postfertilization. Immunofluorescence and immuno-electron microscopic analyses showed localization of the XEEL protein in a typical secretory granule pathway of nonciliated epidermal cells. When tailbud embryos were cultured in the standard medium, XEEL was accumulated in the medium, indicating secretion of XEEL into the environmental water. The rate of XEEL secretion greatly increased at around the hatching stage and stayed at a high level during the first week after hatching. XEEL may have a role in innate immunity to protect embryos and larvae against pathogenic microorganisms in the environmental water.

  7. Decision by division: making cortical maps

    PubMed Central

    Rakic, Pasko; Ayoub, Albert E.; Breunig, Joshua J.; Dominguez, Martin H.

    2013-01-01

    In the past three decades, mounting evidence has revealed that specification of the basic cortical neuronal classes starts at the time of their final mitotic divisions in the embryonic proliferative zones. This early cell determination continues during the migration of the newborn neurons across the widening cerebral wall, and it is in the cortical plate that they attain their final positions and establish species-specific cytoarchitectonic areas. Here, the development and evolutionary expansion of the neocortex is viewed in the context of the radial unit and protomap hypotheses. A broad spectrum of findings gave insight into the pathogenesis of cortical malformations and the biological bases for the evolution of the modern human neocortex. We examine the history and evidence behind the concept of early specification of neurons and provide the latest compendium of genes and signaling molecules involved in neuronal fate determination and specification. PMID:19380167

  8. Electroporation of Embryonic Kidney Explants

    NASA Astrophysics Data System (ADS)

    Haddad, Nicholas; Houle, Daniel; Gupta, Indra R.

    Metanephric kidney development in the mouse begins at embryonic day (E) 10.5, when the ureteric bud (UB), an outgrowth of the epithelial nephric duct, invades the neighboring metanephric mesenchyme (MM). The ureteric bud then undergoes a series of branching events to form the collecting duct network of the adult kidney (Fig. 19.1). As each ureteric bud tip forms, the adjacent undifferentiated mesenchyme is induced to epithelialize and form a nephron, the functional unit of the adult kidney that filters waste. Rodent embryonic kidneys can be dissected and cultured as explants such that branching morphogenesis and nephrogenesis can be observed ex vivo (Rothenpieler and Dressler, 1993; Vega et al., 1996; Piscione et al., 1997; Gupta et al., 2003).

  9. WNT/β-Catenin signaling pathway regulates non-tumorigenesis of human embryonic stem cells co-cultured with human umbilical cord mesenchymal stem cells

    PubMed Central

    Chang, Yu-Hsun; Chu, Tang-Yuan; Ding, Dah-Ching

    2017-01-01

    Human pluripotent stem cells harbor hope in regenerative medicine, but have limited application in treating clinical diseases due to teratoma formation. Our previous study has indicated that human umbilical cord mesenchymal stem cells (HUCMSC) can be adopted as non-teratogenenic feeders for human embryonic stem cells (hESC). This work describes the mechanism of non-tumorigenesis of that feeder system. In contrast with the mouse embryonic fibroblast (MEF) feeder, HUCMSC down-regulates the WNT/β-catenin/c-myc signaling in hESC. Thus, adding β-catenin antagonist (FH535 or DKK1) down-regulates β-catenin and c-myc expressions, and suppresses tumorigenesis (3/14 vs. 4/4, p = 0.01) in hESC fed with MEF, while adding the β-catenin enhancer (LiCl or 6-bromoindirubin-3′-oxime) up-regulates the expressions, and has a trend (p = 0.056) to promote tumorigenesis (2/7 vs. 0/21) in hESC fed with HUCMSC. Furthermore, FH535 supplement does not alter the pluripotency of hESC when fed with MEF, as indicated by the differentiation capabilities of the three germ layers. Taken together, this investigation concludes that WNT/β-catenin/c-myc pathway causes the tumorigenesis of hESC on MEF feeder, and β-catenin antagonist may be adopted as a tumor suppressor. PMID:28157212

  10. Production and validation of a good manufacturing practice grade human fibroblast line for supporting human embryonic stem cell derivation and culture

    PubMed Central

    2012-01-01

    Introduction The development of reproducible methods for deriving human embryonic stem cell (hESC) lines in compliance with good manufacturing practice (GMP) is essential for the development of hESC-based therapies. Although significant progress has been made toward the development of chemically defined conditions for the maintenance and differentiation of hESCs, efficient derivation of new hESCs requires the use of fibroblast feeder cells. However, GMP-grade feeder cell lines validated for hESC derivation are not readily available. Methods We derived a fibroblast cell line (NclFed1A) from human foreskin in compliance with GMP standards. Consent was obtained to use the cells for the production of hESCs and to generate induced pluripotent stem cells (iPSCs). We compared the line with a variety of other cell lines for its ability to support derivation and self-renewal of hESCs. Results NclFed1A supports efficient rates (33%) of hESC colony formation after explantation of the inner cell mass (ICM) of human blastocysts. This compared favorably with two mouse embryonic fibroblast (MEF) cell lines. NclFed1A also compared favorably with commercially available foreskin fibroblasts and MEFs in promoting proliferation and pluripotency of a number of existing and widely used hESCs. The ability of NclFed1A to maintain self-renewal remained undiminished for up to 28 population doublings from the master cell bank. Conclusions The human fibroblast line Ncl1Fed1A, produced in compliance with GMP standards and qualified for derivation and maintenance of hESCs, is a useful resource for the advancement of progress toward hESC-based therapies in regenerative medicine. PMID:22472092

  11. The autism-related gene SNRPN regulates cortical and spine development via controlling nuclear receptor Nr4a1

    PubMed Central

    Li, Huiping; Zhao, Pingping; Xu, Qiong; Shan, Shifang; Hu, Chunchun; Qiu, Zilong; Xu, Xiu

    2016-01-01

    The small nuclear ribonucleoprotein polypeptide N (SNRPN) gene, encoding the RNA-associated SmN protein, duplications or deletions of which are strongly associated with neurodevelopmental disabilities. SNRPN-coding protein is highly expressed in the brain. However, the role of SNRPN protein in neural development remains largely unknown. Here we showed that the expression of SNRPN increased markedly during postnatal brain development. Overexpression or knockdown of SNRPN in cortical neurons impaired neurite outgrowth, neuron migration, and the distribution of dendritic spines. We found that SNRPN regulated the expression level of Nr4a1, a critical nuclear receptor during neural development, in cultured primary cortical neurons. The abnormal spine development caused by SNRPN overexpression could be fully rescued by Nr4a1 co-expression. Importantly, we found that either knockdown of Nr4a1 or 3, 3′- Diindolylmethane (DIM), an Nr4a1 antagonist, were able to rescue the effects of SNRPN knockdown on neurite outgrowth of embryonic cortical neurons, providing the potential therapeutic methods for SNRPN deletion disorders. We thus concluded that maintaining the proper level of SNRPN is critical in cortical neurodevelopment. Finally, Nr4a1 may serve as a potential drug target for SNRPN-related neurodevelopmental disabilities, including Prader-Willi syndrome (PWS) and autism spectrum disorders (ASDs). PMID:27430727

  12. Isolated rat cortical progenitor cells are maintained in division in vitro by membrane-associated factors.

    PubMed

    Temple, S; Davis, A A

    1994-04-01

    Ventricular zone cells in the developing CNS undergo extensive cell division in vivo and under certain conditions in vitro. The culture conditions that promote cell division have been studied to determine the role that contact with cell membrane associated factors play in the proliferation of these cells. Progenitor cells have been taken from the ventricular zone of developing rat cerebral cortex and placed into microwells. Small clusters of these cells can generate large numbers of neurons and non-neuronal progeny. In contrast, single progenitor cells largely cease division, approximately 90% acquiring neuron-like characteristics by 1 day in vitro. DiI-labeled, single cells from embryonic day 14 cortex plated onto clusters of unmarked progenitor cells have a significantly higher probability (approximately 3-fold) of maintaining a progenitor cell phenotype than if plated onto the plastic substratum around 100 microns away from the clusters. Contact with purified astrocytes also promotes the progenitor cell phenotype, whereas contact with meningeal fibroblasts or balb3T3 cells promotes their differentiation. Membrane homogenates from cortical astrocytes stimulate significantly more incorporation of BrdU by E14 cortical progenitor cells than membrane homogenates from meningeal fibroblasts. These data indicate that the proliferation of rat cortical progenitor cells can be maintained by cell-type specific, membrane-associated factors.

  13. Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AM Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.; Wuethrich, A. J.; Hancock, D. L.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    Broiler chickens at 35 days of age were fed 1 ppm clenbuterol for 14 days. This level of dietary clenbuterol led to 5-7% increases in weights of leg and breast muscle tissue. At the end of the 14-day period, serum was prepared from both control and clenbuterol-treated chickens and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and breast muscle groups of twelve-day chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 micron clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 days beginning on the seventh day in culture. Neither the percent fusion nor the number of nuclei in myotubes were significantly affected by any of the treatments. The quantity of MHC was not increased by serum from clenbuterol-treated chickens in either breast and leg muscle cultures; however, MHC quantity was 50- 100% higher in cultures grown in control chicken serum to which 10 nM and 50 nM clenbuterol had also been added. The Beta-AR population was 4,000-7,000 Beta-AR per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the Beta-AR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 18,000-20,000 Beta-AR per cell. Basal concentration of cAMP was not significantly affected by any of the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 micron isoproterenol, limited increases of 12-20% in cAMP concentration above basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 micron isoproterenol, increases of 600

  14. The biology and dynamics of mammalian cortical granules.

    PubMed

    Liu, Min

    2011-11-17

    Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals.

  15. The biology and dynamics of mammalian cortical granules

    PubMed Central

    2011-01-01

    Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals. PMID:22088197

  16. [The comparison of biologic character between mouse embryonic fibroblast and human embryonic fibroblast].

    PubMed

    Zhang, Yi; Zhao, Liansan; Wang, Chengxiao; Lei, Binjun

    2003-06-01

    To evaluate the feasibility of using human embryonic fibroblast(HEF) as feeder layer in the culture of human embryonic stem(ES) cells in vitro, we investigated the morphology, the sensitivity to 0.25% trypsin, the growth curve and cell cycle of HEF with DMEM(low glucose) +10% FBS used as culture medium, and then we compared HEF with mouse embryonic fibroblast (MEF). The results showed that both HEF and MEF are adherent cells in vitro, and HEF has longer life span and better growth ability than MEF. In room temperature, HEF is more sensitive to 0.25% trypsin. Our research suggested that HEF can be used as feeder layer in culture of ES cells. HEF has longer service life than MEF and is worthy to be studied further.

  17. Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia.

    PubMed

    Hisaoka-Nakashima, Kazue; Kajitani, Naoto; Kaneko, Masahiro; Shigetou, Takahiro; Kasai, Miho; Matsumoto, Chie; Yokoe, Toshiki; Azuma, Honami; Takebayashi, Minoru; Morioka, Norimitsu; Nakata, Yoshihiro

    2016-03-01

    A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action.

  18. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    PubMed Central

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  19. Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AMP Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, Kristin Y.; Wuethrich, Andrew J.; Hancock, Deana L.

    2002-01-01

    Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 uM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The B-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum with leg muscle cultures having approximately 25-30% more receptors than breast muscle Culture. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR Population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 uM isoproterenol, limited increases of 12-20% in cAMP Concentration above the. basal levels were observed. However, when cultures grown in the presence of horse serum were

  20. Replacement of serum with sericin in in vitro maturation and culture media: Effects on embryonic developmental competence of Sanjabi sheep embryo during breeding season.

    PubMed

    Hajarian, H; Aghaz, F; Karami Shabankareh, H

    2017-04-01

    Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation, as a serum replacement, in maturation and culture media on the meiotic competence of oocytes or in vitro culture of ovine embryos. In experiment 1, oocytes were matured in the presence of 10% fetal ovine serum (FOS), 0.1% polyvinyl alcohol (PVA) and different concentrations of sericin (0.1, 0.5, 1 and 2.5%), for 24 h. The addition of 0.5% sericin to maturation medium increased the rates of maturation to metaphase II of oocytes compared with those in cultures with 0.1% PVA. Following fertilization, blastocyst development was higher for oocytes matured with 0.5% of sericin compared with 0.1% PVA. However, the rates of nuclear maturation of oocytes and blastocyst development under FOS and 0.5% sericin were not significantly different. In experiment 2, presumptive zygotes were cultured in the presence of 10% FOS, 0.1% PVA and different concentrations of sericin (0.1, 0.5, 1 and 2.5%), for 7-8 days. The addition of 0.5% sericin to culture medium increased the blastocyst rate compared with those in cultures without sericin or addition of 0.1% PVA and 10% FOS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM and IVC in ovine oocytes and zygotes.

  1. Live imaging of mitosis in the developing mouse embryonic cortex.

    PubMed

    Pilaz, Louis-Jan; Silver, Debra L

    2014-06-04

    Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

  2. Serum-free culture of an embryonic cell line from Bombyx mori and reinforcement of susceptibility of a recombinant BmNPV by cooling.

    PubMed

    Imanishi, Shigeo; Kobayashi, Jun; Sekine, Toshiaki

    2012-03-01

    We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67±5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.

  3. Comparative aspects of cortical neurogenesis in vertebrates.

    PubMed

    Cheung, Amanda F P; Pollen, Alexander A; Tavare, Aniket; DeProto, Jamin; Molnár, Zoltán

    2007-08-01

    The mammalian neocortex consists of six layers. By contrast, the reptilian and avian cortices have only three, which are believed to be equivalent to layers I, V and VI of mammals. In mammals, the majority of cortical cell proliferation occurs in the ventricular and subventricular zones, but there are a small number of scattered individual divisions throughout the cortex. Neurogenesis in the cortical subventricular zone is believed to contribute to the supragranular layers. To estimate the proportions of different forms of divisions in reptiles and birds, we examined the site of proliferation in embryonic turtle (stages 18-25) and chick (embryonic days 8-15) brains using phospho-histone H3 (a G2 and M phase marker) immunohistochemistry. In turtle, only few scattered abventricular H3-immunoreactive cells were found outside the ventricular zone; the majority of the H3-immunoreactive cells were located in the ventricular zone throughout the entire turtle brain. Ventricular zone cell proliferation peaks at stages 18 and 20, before an increase of abventricular proliferation at stages 23 and 25. In turtle cortex, however, abventricular proliferation at any given stage never exceeded 17.5+/-2.47% of the total division and the mitotic profiles did not align parallel to the ventricular zone. Phospho-histone H3 immunoreactivity in embryonic chick brains suggests the lack of subventricular zone in the dorsal cortex, but the presence of subventricular zone in the ventral telencephalon. We were able to demonstrate that the avian subventricular zone is present in both pallial and subpallial regions of the ventral telencephalon during embryonic development, and we characterize the spatial and temporal organization of the subventricular zone. Comparative studies suggest that the subventricular zone was involved in the laminar expansion of the cortex to six layers in mammals from the three-layered cortex found in reptiles and birds. Within mammals, the number of neurons in a

  4. Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator

    PubMed Central

    Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Yasuda, Hisataka; Sakamoto, Reiko; Yoshida, Nobuaki

    2016-01-01

    Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs. PMID:27401343

  5. Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator.

    PubMed

    Akiyama, Nobuko; Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Demizu, Yosuke; Yasuda, Hisataka; Yagi, Shintaro; Wu, Guoying; Matsumoto, Mitsuru; Sakamoto, Reiko; Yoshida, Nobuaki; Penninger, Josef M; Kobayashi, Yasuhiro; Inoue, Jun-Ichiro; Akiyama, Taishin

    2016-07-25

    Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs.

  6. Inotropic effect, binding properties, and calcium flux effects of the calcium channel agonist CGP 28392 in intact cultured embryonic chick ventricular cells

    SciTech Connect

    Laurent, S.; Kim, D.; Smith, T.W.; Marsh, J.D.

    1985-05-01

    CGP 28392 is a recently described dihydropyridine derivative with positive inotropic properties. To study the mechanism of action of this putative calcium channel agonist, we have related the effects of CGP 28392 on contraction (measured with an optical video system) and radioactive calcium uptake to ligand-binding studies in cultured, spontaneously beating chick embryo ventricular cells. CGP 28392 produced a concentration-dependent increase in amplitude and velocity of contraction (EC/sub 50/ = 2 x 10(-7) M; maximum contractile effect = 85% of the calcium 3.6 mM response). Nifedipine produced a shift to the right of the concentration-effect curve for CGP 28392 without decreasing the maximum contractile response, suggesting competitive antagonism (pA2 = 8.3). Computer analysis of displacement of (/sup 3/H)nitrendipine binding to intact heart cells by unlabeled CGP 28392 indicated a K /sub D/ = 2.2 +/- 0.95 x 10(-7) M, in good agreement with the EC/sub 50/ for the inotropic effect. CGP 28392 increased the rate of radioactive calcium influx (+39% at 10 seconds) without altering beating rate, while nifedipine decreased radioactive calcium influx and antagonized the CGP 28392-induced increase in calcium influx. Our results indicate that, in intact cultured myocytes, CGP 28392 acts as a calcium channel agonist and competes for the dihydropyridine-binding site of the slow calcium channel. In contrast to calcium channel blockers, CGP 28392 increases calcium influx and enhances the contractile state.

  7. Comparison of the mouse Embryonic Stem cell Test, the rat Whole Embryo Culture and the Zebrafish Embryotoxicity Test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles

    SciTech Connect

    Jong, Esther de; Barenys, Marta; Hermsen, Sanne A.B.; Verhoef, Aart; Ossendorp, Bernadette C.; Bessems, Jos G.M.; Piersma, Aldert H.

    2011-06-01

    The relatively high experimental animal use in developmental toxicity testing has stimulated the search for alternatives that are less animal intensive. Three widely studied alternative assays are the mouse Embryonic Stem cell Test (EST), the Zebrafish Embryotoxicity Test (ZET) and the rat postimplantation Whole Embryo Culture (WEC). The goal of this study was to determine their efficacy in assessing the relative developmental toxicity of six 1,2,4-triazole compounds, flusilazole, hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole. For this purpose, we analyzed effects and relative potencies of the compounds in and among the alternative assays and compared the findings to their known in vivo developmental toxicity. Triazoles are antifungal agents used in agriculture and medicine, some of which are known to induce craniofacial and limb abnormalities in rodents. The WEC showed a general pattern of teratogenic effects, typical of exposure to triazoles, mainly consisting of reduction and fusion of the first and second branchial arches, which are in accordance with the craniofacial malformations reported after in vivo exposure. In the EST all triazole compounds inhibited cardiomyocyte differentiation concentration-dependently. Overall, the ZET gave the best correlation with the relative in vivo developmental toxicities of the tested compounds, closely followed by the EST. The relative potencies observed in the WEC showed the lowest correlation with the in vivo developmental toxicity data. These differences in the efficacy between the test systems might be due to differences in compound kinetics, in developmental stages represented and in the relative complexity of the alternative assays.

  8. Comparison of the mouse Embryonic Stem cell Test, the rat Whole Embryo Culture and the Zebrafish Embryotoxicity Test as alternative methods for developmental toxicity testing of six 1,2,4-triazoles.

    PubMed

    de Jong, Esther; Barenys, Marta; Hermsen, Sanne A B; Verhoef, Aart; Ossendorp, Bernadette C; Bessems, Jos G M; Piersma, Aldert H

    2011-06-01

    The relatively high experimental animal use in developmental toxicity testing has stimulated the search for alternatives that are less animal intensive. Three widely studied alternative assays are the mouse Embryonic Stem cell Test (EST), the Zebrafish Embryotoxicity Test (ZET) and the rat postimplantation Whole Embryo Culture (WEC). The goal of this study was to determine their efficacy in assessing the relative developmental toxicity of six 1,2,4-triazole compounds,(1) flusilazole, hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole. For this purpose, we analyzed effects and relative potencies of the compounds in and among the alternative assays and compared the findings to their known in vivo developmental toxicity. Triazoles are antifungal agents used in agriculture and medicine, some of which are known to induce craniofacial and limb abnormalities in rodents. The WEC showed a general pattern of teratogenic effects, typical of exposure to triazoles, mainly consisting of reduction and fusion of the first and second branchial arches, which are in accordance with the craniofacial malformations reported after in vivo exposure. In the EST all triazole compounds inhibited cardiomyocyte differentiation concentration-dependently. Overall, the ZET gave the best correlation with the relative in vivo developmental toxicities of the tested compounds, closely followed by the EST. The relative potencies observed in the WEC showed the lowest correlation with the in vivo developmental toxicity data. These differences in the efficacy between the test systems might be due to differences in compound kinetics, in developmental stages represented and in the relative complexity of the alternative assays.

  9. Effects of 810 nm laser on mouse primary cortical neurons

    NASA Astrophysics Data System (ADS)

    Kharkwal, Gitika B.; Sharma, Sulbha K.; Huang, Ying-Ying; De Taboada, Luis; McCarthy, Thomas; Hamblin, Michael R.

    2011-03-01

    In the past four decades numerous studies have reported the efficacy of low level light (laser) therapy (LLLT) as a treatment for diverse diseases and injuries. Recent studies have shown that LLLT can biomodulate processes in the central nervous system and has been extensively studied as a stroke treatment. However there is still a lack of knowledge on the effects of LLLT at the cellular level in neurons. The present study aimed to study the effect of 810 nm laser on several cellular processes in primary cortical neurons cultured from mouse embryonic brains. Neurons were irradiated with light dose of 0.03, 0.3, 3, 10 and 30 J/cm2 and intracellular levels of reactive oxygen species, nitric oxide and calcium were measured. The changes in mitochondrial function in response to light were studied in terms of adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP). Light induced a significant increase in calcium, ATP and MMP at lower fluences and a decrease at higher fluence. ROS was induced significantly by light at all light doses. Nitric oxide levels also showed an increase on treatment with light. The results of the present study suggest that LLLT at lower fluences is capable of inducing mediators of cell signaling process which in turn may be responsible for the biomodulatory effects of the low level laser. At higher fluences beneficial mediators are reduced but potentially harmful mediators are increased thus offering an explanation for the biphasic dose response.

  10. carboxypeptidase E-ΔN, a neuroprotein transiently expressed during development protects embryonic neurons against glutamate neurotoxicity.

    PubMed

    Qin, Xiao-Yan; Cheng, Yong; Murthy, Saravana R K; Selvaraj, Prabhuanand; Loh, Y Peng

    2014-01-01

    Neuroprotective proteins expressed in the fetus play a critical role during early embryonic neurodevelopment, especially during maternal exposure to alcohol and drugs that cause stress, glutamate neuroexcitotoxicity, and damage to the fetal brain, if prolonged. We have identified a novel protein, carboxypeptidase E-ΔN (CPE-ΔN), which is a splice variant of CPE that has neuroprotective effects on embryonic neurons. CPE-ΔN is transiently expressed in mouse embryos from embryonic day 5.5 to postnatal day 1. It is expressed in embryonic neurons, but not in 3 week or older mouse brains, suggesting a function primarily in utero. CPE-ΔN expression was up-regulated in embryonic hippocampal neurons in response to dexamethasone treatment. CPE-ΔN transduced into rat embryonic cortical and hippocampal neurons protected them from glutamate- and H2O2-induced cell death. When transduced into embryonic cortical neurons, CPE-ΔN was found in the nucleus and enhanced the transcription of FGF2 mRNA. Embryonic cortical neurons challenged with glutamate resulted in attenuated FGF2 levels and cell death, but CPE-ΔN transduced neurons treated in the same manner showed increased FGF2 expression and normal viability. This neuroprotective effect of CPE-ΔN was mediated by secreted FGF2. Through receptor signaling, FGF2 activated the AKT and ERK signaling pathways, which in turn increased BCL-2 expression. This led to inhibition of caspase-3 activity and cell survival.

  11. Carboxypeptidase E-ΔN, a Neuroprotein Transiently Expressed during Development Protects Embryonic Neurons against Glutamate Neurotoxicity

    PubMed Central

    Murthy, Saravana R. K.; Selvaraj, Prabhuanand; Loh, Y. Peng

    2014-01-01

    Neuroprotective proteins expressed in the fetus play a critical role during early embryonic neurodevelopment, especially during maternal exposure to alcohol and drugs that cause stress, glutamate neuroexcitotoxicity, and damage to the fetal brain, if prolonged. We have identified a novel protein, carboxypeptidase E-ΔN (CPE-ΔN), which is a splice variant of CPE that has neuroprotective effects on embryonic neurons. CPE-ΔN is transiently expressed in mouse embryos from embryonic day 5.5 to postnatal day 1. It is expressed in embryonic neurons, but not in 3 week or older mouse brains, suggesting a function primarily in utero. CPE-ΔN expression was up-regulated in embryonic hippocampal neurons in response to dexamethasone treatment. CPE-ΔN transduced into rat embryonic cortical and hippocampal neurons protected them from glutamate- and H2O2-induced cell death. When transduced into embryonic cortical neurons, CPE-ΔN was found in the nucleus and enhanced the transcription of FGF2 mRNA. Embryonic cortical neurons challenged with glutamate resulted in attenuated FGF2 levels and cell death, but CPE-ΔN transduced neurons treated in the same manner showed increased FGF2 expression and normal viability. This neuroprotective effect of CPE-ΔN was mediated by secreted FGF2. Through receptor signaling, FGF2 activated the AKT and ERK signaling pathways, which in turn increased BCL-2 expression. This led to inhibition of caspase-3 activity and cell survival. PMID:25426952

  12. Effect of diet on ability of Vascular Endothelial Growth Factor A (VEGFA) isoforms to alter follicular progression in bovine ovarian cortical cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to determine the effect of changes in diet on ability of VEGFA isoforms to alter follicle progression in bovine ovarian cortex cultures. Our hypothesis was that diet would affect the magnitude of VEGFA isoform actions on follicular development. Heifers (n = 30) receiv...

  13. Glutamine synthetase in cultured whole retinas from the embryonic chick. Role of protein and RNA syntheses in 4 degrees C storage enhancement.

    PubMed

    Piperberg, J B; Reif-Lehrer, L

    1984-06-01

    Glutamine synthetase (GS) activity is enhanced in cultured whole retinas when a 72 h incubation at 37 degrees C is preceded by storage at 4 degrees C for 2-24 h. This enhancement occurs even in the absence of glucocorticoids and is maximal in retinas from 11 to 14 d embryos. In comparison, cortisol-induced increases in retinal GS activity at 37 degrees C are optimal in retinas from 8 to 12 d embryos. This study, using cycloheximide (an inhibitor of protein synthesis) and cordycepin (an inhibitor of RNA synthesis), indicates that both protein and RNA synthesis are required for the 4 degrees C storage enhancement of GS activity. The necessary RNA synthesis occurs within the first 48 h following transfer to 37 degrees C and does not require concomitant protein synthesis. Uridine uptake, but not incorporation into trichloroacetic acid-precipitable material, is increased by initial 4 degrees C storage when compared with whole retina controls incubated at 37 degrees C for the total time. In contrast, both uptake and incorporation of amino acids are increased in 4 degrees C-stored retinas for as long as 72 h subsequent to transfer from 4 to 37 degrees C. This suggests that enhancement GS activity may arise from a combination of elevated general protein synthesis and specific messenger-RNA synthesis following 4 degrees C storage.

  14. Embryonal cancers in Europe.

    PubMed

    Gatta, Gemma; Ferrari, Andrea; Stiller, Charles A; Pastore, Guido; Bisogno, Gianni; Trama, Annalisa; Capocaccia, Riccardo

    2012-07-01

    Embryonal cancers are a heterogeneous group of rare cancers which mainly occur in children and adolescents. The aim of the present study was to estimate the burden (incidence, prevalence, survival and proportion of cured) for the principal embryonal cancers in Europe (EU27), using population-based data from cancer registries (CRs) participating in RARECARE. We identified 3322 cases diagnosed from 1995 to 2002 (latest period for which data are available): 44% neuroblastoma, 35% nephroblastoma, 13% retinoblastoma and 6% hepatoblastoma. Very few cases of pulmonary blastoma (43 cases) and pancreatoblastoma (seven cases) were diagnosed. About 2000 new embryonal cancers were estimated every year in EU27, for an annual incidence rate of 4 per million (1.8 neuroblastoma, 1.4 nephroblastoma, and 0.5 retinoblastoma); 91% of cases occurred in patients under 15 years. Five-year relative survival for all embryonal cancers was 80% (99% retinoblastoma, 90% nephroblastoma, 71% hepatoblastoma and 68% neuroblastoma). Overall survival was lower in adolescents and adults than in those under 15 years. The cure rate was estimated at 80%. Slightly less than 40,000 persons were estimated alive in EU27 with a diagnosis of embryonal cancer in 2008. Nephroblastoma was the most prevalent (18,150 cases in EU27), followed by neuroblastoma (12,100), retinoblastoma (5200), hepatoblastoma (2700) and pulmonary blastoma (614). This is the first study to delineate the embryonal cancer burden in Europe by age, sex and European region. Survival/cure rate is generally high, but there are considerable gaps in our understanding of the natural histories of these rare diseases particularly in adults.

  15. Ect2, an ortholog of Drosophila Pebble, regulates formation of growth cones in primary cortical neurons

    PubMed Central

    Tsuji, Takahiro; Higashida, Chiharu; Aoki, Yoshihiko; Islam, Mohammad Saharul; Dohmoto, Mitsuko; Higashida, Haruhiro

    2016-01-01

    In collaboration with Marshall Nirenberg, we performed in vivo RNA interference (RNAi) genome-wide screening in Drosophila embryos. Pebble has been shown to be involved in Drosophila neuronal development. We have also reported that depletion of Ect2, a mammalian ortholog of Pebble, induces differentiation in NG108-15 neuronal cells. However, the precise role of Ect2 in neuronal development has yet to be studied. Here, we confirmed in PC12 pheochromocytoma cells that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth, and in the mouse embryonic cortex that Ect2 was accumulated throughout the ventricular and subventricular zones with neuronal progenitor cells. Next, the effects of Ect2 depletion were studied in primary cultures of mouse embryonic cortical neurons: Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Taken together, our results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics. PMID:22366651

  16. Ect2, an ortholog of Drosophila Pebble, regulates formation of growth cones in primary cortical neurons.

    PubMed

    Tsuji, Takahiro; Higashida, Chiharu; Aoki, Yoshihiko; Islam, Mohammad Saharul; Dohmoto, Mitsuko; Higashida, Haruhiro

    2012-11-01

    In collaboration with Marshall Nirenberg, we performed in vivo RNA interference (RNAi) genome-wide screening in Drosophila embryos. Pebble has been shown to be involved in Drosophila neuronal development. We have also reported that depletion of Ect2, a mammalian ortholog of Pebble, induces differentiation in NG108-15 neuronal cells. However, the precise role of Ect2 in neuronal development has yet to be studied. Here, we confirmed in PC12 pheochromocytoma cells that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth, and in the mouse embryonic cortex that Ect2 was accumulated throughout the ventricular and subventricular zones with neuronal progenitor cells. Next, the effects of Ect2 depletion were studied in primary cultures of mouse embryonic cortical neurons: Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Taken together, our results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics.

  17. In Vitro Developmental Neurotoxicity Following Chronic Exposure to 50 Hz Extremely Low-Frequency Electromagnetic Fields in Primary Rat Cortical Cultures.

    PubMed

    de Groot, Martje W G D M; van Kleef, Regina G D M; de Groot, Aart; Westerink, Remco H S

    2016-02-01

    Exposure to 50-60 Hz extremely low-frequency electromagnetic fields (ELF-EMFs) has increased considerably over the last decades. Several epidemiological studies suggested that ELF-EMF exposure is associated with adverse health effects, including neurotoxicity. However, these studies are debated as results are often contradictory and the possible underlying mechanisms are unknown. Since the developing nervous system is particularly vulnerable to insults, we investigate effects of chronic, developmental ELF-EMF exposure in vitro. Primary rat cortical neurons received 7 days developmental exposure to 50 Hz block-pulsed ELF-EMF (0-1000 μT) to assess effects on cell viability (Alamar Blue/CFDA assay), calcium homeostasis (single cell fluorescence microscopy), neurite outgrowth (β(III)-Tubulin immunofluorescent staining), and spontaneous neuronal activity (multi-electrode arrays). Our data demonstrate that cell viability is not affected by developmental ELF-EMF (0-1000 μT) exposure. Depolarization- and glutamate-evoked increases in intracellular calcium concentration ([Ca(2+)]i) are slightly increased at 1 μT, whereas both basal and stimulation-evoked [Ca(2+)]i show a modest inhibition at 1000 μT. Subsequent morphological analysis indicated that neurite length is unaffected up to 100 μT, but increased at 1000 μT. However, neuronal activity appeared largely unaltered following chronic ELF-EMF exposure up to 1000 μT. The effects of ELF-EMF exposure were small and largely restricted to the highest field strength (1000 μT), ie, 10 000 times above background exposure and well above current residential exposure limits. Our combined data therefore indicate that chronic ELF-EMF exposure has only limited (developmental) neurotoxic potential in vitro.

  18. Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture.

    PubMed

    Odawara, A; Katoh, H; Matsuda, N; Suzuki, I

    2016-05-18

    The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20-30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

  19. Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

    PubMed Central

    Verma, Saurabh; Traystman, Richard J.; Herson, Paco S.

    2013-01-01

    Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome. PMID:24378980

  20. Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

    SciTech Connect

    Liang, B.T.

    1989-06-01

    Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).

  1. Cytoprotective effects of vitamin E homologues against glutamate-induced cell death in immature primary cortical neuron cultures: Tocopherols and tocotrienols exert similar effects by antioxidant function.

    PubMed

    Saito, Yoshiro; Nishio, Keiko; Akazawa, Yoko Ogawa; Yamanaka, Kazunori; Miyama, Akiko; Yoshida, Yasukazu; Noguchi, Noriko; Niki, Etsuo

    2010-11-30

    Glutamate plays a critical role in pathological cell death within the nervous system. Vitamin E is known to protect cells from glutamate cytotoxicity, either by direct antioxidant action or by indirect nonantioxidant action. Further, α-tocotrienol (α-T3) has been reported to be more effective against glutamate-induced cytotoxicity than α-tocopherol (α-T). To shed more light on the function of vitamin E against glutamate toxicity, the protective effects of eight vitamin E homologues and related compounds, 2,2,5,7,8-pentamethyl-6-chromanol (PMC) and 2-carboxy-2,5,7,8-pentamethyl-6-chromanol (Trolox), against glutamate-induced cytotoxicity on immature primary cortical neurons were examined using different protocols. Glutamate induced the depletion of glutathione and generation of reactive oxygen species and lipid hydroperoxides, leading to cell death. α-, β-, γ-, and δ-T and -T3; PMC; and Trolox all exerted cytoprotective effects against glutamate-induced cytotoxicity, and a longer preincubation time increased both the cellular content and the cytoprotective effects of T more significantly than those of T3, the effect of preincubation being relatively small for T3 and PMC. The protective effect of Trolox was less potent than that of PMC. The cytoprotective effects of α-T and α-T3 corresponded to their intracellular content. Further, lipid peroxidation products were measured after reduction with triphenylphosphine followed by saponification with potassium hydroxide. It was found that glutamate treatment increased the formation of hydroxyeicosatetraenoic acid, hydroxyoctadecadienoic acid, and 8-F(2)-isoprostane 2α, which was suppressed by α-T. This study shows that vitamin E protects cells from glutamate-induced toxicity primarily by direct antioxidant action and that the apparent higher capacity of T3 compared to T is ascribed to the faster uptake of T3 compared to T into the cells. It is suggested that, considering the bioavailability, α-T should be more

  2. Evolution of cortical neurogenesis.

    PubMed

    Abdel-Mannan, Omar; Cheung, Amanda F P; Molnár, Zoltán

    2008-03-18

    The neurons of the mammalian neocortex are organised into six layers. By contrast, the reptilian and avian dorsal cortices only have three layers which are thought to be equivalent to layers I, V and VI of mammals. Increased repertoire of mammalian higher cognitive functions is likely a result of an expanded cortical surface area. The majority of cortical cell proliferation in mammals occurs in the ventricular zone (VZ) and subventricular zone (SVZ), with a small number of scattered divisions outside the germinal zone. Comparative developmental studies suggest that the appearance of SVZ coincides with the laminar expansion of the cortex to six layers, as well as the tangential expansion of the cortical sheet seen within mammals. In spite of great variation and further compartmentalisation in the mitotic compartments, the number of neurons in an arbitrary cortical column appears to be remarkably constant within mammals. The current challenge is to understand how the emergence and elaboration of the SVZ has contributed to increased cortical cell diversity, tangential expansion and gyrus formation of the mammalian neocortex. This review discusses neurogenic processes that are believed to underlie these major changes in cortical dimensions in vertebrates.

  3. Embryonic and embryonic-like stem cells in heart muscle engineering.

    PubMed

    Zimmermann, Wolfram-Hubertus

    2011-02-01

    Cardiac muscle engineering is evolving rapidly and may ultimately be exploited to (1) model cardiac development, physiology, and pathology; (2) identify and validate drug targets; (3) assess drug safety and efficacy; and (4) provide therapeutic substitute myocardium. The ultimate success in any of these envisioned applications depends on the utility of human cells and their assembly into myocardial equivalents with structural and functional properties of mature heart muscle. Embryonic stem cells appear as a promising cell source in this respect, because they can be cultured reliably and differentiated robustly into cardiomyocytes. Despite their unambiguous cardiogenicity, data on advanced maturation and seamless myocardial integration of embryonic stem cell-derived cardiomyocytes in vivo are sparse. Additional concerns relate to the limited control over cardiomyogenic specification and cardiomyocyte maturation in vitro as well as the risk of teratocarcinoma formation and immune rejection of stem cell implants in vivo. Through the invent of embryonic-like stem cells - such as parthenogenetic stem cells, male germline stem cells, and induced pluripotent stem cells - some but certainly not all of these issues may be addressed, albeit at the expense of additional concerns. This review will discuss the applicability of embryonic and embryonic-like stem cells in myocardial tissue engineering and address issues that require particular attention before the potential of stem cell-based heart muscle engineering may be fully exploited. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

  4. Culture.

    ERIC Educational Resources Information Center

    1997

    Twelve conference papers on cultural aspects of second language instruction include: "Towards True Multiculturalism: Ideas for Teachers" (Brian McVeigh); Comparing Cultures Through Critical Thinking: Development and Interpretations of Meaningful Observations" (Laurel D. Kamada); "Authority and Individualism in Japan and the…

  5. The Kv2.1 K+ channel targets to the axon initial segment of hippocampal and cortical neurons in culture and in situ

    PubMed Central

    Sarmiere, Patrick D; Weigle, Cecile M; Tamkun, Michael M

    2008-01-01

    Background The Kv2.1 delayed-rectifier K+ channel regulates membrane excitability in hippocampal neurons where it targets to dynamic cell surface clusters on the soma and proximal dendrites. In the past, Kv2.1 has been assumed to be absent from the axon initial segment. Results Transfected and endogenous Kv2.1 is now demonstrated to preferentially accumulate within the axon initial segment (AIS) over other neurite processes; 87% of 14 DIV hippocampal neurons show endogenous channel concentrated at the AIS relative to the soma and proximal dendrites. In contrast to the localization observed in pyramidal cells, GAD positive inhibitory neurons within the hippocampal cultures did not show AIS targeting. Photoactivable-GFP-Kv2.1-containing clusters at the AIS were stable, moving <1 μm/hr with no channel turnover. Photobleach studies indicated individual channels within the cluster perimeter were highly mobile (FRAP τ = 10.4 ± 4.8 sec), supporting our model that Kv2.1 clusters are formed by the retention of mobile channels behind a diffusion-limiting perimeter. Demonstrating that the AIS targeting is not a tissue culture artifact, Kv2.1 was found in axon initial segments within both the adult rat hippocampal CA1, CA2, and CA3 layers and cortex. Conclusion In summary, Kv2.1 is associated with the axon initial segment both in vitro and in vivo where it may modulate action potential frequency and back propagation. Since transfected Kv2.1 initially localizes to the AIS before appearing on the soma, it is likely multiple mechanisms regulate Kv2.1 trafficking to the cell surface. PMID:19014551

  6. Gravity and embryonic development

    NASA Technical Reports Server (NTRS)

    Young, R. S.

    1976-01-01

    The relationship between the developing embryo (both plant and animal) and a gravitational field has long been contemplated. The difficulty in designing critical experiments on the surface of the earth because of its background of 1 g, has been an obstacle to a resolution of the problem. Biological responses to gravity (particularly in plants) are obvious in many cases; however, the influence of gravity as an environmental input to the developing embryo is not as obvious and has proven to be extremely difficult to define. In spite of this, over the years numerous attempts have been made using a variety of embryonic materials to come to grips with the role of gravity in development. Three research tools are available: the centrifuge, the clinostat, and the orbiting spacecraft. Experimental results are now available from all three sources. Some tenuous conclusions are drawn, and an attempt at a unifying theory of gravitational influence on embryonic development is made.

  7. [Effect of zuoguiwan on early embryonic development of mice].

    PubMed

    Feng, Q J; Feng, M L; Wang, Y L

    1996-11-01

    Effects of Zuoguiwan (ZGW, a prescription for reinforcing Kidney Yin) on early embryonic development were observed by using embryonic developmental retardation model of mice formed by alcohol. Drug was given in three ways: add ZGW into cultural medium directly (group A), add the serum of mice received ZGW (group B) and cultured the embryo taken from ZGW treated mice (group C). The result was compared with that treated with Bazhen decoction (BZD, a prescription for supplementing Qi and blood). Results showed that the in vitro developmental rate of embryo from 2-cell stage to blastula stage in group B and C, which approached to normal control group, was higher than that in untreated model obviously. While in BZW group, it was higher than in normal control group only in certain stage. However, adding ZGW directly into culture medium didn't reveal marked effect on early embryonic development.

  8. Postpartum cortical blindness.

    PubMed

    Faiz, Shakeel Ahmed

    2008-09-01

    A 30-years-old third gravida with previous normal pregnancies and an unremarkable prenatal course had an emergency lower segment caesarean section at a periphery hospital for failure of labour to progress. She developed bilateral cortical blindness immediately after recovery from anesthesia due to cerebral angiopathy shown by CT and MR scan as cortical infarct cerebral angiopathy, which is a rare complication of a normal pregnancy.

  9. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

    PubMed

    Leke, Renata; Bak, Lasse K; Anker, Malene; Melø, Torun M; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Portela, Luis V; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

    2011-04-01

    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

  10. Fumonisin B1 induces necrotic cell death in BV-2 cells and murine cultured astrocytes and is antiproliferative in BV-2 cells while N2A cells and primary cortical neurons are resistant.

    PubMed

    Osuchowski, Marcin F; Sharma, Raghubir P

    2005-12-01

    Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides, causes equine leukoencephalomalacia, impairs myelination, and inhibits neuronal growth in vitro. Intact mice do not show brain damage after systemic administration of FB1. We recently reported that intracerebroventricular administration of FB1 in mice caused neurodegeneration in the cortex and activation of astrocytes in the hippocampal area; results suggested that the neuronal damage may be secondary to activation of immunocompetent non-neuronal cells. Current study investigated effects of FB1 upon murine microglial (BV-2) and neuroblastoma (N2A) cell lines, and primary astrocytes and cortical neurons. BV-2 and N2A cultures and cells prepared from neonatal and postnatal brains of BALB/c mice were exposed to various concentrations of FB1 for 4 (BV-2 and N2A) or 4 and 8 (astrocytes and cortical neurons) days. FB1 at 25 microM decreased viability in BV-2 cells, whereas at 50 microM caused necrotic but not apoptotic cell death in both BV-2 and primary astrocytes (at day 8 only), assessed by lactic dehydrogenase release, and pripidium iodide and annexin V staining. Thymidine incorporation indicated that 2.5 microM FB1 decreased proliferation in BV-2 cells. DNA analysis by flow cytometry showed that the inhibition was not caused by cell cycle arrest. The mitochondrial activity decreased dose-dependently in BV-2 cells and was significantly elevated at 25 microM FB1, but not at 50 microM at days 4 or 8 in astrocytes. In BV-2 cells and primary astrocytes, the expression of TNFalpha and IL-1beta analyzed by real-time polymerase chain reaction was downregulated at 6 or 24 h. In all cell types tested the FB1 treatment caused accumulation of free sphinganine and decrease in free sphingosine levels at selected time points. Results indicated that primary and established murine brain immunocompetent cells are vulnerable to the FB1-dependent cytotoxicity in vitro whereas neuronal cells are not. The toxic effects

  11. Acamprosate {monocalcium bis(3-acetamidopropane-1-sulfonate)} reduces ethanol-drinking behavior in rats and glutamate-induced toxicity in ethanol-exposed primary rat cortical neuronal cultures.

    PubMed

    Oka, Michiko; Hirouchi, Masaaki; Tamura, Masaru; Sugahara, Seishi; Oyama, Tatsuya

    2013-10-15

    Acamprosate, the calcium salt of bis(3-acetamidopropane-1-sulfonate), contributes to the maintenance of abstinence in alcohol-dependent patients, but its mechanism of action in the central nervous system is unclear. Here, we report the effect of acamprosate on ethanol-drinking behavior in standard laboratory Wistar rats, including voluntary ethanol consumption and the ethanol-deprivation effect. After forced ethanol consumption arranged by the provision of only one drinking bottle containing 10% ethanol, the rats were given a choice between two drinking bottles, one containing water and the other containing 10% ethanol. In rats selected for high ethanol preference, repeated oral administration of acamprosate diminished voluntary ethanol drinking. After three months of continuous access to two bottles, rats were deprived of ethanol for three days and then presented with two bottles again. After ethanol deprivation, ethanol preference was increased, and the increase was largely abolished by acamprosate. After exposure of primary neuronal cultures of rat cerebral cortex to ethanol for four days, neurotoxicity, as measured by the extracellular leakage of lactate dehydrogenase (LDH), was induced by incubation with glutamate for 1h followed by incubation in the absence of ethanol for 24h. The N-methyl-D-aspartate receptor blocker 5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine, the metabotropic glutamate receptor subtype 5 antagonist 6-methyl-2-(phenylethynyl)pyridine and the voltage-gated calcium-channel blocker nifedipine all inhibited glutamate-induced LDH leakage from ethanol-exposed neurons. Acamprosate inhibited the glutamate-induced LDH leakage from ethanol-exposed neurons more strongly than that from intact neurons. In conclusion, acamprosate showed effective reduction of drinking behavior in rats and protected ethanol-exposed neurons by multiple blocking of glutamate signaling.

  12. OCT guided microinjections for mouse embryonic research

    NASA Astrophysics Data System (ADS)

    Larin, Kirill V.; Syed, Saba H.; Coughlin, Andrew J.; Wang, Shang; West, Jennifer L.; Dickinson, Mary E.; Larina, Irina V.

    2013-02-01

    Optical coherence tomography (OCT) is gaining popularity as live imaging tool for embryonic research in animal models. Recently we have demonstrated that OCT can be used for live imaging of cultured early mouse embryos (E7.5-E10) as well as later stage mouse embryos in utero (E12.5 to the end of gestation). Targeted delivery of signaling molecules, drugs, and cells is a powerful approach to study normal and abnormal development, and image guidance is highly important for such manipulations. Here we demonstrate that OCT can be used to guide microinjections of gold nanoshell suspensions in live mouse embryos. This approach can potentially be used for variety of applications such as guided injections of contrast agents, signaling molecules, pharmacological agents, cell transplantation and extraction, as well as other image-guided micromanipulations. Our studies also reveal novel potential for gold nanoshells in embryonic research.

  13. Methylmalonate toxicity in primary neuronal cultures.

    PubMed

    McLaughlin, B A; Nelson, D; Silver, I A; Erecinska, M; Chesselet, M F

    1998-09-01

    Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to

  14. Influence of the embryonic preplate on the organization of the cerebral cortex: a targeted ablation model.

    PubMed

    Xie, Y; Skinner, E; Landry, C; Handley, V; Schonmann, V; Jacobs, E; Fisher, R; Campagnoni, A

    2002-10-15

    Transgenic mice were generated to permit the targeted ablation of cortical preplate cells at the time they are born. In these mice, the 1.3 kb golli promoter of the myelin basic protein gene was used to drive the herpes simplex virus thymidine kinase (TK) transgene in cortical preplate cells. Heterozygous transgenic pairs were bred, and pregnant dams were treated with ganciclovir at embryonic days 11-12 to ablate preplate cells at the time the preplate was forming. This paradigm exposed control (TK-) and experimental (TK+) littermates to exactly the same conditions. Embryological ablation of preplate cells led to an early disruption of the radial glial framework and subplate structure in the developing cortex and dramatically altered the cellular lamination and connectivity of the cortical plate. The disturbed radial glial network contributed to an impaired radial migration of neurons into the cortical plate from the ventricular zone. The cortical plate became dyslaminated, and there was a substantial reduction in short- and long-range cortical projections within the cortex and to subcortical regions. Cell death within the cortical plate and the proliferative zones was substantially increased in the ablated animals. After birth, a cortical lesion developed, which became exacerbated with the secondary onset of hydrocephaly in the second postnatal week. The results underscore the critical importance of the preplate in cortex formation, mediated through its guidance of the formation of radial glial scaffolding, subsequent neuronal migration into the incipient cortical plate, and the final arrangement of its vertical organization and cellular connectivity.

  15. System N transporters are critical for glutamine release and modulate metabolic fluxes of glucose and acetate in cultured cortical astrocytes: changes induced by ammonia.

    PubMed

    Zielińska, Magdalena; Dąbrowska, Katarzyna; Hadera, Mussie Ghezu; Sonnewald, Ursula; Albrecht, Jan

    2016-01-01

    Glutamine (Gln) is synthesized in astrocytes from glutamate (Glu) and ammonia, whereupon it can be released to be transferred to neurons. This study evaluated the as yet not definitely established role of the astrocytic Gln transporters SN1 and SN2 (Slc38a3 and Slc38a5 respectively) in Gln release and metabolic fluxes of glucose and acetate, the canonical precursors of Glu. Cultured neocortical astrocytes were grown in the absence or presence of ammonia (5 mM NH4 Cl, 24 h), which deregulates astrocytic metabolism in hyperammonemic encephalopathies. HPLC analyses of cell extracts of SN1/SN2 siRNA-treated (SN1/SN2-) astrocytes revealed a ~ 3.5-fold increase in Gln content and doubling of glutathione, aspartate, alanine and glutamate contents, as compared to SN1/SN2+ astrocytes. Uptake and efflux of preloaded [(3) H]Gln was likewise significantly decreased in SN1/SN2- astrocytes. The atom percent excess (13) C values (given as M + 1) for alanine, aspartate and glutamate were decreased when the SN1/SN2- cells were incubated with [1-(13) C] glucose, while Gln consumption was not changed. No difference was seen in M + 1 values in SN1/SN2- cells incubated with [2-(13) C] acetate, which were not treated with ammonia. In SN1/SN2- astrocytes, the increase in Gln content and the decrease in radiolabeled Gln release upon exposure to ammonia were found abrogated, and glutamate labeling from [2-(13) C]acetate was decreased as compared to SN1/SN2+ astrocytes. The results underscore a profound role of SN1 and/or SN2 in Gln release from astrocytes under physiological conditions, but less so in ammonia-overexposed astrocytes, and appear to manifest dependence of astrocytic glucose metabolism to Glu/Gln on unimpaired SN1/SN2- mediated Gln release from astrocytes. The astrocytic N system transporters SN1 and SN2 show preponderance to mediate glutamine (Gln) efflux. Under hyperammonemic conditions, accumulation of Gln, a direct product of ammonia detoxification, may deregulate

  16. Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods

    PubMed Central

    González, Sheyla; Ibáñez, Elena

    2010-01-01

    Purpose The aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process. Methods Three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages. Results A total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%. Conclusions Using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process. PMID:20862536

  17. Gene Expression in Cortical Interneuron Precursors is Prescient of their Mature Function

    PubMed Central

    Batista-Brito, Renata; Machold, Robert; Klein, Corinna

    2008-01-01

    At present little is known about the developmental mechanisms that give rise to inhibitory γ-aminobutyric acidergic interneurons of the neocortex or the timing of their subtype specification. As such, we performed a gene expression microarray analysis on cortical interneuron precursors isolated through their expression of a Dlx5/6Cre-IRES-EGFP transgene. We purified these precursors from the embryonic mouse neocortex at E13.5 and E15.5 by sorting of enhanced green fluorescent protein-expressing cells. We identified novel transcription factors, neuropeptides, and cell surface genes whose expression is highly enriched in embryonic cortical interneuron precursors. Our identification of many of the genes known to be selectively enriched within cortical interneurons validated the efficacy of our approach. Surprisingly, we find that subpopulations of migrating cortical interneurons express genes encoding for proteins characteristic of mature interneuron subtypes as early as E13.5. These results provide support for the idea that many of the genes characteristic of specific cortical interneuron subtypes are evident prior to their functional integration into cortical microcircuitry. They suggest interneurons are already relegated to specific genetic subtypes shortly after they become postmitotic. Moreover, our work has revealed that many of the genes expressed in cortical interneuron precursors have been independently linked to neurological disorders in both mice and humans PMID:18250082

  18. Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia

    PubMed Central

    Bradley, Ryan M.; Mardian, Emily B.; Marvyn, Phillip M.; Vasefi, Maryam S.; Beazely, Michael A.; Mielke, John G.; Duncan, Robin E.

    2015-01-01

    Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1]. PMID:26759825

  19. Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia.

    PubMed

    Bradley, Ryan M; Mardian, Emily B; Marvyn, Phillip M; Vasefi, Maryam S; Beazely, Michael A; Mielke, John G; Duncan, Robin E

    2016-03-01

    Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1].

  20. Prenatal thalamic waves regulate cortical area size prior to sensory processing.

    PubMed

    Moreno-Juan, Verónica; Filipchuk, Anton; Antón-Bolaños, Noelia; Mezzera, Cecilia; Gezelius, Henrik; Andrés, Belen; Rodríguez-Malmierca, Luis; Susín, Rafael; Schaad, Olivier; Iwasato, Takuji; Schüle, Roland; Rutlin, Michael; Nelson, Sacha; Ducret, Sebastien; Valdeolmillos, Miguel; Rijli, Filippo M; López-Bendito, Guillermina

    2017-02-03

    The cerebral cortex is organized into specialized sensory areas, whose initial territory is determined by intracortical molecular determinants. Yet, sensory cortical area size appears to be fine tuned during development to respond to functional adaptations. Here we demonstrate the existence of a prenatal sub-cortical mechanism that regulates the cortical areas size in mice. This mechanism is mediated by spontaneous thalamic calcium waves that propagate among sensory-modality thalamic nuclei up to the cortex and that provide a means of communication among sensory systems. Wave pattern alterations in one nucleus lead to changes in the pattern of the remaining ones, triggering changes in thalamic gene expression and cortical area size. Thus, silencing calcium waves in the auditory thalamus induces Rorβ upregulation in a neighbouring somatosensory nucleus preluding the enlargement of the barrel-field. These findings reveal that embryonic thalamic calcium waves coordinate cortical sensory area patterning and plasticity prior to sensory information processing.

  1. Prenatal thalamic waves regulate cortical area size prior to sensory processing

    PubMed Central

    Moreno-Juan, Verónica; Filipchuk, Anton; Antón-Bolaños, Noelia; Mezzera, Cecilia; Gezelius, Henrik; Andrés, Belen; Rodríguez-Malmierca, Luis; Susín, Rafael; Schaad, Olivier; Iwasato, Takuji; Schüle, Roland; Rutlin, Michael; Nelson, Sacha; Ducret, Sebastien; Valdeolmillos, Miguel; Rijli, Filippo M.; López-Bendito, Guillermina

    2017-01-01

    The cerebral cortex is organized into specialized sensory areas, whose initial territory is determined by intracortical molecular determinants. Yet, sensory cortical area size appears to be fine tuned during development to respond to functional adaptations. Here we demonstrate the existence of a prenatal sub-cortical mechanism that regulates the cortical areas size in mice. This mechanism is mediated by spontaneous thalamic calcium waves that propagate among sensory-modality thalamic nuclei up to the cortex and that provide a means of communication among sensory systems. Wave pattern alterations in one nucleus lead to changes in the pattern of the remaining ones, triggering changes in thalamic gene expression and cortical area size. Thus, silencing calcium waves in the auditory thalamus induces Rorβ upregulation in a neighbouring somatosensory nucleus preluding the enlargement of the barrel-field. These findings reveal that embryonic thalamic calcium waves coordinate cortical sensory area patterning and plasticity prior to sensory information processing. PMID:28155854

  2. Merlin/ERM proteins establish cortical asymmetry and centrosome position

    PubMed Central

    Hebert, Alan M.; DuBoff, Brian; Casaletto, Jessica B.; Gladden, Andrew B.; McClatchey, Andrea I.

    2012-01-01

    The ability to generate asymmetry at the cell cortex underlies cell polarization and asymmetric cell division. Here we demonstrate a novel role for the tumor suppressor Merlin and closely related ERM proteins (Ezrin, Radixin, and Moesin) in generating cortical asymmetry in the absence of external cues. Our data reveal that Merlin functions to restrict the cortical distribution of the actin regulator Ezrin, which in turn positions the interphase centrosome in single epithelial cells and three-dimensional organotypic cultures. In the absence of Merlin, ectopic cortical Ezrin yields mispositioned centrosomes, misoriented spindles, and aberrant epithelial architecture. Furthermore, in tumor cells with centrosome amplification, the failure to restrict cortical Ezrin abolishes centrosome clustering, yielding multipolar mitoses. These data uncover fundamental roles for Merlin/ERM proteins in spatiotemporally organizing the cell cortex and suggest that Merlin's role in restricting cortical Ezrin may contribute to tumorigenesis by disrupting cell polarity, spindle orientation, and, potentially, genome stability. PMID:23249734

  3. Mapping cortical mesoscopic networks of single spiking cortical or sub-cortical neurons.

    PubMed

    Xiao, Dongsheng; Vanni, Matthieu P; Mitelut, Catalin C; Chan, Allen W; LeDue, Jeffrey M; Xie, Yicheng; Chen, Andrew Cn; Swindale, Nicholas V; Murphy, Timothy H

    2017-02-04

    Understanding the basis of brain function requires knowledge of cortical operations over wide-spatial scales, but also within the context of single neurons. In vivo, wide-field GCaMP imaging and sub-cortical/cortical cellular electrophysiology were used in mice to investigate relationships between spontaneous single neuron spiking and mesoscopic cortical activity. We make use of a rich set of cortical activity motifs that are present in spontaneous activity in anesthetized and awake animals. A mesoscale spike-triggered averaging procedure allowed the identification of motifs that are preferentially linked to individual spiking neurons by employing genetically targeted indicators of neuronal activity. Thalamic neurons predicted and reported specific cycles of wide-scale cortical inhibition/excitation. In contrast, spike-triggered maps derived from single cortical neurons yielded spatio-temporal maps expected for regional cortical consensus function. This approach can define network relationships between any point source of neuronal spiking and mesoscale cortical maps.

  4. Medaka haploid embryonic stem cells.

    PubMed

    Hong, Yunhan

    2010-01-01

    The appearance of diploidy, the presence of two genomes or chromosome sets, is a fundamental hallmark of eukaryotic evolution and bisexual reproduction, because diploidy offers the basis for the bisexual life cycle, allowing for oscillation between diploid and haploid phases. Meiosis produces haploid gametes. At fertilization, male and female gametes fuse to restore diploidy in a zygote, which develops into a new life. At sex maturation, diploid cells enter into meiosis, culminating in the production of haploid gametes. Therefore, diploidy ensures pluripotency, cell proliferation, and functions, whereas haploidy is restricted only to the post-meiotic gamete phase of germline development and represents the end point of cell growth. Diploidy is advantageous for evolution. Haploidy is ideal for genetic analyses, because any recessive mutations of essential genes will show a clear phenotype in the absence of a second gene copy. Recently, my laboratory succeeded in the generation of medaka haploid embryonic stem (ES) cells capable of whole animal production. Therefore, haploidy in a vertebrate is able to support stable cell culture and pluripotency. This finding anticipates the possibility to generate haploid ES cells in other vertebrate species such as zebrafish. These medaka haploid ES cells elegantly combine haploidy and pluripotency, offering a unique yeast-like system for in vitro genetic analyses of molecular, cellular, and developmental events in various cell lineages. This chapter is aimed to describe the strategy of haploid ES cell derivation and their characteristics, and illustrate the perspectives of haploid ES cells for infertility treatment, genetic screens, and analyses.

  5. Do embryonic polar bodies commit suicide?

    PubMed

    Fabian, Dušan; Čikoš, Štefan; Rehák, Pavol; Koppel, Juraj

    2014-02-01

    The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

  6. [Heart tissue from embryonic stem cells].

    PubMed

    Zimmermann, W-H

    2008-09-01

    Embryonic stem cells can give rise to all somatic cells, making them an attractive cell source for tissue engineering applications. The propensity of cells to form tissue-like structures in a culture dish has been well documented. We and others made use of this intrinsic property to generate bioartificial heart muscle. First proof-of-concept studies involved immature heart cells mainly from fetal chicken, neonatal rats and mice. They eventually provided evidence that force-generating heart muscle can be engineered in vitro. Recently, the focus shifted to the application of stem cells to eventually enable the generation of human heart muscle and reach following long-term goals: (1) development of a simplified in vitro model of heart muscle development; (2) generation of a human test-bed for drug screening and development; (3) allocation of surrogate heart tissue to myocardial repair applications. This overview will provide the background for cell-based myocardial repair, introduce the main myocardial tissue engineering concepts, discuss the use of embryonic and non-embryonic stem cells, and lays out the potential direct and indirect therapeutic use of human tissue engineered myocardium.

  7. Human embryonic stem cells and lung regeneration.

    PubMed

    Varanou, A; Page, C P; Minger, S L

    2008-10-01

    Human embryonic stem cells are pluripotent cells derived from the inner cell mass of preimplantation stage embryos. Their unique potential to give rise to all differentiated cell types has generated great interest in stem cell research and the potential that it may have in developmental biology, medicine and pharmacology. The main focus of stem cell research has been on cell therapy for pathological conditions with no current methods of treatment, such as neurodegenerative diseases, cardiac pathology, retinal dysfunction and lung and liver disease. The overall aim is to develop methods of application either of pure cell populations or of whole tissue parts to the diseased organ under investigation. In the field of pulmonary research, studies using human embryonic stem cells have succeeded in generating enriched cultures of type II pneumocytes in vitro. On account of their potential of indefinite proliferation in vitro, embryonic stem cells could be a source of an unlimited supply of cells available for transplantation and for use in gene therapy. Uncovering the ability to generate such cell types will expand our understanding of biological processes to such a degree that disease understanding and management could change dramatically.

  8. Mechanical signaling coordinates the embryonic heartbeat

    PubMed Central

    Chiou, Kevin K.; Rocks, Jason W.; Chen, Christina Yingxian; Cho, Sangkyun; Merkus, Koen E.; Rajaratnam, Anjali; Robison, Patrick; Tewari, Manorama; Vogel, Kenneth; Majkut, Stephanie F.; Prosser, Benjamin L.; Discher, Dennis E.; Liu, Andrea J.

    2016-01-01

    In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats. PMID:27457951

  9. Peptides Regulate Cortical Thymocytes Differentiation, Proliferation, and Apoptosis

    PubMed Central

    Khavinson, V. Kh.; Polyakova, V. O.; Linkova, N. S.; Dudkov, A. V.; Kvetnoy, I. M.

    2011-01-01

    The processes of differentiation, proliferation, and apoptosis were studied in a cell culture of human cortical thymocytes under the influence of short peptides T-32 (Glu-Asp-Ala) and T-38 (Lys-Glu-Asp). Peptides T-32 and T-38 amplified cortical thymocytes differentiation towards regulatory T cells, increased their proliferative activity, and decreased the level of apoptosis. Moreover, peptides under study stimulated proliferative and antiapoptotic activity of the mature regulatory T cells. PMID:22312461

  10. Concise review: no breakthroughs for human mesenchymal and embryonic stem cell culture: conditioned medium, feeder layer, or feeder-free; medium with fetal calf serum, human serum, or enriched plasma; serum-free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold!

    PubMed

    Mannello, Ferdinando; Tonti, Gaetana A

    2007-07-01

    The choice of an optimal strategy of stem cell culture is at the moment an impossible task, and the elaboration of a culture medium adapted to the production of embryonic and adult mesenchymal stem cells for the clinical application of cell therapy remains a crucial matter. To make an informed choice, it is crucial to not underestimate the theoretical health risk of using xenogenic compounds, to limit the immunological reactions once stem cells are transplanted, to not overestimate the controversial results obtained with human serum, plasma, and blood derivatives, as well as to carefully examine the pros and cons of serum-free and ad hoc formulation strategies; besides that, to also maintain multipotentiality, self-renewal, and transplantability. The extent to which we are able to achieve effective cell therapies will depend on assimilating a rapidly developing base of scientific knowledge with the practical considerations of design, delivery, and host response. Although clinical studies have already started, many questions remain unsolved, and concomitantly even more evidence on suitable and safe off-the-shelf products (mainly xeno-free) for embryonic and mesenchymal stem cells is cropping up, even though there should be no rush to enter the clinical stage while the underlying basic research is still not so solid; this solely will lead to high-quality translational research, without making blunders stemming from the assumption that all that glitters is not gold. Disclosure of potential conflicts of interest is found at the end of this article.

  11. Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke.

    PubMed

    Shimada, Issei S; LeComte, Matthew D; Granger, Jerrica C; Quinlan, Noah J; Spees, Jeffrey L

    2012-06-06

    In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.

  12. The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures.

    PubMed

    Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W

    2014-03-01

    Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to

  13. Sparse and powerful cortical spikes.

    PubMed

    Wolfe, Jason; Houweling, Arthur R; Brecht, Michael

    2010-06-01

    Activity in cortical networks is heterogeneous, sparse and often precisely timed. The functional significance of sparseness and precise spike timing is debated, but our understanding of the developmental and synaptic mechanisms that shape neuronal discharge patterns has improved. Evidence for highly specialized, selective and abstract cortical response properties is accumulating. Singe-cell stimulation experiments demonstrate a high sensitivity of cortical networks to the action potentials of some, but not all, single neurons. It is unclear how this sensitivity of cortical networks to small perturbations comes about and whether it is a generic property of cortex. The unforeseen sensitivity to cortical spikes puts serious constraints on the nature of neural coding schemes.

  14. Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production

    PubMed Central

    Limaye, Advait; Hall, Bradford; Kulkarni, Ashok B

    2009-01-01

    The establishment of mouse embryonic stem (ES) cell liness has allowed for the generation of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene targeting experiment. This unit describes detailed step-by-step protocols, reagents, equipment, and strategies needed for the successful generation of gene knockout embryonic stem cells using homologous recombination technologies. PMID:19731225

  15. Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

    PubMed

    Ma, W; Maric, D; Li, B S; Hu, Q; Andreadis, J D; Grant, G M; Liu, Q Y; Shaffer, K M; Chang, Y H; Zhang, L; Pancrazio, J J; Pant, H C; Stenger, D A; Barker, J L

    2000-04-01

    Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

  16. Myosin synthesis in embryonic chicken fibroblasts

    PubMed Central

    1979-01-01

    The rate of constitutive myosin synthesis was measured in cultures of replicating embryonic chicken skin fibroblasts by pulse labeling with [3H]leucine. These cells synthesized the 200,000-dalton heavy chain of myosin (MHC) at a rate of 3.2 x 10(3) molecules/cell/min. Additionally, an independent estimate of the MHC synthesis rate needed to maintain a constant level of constitutive MHC/cell was calculated from total protein content, percentage MHC, fibroblast doubling time, and MHC half- life. This calculated rate of approximately 2.9 x 10(3) molecules/cell/min was in close agreement with the measured rate. By comparison, the synthesis rate of myofibrillar MHC in fully activated muscle cell cultures was approximately 2.9 x 10(4) molecules/nucleus/min. PMID:479285

  17. Posterior Cortical Atrophy

    PubMed Central

    Crutch, Sebastian J; Lehmann, Manja; Schott, Jonathan M; Rabinovici, Gil D; Rossor, Martin N; Fox, Nick C

    2013-01-01

    Posterior cortical atrophy (PCA) is a neurodegenerative syndrome that is characterized by a progressive decline in visuospatial, visuoperceptual, literacy and praxic skills. The progressive neurodegeneration affecting parietal, occipital and occipito-temporal cortices which underlies PCA is attributable to Alzheimer's disease (AD) in the majority of patients. However, alternative underlying aetiologies including Dementia with Lewy Bodies (DLB), corticobasal degeneration (CBD) and prion disease have also been identified, and not all PCA patients have atrophy on clinical imaging. This heterogeneity has led to diagnostic and terminological inconsistencies, caused difficulty comparing studies from different centres, and limited the generalizability of clinical trials and investigations of factors driving phenotypic variability. Significant challenges remain in identifying the factors associated with both the selective vulnerability of posterior cortical regions and the young age of onset seen in PCA. Greater awareness of the syndrome and agreement over the correspondence between syndrome-and disease-level classifications are required in order to improve diagnostic accuracy, research study design and clinical management. PMID:22265212

  18. Bioengineered functional brain-like cortical tissue

    PubMed Central

    Tang-Schomer, Min D.; White, James D.; Tien, Lee W.; Schmitt, L. Ian; Valentin, Thomas M.; Graziano, Daniel J.; Hopkins, Amy M.; Omenetto, Fiorenzo G.; Haydon, Philip G.; Kaplan, David L.

    2014-01-01

    The brain remains one of the most important but least understood tissues in our body, in part because of its complexity as well as the limitations associated with in vivo studies. Although simpler tissues have yielded to the emerging tools for in vitro 3D tissue cultures, functional brain-like tissues have not. We report the construction of complex functional 3D brain-like cortical tissue, maintained for months in vitro, formed from primary cortical neurons in modular 3D compartmentalized architectures with electrophysiological function. We show that, on injury, this brain-like tissue responds in vitro with biochemical and electrophysiological outcomes that mimic observations in vivo. This modular 3D brain-like tissue is capable of real-time nondestructive assessments, offering previously unidentified directions for studies of brain homeostasis and injury. PMID:25114234

  19. Microfluidics for gametes, embryos, and embryonic stem cells.

    PubMed

    Smith, G D; Swain, J E; Bormann, C L

    2011-01-01

    Microfluidics is a young but established field that holds significant potential for scientific discovery. The utility of microfluidics can improve our knowledge of basic biology as well as expand our understanding in specialized areas such as assisted reproduction and stem cell developmental biology. This review describes the technology of microfluidics and discusses applications within assisted reproduction technology and embryonic stem cell growth and directed differentiation. Development of an integrated microfluidic platform for assisted reproduction, which can manipulate gametes, embryos, embryonic stem cells, their culture environment, and incorporate biomarker analysis, could have a dramatic impact on the basic understanding of embryo/embryonic stem cell development, as well as provide significant improvements in current technologies used to treat infertility, preserve fertility, and derive therapeutic cells from stem cells.

  20. Cortical vesicle breakdown in fertilized eggs of Fundulus heteroclitus

    SciTech Connect

    Brummett, A.R.; Dumont, J.N.

    1981-01-01

    A scanning and transmission electron microscope study has been made of the cortical alveoli of the egg of Fundulus heteroclitus. The study includes both unactivated eggs and fertilized eggs fixed at intervals of 1 second to 10 minutes after insemination. The alveoli appear to vary considerably in size, in contents, and in morphological aspects of their breakdown. As it undergoes dehiscence, each vesicle may form one or several openings in the egg suface; dense granules and particulate, fibrous, or membranous material, apparently in any combination, are liberated to the nascent perivitelline space. It appears that much of the excess membrane externalized during the reaction is strung out in threads and probably lost to the perivitelline space. The evidence does not suggest that the excess membrane either ''dissolves'' or is retrieved by the egg cytoplasm. That part of the cortical vesical membrane which remains continuous with the oolemma gradually becomes microvillous and loses its morphological identity. Granules and particulate matter, presumably liberated from the cortical alveoli, are seen adhering to the inner surface of chorions removed from activated eggs. The micropyle appears to be sealed with similar material. Supernumerary sperm are observed inside the chorion in some instances. The cortical reaction appears to be somehow related to the subsequent formation of a normal embryonic blastodisc.

  1. The cortical hem regulates the size and patterning of neocortex.

    PubMed

    Caronia-Brown, Giuliana; Yoshida, Michio; Gulden, Forrest; Assimacopoulos, Stavroula; Grove, Elizabeth A

    2014-07-01

    The cortical hem, a source of Wingless-related (WNT) and bone morphogenetic protein (BMP) signaling in the dorsomedial telencephalon, is the embryonic organizer for the hippocampus. Whether the hem is a major regulator of cortical patterning outside the hippocampus has not been investigated. We examined regional organization across the entire cerebral cortex in mice genetically engineered to lack the hem. Indicating that the hem regulates dorsoventral patterning in the cortical hemisphere, the neocortex, particularly dorsomedial neocortex, was reduced in size in late-stage hem-ablated embryos, whereas cortex ventrolateral to the neocortex expanded dorsally. Unexpectedly, hem ablation also perturbed regional patterning along the rostrocaudal axis of neocortex. Rostral neocortical domains identified by characteristic gene expression were expanded, and caudal domains diminished. A similar shift occurs when fibroblast growth factor (FGF) 8 is increased at the rostral telencephalic organizer, yet the FGF8 source was unchanged in hem-ablated brains. Rather we found that hem WNT or BMP signals, or both, have opposite effects to those of FGF8 in regulating transcription factors that control the size and position of neocortical areas. When the hem is ablated a necessary balance is perturbed, and cerebral cortex is rostralized. Our findings reveal a much broader role for the hem in cortical development than previously recognized, and emphasize that two major signaling centers interact antagonistically to pattern cerebral cortex.

  2. Cortical Clefts and Cortical Bumps: A Continuous Spectrum

    PubMed Central

    Furruqh, Farha; Thirunavukarasu, Suresh; Vivekandan, Ravichandran

    2016-01-01

    Cortical ‘clefts’ (schizencephaly) and cortical ‘bumps’ (polymicrogyria) are malformations arising due to defects in postmigrational development of neurons. They are frequently encountered together, with schizencephalic clefts being lined by polymicrogyria. We present the case of an eight-year-old boy who presented with seizures. Imaging revealed closed lip schizencephaly, polymicrogyria and a deep ‘incomplete’ cleft lined by polymicrogyria not communicating with the lateral ventricle. We speculate that hypoperfusion or ischaemic cortical injury during neuronal development may lead to a spectrum of malformations ranging from polymicrogyria to incomplete cortical clefts to schizencephaly. PMID:27630923

  3. Use of murine embryonic stem cells in embryotoxicity assays: the embryonic stem cell test.

    PubMed

    Seiler, Andrea E M; Buesen, Roland; Visan, Anke; Spielmann, Horst

    2006-01-01

    The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for embryotoxicity testing based on the most important mechanisms in embryotoxicity-cytotoxicity and differentiation--as well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and alpha-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process.

  4. Inhibitor of Differentiation-1 and Hypoxia-Inducible Factor-1 Mediate Sonic Hedgehog Induction by Amyloid Beta-Peptide in Rat Cortical Neurons.

    PubMed

    Hung, Yu-Hsing; Chang, Shih-Hsin; Huang, Chao-Tzu; Yin, Jiu-Haw; Hwang, Chi-Shin; Yang, Liang-Yo; Yang, Ding-I

    2016-03-01

    One major pathological hallmark of Alzheimer's disease (AD) is the accumulation of senile plaques mainly composed of neurotoxic amyloid beta-peptide (Aβ) in the patients' brains. Sonic hedgehog (SHH) is a morphogen critically involved in the embryonic development of the central nervous system (CNS). In the present study, we tested whether Aβ may induce SHH expression and explored its underlying mechanisms. We found that both Aβ25-35 and Aβ1-42 enhanced SHH expression in the primary cortical neurons derived from fetal rat brains. Immunohistochemistry revealed heightened expression of SHH in the cortex and hippocampus of aged (9 and 12 months old) AD transgenic mouse brains as compared to age-matched littermate controls. Chromatin immunoprecipitation (ChIP) assay demonstrated that Aβ25-35 enhanced binding of hypoxia-inducible factor-1 (HIF-1) to the promoter of the Shh gene in primary cortical cultures; consistently, Aβ25-35 induction of SHH was abolished by HIF-1α small interfering RNA (siRNA). Aβ25-35 also time-dependently induced inhibitor of differentiation-1 (Id1) that has been shown to stabilize HIF-1α; further, Aβ25-35-mediated induction of HIF-1α and SHH was both suppressed by Id1 siRNA. Pharmacological induction of HIF-1α by cobalt chloride and application of the cell-permeable recombinant Id1 proteins were both sufficient to induce SHH expression. Finally, both the SHH pathway inhibitor cyclopamine and its neutralizing antibody attenuated Aβ cytotoxicity, albeit to a minor extent. These results thus established a signaling cascade of "Aβ → Id1 → HIF-1 → SHH" in primary rat cortical cultures; furthermore, SHH may in part contribute to Aβ neurotoxicity.

  5. Cortical commands in active touch.

    PubMed

    Brecht, Michael

    2006-01-01

    The neocortex is an enormous network of extensively interconnected neurons. It has become clear that the computations performed by individual cortical neurons will critically depend on the quantitative composition of cortical activity. Here we discuss quantitative aspects of cortical activity and modes of cortical processing in the context of rodent active touch. Through in vivo whole-cell recordings one observes widespread subthreshold and very sparse evoked action potential (AP) activity in the somatosensory cortex both for passive whisker deflection in anaesthetized animals and during active whisker movements in awake animals. Neurons of the somatosensory cortex become either suppressed during whisking or activated by an efference copy of whisker movement signal that depolarize cells at certain phases of the whisking cycle. To probe the read out of cortical motor commands we applied intracellular stimulation in rat whisker motor cortex. We find that APs in individual cortical neurons can evoke long sequences of small whisker movements. The capacity of an individual neuron to evoke movements is most astonishing given the large number of neurons in whisker motor cortex. Thus, few cortical APs may suffice to control motor behaviour and such APs can be translated into action with the utmost precision. We conclude that there is very widespread subthreshold cortical activity and very sparse, highly specific cortical AP activity.

  6. Chemically induced bidirectional differentiation of embryonal carcinoma cells in vitro.

    PubMed Central

    Speers, W. C.; Birdwell, C. R.; Dixon, F. J.

    1979-01-01

    N,N-dimethylacetamide, hexamethylene bisacetamide, and Polybrene induced rapid and extensive differentiation in vitro in an otherwise slowly differentiating subline of embryonal carcinoma cells. The type of differentiated cell induced was dependent on the spatial organization of the stem cells during drug treatment. In monalayer culture "epithelial" cells were produced exclusively. However, treatment of aggregated suspension cultures yielded predominantly "fibroblast-like" cells. The undifferentiated embryonal carcinoma cells and the two differentiated cell types were morphologically distinct when examined by light microscopy, scanning electron microscopy, and transmission electron microscopy; and they had differences in cell surface antigens. Both differential cell types produced large amounts of fibronectin, whereas the embryonal carcinoma cells produced only minimal amounts. This system provides a convenient way to induce relatively synchronous differentiation of embryonal carcinoma cells into specific differentiated cell types. Images Figure 5 Figure 6 Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 PMID:507191

  7. Endothelial cells derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  8. Mechanotransduction in Embryonic Vascular Development

    PubMed Central

    Roman, Beth L.; Pekkan, Kerem

    2015-01-01

    A plethora of biochemical signals provides spatial and temporal cues that carefully orchestrate the complex process of vertebrate embryonic development. The embryonic vasculature develops not only in the context of these biochemical cues, but also in the context of the biomechanical forces imparted by blood flow. In the mature vasculature, different blood flow regimes induce distinct genetic programs, and significant progress has been made toward understanding how these forces are perceived by endothelial cells and transduced into biochemical signals. However, it cannot be assumed that paradigms that govern the mature vasculature are pertinent to the developing embryonic vasculature. The embryonic vasculature can respond to the mechanical forces of blood flow, and these responses are critical in vascular remodeling, certain aspects of sprouting angiogenesis, and maintenance of arterial-venous identity. Here, we review data regarding mechanistic aspects of endothelial cell mechanotransduction, with a focus on the response to shear stress, and elaborate upon the multifarious effects of shear stress on the embryonic vasculature. In addition, we discuss emerging predictive vascular growth models and highlight the prospect of combining signaling pathway information with computational modeling. We assert that correlation of precise measurements of hemodynamic parameters with effects on endothelial cell gene expression and cell behavior is required for fully understanding how blood flow-induced loading governs normal vascular development and shapes congenital cardiovascular abnormalities. PMID:22744845

  9. Embryonic death and the creation of human embryonic stem cells.

    PubMed

    Landry, Donald W; Zucker, Howard A

    2004-11-01

    The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.

  10. Embryonic stem cells: from markers to market.

    PubMed

    Deb, Kaushik Dilip; Jayaprakash, Anitha Devi; Sharma, Vijay; Totey, Satish

    2008-02-01

    ABSTRACT Embryonic stem cells are considered the mother of all kinds of tissues and cells and it is envisioned as the holy grail of regenerative medicine. However, their use in cell replacement therapies (CRT) has so far been limited and their potentials are yet to be fully realized. The use of human embryonic stem cells (hESC) involves many safety issues pertaining to culture conditions and epigenetic changes. The role and importance of an epigenomic signature in derivation and maintenance of hESC are discussed. We provide a list of important epigenetic markers, which should be studied for evaluation of safety in hESC-based cell replacement therapies. These genes also need to be screened to determine an epigenetic signature for pluripotency in the hESCs. Finally a comprehensive list of all known stemness signature genes and the marker genes for different germ line lineages are presented. This review aims at summing up most of the intriguing molecules that can play a role in the maintenance of pluripotency and can help in determining hESC differentiation to various lineages. Extensive understanding of these markers will eventually help the researchers to transform the hESC research from bench to the bedside. The use of hESCs in CRTs is still in its infancy; much effort is warranted to turn them into the much dreamed about magic wand of regenerative medicine.

  11. Mapping cortical mesoscopic networks of single spiking cortical or sub-cortical neurons

    PubMed Central

    Xiao, Dongsheng; Vanni, Matthieu P; Mitelut, Catalin C; Chan, Allen W; LeDue, Jeffrey M; Xie, Yicheng; Chen, Andrew CN; Swindale, Nicholas V; Murphy, Timothy H

    2017-01-01

    Understanding the basis of brain function requires knowledge of cortical operations over wide-spatial scales, but also within the context of single neurons. In vivo, wide-field GCaMP imaging and sub-cortical/cortical cellular electrophysiology were used in mice to investigate relationships between spontaneous single neuron spiking and mesoscopic cortical activity. We make use of a rich set of cortical activity motifs that are present in spontaneous activity in anesthetized and awake animals. A mesoscale spike-triggered averaging procedure allowed the identification of motifs that are preferentially linked to individual spiking neurons by employing genetically targeted indicators of neuronal activity. Thalamic neurons predicted and reported specific cycles of wide-scale cortical inhibition/excitation. In contrast, spike-triggered maps derived from single cortical neurons yielded spatio-temporal maps expected for regional cortical consensus function. This approach can define network relationships between any point source of neuronal spiking and mesoscale cortical maps. DOI: http://dx.doi.org/10.7554/eLife.19976.001 PMID:28160463

  12. Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase.

    PubMed

    Bembenek, Joshua N; Richie, Christopher T; Squirrell, Jayne M; Campbell, Jay M; Eliceiri, Kevin W; Poteryaev, Dmitry; Spang, Anne; Golden, Andy; White, John G

    2007-11-01

    In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

  13. Evaluating Mandibular Cortical Index Quantitatively

    PubMed Central

    Yasar, Fusun; Akgunlu, Faruk

    2008-01-01

    Objectives The aim was to assess whether Fractal Dimension and Lacunarity analysis can discriminate patients having different mandibular cortical shape. Methods Panoramic radiographs of 52 patients were evaluated for mandibular cortical index. Weighted Kappa between the observations were varying between 0.718–0.805. These radiographs were scanned and converted to binary images. Fractal Dimension and Lacunarity were calculated from the regions where best represents the cortical morphology. Results It was found that there were statistically significant difference between the Fractal Dimension and Lacunarity of radiographs which were classified as having Cl 1 and Cl 2 (Fractal Dimension P:0.000; Lacunarity P:0.003); and Cl 1 and Cl 3 cortical morphology (Fractal Dimension P:0.008; Lacunarity P:0.001); but there was no statistically significant difference between Fractal Dimension and Lacunarity of radiographs which were classified as having Cl 2 and Cl 3 cortical morphology (Fractal Dimension P:1.000; Lacunarity P:0.758). Conclusions FD and L can differentiate Cl 1 mandibular cortical shape from both Cl 2 and Cl 3 mandibular cortical shape but cannot differentiate Cl 2 from Cl 3 mandibular cortical shape on panoramic radiographs. PMID:19212535

  14. Cortico-cortical communication dynamics

    PubMed Central

    Roland, Per E.; Hilgetag, Claus C.; Deco, Gustavo

    2014-01-01

    In principle, cortico-cortical communication dynamics is simple: neurons in one cortical area communicate by sending action potentials that release glutamate and excite their target neurons in other cortical areas. In practice, knowledge about cortico-cortical communication dynamics is minute. One reason is that no current technique can capture the fast spatio-temporal cortico-cortical evolution of action potential transmission and membrane conductances with sufficient spatial resolution. A combination of optogenetics and monosynaptic tracing with virus can reveal the spatio-temporal cortico-cortical dynamics of specific neurons and their targets, but does not reveal how the dynamics evolves under natural conditions. Spontaneous ongoing action potentials also spread across cortical areas and are difficult to separate from structured evoked and intrinsic brain activity such as thinking. At a certain state of evolution, the dynamics may engage larger populations of neurons to drive the brain to decisions, percepts and behaviors. For example, successfully evolving dynamics to sensory transients can appear at the mesoscopic scale revealing how the transient is perceived. As a consequence of these methodological and conceptual difficulties, studies in this field comprise a wide range of computational models, large-scale measurements (e.g., by MEG, EEG), and a combination of invasive measurements in animal experiments. Further obstacles and challenges of studying cortico-cortical communication dynamics are outlined in this critical review. PMID:24847217

  15. Reprogramming Malignant Cancer Cells toward a Benign Phenotype following Exposure to Human Embryonic Stem Cell Microenvironment

    PubMed Central

    Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio

    2017-01-01

    The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells. PMID:28068409

  16. Reprogramming Malignant Cancer Cells toward a Benign Phenotype following Exposure to Human Embryonic Stem Cell Microenvironment.

    PubMed

    Zhou, Shufeng; Abdouh, Mohamed; Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio

    2017-01-01

    The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells.

  17. Generation of Corneal Keratocytes from Human Embryonic Stem Cells

    PubMed Central

    Hertsenberg, Andrew J.; Funderburgh, James L.

    2017-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype. PMID:26026882

  18. Embryonic amygdalar transplants in adult rats with motor cortex lesions: a molecular and electrophysiological analysis.

    PubMed

    Jiménez-Díaz, Lydia; Nava-Mesa, Mauricio O; Heredia, Margarita; Riolobos, Adelaida S; Gómez-Álvarez, Marcelo; Criado, José María; de la Fuente, Antonio; Yajeya, Javier; Navarro-López, Juan D

    2011-01-01

    Transplants of embryonic nervous tissue ameliorate motor deficits induced by motor cortex lesions in adult animals. Restoration of lost brain functions has been recently shown in grafts of homotopic cortical origin, to be associated with a functional integration of the transplant after development of reciprocal host-graft connections. Nevertheless little is known about physiological properties or gene expression profiles of cortical implants with functional restorative capacity but no cortical origin. In this study, we show molecular and electrophysiological evidence supporting the functional development and integration of heterotopic transplants of embryonic amygdalar tissue placed into pre-lesioned motor cortex of adult rats. Grafts were analyzed 3 months post-transplantation. Using reverse transcriptase quantitative polymerase chain reaction, we found that key glutamatergic, GABAergic, and muscarinic receptors transcripts were expressed at different quantitative levels both in grafted and host tissues, but were all continuously present in the graft. Parallel sharp electrode recordings of grafted neurons in brain slices showed a regular firing pattern of transplanted neurons similar to host amygdalar pyramidal neurons. Synaptic connections from the adjacent host cortex on grafted neurons were electrophysiologically investigated and confirmed our molecular results. Taken together, our findings indicate that grafted neurons from a non-cortical, non-motor-related, but ontogenetical similar source, not only received functionally effective contacts from the adjacent motor cortex, but also developed electrophysiological and gene expression patterns comparable to host pyramidal neurons; suggesting an interesting tool for the field of neural repair and donor tissue in adults.

  19. Modeling cortical circuits.

    SciTech Connect

    Rohrer, Brandon Robinson; Rothganger, Fredrick H.; Verzi, Stephen J.; Xavier, Patrick Gordon

    2010-09-01

    The neocortex is perhaps the highest region of the human brain, where audio and visual perception takes place along with many important cognitive functions. An important research goal is to describe the mechanisms implemented by the neocortex. There is an apparent regularity in the structure of the neocortex [Brodmann 1909, Mountcastle 1957] which may help simplify this task. The work reported here addresses the problem of how to describe the putative repeated units ('cortical circuits') in a manner that is easily understood and manipulated, with the long-term goal of developing a mathematical and algorithmic description of their function. The approach is to reduce each algorithm to an enhanced perceptron-like structure and describe its computation using difference equations. We organize this algorithmic processing into larger structures based on physiological observations, and implement key modeling concepts in software which runs on parallel computing hardware.

  20. Sonic Hedgehog Promotes Neurite Outgrowth of Primary Cortical Neurons Through Up-Regulating BDNF Expression.

    PubMed

    He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao

    2016-04-01

    Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression.

  1. A Concise Protocol for siRNA-Mediated Gene Suppression in Human Embryonic Stem Cells.

    PubMed

    Renz, Peter F; Beyer, Tobias A

    2016-01-01

    Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA).

  2. Paroxysmal kinesigenic dyskinesia: cortical or non-cortical origin.

    PubMed

    van Strien, Teun W; van Rootselaar, Anne-Fleur; Hilgevoord, Anthony A J; Linssen, Wim H J P; Groffen, Alexander J A; Tijssen, Marina A J

    2012-06-01

    Paroxysmal kinesigenic dyskinesia (PKD) is characterized by involuntary dystonia and/or chorea triggered by a sudden movement. Cases are usually familial with an autosomal dominant inheritance. Hypotheses regarding the pathogenesis of PKD focus on the controversy whether PKD has a cortical or non-cortical origin. A combined familial trait of PKD and benign familial infantile seizures has been reported as the infantile convulsions and paroxysmal choreoathetosis (ICCA) syndrome. Here, we report a family diagnosed with ICCA syndrome with an Arg217STOP mutation. The index patient showed interictal EEG focal changes compatible with paroxysmal dystonic movements of his contralateral leg. This might support cortical involvement in PKD.

  3. Avian influenza virus isolation, propagation and titration in embryonated chicken eggs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian influenza (AI) virus is usually isolated, propagated, and titrated in embryonated chickens eggs (ECE). Most any sample type can be accommodated for culture with appropriate processing. Isolation may also be accomplished in cell culture particularly if mammalian lineage isolates are suspected, ...

  4. Shrink-film configurable multiscale wrinkles for functional alignment of human embryonic stem cells and their cardiac derivatives.

    PubMed

    Chen, Aaron; Lieu, Deborah K; Freschauf, Lauren; Lew, Valerie; Sharma, Himanshu; Wang, Jiaxian; Nguyen, Diep; Karakikes, Ioannis; Hajjar, Roger J; Gopinathan, Ajay; Botvinick, Elliot; Fowlkes, Charless C; Li, Ronald A; Khine, Michelle

    2011-12-22

    A biomimetic substrate for cell-culture is fabricated by plasma treatment of a prestressed thermoplastic shrink film to create tunable multiscaled alignment "wrinkles". Using this substrate, the functional alignment of human embryonic stem cell derived cardiomyocytes is demonstrated.

  5. Embryonic cerebrospinal fluid collaborates with the isthmic organizer to regulate mesencephalic gene expression.

    PubMed

    Parada, Carolina; Martín, Cristina; Alonso, María I; Moro, José A; Bueno, David; Gato, Angel

    2005-11-01

    Early in development, the behavior of neuroepithelial cells is controlled by several factors acting in a developmentally regulated manner. Recently it has been shown that diffusible factors contained within embryonic cerebrospinal fluid (CSF) promote neuroepithelial cell survival, proliferation, and neurogenesis in mesencephalic explants lacking any known organizing center. In this paper, we show that mesencephalic and mesencephalic+isthmic organizer explants cultured only with basal medium do not express the typically expressed mesencephalic or isthmic organizer genes analyzed (otx2 and fgf8, respectively) and that mesencephalic explants cultured with embryonic CSF-supplemented medium do effect such expression, although they exhibit an altered pattern of gene expression, including ectopic shh expression domains. Other trophic sources that are able to maintain normal neuroepithelial cell behavior, i.e., fibroblast growth factor-2, fail to activate this ectopic shh expression. Conversely, the expression pattern of the analyzed genes in mesencephalic+isthmic organizer explants cultured with embryonic cerebrospinal fluid-supplemented medium mimics the pattern for control embryos developed in ovo. We demonstrate that embryonic CSF collaborates with the isthmic organizer in regulation of the expression pattern of some characteristic neuroectodermal genes during early stages of central nervous system (CNS) development, and we suggest that this collaboration is not restricted to the maintenance of neuroepithelial cell survival. Data reported in this paper corroborate the hypothesis that factors contained within embryonic CSF contribute to the patterning of the CNS during early embryonic development.

  6. Properties of persistent postnatal cortical subplate neurons.

    PubMed

    Torres-Reveron, Juan; Friedlander, Michael J

    2007-09-12

    Subplate (SP) neurons are important for the proper development of thalamocortical innervation. They are necessary for formation of ocular dominance and orientation columns in visual cortex. During the perinatal period, many SP neurons die. The surviving cohort forms interstitial cells in the white matter (WM) and a band of horizontally oriented cells below layer VI (layer VIb, layer VII, or subplate cells). Although the function of embryonic SP neurons has been well established, the functional roles of WM and postnatal SP cells are not known. We used a combination of anatomical, immunohistochemical, and electrophysiological techniques to explore the dendritic morphology, neurotransmitter phenotype, intrinsic electrophysiological, and synaptic input properties of these surviving cells in the rat visual cortex. The density of SP and WM cells significantly decreases during the first month of life. Both populations express neuronal markers and have extensive dendritic arborizations within the SP, WM, and to the overlying visual cortex. Some intrinsic electrophysiological properties of SP and WM cells are similar: each generates high-frequency slowly adapting trains of action potentials in response to a sustained depolarization. However, SP cells exhibit greater frequency-dependent action potential broadening than WM neurons. Both cell types receive predominantly AMPA/kainate receptor-mediated excitatory synaptic input that undergoes paired-pulse facilitation as well as NMDA receptor and GABAergic input. Synaptic inputs to these cells can also undergo long-term synaptic plasticity. Thus, surviving SP and WM cells are functional electrogenic neurons integrated within the postnatal visual cortical circuit.

  7. Use of cortical neuronal networks for in vitro material biocompatibility testing.

    PubMed

    Charkhkar, Hamid; Frewin, Christopher; Nezafati, Maysam; Knaack, Gretchen L; Peixoto, Nathalia; Saddow, Stephen E; Pancrazio, Joseph J

    2014-03-15

    Neural interfaces aim to restore neurological function lost during disease or injury. Novel implantable neural interfaces increasingly capitalize on novel materials to achieve microscale coupling with the nervous system. Like any biomedical device, neural interfaces should consist of materials that exhibit biocompatibility in accordance with the international standard ISO10993-5, which describes in vitro testing involving fibroblasts where cytotoxicity serves as the main endpoint. In the present study, we examine the utility of living neuronal networks as functional assays for in vitro material biocompatibility, particularly for materials that comprise implantable neural interfaces. Embryonic mouse cortical tissue was cultured to form functional networks where spontaneous action potentials, or spikes, can be monitored non-invasively using a substrate-integrated microelectrode array. Taking advantage of such a platform, we exposed established positive and negative control materials to the neuronal networks in a consistent method with ISO 10993-5 guidance. Exposure to the negative controls, gold and polyethylene, did not significantly change the neuronal activity whereas the positive controls, copper and polyvinyl chloride (PVC), resulted in reduction of network spike rate. We also compared the functional assay with an established cytotoxicity measure using L929 fibroblast cells. Our findings indicate that neuronal networks exhibit enhanced sensitivity to positive control materials. In addition, we assessed functional neurotoxicity of tungsten, a common microelectrode material, and two conducting polymer formulations that have been used to modify microelectrode properties for in vivo recording and stimulation. These data suggest that cultured neuronal networks are a useful platform for evaluating the functional toxicity of materials intended for implantation in the nervous system.

  8. Effects of exposing rat embryos in utero to physical or chemical teratogens are expressed later as enhanced induction of heat-shock proteins when embryonic hearts are cultured in vitro

    SciTech Connect

    Higo, H.; Higo, K.; Lee, J.Y.; Hori, H.; Satow, Y.

    1988-01-01

    In order to get more insight into the effects of teratogens on developing embryos, we investigated the protein synthesis patterns of the target organs isolated from teratogen-treated embryos. Rat embryos were either irradiated in utero with either 252Cf fission neutrons or 60Co gamma rays on day 8 of gestation or treated in utero with a bis(dichloroacetyl)diamine (a chemical teratogen) on days 9 and 10. Hearts were removed from the embryos on day 12 and were incubated in vitro at 37 degrees C in the presence of (35S)methionine for up to 8 hr. The newly synthesized labeled proteins were then analyzed qualitatively by two-dimensional polyacrylamide gel electrophoresis. Enhanced and prolonged induction of a family of heat-shock (stress) proteins with a molecular weight of about 70,000 (SP70s) was observed as compared with those of controls. Among the teratogen-treated hearts, those with gross malformations already detectable at this early stage showed especially higher inductions of SP70s than did the others. The abnormal expression of SP70s observed in the present study appears to be a reflection of persisting cellular (tissue) damage inflicted by the teratogens, and the extent of the induction may be indicative of the degree and/or type of the damage. Such persisting defects in surviving cells, manifested by abnormal induction of SP70s in the present study, might be related to malformation of embryonic hearts.

  9. Comparative Analysis of Whole-Genome Gene Expression Changes in Cultured Human Embryonic Stem Cells in Response to Low, Clinical Diagnostic Relevant, and High Doses of Ionizing Radiation Exposure.

    PubMed

    Sokolov, Mykyta; Nguyen, Van; Neumann, Ronald

    2015-06-30

    The biological effects of low-dose ionizing radiation (LDIR) exposure in humans are not comprehensively understood, generating a high degree of controversy in published literature. The earliest stages of human development are known to be among the most sensitive to stress exposures, especially genotoxic stresses. However, the risks stemming from exposure to LDIR, particularly within the clinical diagnostic relevant dose range, have not been directly evaluated in human embryonic stem cells (hESCs). Here, we describe the dynamics of the whole genome transcriptional responses of different hESC lines to both LDIR and, as a reference, high-dose IR (HDIR). We found that even doses as low as 0.05 Gy could trigger statistically significant transient changes in a rather limited subset of genes in all hESCs lines examined. Gene expression signatures of hESCs exposed to IR appear to be highly dose-, time-, and cell line-dependent. We identified 50 genes constituting consensus gene expression signature as an early response to HDIR across all lines of hESC examined. We observed substantial differences in biological pathways affected by either LDIR or HDIR in hESCs, suggesting that the molecular mechanisms underpinning the responses of hESC may fundamentally differ depending on radiation doses.

  10. Pre-flight report on cultured human embryonic kidney cell handling and cell electrophoresis. Prepared prior to continuous-flow electrophoretic separation experiments aboard space shuttle flight STS-8

    NASA Technical Reports Server (NTRS)

    Todd, P. W.; Sarnoff, B. E.; Li, Z. K.

    1985-01-01

    Studies of the physical properties of continuous-flow zero-G electrophoretic separator (CFES) buffer, the electrokinetic properties of human erythrocytes in the CFES buffer, the electrokinetic properties of human embryonic kidney cells in the CFES buffer, and the viability and yield of human embryonc kidney cells subjected to flight handling procedures are discussed. In general, the procedure for cell handling and electrophoresis of HEK-8514 cells in 1st or 2nd passage on STS-8 is acceptable if executed properly. The CFES buffer has ionic strength that is barely compatible with cell viability and membrane stability, as seen in experiments with human erythrocytes and trypan-blue staining of human kidney cells. Cells suspended in 10% dialysed horse serum for 3 days in the cold appear to be more stable than freshly trypsinized cells. 10% horse serum appears to be superior to 5% horse serum for this purpose. The mean absolute raw mobility of HEK-8514 cells in CFES buffer at 6 degrees, conductivity 0.055 mmho/cm, is 1.1 to 1.4 um-cm/V-sec, with a range of nearly a whole mobility unit.

  11. Embryonic stem cell lines of nonhuman primates.

    PubMed

    Nakatsuji, Norio; Suemori, Hirofumi

    2002-06-26

    Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics. Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases. So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice. The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is

  12. Shockwaves Cause Synaptic Degeneration in Cultured Neurons

    DTIC Science & Technology

    2009-11-02

    constructed of delrin. A piezoresistive pressure sensor (Endevco Model 8530C) was mounted flush with the plate, coaxial with the center of the gene gun ...biolostic gene gun to deliver shockwaves to cultured hippocampal or cortical neurons. These cultured cells form abundant synapses in vitro, and after a 24-48...neurons, we used a biolostic gene gun to deliver shockwaves to cultured hippocampal or cortical neurons. These cultured cells form abundant synapses in

  13. Transplanted embryonic neurons integrate into adult neocortical circuits.

    PubMed

    Falkner, Susanne; Grade, Sofia; Dimou, Leda; Conzelmann, Karl-Klaus; Bonhoeffer, Tobias; Götz, Magdalena; Hübener, Mark

    2016-11-10

    The ability of the adult mammalian brain to compensate for neuronal loss caused by injury or disease is very limited. Transplantation aims to replace lost neurons, but the extent to which new neurons can integrate into existing circuits is unknown. Here, using chronic in vivo two-photon imaging, we show that embryonic neurons transplanted into the visual cortex of adult mice mature into bona fide pyramidal cells with selective pruning of basal dendrites, achieving adult-like densities of dendritic spines and axonal boutons within 4-8 weeks. Monosynaptic tracing experiments reveal that grafted neurons receive area-specific, afferent inputs matching those of pyramidal neurons in the normal visual cortex, including topographically organized geniculo-cortical connections. Furthermore, stimulus-selective responses refine over the course of many weeks and finally become indistinguishable from those of host neurons. Thus, grafted neurons can integrate with great specificity into neocortical circuits that normally never incorporate new neurons in the adult brain.

  14. Embryonic markers of cone differentiation

    PubMed Central

    Rodgers, Helen M.; Belcastro, Marycharmain; Sokolov, Maxim

    2016-01-01

    Purpose Photoreceptor cells are born in two distinct phases of vertebrate retinogenesis. In the mouse retina, cones are born primarily during embryogenesis, while rod formation occurs later in embryogenesis and early postnatal ages. Despite this dichotomy in photoreceptor birthdates, the visual pigments and phototransduction machinery are not reactive to visual stimulus in either type of photoreceptor cell until the second postnatal week. Several markers of early cone formation have been identified, including Otx2, Crx, Blimp1, NeuroD, Trβ2, Rorβ, and Rxrγ, and all are thought to be involved in cellular determination. However, little is known about the expression of proteins involved in cone visual transduction during early retinogenesis. Therefore, we sought to characterize visual transduction proteins that are expressed specifically in photoreceptors during mouse embryogenesis. Methods Eye tissue was collected from control and phosducin-null mice at embryonic and early postnatal ages. Immunohistochemistry and quantitative reverse transcriptase-PCR (qPCR) were used to measure the spatial and temporal expression patterns of phosducin (Pdc) and cone transducin γ (Gngt2) proteins and transcripts in the embryonic and early postnatal mouse retina. Results We identified the embryonic expression of phosducin (Pdc) and cone transducin γ (Gngt2) that coincides temporally and spatially with the earliest stages of cone histogenesis. Using immunohistochemistry, the phosducin protein was first detected in the retina at embryonic day (E)12.5, and cone transducin γ was observed at E13.5. The phosducin and cone transducin γ proteins were seen only in the outer neuroblastic layer, consistent with their expression in photoreceptors. At the embryonic ages, phosducin was coexpressed with Rxrγ, a known cone marker, and with Otx2, a marker of photoreceptors. Pdc and Gngt2 mRNAs were detected as early as E10.5 with qPCR, although at low levels. Conclusions Visual transduction

  15. Infiltrating cells from host brain restore the microglial population in grafted cortical tissue

    PubMed Central

    Wang, Cong; Tao, Sijue; Fang, Yukun; Guo, Jing; Zhu, Lirui; Zhang, Shengxiang

    2016-01-01

    Transplantation of embryonic cortical tissue is considered as a promising therapy for brain injury. Grafted neurons can reestablish neuronal network and improve cortical function of the host brain. Microglia is a key player in regulating neuronal survival and plasticity, but its activation and dynamics in grafted cortical tissue remain unknown. Using two-photon intravital imaging and parabiotic model, here we investigated the proliferation and source of microglia in the donor region by transplanting embryonic cortical tissue into adult cortex. Live imaging showed that the endogenous microglia of the grafted tissue were rapidly lost after transplantation. Instead, host-derived microglia infiltrated and colonized the graft. Parabiotic model suggested that the main source of infiltrating cells is the parenchyma of the host brain. Colonized microglia proliferated and experienced an extensive morphological transition and eventually differentiated into resting ramified morphology. Collectively, these results demonstrated that donor tissue has little contribution to the activated microglia and host brain controls the microglial population in the graft. PMID:27615195

  16. Further Work on the Shaping of Cortical Development and Function by Synchrony and Metabolic Competition

    PubMed Central

    Wright, James J.; Bourke, Paul D.

    2016-01-01

    This paper furthers our attempts to resolve two major controversies—whether gamma synchrony plays a role in cognition, and whether cortical columns are functionally important. We have previously argued that the configuration of cortical cells that emerges in development is that which maximizes the magnitude of synchronous oscillation and minimizes metabolic cost. Here we analyze the separate effects in development of minimization of axonal lengths, and of early Hebbian learning and selective distribution of resources to growing synapses, by showing in simulations that these effects are partially antagonistic, but their interaction during development produces accurate anatomical and functional properties for both columnar and non-columnar cortex. The resulting embryonic anatomical order can provide a cortex-wide scaffold for postnatal learning that is dimensionally consistent with the representation of moving sensory objects, and, as learning progressively overwrites the embryonic order, further associations also occur in a dimensionally consistent framework. The role ascribed to cortical synchrony does not demand specific frequency, amplitude or phase variation of pulses to mediate “feature linking.” Instead, the concerted interactions of pulse synchrony with short-term synaptic dynamics, and synaptic resource competition can further explain cortical information processing in analogy to Hopfield networks and quantum computation. PMID:28018202

  17. Analysis of Cortical Flow Models In Vivo

    PubMed Central

    Benink, Hélène A.; Mandato, Craig A.; Bement, William M.

    2000-01-01

    Cortical flow, the directed movement of cortical F-actin and cortical organelles, is a basic cellular motility process. Microtubules are thought to somehow direct cortical flow, but whether they do so by stimulating or inhibiting contraction of the cortical actin cytoskeleton is the subject of debate. Treatment of Xenopus oocytes with phorbol 12-myristate 13-acetate (PMA) triggers cortical flow toward the animal pole of the oocyte; this flow is suppressed by microtubules. To determine how this suppression occurs and whether it can control the direction of cortical flow, oocytes were subjected to localized manipulation of either the contractile stimulus (PMA) or microtubules. Localized PMA application resulted in redirection of cortical flow toward the site of application, as judged by movement of cortical pigment granules, cortical F-actin, and cortical myosin-2A. Such redirected flow was accelerated by microtubule depolymerization, showing that the suppression of cortical flow by microtubules is independent of the direction of flow. Direct observation of cortical F-actin by time-lapse confocal analysis in combination with photobleaching showed that cortical flow is driven by contraction of the cortical F-actin network and that microtubules suppress this contraction. The oocyte germinal vesicle serves as a microtubule organizing center in Xenopus oocytes; experimental displacement of the germinal vesicle toward the animal pole resulted in localized flow away from the animal pole. The results show that 1) cortical flow is directed toward areas of localized contraction of the cortical F-actin cytoskeleton; 2) microtubules suppress cortical flow by inhibiting contraction of the cortical F-actin cytoskeleton; and 3) localized, microtubule-dependent suppression of actomyosin-based contraction can control the direction of cortical flow. We discuss these findings in light of current models of cortical flow. PMID:10930453

  18. Cortical granule complements in human oocytes undergoing partial zona dissection.

    PubMed

    Lanzendorf, S E; Kazer, R R; Patton, P E; Wolf, D P

    1992-02-01

    This study was performed to evaluate the effects of mechanical stimulation and sucrose treatment on the oocyte activation process. Fresh and aged human oocytes were exposed to sucrose and zonae were dissected with microneedles before fixation and quantitative analysis of cortical granules by transmission electron microscopy. Examination of the mean number of cortical granules/analyzed segment revealed no significant differences between control oocytes or oocytes treated with sucrose or sucrose treatment followed by zona dissection. A significant decline in the number of cortical granules/segment was observed for oocytes undergoing prolonged culture after dissection (P less than 0.05). Thus, zona dissection and sucrose exposure of freshly aspirated mature human oocytes do not result in classical oocyte activation.

  19. Embryonic development during chronic acceleration

    NASA Technical Reports Server (NTRS)

    Smith, A. H.; Abbott, U. K.

    1982-01-01

    Experiments carried out on chicken eggs indicate that the embryo is affected during very early development, especially over the first four days, and during hatching. In the first four days, the brain develops as well as the anlage for all other organs. In addition, the heart commences to function and the extraembryonic membranes that compartmentalize the egg contents form. The latter require an appreciable extension and folding of tissue which may be disrupted by the mechanical load. Observations of embryonic abnormalities that occur during chronic acceleration suggest an inhibition of development of the axial skeleton, which is rarely seen otherwise, a general retardation of embryonic growth, and circulatory problems. The final stages of development (after 18 days) involve the uptake of fluids, the transition to aerial respiration, and the reorientation of the embryo into a normal hatching position. At 4 G mortality is very high during this period, with a majority of embryos failing to reorient into the normal hatching position.

  20. Nicotine alters bovine oocyte meiosis and affects subsequent embryonic development.

    PubMed

    Liu, Ying; Li, Guang-Peng; White, Kenneth L; Rickords, Lee F; Sessions, Benjamin R; Aston, Kenneth I; Bunch, Thomas D

    2007-11-01

    The effects of nicotine on nuclear maturation and meiotic spindle dynamics of bovine oocytes and subsequent embryonic development were investigated. Maturation rates (85%-94%) derived from nicotine treatments at 0.01 to 1.0 mM were similar to the control (86%), but significantly decreased at 2.0 to 6.0 mM. Haploid complements of metaphase II oocytes in 0.01 to 1.0 mM nicotine (approximately 90%) were similar to the control, while lower (ranged from 63% to 76%, P < 0.05 or P < 0.01) haploid oocytes were observed in the 2.0 to 6.0 mM nicotine groups. The majority of the PB1-free oocytes derived from 3.0 to 6.0 mM nicotine treatments were diploidy (2n = 60). Spindle microtubules changed from characteristically being asymmetrical in the controls to being equally distributed into two separate chromosome groups in the nicotine treatments. Nicotine disorganized the microfilament organization and inhibited the movement of anaphase or telophase chromosomes to the cortical area. The inhibited two chromosome groups became two spindles that either moved close in proximity or merged entirely together resulting in diploidy within the affected oocyte. Nicotine treatment significantly reduced the rate of cleavage and blastocyst development after parthenogenetic activation. Diploidy and cell number were drastically reduced in the resultant blastocysts. In conclusion, nicotine can alter the normal process of bovine oocyte meiosis and affects subsequent embryonic development.

  1. ADAM17 Transactivates EGFR Signaling during Embryonic Eyelid Closure

    PubMed Central

    Hassemer, Eryn L.; Endres, Bradley; Toonen, Joseph A.; Ronchetti, Adam; Dubielzig, Richard; Sidjanin, Duska J.

    2013-01-01

    Purpose. During mammalian embryonic eyelid closure ADAM17 has been proposed to play a role as a transactivator of epidermal growth factor receptor (EGFR) signaling by shedding membrane bound EGFR ligands. However, ADAM17 also sheds numerous other ligands, thus implicating ADAM17 in additional molecular pathways. The goal of this study was to experimentally establish the role of ADAM17 and determine ADAM17-mediated pathways essential for the embryonic eyelid closure. Methods. Wild-type (WT) and woe mice, carrying a hypomorphic mutation in Adam17, were evaluated using H&E and scanning electron microscopy. Expressions of ADAM17, EGFR, and the phosphorylated form EGFR-P were evaluated using immunohistochemistry. BrdU and TUNEL assays were used to evaluate cell proliferation and apoptosis, respectively. In vitro scratch assays of primary cultures were used to evaluate cell migration. Clinical and histologic analyses established if the hypermorphic EgfrDsk5 allele can rescue the woe embryonic eyelid closure. Results. woe mice exhibited a failure to develop the leading edge of the eyelid and consequently failure of the embryonic eyelid closure. Expression of ADAM17 was identified in the eyelid epithelium in the cells of the leading edge. ADAM17 is essential for epithelial cell migration, but does not play a role in proliferation and apoptosis. EGFR was expressed in both WT and woe eyelid epithelium, but the phosphorylated EGFR-P form was detected only in WT. The EgfrDsk5 allele rescued woe eyelid closure defects, but also rescued woe anterior segment defects and the absence of meibomian glands. Conclusions. We provide in vivo genetic evidence that the role of ADAM17 during embryonic eyelid closure is to transactivate EGFR signaling. PMID:23211830

  2. Nogo-a regulates neural precursor migration in the embryonic mouse cortex.

    PubMed

    Mathis, Carole; Schröter, Aileen; Thallmair, Michaela; Schwab, Martin E

    2010-10-01

    Although Nogo-A has been intensively studied for its inhibitory effect on axonal regeneration in the adult central nervous system, little is known about its function during brain development. In the embryonic mouse cortex, Nogo-A is expressed by radial precursor/glial cells and by tangentially migrating as well as postmigratory neurons. We studied radially migrating neuroblasts in wild-type and Nogo-A knockout (KO) mouse embryos. In vitro analysis showed that Nogo-A and its receptor components NgR, Lingo-1, TROY, and p75 are expressed in cells emigrating from embryonic forebrain-derived neurospheres. Live imaging revealed an increased cell motility when Nogo-A was knocked out or blocked with antibodies. Antibodies blocking NgR or Lingo-1 showed the same motility-enhancing effect supporting a direct role of surface Nogo-A on migration. Bromodeoxyuridine (BrdU) labeling of embryonic day (E)15.5 embryos demonstrated that Nogo-A influences the radial migration of neuronal precursors. At E17.5, the normal transient accumulation of radially migrating precursors within the subventricular zone was not detectable in the Nogo-A KO mouse cortex. At E19, migration to the upper cortical layers was disturbed. These findings suggest that Nogo-A and its receptor complex play a role in the interplay of adhesive and repulsive cell interactions in radial migration during cortical development.

  3. Cryopreservation of somatic embryos and embryonic axes of Camellia japonica L.

    PubMed

    Janeiro, L V; Vieitez, A M; Ballester, A

    1996-05-01

    Cryopreservation in liquid nitrogen was attempted with both somatic embryos and zygotic embryonic axes of the ornamental Camellia japonica L. Several protective measures were applied to somatic embryos (desiccation, chemical protectors, hardening by culture at low temperatures, encapsulation in alginate beads), but none allowed somatic embryos cultures to survive after 24 h in liquid nitrogen. Embryonic axes, however, were easily cryopreserved by means of the simplest technique: desiccation in a laminar flow hood and direct immersion in liquid nitrogen. Although the causes of the difference in cryopreservability between the two types of material are not known, one might be the difference between their degrees of differentiation and water content.

  4. Culturing rat hippocampal neurons.

    PubMed

    Audesirk, G; Audesirk, T; Ferguson, C

    2001-01-01

    Cultured neurons are widely used to investigate the mechanisms of neurotoxicity. Embryonic rat hippocampal neurons may be grown as described under a wide variety of conditions to suit differing experimental procedures, including electrophysiology, morphological analysis of neurite development, and various biochemical and molecular analyses.

  5. A Circuit for Motor Cortical Modulation of Auditory Cortical Activity

    PubMed Central

    Nelson, Anders; Schneider, David M.; Takatoh, Jun; Sakurai, Katsuyasu; Wang, Fan

    2013-01-01

    Normal hearing depends on the ability to distinguish self-generated sounds from other sounds, and this ability is thought to involve neural circuits that convey copies of motor command signals to various levels of the auditory system. Although such interactions at the cortical level are believed to facilitate auditory comprehension during movements and drive auditory hallucinations in pathological states, the synaptic organization and function of circuitry linking the motor and auditory cortices remain unclear. Here we describe experiments in the mouse that characterize circuitry well suited to transmit motor-related signals to the auditory cortex. Using retrograde viral tracing, we established that neurons in superficial and deep layers of the medial agranular motor cortex (M2) project directly to the auditory cortex and that the axons of some of these deep-layer cells also target brainstem motor regions. Using in vitro whole-cell physiology, optogenetics, and pharmacology, we determined that M2 axons make excitatory synapses in the auditory cortex but exert a primarily suppressive effect on auditory cortical neuron activity mediated in part by feedforward inhibition involving parvalbumin-positive interneurons. Using in vivo intracellular physiology, optogenetics, and sound playback, we also found that directly activating M2 axon terminals in the auditory cortex suppresses spontaneous and stimulus-evoked synaptic activity in auditory cortical neurons and that this effect depends on the relative timing of motor cortical activity and auditory stimulation. These experiments delineate the structural and functional properties of a corticocortical circuit that could enable movement-related suppression of auditory cortical activity. PMID:24005287

  6. A circuit for motor cortical modulation of auditory cortical activity.

    PubMed

    Nelson, Anders; Schneider, David M; Takatoh, Jun; Sakurai, Katsuyasu; Wang, Fan; Mooney, Richard

    2013-09-04

    Normal hearing depends on the ability to distinguish self-generated sounds from other sounds, and this ability is thought to involve neural circuits that convey copies of motor command signals to various levels of the auditory system. Although such interactions at the cortical level are believed to facilitate auditory comprehension during movements and drive auditory hallucinations in pathological states, the synaptic organization and function of circuitry linking the motor and auditory cortices remain unclear. Here we describe experiments in the mouse that characterize circuitry well suited to transmit motor-related signals to the auditory cortex. Using retrograde viral tracing, we established that neurons in superficial and deep layers of the medial agranular motor cortex (M2) project directly to the auditory cortex and that the axons of some of these deep-layer cells also target brainstem motor regions. Using in vitro whole-cell physiology, optogenetics, and pharmacology, we determined that M2 axons make excitatory synapses in the auditory cortex but exert a primarily suppressive effect on auditory cortical neuron activity mediated in part by feedforward inhibition involving parvalbumin-positive interneurons. Using in vivo intracellular physiology, optogenetics, and sound playback, we also found that directly activating M2 axon terminals in the auditory cortex suppresses spontaneous and stimulus-evoked synaptic activity in auditory cortical neurons and that this effect depends on the relative timing of motor cortical activity and auditory stimulation. These experiments delineate the structural and functional properties of a corticocortical circuit that could enable movement-related suppression of auditory cortical activity.

  7. Dynamic microtubules at the vegetal cortex predict the embryonic axis in zebrafish.

    PubMed

    Tran, Long Duc; Hino, Hiromu; Quach, Helen; Lim, Shimin; Shindo, Asako; Mimori-Kiyosue, Yuko; Mione, Marina; Ueno, Naoto; Winkler, Christoph; Hibi, Masahiko; Sampath, Karuna

    2012-10-01

    In zebrafish, as in many animals, maternal dorsal determinants are vegetally localized in the egg and are transported after fertilization in a microtubule-dependent manner. However, the organization of early microtubules, their dynamics and their contribution to axis formation are not fully understood. Using live imaging, we identified two populations of microtubules, perpendicular bundles and parallel arrays, which are directionally oriented and detected exclusively at the vegetal cortex before the first cell division. Perpendicular bundles emanate from the vegetal cortex, extend towards the blastoderm, and orient along the animal-vegetal axis. Parallel arrays become asymmetric on the vegetal cortex, and orient towards dorsal. We show that the orientation of microtubules at 20 minutes post-fertilization can predict where the embryonic dorsal structures in zebrafish will form. Furthermore, we find that parallel microtubule arrays colocalize with wnt8a RNA, the candidate maternal dorsal factor. Vegetal cytoplasmic granules are displaced with parallel arrays by ~20°, providing in vivo evidence of a cortical rotation-like process in zebrafish. Cortical displacement requires parallel microtubule arrays, and probably contributes to asymmetric transport of maternal determinants. Formation of parallel arrays depends on Ca(2+) signaling. Thus, microtubule polarity and organization predicts the zebrafish embryonic axis. In addition, our results suggest that cortical rotation-like processes might be more common in early development than previously thought.

  8. Components of vestibular cortical function.

    PubMed

    Klingner, Carsten M; Volk, Gerd F; Flatz, Claudia; Brodoehl, Stefan; Dieterich, Marianne; Witte, Otto W; Guntinas-Lichius, Orlando

    2013-01-01

    It is known that the functional response (e.g., nystagmus) to caloric vestibular stimulation is delayed and prolonged compared with the stimulus-response timing of other sensory systems. Imaging studies have used different models to predict cortical responses and to determine the areas of the brain that are involved. These studies have revealed a widespread network of vestibular brain regions. However, there is some disagreement regarding the brain areas involved, which may partly be caused by differences in the models used. This disagreement indicates the possible existence of multiple cortical components with different temporal characteristics that underlie cortical vestibular processing. However, data-driven methods have yet to be used to analyze the underlying hemodynamic components during and after vestibular stimulation. We performed functional magnetic resonance imaging (fMRI) on 12 healthy subjects during caloric stimulation and analyzed these data using a model-free analysis method (ICA). We found seven independent stimulus-induced components that outline a robust pattern of cortical activation and deactivation. These independent components demonstrated significant differences in their time courses. No single-modeled response function was able to cover the entire range of these independent components. The response functions determined in the present study should improve model-based studies investigating vestibular cortical processing.

  9. Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

    PubMed

    Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

    2016-10-01

    The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface.

  10. Cortical myoclonus in Huntington's disease.

    PubMed

    Thompson, P D; Bhatia, K P; Brown, P; Davis, M B; Pires, M; Quinn, N P; Luthert, P; Honovar, M; O'Brien, M D; Marsden, C D

    1994-11-01

    We describe three patients with Huntington's disease, from two families, in whom myoclonus was the predominant clinical feature. The diagnosis was confirmed at autopsy in two cases and by DNA analysis in all three. These patients all presented before the age of 30 years and were the offspring of affected fathers. Neurophysiological studies documented generalised and multifocal action myoclonus of cortical origin that was strikingly stimulus sensitive, without enlargement of the cortical somatosensory evoked potential. The myoclonus improved with piracetam therapy in one patient and a combination of sodium valproate and clonazepam in the other two. Cortical reflex myoclonus is a rare but disabling component of the complex movement disorder of Huntington's disease, which may lead to substantial diagnostic difficulties.

  11. Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.

    PubMed

    Handel, Adam E; Chintawar, Satyan; Lalic, Tatjana; Whiteley, Emma; Vowles, Jane; Giustacchini, Alice; Argoud, Karene; Sopp, Paul; Nakanishi, Mahito; Bowden, Rory; Cowley, Sally; Newey, Sarah; Akerman, Colin; Ponting, Chris P; Cader, M Zameel

    2016-03-01

    Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.

  12. Dose Response Effects of 810 nm Laser Light on Mouse Primary Cortical Neurons

    PubMed Central

    Sharma, Sulbha K.; Kharkwal, Gitika B.; Sajo, Mari; Huang, Ying-Ying; De Taboada, Luis; McCarthy, Thomas; Hamblin, Michael R.

    2011-01-01

    Background and Objectives In the past four decades numerous studies have reported the efficacy of low level light (laser) therapy (LLLT) as a treatment for diverse diseases and injuries. Recent studies have shown that LLLT can biomodulate processes in the central nervous system and has been extensively studied as a stroke treatment. However there is still a lack of knowledge on the effects of LLLT at the cellular level in neurons. The present study aimed to study the effect of 810 nm laser on several cellular processes in primary cortical neurons cultured from embryonic mouse brains. Study Design/Materials and Methods Neurons were irradiated with fluences of 0.03, 0.3, 3, 10, or 30 J/cm2 of 810-nm laser delivered over varying times at 25 mW/cm2 and intracellular levels of reactive oxygen species (ROS), nitric oxide and calcium were measured using fluorescent probes within 5 minutes of the end of irradiation. The changes in mitochondrial function in response to light were studied in terms of adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP). Results Light induced a significant increase in calcium, ATP and MMP at lower fluences and a decrease at higher fluences. ROS was significantly induced at low fluences, followed by a decrease and a second larger increase at 30 J/cm2. Nitric oxide levels showed a similar pattern of a double peak but values were less significant compared to ROS. Conclusions The results suggest that LLLT at lower fluences is capable of inducing mediators of cell signaling processes which in turn may be responsible for the beneficial stimulatory effects of the low level laser. At higher fluences beneficial mediators are reduced and high levels of Janus-type mediators such as ROS and NO (beneficial at low concentrations and harmful at high concentrations) may be responsible for the damaging effects of high-fluence light and the overall biphasic dose response. PMID:21956634

  13. High titers of ecdysteroids are associated with the secretory process of embryonic envelopes in the european lobster.

    PubMed

    Goudeau, M; Lachaise, F; Carpentier, G; Goxe, B

    1990-01-01

    The newly laid egg of the lobster Homarus gammarus is surrounded by a vitelline coat. Just after fertilization, a new subjacent envelope (2), originating from the cortical reaction, is deposited beneath the vitelline coat. In the course of embryonic development, five new coatings (envelopes 3 to 7) are secreted successively from the ectodermal embryonic cells. These will remain until hatching, freeing the mysis larva in concentric order without exuviation. The concentration of both the two major ecdysteroids (ponasterone A and 20-hydroxyecdysone) and their respective precursors (25-deoxyecdysone and ecdysone) were determined as a function of the secretory phase for three embryonic envelopes (2, 3 and 6). We determined that the secretory processes proceed in the presence of high titers of 20-hydroxyecdysone during the onset of envelope secretion and of ponasterone A in the last phase of secretion.

  14. Cell autonomous defects in cortical development revealed by two-color chimera analysis.

    PubMed

    Kwiatkowski, Adam V; Garner, Craig C; Nelson, W James; Gertler, Frank B

    2009-05-01

    A complex program of cell intrinsic and extrinsic signals guide cortical development. Although genetic studies in mice have uncovered roles for numerous genes and gene families in multiple aspects of corticogenesis, determining their cell autonomous functions is often complicated by pleiotropic defects. Here we describe a novel lentiviral-based method to analyze cell autonomy by generating two-color chimeric mouse embryos. Ena/VASP-deficient mutant and control embryonic stem (ES) cells were labeled with different fluorescent chimeric proteins (EGFP and mCherry) that were modified to bind to the plasma membrane. These labeled ES cells were used to generate two-color chimeric embryos possessing two genetically distinct populations of cortical cells, permitting multiple aspects of neuronal morphogenesis to be analyzed and compared between the two cell populations. We observed little difference between the ability of control and Ena/VASP-deficient cells to contribute to cortical organization during development. In contrast, we observed axon fiber tracts originating from control neurons but not Ena/VASP-deficient neurons, indicating that loss of Ena/VASP causes a cell autonomous defect in cortical axon formation. This technique could be applied to determine other cell autonomous functions in different stages of cortical development.

  15. A Stem Cell Niche for Intermediate Progenitor Cells of the Embryonic Cortex

    PubMed Central

    Kriegstein, Arnold

    2009-01-01

    The excitatory neurons of the mammalian cerebral cortex arise from asymmetric divisions of radial glial cells in the ventricular zone and symmetric division of intermediate progenitor cells (IPCs) in the subventricular zone (SVZ) of the embryonic cortex. Little is known about the microenvironment in which IPCs divide or whether a stem cell niche exists in the SVZ of the embryonic cortex. Recent evidence suggests that vasculature may provide a niche for adult stem cells but its role in development is less clear. We have investigated the vasculature in the embryonic cortex during neurogenesis and find that IPCs are spatially and temporally associated with blood vessels during cortical development. Intermediate progenitors mimic the pattern of capillaries suggesting patterns of angiogenesis and neurogenesis are coordinated during development. More importantly, we find that IPCs divide near blood vessel branch points suggesting that cerebral vasculature establishes a stem cell niche for intermediate progenitors in the SVZ. These data provide novel evidence for the presence of a neurogenic niche for intermediate progenitors in the embryonic SVZ and suggest blood vessels are important for proper patterning of neurogenesis. PMID:19346271

  16. Cortical Interneurons Require Jnk1 to Enter and Navigate the Developing Cerebral Cortex

    PubMed Central

    Myers, Abigail K.; Meechan, Daniel W.; Adney, Danielle R.

    2014-01-01

    Proper assembly of cortical circuitry relies on the correct migration of cortical interneurons from their place of birth in the ganglionic eminences to their place of terminal differentiation in the cerebral cortex. Although molecular mechanisms mediating cortical interneuron migration have been well studied, intracellular signals directing their migration are largely unknown. Here we illustrate a novel and essential role for c-Jun N-terminal kinase (JNK) signaling in guiding the pioneering population of cortical interneurons into the mouse cerebral cortex. Migrating cortical interneurons express Jnk proteins at the entrance to the cortical rudiment and have enriched expression of Jnk1 relative to noninterneuronal cortical cells. Pharmacological blockade of JNK signaling in ex vivo slice cultures resulted in dose-dependent and highly specific disruption of interneuron migration into the nascent cortex. Time-lapse imaging revealed that JNK-inhibited cortical interneurons advanced slowly and assumed aberrant migratory trajectories while traversing the cortical entry zone. In vivo analyses of JNK-deficient embryos supported our ex vivo pharmacological data. Deficits in interneuron migration were observed in Jnk1 but not Jnk2 single nulls, and those migratory deficits were further exacerbated when homozygous loss of Jnk1 was combined with heterozygous reduction of Jnk2. Finally, genetic ablation of Jnk1 and Jnk2 from cortical interneurons significantly perturbed migration in vivo, but not in vitro, suggesting JNK activity functions to direct their guidance rather than enhance their motility. These data suggest JNK signaling, predominantly mediated by interneuron expressed Jnk1, is required for guiding migration of cortical interneurons into and within the developing cerebral cortex. PMID:24899703

  17. Generating Inner Ear Organoids from Mouse Embryonic Stem Cells.

    PubMed

    Longworth-Mills, Emma; Koehler, Karl R; Hashino, Eri

    2016-01-01

    This protocol describes a three-dimensional culture method for generating inner ear sensory epithelia, which comprises sensory hair cells and a concurrently arising neuronal population. Mouse embryonic stem cells are initially plated in 96-well plates with differentiation media; following aggregation, Matrigel is added in order to promote epithelialization. A series of small molecule applications is then used over the first 14 days of culture to guide differentiation towards an otic lineage. After 16-20 days, vesicles containing inner ear sensory hair cells and supporting cells arise from the cultured aggregates. Aggregates may be analyzed using immunohistochemistry and electrophysiology techniques. This system serves as a simple and relatively inexpensive in vitro model of inner ear development.

  18. Pharmacological characterization of the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) as a potent NCX3 inhibitor that worsens anoxic injury in cortical neurons, organotypic hippocampal cultures, and ischemic brain.

    PubMed

    Secondo, Agnese; Pignataro, Giuseppe; Ambrosino, Paolo; Pannaccione, Anna; Molinaro, Pasquale; Boscia, Francesca; Cantile, Maria; Cuomo, Ornella; Esposito, Alba; Sisalli, Maria Josè; Scorziello, Antonella; Guida, Natascia; Anzilotti, Serenella; Fiorino, Ferdinando; Severino, Beatrice; Santagada, Vincenzo; Caliendo, Giuseppe; Di Renzo, Gianfranco; Annunziato, Lucio

    2015-08-19

    The Na(+)/Ca(2+) exchanger (NCX), a 10-transmembrane domain protein mainly involved in the regulation of intracellular Ca(2+) homeostasis, plays a crucial role in cerebral ischemia. In the present paper, we characterized the effect of the newly synthesized compound 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) on the activity of the three NCX isoforms and on the evolution of cerebral ischemia. BED inhibited NCX isoform 3 (NCX3) activity (IC50 = 1.9 nM) recorded with the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration. Furthermore, this drug displayed negligible effect on NCX2, the other isoform expressed within the CNS, and it failed to modulate the ubiquitously expressed NCX1 isoform. Concerning the molecular site of action, the use of chimera strategy and deletion mutagenesis showed that α1 and α2 repeats of NCX3 represented relevant molecular determinants for BED inhibitory action, whereas the intracellular regulatory f-loop was not involved. At 10 nM, BED worsened the damage induced by oxygen/glucose deprivation (OGD) followed by reoxygenation in cortical neurons through a dysregulation of [Ca(2+)]i. Furthermore, at the same concentration, BED significantly enhanced cell death in CA3 subregion of hippocampal organotypic slices exposed to OGD and aggravated infarct injury after transient middle cerebral artery occlusion in mice. These results showed that the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride is one of the most potent inhibitor of NCX3 so far identified, representing an useful tool to dissect the role played by NCX3 in the control of Ca(2+) homeostasis under physiological and pathological conditions.

  19. Plasticity of recurring spatiotemporal activity patterns in cortical networks

    NASA Astrophysics Data System (ADS)

    Madhavan, Radhika; Chao, Zenas C.; Potter, Steve M.

    2007-09-01

    How do neurons encode and store information for long periods of time? Recurring patterns of activity have been reported in various cortical structures and were suggested to play a role in information processing and memory. To study the potential role of bursts of action potentials in memory mechanisms, we investigated patterns of spontaneous multi-single-unit activity in dissociated rat cortical cultures in vitro. Spontaneous spikes were recorded from networks of approximately 50 000 neurons and glia cultured on a grid of 60 extracellular substrate- embedded electrodes (multi-electrode arrays). These networks expressed spontaneous culture- wide bursting from approximately one week in vitro. During bursts, a large portion of the active electrodes showed elevated levels of firing. Spatiotemporal activity patterns within spontaneous bursts were clustered using a correlation-based clustering algorithm, and the occurrences of these burst clusters were tracked over several hours. This analysis revealed spatiotemporally diverse bursts occurring in well-defined patterns, which remained stable for several hours. Activity evoked by strong local tetanic stimulation resulted in significant changes in the occurrences of spontaneous bursts belonging to different clusters, indicating that the dynamical flow of information in the neuronal network had been altered. The diversity of spatiotemporal structure and long-term stability of spontaneous bursts together with their plastic nature strongly suggests that such network patterns could be used as codes for information transfer and the expression of memories stored in cortical networks.

  20. Endocannabinoid modulation of cortical up-states and NREM sleep.

    PubMed

    Pava, Matthew J; den Hartog, Carolina R; Blanco-Centurion, Carlos; Shiromani, Priyattam J; Woodward, John J

    2014-01-01

    Up-/down-state transitions are a form of network activity observed when sensory input into the cortex is diminished such as during non-REM sleep. Up-states emerge from coordinated signaling between glutamatergic and GABAergic synapses and are modulated by systems that affect the balance between inhibition and excitation. We hypothesized that the endocannabinoid (EC) system, a neuromodulatory system intrinsic to the cortical microcircuitry, is an important regulator of up-states and sleep. To test this hypothesis, up-states were recorded from layer V/VI pyramidal neurons in organotypic cultures of wild-type or CB1R knockout (KO) mouse prefrontal cortex. Activation of the cannabinoid 1 receptor (CB1) with exogenous agonists or by blocking metabolism of endocannabinoids, anandamide or 2-arachidonoyl glycerol, increased up-state amplitude and facilitated action potential discharge during up-states. The CB1 agonist also produced a layer II/III-selective reduction in synaptic GABAergic signaling that may underlie its effects on up-state amplitude and spiking. Application of CB1 antagonists revealed that an endogenous EC tone regulates up-state duration. Paradoxically, the duration of up-states in CB1 KO cultures was increased suggesting that chronic absence of EC signaling alters cortical activity. Consistent with increased cortical excitability, CB1 KO mice exhibited increased wakefulness as a result of reduced NREM sleep and NREM bout duration. Under baseline conditions, NREM delta (0.5-4 Hz) power was not different in CB1 KO mice, but during recovery from forced sleep deprivation, KO mice had reduced NREM delta power and increased sleep fragmentation. Overall, these findings demonstrate that the EC system actively regulates cortical up-states and important features of NREM sleep such as its duration and low frequency cortical oscillations.

  1. Membrane-Associated Molecules Regulate the Formation of Layer-Specific Cortical Circuits

    NASA Astrophysics Data System (ADS)

    Castellani, Valerie; Bolz, Jurgen

    1997-06-01

    The columnar organization of the mammalian neocortex is based on radially oriented axon collaterals which precisely link cells from distinct cortical layers. During development, these interlaminar connections are specific from their initial outgrowth: collaterals form only in the target layers and there are no transient axonal collaterals in the nontarget layers. To examine whether positional cues within individual cortical layers regulate the laminar specificity of collateral formation, explants of cells destined for different cortical layers were cultured on membranes prepared from target and nontarget layers. Axonal growth and branching were examined on homogeneous membrane substrates and on alternating stripes of membranes from different layers. Results show that axons branch preferentially on membrane substrates from those layers that they would target in vivo. In addition, when cortical axons were given a choice to grow on membranes from either their target or their nontarget layer, they exhibited a clear preference for the target layers. This indicates that membrane-associated cues confined to individual layers regulate the formation of collaterals of cortical axons and restrict their growth to their target layers. Heat inactivation of membranes from target layers resulted in reduced axonal branching. The same manipulation of membranes from nontarget layers increased axonal branching for one population of cortical neurons. Taken together, these results suggest that membrane-associated molecules confined to individual layers induce and prevent the formation of axon collaterals in distinct populations of cortical neurons. Thus, the expression of layer-specific cues provides important constraints for the remodeling of local circuits during cortical development.

  2. Isolating specific embryonic cells of the sea urchin by FACS.

    PubMed

    Juliano, Celina; Swartz, S Zachary; Wessel, Gary

    2014-01-01

    Isolating cells based on specific gene expression enables a focused biochemical and molecular analysis. While cultured cells and hematopoietic cells, for example, are routinely isolated by fluorescence activated cell sorting (FACS), early embryonic cells are a relatively untapped source for FACS applications often because the embryos of many animals are quite limiting. Furthermore, many applications require genetic model organisms in which cells can be labeled by fluorescent transgenes, or antibodies against cell surface antigens. Here we define conditions in the sea urchin embryo for isolation of embryonic cells based on expression of specific proteins. We use the sea urchin embryo for which a nearly unlimited supply of embryonic cells is available and demonstrate the conditions for separation of the embryo into single cells, fixation of the cells for antibody penetration into the cells, and conditions for FACS of a rare cell type in the embryo. This protocol may be adapted for analysis of mRNA, chromatin, protein, or carbohydrates and depends only on the probe availability for the cell of interest. We anticipate that this protocol will be broadly applicable to embryos of other species.

  3. Cell Labeling and Injection in Developing Embryonic Mouse Hearts

    PubMed Central

    Dirschinger, Ralf J.; Evans, Sylvia M.; Puceat, Michel

    2014-01-01

    Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development.  Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development. PMID:24797676

  4. [Infantile cortical hyperostosis: Case report].

    PubMed

    Rodríguez, Mónica; Martínez, Luz Elena; Cortés, José; de Uña, Armando; Vega, Valentina; Acosta, Mario

    Infantile Cortical Hyperostosis, or Caffey-Silverman disease, is a rare condition characterised by generalised bone proliferation mediated by an acute inflammatory process. Diagnosis can be made through clinical evaluation and X-ray studies. The course is generally self-limiting and prognosis is excellent.

  5. Biomechanics of Single Cortical Neurons

    PubMed Central

    Bernick, Kristin B.; Prevost, Thibault P.; Suresh, Subra; Socrate, Simona

    2011-01-01

    This study presents experimental results and computational analysis of the large strain dynamic behavior of single neurons in vitro with the objective of formulating a novel quantitative framework for the biomechanics of cortical neurons. Relying on the atomic force microscopy (AFM) technique, novel testing protocols are developed to enable the characterization of neural soma deformability over a range of indentation rates spanning three orders of magnitude – 10, 1, and 0.1 μm/s. Modified spherical AFM probes were utilized to compress the cell bodies of neonatal rat cortical neurons in load, unload, reload and relaxation conditions. The cell response showed marked hysteretic features, strong non-linearities, and substantial time/rate dependencies. The rheological data were complemented with geometrical measurements of cell body morphology, i.e. cross-diameter and height estimates. A constitutive model, validated by the present experiments, is proposed to quantify the mechanical behavior of cortical neurons. The model aimed to correlate empirical findings with measurable degrees of (hyper-) elastic resilience and viscosity at the cell level. The proposed formulation, predicated upon previous constitutive model developments undertaken at the cortical tissue level, was implemented into a three-dimensional finite element framework. The simulated cell response was calibrated to the experimental measurements under the selected test conditions, providing a novel single cell model that could form the basis for further refinements. PMID:20971217

  6. Human embryonic stem cell derivation and directed differentiation.

    PubMed

    Trounson, A

    2005-01-01

    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

  7. YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

    SciTech Connect

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung

    2015-02-27

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.

  8. [Differentiation of human amniotic fluid stem cells into cardiomyocytes through embryonic body formation].

    PubMed

    Wang, Han; Chen, Shuai; Cheng, Xiang; Dou, Zhongying; Wang, Huayan

    2008-09-01

    To isolate human amniotic fluid stem cells (hASCs) and induce hASCs into cardiomyocytes after forming the embryonic bodies. We cultivated hASCs isolated from the amniotic fluid continually for over 42 passages. The biological characteristics of hASCs were detected by immunocytochemistry, RT-PCR and flow cytometer, hASCs at 10-15th passage were suspension cultured to form embryonic bodies that were induced to cardiomyocytes. Fibroblastoid-type hASCs were obtained. Immunocytochemistry, RT-PCR and flow cytometry analysis demonstrated that hASCs were positive for some specific makers of the embryonic stem cell. hASCs could form embryonic bodies that were alkaline-phosphatase positive and expressed fgf5, zeta-globin and alpha-fetoprotein. The embryonic bodies could differentiate into cardiomyocytes showing alpha-actin positive and Tbx5, Nkx2.5, GATA4 and alpha-MHC positive. We conclued that hASCs obtained from human amniotic fluid could differentiate into cardiomyocytes through the formation of embryonic bodies.

  9. Cortical neurons gradually attain a post-mitotic state.

    PubMed

    Anda, Froylan Calderon de; Madabhushi, Ram; Rei, Damien; Meng, Jia; Gräff, Johannes; Durak, Omer; Meletis, Konstantinos; Richter, Melanie; Schwanke, Birgit; Mungenast, Alison; Tsai, Li-Huei

    2016-09-01

    Once generated, neurons are thought to permanently exit the cell cycle and become irreversibly differentiated. However, neither the precise point at which this post-mitotic state is attained nor the extent of its irreversibility is clearly defined. Here we report that newly born neurons from the upper layers of the mouse cortex, despite initiating axon and dendrite elongation, continue to drive gene expression from the neural progenitor tubulin α1 promoter (Tα1p). These observations suggest an ambiguous post-mitotic neuronal state. Whole transcriptome analysis of sorted upper cortical neurons further revealed that neurons continue to express genes related to cell cycle progression long after mitotic exit until at least post-natal day 3 (P3). These genes are however down-regulated thereafter, associated with a concomitant up-regulation of tumor suppressors at P5. Interestingly, newly born neurons located in the cortical plate (CP) at embryonic day 18-19 (E18-E19) and P3 challenged with calcium influx are found in S/G2/M phases of the cell cycle, and still able to undergo division at E18-E19 but not at P3. At P5 however, calcium influx becomes neurotoxic and leads instead to neuronal loss. Our data delineate an unexpected flexibility of cell cycle control in early born neurons, and describe how neurons transit to a post-mitotic state.

  10. A decrease of intracellular ATP is compensated by increased respiration and acidification at sub-lethal parathion concentrations in murine embryonic neuronal cells: measurements in metabolic cell-culture chips.

    PubMed

    Buehler, S M; Stubbe, M; Gimsa, U; Baumann, W; Gimsa, J

    2011-11-30

    We present a label-free in vitro method for testing the toxic potentials of chemical substances using primary neuronal cells. The cells were prepared from 16-day-old NMRI mouse embryos and cultured on silicon chips (www.bionas.de) under the influence of different parathion concentrations with sensors for respiration (Clark-type oxygen electrodes), acidification (pH-ISFETs) and cell adhesion (interdigitated electrode structures, IDES). After 12 days in vitro, the sensor readouts were simultaneously recorded for 350 min in the presence of parathion applying a serial 1:3 dilution. The parathion-dependent data was fitted by logistic functions. IC(50) values of approximately 105 μM, 65 μM, and 54 μM were found for respiration, acidification, and adhesion, respectively. An IC(50) value of approximately 36 μM was determined from the intracellular ATP-levels of cells, which were detected by an ATP-luminescence assay using micro-well plates. While the intracellular ATP level and cell adhesion showed no deviation from a simple logistic decay, increases of approximately 29% in the respiration and 15% in the acidification rates above the control values were found at low parathion concentrations, indicating hormesis. These increases could be fitted by a modified logistic function. We believe that the label-free, continuous, multi-parametric monitoring of cell-metabolic processes may have applications in systems-biology and biomedical research, as well as in environmental monitoring. The parallel characterization of IC(50) values and hormetic effects may provide new insights into the metabolic mechanisms of toxic challenges to the cell.

  11. Cardiac specific ATP-sensitive K+ channel (KATP) overexpression results in embryonic lethality.

    PubMed

    Toib, Amir; Zhang, Hai Xia; Broekelmann, Thomas J; Hyrc, Krzysztof L; Guo, Qiusha; Chen, Feng; Remedi, Maria S; Nichols, Colin G

    2012-09-01

    Transgenic mice overexpressing SUR1 and gain of function Kir6.2[∆N30, K185Q] K(ATP) channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K(ATP) overexpression, wild type (WT) and K(ATP) overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K(ATP) overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K(ATP) currents were detectable in both WT and K(ATP)-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K(ATP)-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K(ATP) current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K(ATP) channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K(ATP) channel may have an important physiological role during early cardiac development.

  12. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

    EPA Science Inventory

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultur...

  13. Embryonic and postnatal development of the layer I-directed ("matrix") thalamocortical system in the rat.

    PubMed

    Galazo, Maria J; Martinez-Cerdeño, Verónica; Porrero, César; Clascá, Francisco

    2008-02-01

    Inputs to the layer I apical dendritic tufts of pyramidal cells are crucial in "top-down" interactions in the cerebral cortex. A large population of thalamocortical cells, the "matrix" (M-type) cells, provides a direct robust input to layer I that is anatomically and functionally different from the thalamocortical input to layer VI. The developmental timecourse of M-type axons is examined here in rats aged E (embryonic day) 16 to P (postnatal day) 30. Anterograde techniques were used to label axons arising from 2 thalamic nuclei mainly made up of M-type cells, the Posterior and the Ventromedial. The primary growth cones of M-type axons rapidly reached the subplate of dorsally situated cortical areas. After this, interstitial branches would sprout from these axons under more lateral cortical regions to invade the overlying cortical plate forming secondary arbors. Moreover, retrograde labeling of M-type cell somata in the thalamus after tracer deposits confined to layer I revealed that large numbers of axons from multiple thalamic nuclei had already converged in a given spot of layer I by P3. Because of early ingrowth in such large numbers, interactions of M-type axons may significantly influence the early development of cortical circuits.

  14. Embryonic mosaic deletion of APP results in displaced Reelin-expressing cells in the cerebral cortex.

    PubMed

    Callahan, D G; Taylor, W M; Tilearcio, M; Cavanaugh, T; Selkoe, D J; Young-Pearse, T L

    2017-03-08

    It is widely accepted that amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease. In addition, APP has been proposed to have functions in numerous biological processes including neuronal proliferation, differentiation, migration, axon guidance, and neurite outgrowth, as well as in synapse formation and function. However, germline knockout of APP yields relatively subtle phenotypes, and brain development appears grossly normal. This is thought to be due in part to functional compensation by APP family members and other type I transmembrane proteins. Here, we have generated a conditional mouse knockout for APP that is controlled temporally using Cre(ER) and tamoxifen administration. We show that total cortical expression of APP is reduced following tamoxifen administration during embryonic time points critical for cortical lamination, and that this results in displacement of Reelin-positive cells below the cortical plate with a concurrent elevation in Reelin protein levels. These results support a role for APP in cortical lamination and demonstrate the utility of a conditional knockout approach in which APP can be deleted with temporal control in vivo. This new tool should be useful for many different applications in the study of APP function across the mammalian life span.

  15. Calmodulin immunolocalization to cortical microtubules is calcium independent

    SciTech Connect

    Fisher, D.D.; Cyr, R.J.

    1992-12-31

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  16. Calmodulin immunolocalization to cortical microtubules is calcium independent

    SciTech Connect

    Fisher, D.D.; Cyr, R.J.

    1992-01-01

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  17. Infrared inhibition of embryonic hearts

    NASA Astrophysics Data System (ADS)

    Wang, Yves T.; Rollins, Andrew M.; Jenkins, Michael W.

    2016-06-01

    Infrared control is a new technique that uses pulsed infrared lasers to thermally alter electrical activity. Originally developed for nerves, we have applied this technology to embryonic hearts using a quail model, previously demonstrating infrared stimulation and, here, infrared inhibition. Infrared inhibition enables repeatable and reversible block, stopping cardiac contractions for several seconds. Normal beating resumes after the laser is turned off. The block can be spatially specific, affecting propagation on the ventricle or initiation on the atrium. Optical mapping showed that the block affects action potentials and not just calcium or contraction. Increased resting intracellular calcium was observed after a 30-s exposure to the inhibition laser, which likely resulted in reduced mechanical function. Further optimization of the laser illumination should reduce potential damage. Stopping cardiac contractions by disrupting electrical activity with infrared inhibition has the potential to be a powerful tool for studying the developing heart.

  18. Suppression of BDNF-induced expression of neuropeptide Y (NPY) in cortical cultures by oxygen-glucose deprivation: a model system to study ischemic mechanisms in the perinatal brain.

    PubMed

    Barnea, Ayalla; Roberts, Jodie

    2002-04-15

    The aim of this study was to establish a culture system that can serve as a model to study hypoxic-ischemic mechanisms regulating the functional expression of NPY neurons in the perinatal brain. Using an aggregate culture system derived from the rat fetal cortex, we defined the effects of oxygen and glucose deprivation on NPY expression, using BDNF-induced production of NPY as a functional criterion. NPY neurons exhibited a differential susceptibility to oxygen and glucose deprivation. Although the neurons could withstand oxygen deprivation for 16 hr, they were dramatically damaged by 8 hr of glucose deprivation and by 1-4 hr of deprivation of both oxygen and glucose (N+Glu-). One-hour exposure to N+Glu- led to a transient inhibition ( approximately 50%) of NPY production manifesting within 24 hr and recovering by 5 days thereafter, a 2-hr exposure to N+Glu- led to a sustained inhibition (50-75%) manifesting 1-5 days thereafter, and a 4-hr exposure to N+Glu- led to a total irreversible suppression of BDNF-induced production of NPY manifesting within 24 hr and lasting 8 days after re-supply of oxygen and glucose. Moreover, 1-hr exposure to N+Glu- led to a substantial and 4-hr exposure led to a total disappearance of immunostaining for MAP-2 and NPY but not for GFAP; indicating that neurons are the primary cell-type damaged by oxygen-glucose deprivation. Analysis of cell viability (LDH, MTT) indicated that progressive changes in cell integrity take place during the 4-hr exposure to N+Glu- followed by massive cell death 24 hr thereafter. Thus, we defined a culture system that can serve as a model to study mechanisms by which ischemic insult leads to suppression and eventually death of NPY neurons. Importantly, changes in NPY neurons can be integrated into the overall scheme of ischemic injury in the perinatal brain.

  19. Prox1 Regulates the Subtype-Specific Development of Caudal Ganglionic Eminence-Derived GABAergic Cortical Interneurons

    PubMed Central

    Young, Allison; Petros, Timothy; Karayannis, Theofanis; McKenzie Chang, Melissa; Lavado, Alfonso; Iwano, Tomohiko; Nakajima, Miho; Taniguchi, Hiroki; Huang, Z. Josh; Heintz, Nathaniel; Oliver, Guillermo; Matsuzaki, Fumio; Machold, Robert P.

    2015-01-01

    Neurogliaform (RELN+) and bipolar (VIP+) GABAergic interneurons of the mammalian cerebral cortex provide critical inhibition locally within the superficial layers. While these subtypes are known to originate from the embryonic caudal ganglionic eminence (CGE), the specific genetic programs that direct their positioning, maturation, and integration into the cortical network have not been elucidated. Here, we report that in mice expression of the transcription factor Prox1 is selectively maintained in postmitotic CGE-derived cortical interneuron precursors and that loss of Prox1 impairs the integration of these cells into superficial layers. Moreover, Prox1 differentially regulates the postnatal maturation of each specific subtype originating from the CGE (RELN, Calb2/VIP, and VIP). Interestingly, Prox1 promotes the maturation of CGE-derived interneuron subtypes through intrinsic differentiation programs that operate in tandem with extrinsically driven neuronal activity-dependent pathways. Thus Prox1 represents the first identified transcription factor specifically required for the embryonic and postnatal acquisition of CGE-derived cortical interneuron properties. SIGNIFICANCE STATEMENT Despite the recognition that 30% of GABAergic cortical interneurons originate from the caudal ganglionic eminence (CGE), to date, a specific transcriptional program that selectively regulates the development of these populations has not yet been identified. Moreover, while CGE-derived interneurons display unique patterns of tangential and radial migration and preferentially populate the superficial layers of the cortex, identification of a molecular program that controls these events is lacking. Here, we demonstrate that the homeodomain transcription factor Prox1 is expressed in postmitotic CGE-derived cortical interneuron precursors and is maintained into adulthood. We found that Prox1 function is differentially required during both embryonic and postnatal stages of development to

  20. Characterization of Early Cortical Neural Network ...

    EPA Pesticide Factsheets

    We examined the development of neural network activity using microelectrode array (MEA) recordings made in multi-well MEA plates (mwMEAs) over the first 12 days in vitro (DIV). In primary cortical cultures made from postnatal rats, action potential spiking activity was essentially absent on DIV 2 and developed rapidly between DIV 5 and 12. Spiking activity was primarily sporadic and unorganized at early DIV, and became progressively more organized with time in culture, with bursting parameters, synchrony and network bursting increasing between DIV 5 and 12. We selected 12 features to describe network activity and principal components analysis using these features demonstrated a general segregation of data by age at both the well and plate levels. Using a combination of random forest classifiers and Support Vector Machines, we demonstrated that 4 features (CV of within burst ISI, CV of IBI, network spike rate and burst rate) were sufficient to predict the age (either DIV 5, 7, 9 or 12) of each well recording with >65% accuracy. When restricting the classification problem to a binary decision, we found that classification improved dramatically, e.g. 95% accuracy for discriminating DIV 5 vs DIV 12 wells. Further, we present a novel resampling approach to determine the number of wells that might be needed for conducting comparisons of different treatments using mwMEA plates. Overall, these results demonstrate that network development on mwMEA plates is similar to

  1. Complement inhibition and statins prevent fetal brain cortical abnormalities in a mouse model of preterm birth.

    PubMed

    Pedroni, Silvia M A; Gonzalez, Juan M; Wade, Jean; Jansen, Maurits A; Serio, Andrea; Marshall, Ian; Lennen, Ross J; Girardi, Guillermina

    2014-01-01

    Premature babies are particularly vulnerable to brain injury. In this study we focus on cortical brain damage associated with long-term cognitive, behavioral, attentional or socialization deficits in children born preterm. Using a mouse model of preterm birth (PTB), we demonstrated that complement component C5a contributes to fetal cortical brain injury. Disruption of cortical dendritic and axonal cytoarchitecture was observed in PTB-mice. Fetuses deficient in C5aR (-/-) did not show cortical brain damage. Treatment with antibody anti-C5, that prevents generation of C5a, also prevented cortical fetal brain injury in PTB-mice. C5a also showed a detrimental effect on fetal cortical neuron development and survival in vitro. Increased glutamate release was observed in cortical neurons in culture exposed to C5a. Blockade of C5aR prevented glutamate increase and restored neurons dendritic and axonal growth and survival. Similarly, increased glutamate levels - measured by (1)HMRS - were observed in vivo in PTB-fetuses compared to age-matched controls. The blockade of glutamate receptors prevented C5a-induced abnormal growth and increased cell death in isolated fetal cortical neurons. Simvastatin and pravastatin prevented cortical fetal brain developmental and metabolic abnormalities -in vivo and in vitro. Neuroprotective effects of statins were mediated by Akt/PKB signaling pathways. This study shows that complement activation plays a crucial role in cortical fetal brain injury in PTL and suggests that complement inhibitors and statins might be good therapeutic options to improve neonatal outcomes in preterm birth.

  2. Neuropeptide Y protects cerebral cortical neurons by regulating microglial immune function

    PubMed Central

    Li, Qijun; Dong, Changzheng; Li, Wenling; Bu, Wei; Wu, Jiang; Zhao, Wenqing

    2014-01-01

    Neuropeptide Y has been shown to inhibit the immunological activity of reactive microglia in the rat cerebral cortex, to reduce N-methyl-D-aspartate current (INMDA) in cortical neurons, and protect neurons. In this study, after primary cultured microglia from the cerebral cortex of rats were treated with lipopolysaccharide, interleukin-1β and tumor necrosis factor-α levels in the cell culture medium increased, and mRNA expression of these cytokines also increased. After primary cultured cortical neurons were incubated with the lipopolysaccharide-treated microglial conditioned medium, peak INMDA in neurons increased. These effects of lipopolysaccharide were suppressed by neuropeptide Y. After addition of the neuropeptide Y Y1 receptor antagonist BIBP3226, the effects of neuropeptide Y completely disappeared. These results suggest that neuropeptide Y prevents excessive production of interleukin-1β and tumor necrosis factor-α by inhibiting microglial reactivity. This reduces INMDA in rat cortical neurons, preventing excitotoxicity, thereby protecting neurons. PMID:25206918

  3. Functional calcium imaging in developing cortical networks.

    PubMed

    Dawitz, Julia; Kroon, Tim; Hjorth, J J Johannes; Meredith, Rhiannon M

    2011-10-22

    A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network. A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz & Shatz, 1996; Khaziphov & Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation

  4. Female parthenogenetic apomixis and androsporogenesis in Douglas-fir embryonal initials in an artificial sporangium.

    PubMed

    Durzan, Don J

    2011-12-01

    Control of female parthenogenetic apomixis and androsporogenesis of Douglas-fir embryonal initials was studied using an experimental culture system in which changes in growth condition can mediate changes in cell identity and outcomes. This culture system constitutes an artificial sporangium in which myriad culture conditions can be simulated and should be applicable for research on a variety of gymnosperms. In this study, embryonal initials from developing seeds from two Douglas-fir trees were rescued and became reprogrammed for female parthenogenetic apomixis (fPA) and parthenogenetic androsporogenesis (mPA). Female PA was initiated by endomitosis forming a binucleate cell with a diploid egg-equivalent and an apoptotic ventral canal nucleus in an archegonial tube. Egg-equivalent nuclei formed cells (parthenotes) that were discharged into an aqueous culture medium. Parthenotes developed axial tiers atypical of early embryogenesis in seeds. Earlier in the year, androsporangial tubes were parthenogenetically differentiated and released monads, dyads, triads, and tetrads into the culture medium. Spores showed chromosomal aberrations. PA demonstrated a temporal separation in gender expression (dichogamy). Embryonal initials brought forward and by-passed the long juvenile phases normally needed for cells to develop into trees and express reproductive maturity. Expressions of fPA and mPA indicated that the specialized culture flasks served as an artificial sporangium (AS). Awareness is raised for the value of an AS for research in gymnosperm life cycles and as a teaching and research laboratory.

  5. FMRP regulates multipolar to bipolar transition affecting neuronal migration and cortical circuitry.

    PubMed

    La Fata, Giorgio; Gärtner, Annette; Domínguez-Iturza, Nuria; Dresselaers, Tom; Dawitz, Julia; Poorthuis, Rogier B; Averna, Michele; Himmelreich, Uwe; Meredith, Rhiannon M; Achsel, Tilmann; Dotti, Carlos G; Bagni, Claudia

    2014-12-01

    Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.

  6. Reelin expression during embryonic brain development in Crocodylus niloticus.

    PubMed

    Tissir, F; Lambert De Rouvroit, C; Sire, J-Y; Meyer, G; Goffinet, A M

    2003-03-10

    The expression of reelin mRNA and protein was studied during embryonic brain development in the Nile crocodile Crocodylus niloticus, using in situ hybridization and immunohistochemistry. In the forebrain, reelin was highly expressed in the olfactory bulb, septal nuclei, and subpial neurons in the marginal zone of the cerebral cortex, dorsal ventricular ridge, and basal forebrain. At early stages, reelin mRNA was also detected in subventricular zones. In the diencephalon, the ventral lateral geniculate nuclei and reticular nuclei were strongly positive, with moderate expression in the habenula and focal expression in the hypothalamus. High expression levels were noted in the retina, the tectum, and the external granule cell layer of the cerebellum. In the brainstem, there was a high level of signal in cochleovestibular, sensory trigeminal, and some reticular nuclei. No expression was observed in the cortical plate or Purkinje cells. Comparison with reelin expression during brain development in mammals, birds, turtles, and lizards reveals evolutionarily conserved, homologous features that presumably define the expression profile in stem amniotes. The crocodilian cortex contains subpial reelin-positive cells that are also p73 positive, suggesting that they are homologous to mammalian Cajal-Retzius cells, although they express the reelin gene less intensely. Furthermore, the crocodilian cortex does not contain the subcortical reelin-positive cells that are typical of lizards but expresses reelin in subventricular zones at early stages. These observations confirm that reelin is prominently expressed in many structures of the embryonic brain in all amniotes and further emphasize the unique amplification of reelin expression in mammalian Cajal-Retzius cells and its putative role in the evolution of the cerebral cortex.

  7. Extrathalamic Modulation of Cortical Function

    DTIC Science & Technology

    1990-07-27

    and c7rtico-cortical systems. For example, we have shown that primate LC-NA neurons are more acti during waking than sleep and exhibit bursts of...infusion needle. Infusion of the alpha-adrenergic agonist clonidine (CLON), in concentrations ranging from 5-20 uM (67-270pg/50 nl injection...ind hippocampal EEG (HEEG) typically exhibit activity similar to that of a lightly sleeping animal. However, periods of "waking" EEG are sometimes

  8. Role of microglia in embryonic neurogenesis

    PubMed Central

    Tong, Chih Kong

    2016-01-01

    Microglia begin colonizing the developing brain as early as embryonic day 9, prior to the emergence of neurons and other glia. Their ontogeny is also distinct from other central nervous system cells, as they derive from yolk sac hematopoietic progenitors and not neural progenitors. In this review, we feature these unique characteristics of microglia and assess the spatiotemporal similarities between microglia colonization of the central nervous system and embryonic neurogenesis. We also infer to existing evidence for microglia function from embryonic through to postnatal neurodevelopment to postulate roles for microglia in neurogenesis. PMID:27555616

  9. [Parietal Cortices and Body Information].

    PubMed

    Naito, Eiichi; Amemiya, Kaoru; Morita, Tomoyo

    2016-11-01

    Proprioceptive signals originating from skeletal muscles and joints contribute to the formation of both the human body schema and the body image. In this chapter, we introduce various types of bodily illusions that are elicited by proprioceptive inputs, and we discuss distinct functions implemented by different parietal cortices. First, we illustrate the primary importance of the motor network in the processing of proprioceptive (kinesthetic) signals originating from muscle spindles. Next, we argue that the right inferior parietal cortex, in concert with the inferior frontal cortex (both regions connected by the inferior branch of the superior longitudinal fasciculus-SLF III), may be involved in the conscious experience of body image. Further, we hypothesize other functions of distinct parietal regions: the association between internal hand motor representation with external object representation in the left inferior parietal cortex, visuo-kinesthetic processing in the bilateral posterior parietal cortices, and the integration of somatic signals from different body parts in the higher-order somatosensory parietal cortices. Our results indicate that a distinct parietal region, in concert with its anatomically and functionally connected frontal regions, probably plays specialized roles in the processing of body-related information.

  10. IgLON Cell Adhesion Molecules Are Shed from the Cell Surface of Cortical Neurons to Promote Neuronal Growth*

    PubMed Central

    Sanz, Ricardo; Ferraro, Gino B.; Fournier, Alyson E.

    2015-01-01

    Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature cortical neurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature cortical neurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature cortical neurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from cortical neurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature cortical neurons. PMID:25538237

  11. Transcriptomic and anatomic parcellation of 5-HT3AR expressing cortical interneuron subtypes revealed by single-cell RNA sequencing

    PubMed Central

    Frazer, Sarah; Prados, Julien; Niquille, Mathieu; Cadilhac, Christelle; Markopoulos, Foivos; Gomez, Lucia; Tomasello, Ugo; Telley, Ludovic; Holtmaat, Anthony; Jabaudon, Denis; Dayer, Alexandre

    2017-01-01

    Cortical GABAergic interneurons constitute a highly diverse population of inhibitory neurons that are key regulators of cortical microcircuit function. An important and heterogeneous group of cortical interneurons specifically expresses the serotonin receptor 3A (5-HT3AR) but how this diversity emerges during development is poorly understood. Here we use single-cell transcriptomics to identify gene expression patterns operating in Htr3a-GFP+ interneurons during early steps of cortical circuit assembly. We identify three main molecular types of Htr3a-GFP+ interneurons, each displaying distinct developmental dynamics of gene expression. The transcription factor Meis2 is specifically enriched in a type of Htr3a-GFP+ interneurons largely confined to the cortical white matter. These MEIS2-expressing interneurons appear to originate from a restricted region located at the embryonic pallial–subpallial boundary. Overall, this study identifies MEIS2 as a subclass-specific marker for 5-HT3AR-containing interstitial interneurons and demonstrates that the transcriptional and anatomical parcellation of cortical interneurons is developmentally coupled. PMID:28134272

  12. IgLON cell adhesion molecules are shed from the cell surface of cortical neurons to promote neuronal growth.

    PubMed

    Sanz, Ricardo; Ferraro, Gino B; Fournier, Alyson E

    2015-02-13

    Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature cortical neurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature cortical neurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature cortical neurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from cortical neurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature cortical neurons.

  13. Vitamin B-complex initiates growth and development of human embryonic brain cells in vitro.

    PubMed

    Danielyan, K E; Abramyan, R A; Galoyan, A A; Kevorkian, G A

    2011-09-01

    We studied a combined effect of subcomponents of vitamin B complex on the growth, development, and death of human embryonic brain-derived cells (E90) cultured using a modified method of Matson. Cell death was detected by trypan blue staining. According to our results, vitamin B-complex in low-doses promote the development, maturation, and enlargement of human embryonic brain cells, on the one hand, and increases the percent of cell death, which attests to accelerated maturation and metabolism, on the other.

  14. Intermediate Progenitor Cohorts Differentially Generate Cortical Layers and Require Tbr2 for Timely Acquisition of Neuronal Subtype Identity.

    PubMed

    Mihalas, Anca B; Elsen, Gina E; Bedogni, Francesco; Daza, Ray A M; Ramos-Laguna, Kevyn A; Arnold, Sebastian J; Hevner, Robert F

    2016-06-28

    Intermediate progenitors (IPs) amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

  15. Generation of stomach tissue from mouse embryonic stem cells.

    PubMed

    Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

    2015-08-01

    Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

  16. In vitro cultivation and cryopreservation of duck embryonic hepatocytes.

    PubMed

    Schacke, M; Glück, B; Wutzler, P; Sauerbrei, A

    2009-04-01

    Hepatitis B-virucidal testing of biocides in quantitative suspension tests using duck hepatitis B virus (DHBV) requires primary duck embryonic hepatocytes for viral propagation. To improve the test system and availability of these cells, commercial culture plates with different growth surfaces were tested for cell cultivation and different approaches for cryopreservation of hepatocyte suspension were examined. After 12 days of culture, the largest amounts of hepatocytes were grown in CellBIND and TTP plates and CellBIND surface showed the lowest tendency of monolayer detachment nearly comparable with collagen 1-coated CELLCOAT plates. For cryopreservation of hepatocyte suspension, the use of growth medium supplemented with fetal calf serum (FCS) and dimethyl sulfoxide (ME(2)SO), FCS supplemented with ME(2)SO or cryosafe-1 as cryoprotective agents provided the highest rates of surviving cells after thawing. The freezing-thawing process did not significantly reduce the susceptibility of hepatocytes to infection with DHBV. In conclusion, plates without collagen 1 such as CellBIND are recommended for cultivation of primary duck embryonic hepatocytes in infectivity experiments of DHBV for virucidal testing of biocides. The use of cryopreserved hepatocytes is possible when freshly isolated cells from the liver of duck embryos are not available.

  17. Assembly of embryonic and extra-embryonic stem cells to mimic embryogenesis in vitro.

    PubMed

    Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena

    2017-03-02

    Mammalian embryogenesis requires intricate interactions between embryonic and extra-embryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combine mouse embryonic stem cells (ESCs) and extra-embryonic trophoblast stem cells (TSCs) in a 3D-scaffold to generate structures whose morphogenesis is remarkably similar to natural embryos. By using genetically-modified stem cells and specific inhibitors, we show embryogenesis of ESC- and TSC-derived embryos, ETS-embryos, depends on crosstalk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extra-embryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell-types resemble natural embryos.

  18. Derivation and spontaneous differentiation of human embryonic stem cells*

    PubMed Central

    Amit, Michal; Itskovitz-Eldor, Joseph

    2002-01-01

    Abstract Embryonic stem (ES) cells are unique cells derived from the inner cell mass of the mammalian blastocyst. These cells are immortal and pluripotent, retain their developmental potential after prolonged culture, and can be continuously cultured in an undifferentiated state. Many in vitro differentiation systems have been developed for mouse ES cells, including reproducible methods for mouse ES cell differentiation into haematopoietic and neural precursors, cardiomyocytes, insulin-secreting cells, endothelial cells and various other cell types. The derivation of new human ES cell lines provides the opportunity to develop unique models for developmental research and for cell therapies. In this review we consider the derivation and spontaneous differentiation of human ES cells. PMID:12033726

  19. Glioactive ATP controls BDNF recycling in cortical astrocytes

    PubMed Central

    Vignoli, Beatrice; Canossa, Marco

    2017-01-01

    ABSTRACT We have recently reported that long-term memory retention requires synaptic glia for proBDNF uptake and recycling. Through the recycling course, glial cells release endocytic BDNF, a mechanism that is activated in response to glutamate via AMPA and mGluRI/II receptors. Cortical astrocytes express receptors for many different transmitters suggesting for a complex signaling controlling endocytic BDNF secretion. Here, we demonstrated that the extracellular nucleotide ATP, activating P2X and P2Y receptors, regulates endocytic BDNF secretion in cultured astrocytes. Our data indicate that distinct glioactive molecules can participate in BDNF glial recycling and suggest that cortical astrocytes contributing to neuronal plasticity can be influenced by neurotransmitters in tune with synaptic needs. PMID:28289489

  20. Alcohol promotes in vitro chondrogenesis in embryonic facial mesenchyme.

    PubMed

    Hoffman, L M; Kulyk, W M

    1999-03-01

    Ethanol is a well-recognized teratogen in vertebrates that can perturb the development of the facial primordia and various other embryonic structures. However,the mechanisms underlying alcohol's effects on embryogenesis are currently unclear. Recent evidence suggests that the cranial neural crest, which forms the entire facial skeleton, may be a particularly sensitive target of ethanol teratogenicity. In the present study we have examined the influence of in vitro ethanol exposure on cartilage differentiation in micromass cultures of mesenchymal cells isolated from the various facial primordia (maxillary, mandibular, frontonasal, and hyoid processes) of the stage 24 chick embryo. In all four populations of facial mesenchyme, exposure to 1-1.5% ethanol promoted marked increases in Alcian blue-positive cartilage matrix formation, a rise in 35SO4 accumulation into matrix glycosaminoglycans, and enhanced expression of cartilage-characteristic type II collagen and aggrecan gene transcripts. In frontonasal and mandibular mesenchyme cultures, which undergo extensive spontaneous cartilage formation, ethanol treatment quantitatively elevated both matrix production and cartilage-specific gene transcript expression. In cultures of maxillary process and hyoid arch mesenchyme, which form little or no cartilage spontaneously, ethanol exposure induced the formation of chondrogenic cell aggregates and the appearance of aggrecan and type II collagen mRNAs. These actions were not restricted to ethanol, since tertiary butanol treatment also enhanced cartilage differentiation in facial mesenchyme cultures. Our findings demonstrate a potent stimulatory effect of alcohol on the differentiation of prechondrogenic mesenchyme of the facial primordia. Further analysis of this phenomenon might yield insight into the developmental mechanisms underlying the facial dysmorphologies associated with embryonic ethanol exposure.

  1. Embryonic development in Zungaro jahu.

    PubMed

    Marques, Camila; Faustino, Francine; Bertolucci, Bruno; Paes, Maria do Carmo Faria; Silva, Regiane Cristina da; Nakaghi, Laura Satiko Okada

    2017-02-01

    The aim of this study was to characterize the embryonic development of Zungaro jahu, a fresh water teleostei commonly known as 'jaú'. Samples were collected at pre-determined times from oocyte release to larval hatching and analysed under light microscopy, transmission electron microscopy and scanning electron microscopy. At the first collection times, the oocytes and eggs were spherical and yellowish, with an evident micropyle. Embryo development took place at 29.4 ± 1.5°C and was divided into seven stages: zygote, cleavage, morula, blastula, gastrula, organogenesis, and hatching. The differentiation of the animal and vegetative poles occured during the zygote stage, at 10 min post-fertilization (mpf), leading to the development of the egg cell at 15 mpf. From 20 to 75 mpf, successive cleavages resulted in the formation of 2, 4, 8, 16, 32 and 64 blastomeres. The morula stage was observed between 90 and 105 mpf, and the blastula and gastrula stage at 120 and 180 mpf; respectively. The end of the gastrula stage was characterized by the presence of the yolk plug at 360 mpf. Organogenesis followed, with differentiation of the cephalic and caudal regions, elongation of the embryo by the cephalo-caudal axis, and somitogenesis. Hatching occurred at 780 mpf, with mean larval total length of 3.79 ± 0.11 mm.

  2. Embryonal rhabdomyosarcoma: A rare oral tumor

    PubMed Central

    Datta, Sila; Ray, Jay Gopal; Deb, Tushar; Patsa, Santanu

    2016-01-01

    Rhabdomyosarcoma is the malignant neoplasm of striated muscle and a relatively uncommon tumor of the oral cavity. Embryonal variety is the most common subtype, observed in children below 10 years of age but occasionally seen in adolescents and young adults. The present report describes a case of embryonal rhabdomyosarcoma in the left posterior buccal mucosa, with extension in the adjacent alveolus, soft palate, oropharynx and nasopharynx of a 17-year-old female. PMID:27721622

  3. Expression of gonadotropin-releasing hormone receptor in cerebral cortical neurons of embryos and adult rats.

    PubMed

    Quintanar, J Luis; Salinas, Eva; González, Rodolfo

    2007-01-03

    Mammalian gonadotropin-releasing hormone (GnRH) was initially isolated from hypothalamus and its receptor from anterior pituitary, although extrapituitary GnRH receptors have been reported. The aim of the present study was to investigate whether GnRH receptor and its mRNA are expressed in cerebral cortical neurons of rat embryos and adult rats using immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR) techniques. The immunohistochemistry and RT-PCR analysis showed expression of GnRH receptor and presence of its mRNA, in both cerebral cortical neurons of rat embryos and cerebral cortical tissues of adult rats. Additional experiments showed a decrease in the receptor mRNA expression when cultured neurons of rat embryos were treated with GnRH. It is possible that the presence of GnRH receptors in cortical neurons of rat may be involved in other physiological roles such as neurohormone or neuromodulator.

  4. Tangential migration of glutamatergic neurons and cortical patterning during development: Lessons from Cajal-Retzius cells.

    PubMed

    Barber, Melissa; Pierani, Alessandra

    2016-08-01

    Tangential migration is a mode of cell movement, which in the developing cerebral cortex, is defined by displacement parallel to the ventricular surface and orthogonal to the radial glial fibers. This mode of long-range migration is a strategy by which distinct neuronal classes generated from spatially and molecularly distinct origins can integrate to form appropriate neural circuits within the cortical plate. While it was previously believed that only GABAergic cortical interneurons migrate tangentially from their origins in the subpallial ganglionic eminences to integrate in the cortical plate, it is now known that transient populations of glutamatergic neurons also adopt this mode of migration. These include Cajal-Retzius cells (CRs), subplate neurons (SPs), and cortical plate transient neurons (CPTs), which have crucial roles in orchestrating the radial and tangential development of the embryonic cerebral cortex in a noncell-autonomous manner. While CRs have been extensively studied, it is only in the last decade that the molecular mechanisms g