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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. PMID:25804737

  2. Evidence that embryonic neurons regulate the onset of cortical gliogenesis via cardiotrophin-1.

    PubMed

    Barnabé-Heider, Fanie; Wasylnka, Julie A; Fernandes, Karl J L; Porsche, Christian; Sendtner, Michael; Kaplan, David R; Miller, Freda D

    2005-10-20

    Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete. PMID:16242406

  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. FoxP2 regulates neurogenesis during embryonic cortical development.

    PubMed

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

    2013-01-01

    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.

  5. Automated maintenance of embryonic stem cell cultures.

    PubMed

    Terstegge, Stefanie; Laufenberg, Iris; Pochert, Jörg; Schenk, Sabine; Itskovitz-Eldor, Joseph; Endl, Elmar; Brüstle, Oliver

    2007-01-01

    Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.

  6. Controlled, scalable embryonic stem cell differentiation culture.

    PubMed

    Dang, Stephen M; Gerecht-Nir, Sharon; Chen, Jinny; Itskovitz-Eldor, Joseph; Zandstra, Peter W

    2004-01-01

    Embryonic stem (ES) cells are of significant interest as a renewable source of therapeutically useful cells. ES cell aggregation is important for both human and mouse embryoid body (EB) formation and the subsequent generation of ES cell derivatives. Aggregation between EBs (agglomeration), however, inhibits cell growth and differentiation in stirred or high-cell-density static cultures. We demonstrate that the agglomeration of two EBs is initiated by E-cadherin-mediated cell attachment and followed by active cell migration. We report the development of a technology capable of controlling cell-cell interactions in scalable culture by the mass encapsulation of ES cells in size-specified agarose capsules. When placed in stirred-suspension bioreactors, encapsulated ES cells can be used to produce scalable quantities of hematopoietic progenitor cells in a controlled environment.

  7. Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans

    PubMed Central

    1993-01-01

    We have examined the cortex of Caenorhabditis elegans eggs during pseudocleavage (PC), a period of the first cell cycle which is important for the generation of asymmetry at first cleavage (Strome, S. 1989. Int. Rev. Cytol. 114: 81-123). We have found that directed, actin dependent, cytoplasmic, and cortical flow occurs during this period coincident with a rearrangement of the cortical actin cytoskeleton (Strome, S. 1986. J. Cell Biol. 103: 2241-2252). The flow velocity (4-7 microns/min) is similar to previously determined particle movements driven by cortical actin flows in motile cells. We show that directed flows occur in one of the daughters of the first division that itself divides asymmetrically, but not in its sister that divides symmetrically. The cortical and cytoplasmic events of PC can be mimicked in other cells during cytokinesis by displacing the mitotic apparatus with the microtubule polymerization inhibitor nocodazole. In all cases, the polarity of the resulting cortical and cytoplasmic flows correlates with the position of the attenuated mitotic spindle formed. These cortical flows are also accompanied by a change in the distribution of the cortical actin network. The polarity of this redistribution is similarly correlated with the location of the attenuated spindle. These observations suggest a mechanism for generating polarized flows of cytoplasmic and cortical material during embryonic cleavages. We present a model for the events of PC and suggest how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans. PMID:8509454

  8. 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

  9. Ibuprofen augments bilirubin toxicity in rat cortical neuronal culture.

    PubMed

    Berns, Monika; Toennessen, Margit; Koehne, Petra; Altmann, Rodica; Obladen, Michael

    2009-04-01

    Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 microg/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 microM unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 microg/mL ibuprofen caused additional loss of cell viability and increased LDH release (p < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 microM reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism.

  10. 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.

  11. Rbm8a Haploinsufficiency Disrupts Embryonic Cortical Development Resulting in Microcephaly

    PubMed Central

    Mao, Hanqian; Pilaz, Louis-Jan; McMahon, John J.; Golzio, Christelle; Wu, Danwei; Shi, Lei; Katsanis, Nicholas

    2015-01-01

    The cerebral cortex is built during embryonic neurogenesis, a period when excitatory neurons are generated from progenitors. Defects in neurogenesis can cause acute neurodevelopmental disorders, such as microcephaly (reduced brain size). Altered dosage of the 1q21.1 locus has been implicated in the etiology of neurodevelopmental phenotypes; however, the role of 1q21.1 genes in neurogenesis has remained elusive. Here, we show that haploinsufficiency for Rbm8a, an exon junction complex (EJC) component within 1q21.1, causes severe microcephaly and defective neurogenesis in the mouse. At the onset of neurogenesis, Rbm8a regulates radial glia proliferation and prevents premature neuronal differentiation. Reduced Rbm8a levels result in subsequent apoptosis of neurons, and to a lesser extent, radial glia. Hence, compared to control, Rbm8a-haploinsufficient brains have fewer progenitors and neurons, resulting in defective cortical lamination. To determine whether reciprocal dosage change of Rbm8a alters embryonic neurogenesis, we overexpressed human RBM8A in two animal models. Using in utero electroporation of mouse neocortices as well as zebrafish models, we find RBM8A overexpression does not significantly perturb progenitor number or head size. Our findings demonstrate that Rbm8a is an essential neurogenesis regulator, and add to a growing literature highlighting roles for EJC components in cortical development and neurodevelopmental pathology. Our results indicate that disruption of RBM8A may contribute to neurodevelopmental phenotypes associated with proximal 1q21.1 microdeletions. PMID:25948253

  12. 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

  13. Primary culture of embryonic rat olfactory receptor neurons.

    PubMed

    Micholt, Evelien; Jans, Danny; Callewaert, Geert; Bartic, Carmen; Lammertyn, Jeroen; Nicolai, Bart

    2012-12-01

    Embryonic cells are very robust in surviving dissection and culturing protocols and easily adapt to their in vitro environment. Despite these advantages, research in the olfactory field on cultured embryonic olfactory neurons is sparse. In this study, two primary rat olfactory explant cultures of different embryonic d (E17 and E20) were established, comprising epithelium and bulb. The functionality of these neurons was tested by measuring intracellular calcium responses to cAMP-inducing agents forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) with fluorescence microscopy. For E17, the responsive cell fraction increased over time, from an initial 3% at the 1 d in vitro (DIV) to a maximum of 19% at 11 DIV. The response of E20 neurons fluctuated over time around a more or less stable 13%. A logistic regression analysis indicated a significant difference between both embryonic d in the response to FSK + IBMX. In addition, of these functional neurons, 23.3% of E17 and 54.3% of E20 cultures were responsive to the odorant isoamyl acetate. PMID:23150136

  14. Immunodissection and culture of rabbit cortical collecting tubule cells

    SciTech Connect

    Spielman, W.S.; Sonnenburg, W.K.; Allen, M.L.; Arend, L.J.; Gerozissis, K.; Smith, W.L.

    1986-08-01

    A mouse monoclonal antibody designated IgG3 (rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3 (rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10W rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10X RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approx.32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. The results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.

  15. Ethanol upregulates NMDA receptor subunit gene expression in human embryonic stem cell-derived cortical neurons.

    PubMed

    Xiang, Yangfei; Kim, Kun-Yong; Gelernter, Joel; Park, In-Hyun; Zhang, Huiping

    2015-01-01

    Chronic alcohol consumption may result in sustained gene expression alterations in the brain, leading to alcohol abuse or dependence. Because of ethical concerns of using live human brain cells in research, this hypothesis cannot be tested directly in live human brains. In the present study, we used human embryonic stem cell (hESC)-derived cortical neurons as in vitro cellular models to investigate alcohol-induced expression changes of genes involved in alcohol metabolism (ALDH2), anti-apoptosis (BCL2 and CCND2), neurotransmission (NMDA receptor subunit genes: GRIN1, GRIN2A, GRIN2B, and GRIN2D), calcium channel activity (ITPR2), or transcriptional repression (JARID2). hESCs were differentiated into cortical neurons, which were characterized by immunostaining using antibodies against cortical neuron-specific biomarkers. Ethanol-induced gene expression changes were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). After a 7-day ethanol (50 mM) exposure followed by a 24-hour ethanol withdrawal treatment, five of the above nine genes (including all four NMDA receptor subunit genes) were highly upregulated (GRIN1: 1.93-fold, P = 0.003; GRIN2A: 1.40-fold, P = 0.003; GRIN2B: 1.75-fold, P = 0.002; GRIN2D: 1.86-fold, P = 0.048; BCL2: 1.34-fold, P = 0.031), and the results of GRIN1, GRIN2A, and GRIN2B survived multiple comparison correction. Our findings suggest that alcohol responsive genes, particularly NMDA receptor genes, play an important role in regulating neuronal function and mediating chronic alcohol consumption-induced neuroadaptations.

  16. 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.

  17. 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.

  18. 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.

  19. Isolation and propagation of primary human and rodent embryonic neural progenitor cells and cortical neurons

    PubMed Central

    Darbinyan, Armine; Kaminski, Rafal; White, Martyn K; Darbinian, Nune; Khalili, Kamel

    2014-01-01

    Summary The research on human neural progenitor cells holds great potential for the understanding the molecular programs that control differentiation of cells of glial and neuronal lineages and pathogenetic mechanisms of neurological diseases. Stem cell technologies provide also opportunities for pharmaceutical industry to develop new approaches for regenerative medicine. Here we describe the protocol for isolation and maintenance of neural progenitor cells and cortical neurons using human fetal brain tissue. This protocol can be successfully adapted for preparation of rodent neural and oligodendrocyte progenitor cells. While several methods for isolation of neural and ologodendrocyte progenitors from rodent brain tissue have been described, including techniques which use gene transfer and magnetisc resonsnce beads, few methods are focused on derivation of human oligodendrocyte progenitor cells. Development of human culture provides the most physiologically relevent system for investigation of mechanisms which regulate function of oligodendrocyte, specifically of human origin. PMID:23975820

  20. 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.

  1. Optimization of Buffalo (Bubalus bubalis) Embryonic Stem Cell Culture System

    PubMed Central

    Zandi, Mohammad; Muzaffar, Musharifa; Shah, Syed Mohmad; Kumar Singh, Manoj; Palta, Prabhat; Kumar Singla, Suresh; Manik, Radheysham; Chauhan, Manmohan Singh

    2015-01-01

    Objective In order to retain an undifferentiated pluripotent state, embryonic stem (ES) cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli. Materials and Methods In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase (AP) and immunoflourescence staining of various pluripotency markers. We examined the effect of various factors like fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF) and Y-27632 to support the growth and maintenance of bubaline ES cells on gelatin coated dishes, in order to establish feeder free culture systems. We also analyzed the effect of feeder-conditioned media on stem cell growth in gelatin based cultures both in the presence as well as in the absence of the growth factors. Results The results showed that Y-27632, in the presence of FGF-2 and LIF, resulted in higher colony growth and increased expression of Nanog gene. Feeder-Conditioned Medium resulted in a significant increase in growth of buffalo ES cells on gelatin coated plates, however, feeder layer based cultures produced better results than gelatin based cultures. Feeder layers from buffalo fetal fibroblast cells can support buffalo ES cells for more than two years. Conclusion We developed a feeder free culture system that can maintain buffalo ES cells in the short term, as well as feeder layer based culture that can support the long term maintenance of buffalo ES cells. PMID:26199905

  2. In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.

    PubMed

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

    2010-10-08

    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. 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.

  3. [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. PMID:18837407

  4. [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.

  5. Processing of Reelin by embryonic neurons is important for function in tissue but not in dissociated cultured neurons.

    PubMed

    Jossin, Yves; Gui, Lanrun; Goffinet, André M

    2007-04-18

    Reelin, the protein defective in reeler mutant mice, plays a key role during brain development. Reelin is processed proteolytically at two sites, and the central fragment mimics function in vitro. Here, we show that processing is functionally important in vivo, a question that could not be addressed in our previous study. New monoclonal antibodies directed against central Reelin block its binding to lipoprotein receptors and perturb cortical development in vitro, confirming the importance of the central fragment that is detected in tissue and body fluids. Processing occurs when Reelin is incubated with embryonic neurons in culture or with their supernatant, but inhibition of processing by a metalloproteinase blocker does not prevent Reelin signaling in neurons. Furthermore, neurons internalize similarly full-length or central Reelin. In contrast, inhibition of processing prevents signaling and perturbs cortical development in cultured embryonic brain slices. Moreover, in vivo, the concentration of central Reelin is dramatically and selectively increased in receptor-deficient tissue, suggesting its specific downregulation after binding to receptors and internalization. We propose that processing by end-migration neurons is required in tissue (where Reelin is likely anchored to the extracellular matrix) to release the central fragment that diffuses locally and signals to target cells, whereas, in vitro, all Reelin forms have indiscriminate access to cells, so that cleavage is not necessary for signaling.

  6. Adherence of Bilophila wadsworthia to cultured human embryonic intestinal cells.

    PubMed

    Gerardo, S H; Garcia, M M; Wexler, H M; Finegold, S M

    1998-02-01

    Adherence of Bilophila wadsworthia to the cultured human embryonic intestinal cell line, Intestine 407 (Int 407), varied among the strains tested from strongly adherent (76-100% cells positive for one or more adherent bacteria) to non- or weakly adherent (0-25% positive cells). Although negative staining revealed that infrequent cells of an adherent strain, WAL 9077, the adherent type-strain, WAL 7959, and a non-adherent strain, WAL 8448, expressed loosely associated fimbrial structures, a role for these structures in adhesion could not be confirmed with either scanning or thin-section electron micrography. Ruthenium red staining of thin-section preparations and subsequent electron microscopy failed to reveal an extensive extracellular polysaccharide layer. SDS-PAGE analysis of crude outer membrane fractions of WAL 9077 and WAL 8448 demonstrated clear differences in their major and minor outer membrane protein components. Thus, we postulate that the adherence of B. wadsworthia to Int 407 cells is mediated by an outer membrane or cell wall component. PMID:16887620

  7. 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

  8. Magnetic field-magnetic nanoparticle culture system used to grow in vitro murine embryonic stem cells.

    PubMed

    de Freitas, Erika Regina Leal; Soares, Paula Roberta Otaviano; de Santos, Rachel Paula; dos Santos, Regiane Lopes; Porfírio, Elaine Paulucio; Báo, Sônia N; Lima, Emília Celma Oliveira; Guillo, Lídia Andreu

    2011-01-01

    The in vitro growth of embryonic stem cells (ESCs) is usually obtained in the presence of murine embryonic fibroblasts (MEF), but new methods for in vitro expansion of ESCs should be developed due to their potential clinical use. This study aims to establish a culture system to expand and maintain ESCs in the absence of MEF by using murine embryonic stem cells (mECS) as a model of embryonic stem cell. Magnetic nanoparticles (MNPs) were used for growing mESCs in the presence of an external magnetic field, creating the magnetic field-magnetic nanoparticle (MF-MNP) culture system. The growth characteristics were evaluated showing a doubling time slightly higher for mESCs cultivated in the presence of the system than in the presence of the MEF. The undifferentiated state was characterized by RT-PCR, immunofluorescence, alkaline phosphatase activity and electron microscopy. Murine embryonic stem cells cultivated in presence of the MF-MNP culture system exhibited Oct-4 and Nanog expression and high alkaline phosphatase activity. Ultrastructural morphology showed that the MF-MNP culture system did not interfere with processes that cause structural changes in the cytoplasm or nucleus. The MF-MNP culture system provides a tool for in vitro expansion of mESCs and could contribute to studies that aim the therapeutic use of embryonic stem cells. PMID:21446404

  9. Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development.

    PubMed

    Peterson, Suzanne E; Yang, Amy H; Bushman, Diane M; Westra, Jurjen W; Yung, Yun C; Barral, Serena; Mutoh, Tetsuji; Rehen, Stevens K; Chun, Jerold

    2012-11-14

    Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cell's karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions. PMID:23152605

  10. The type 1 cannabinoid receptor is highly expressed in embryonic cortical projection neurons and negatively regulates neurite growth in vitro.

    PubMed

    Vitalis, Tania; Lainé, Jeanne; Simon, Anne; Roland, Alexandre; Leterrier, Christophe; Lenkei, Zsolt

    2008-11-01

    In the rodent and human embryonic brains, the cerebral cortex and hippocampus transiently express high levels of type 1 cannabinoid receptors (CB(1)Rs), at a developmental stage when these areas are composed mainly of glutamatergic neurons. However, the precise cellular and subcellular localization of CB(1)R expression as well as effects of CB(1)R modulation in this cell population remain largely unknown. We report that, starting from embryonic day 12.5, CB(1)Rs are strongly expressed in both reelin-expressing Cajal-Retzius cells and newly differentiated postmitotic glutamatergic neurons of the mouse telencephalon. CB(1)R protein is localized first to somato-dendritic endosomes and at later developmental stages it localizes mostly to developing axons. In young axons, CB(1)Rs are localized both to the axolemma and to large, often multivesicular endosomes. Acute maternal injection of agonist CP-55940 results in the relocation of receptors from axons to somato-dendritic endosomes, indicating the functional competence of embryonic CB(1)Rs. The adult phenotype of CB(1)R expression is established around postnatal day 5. By using pharmacological and mutational modulation of CB(1)R activity in isolated cultured rat hippocampal neurons, we also show that basal activation of CB(1)R acts as a negative regulatory signal for dendritogenesis, dendritic and axonal outgrowth, and branching. Together, the overall negative regulatory role in neurite development suggests that embryonic CB(1)R signaling may participate in the correct establishment of neuronal connectivity and suggests a possible mechanism for the development of reported glutamatergic dysfunction in the offspring following maternal cannabis consumption.

  11. 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...

  12. Arctigenin protects cultured cortical neurons from glutamate-induced neurodegeneration by binding to kainate receptor.

    PubMed

    Jang, Young P; Kim, So R; Choi, Young H; Kim, Jinwoong; Kim, Sang G; Markelonis, George J; Oh, Tae H; Kim, Young C

    2002-04-15

    We previously reported that arctigenin, a lignan isolated from the bark of Torreya nucifera, showed significant neuroprotective activity against glutamate-induced toxicity in primary cultured rat cortical cells. In this study, the mode of action of arctigenin was investigated using primary cultures of rat cortical cells as an in vitro system. Arctigenin significantly attenuated glutamate-induced neurotoxicity when added prior to or after an excitotoxic glutamate challenge. The lignan protected cultured neuronal cells more selectively from neurotoxicity induced by kainic acid than by N-methyl-D-aspartate. The binding of [(3)H]-kainate to its receptors was significantly inhibited by arctigenin in a competitive manner. Furthermore, arctigenin directly scavenged free radicals generated by excess glutamate and successfully reduced the level of cellular peroxide in cultured neurons. These results suggest that arctigenin exerted significant neuroprotective effects on glutamate-injured primary cultures of rat cortical cells by directly binding to kainic acid receptors and partly scavenging of free radicals.

  13. 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.

  14. Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

    PubMed Central

    Conrad, Rebecca; Jablonka, Sibylle; Sczepan, Teresa; Sendtner, Michael; Wiese, Stefan; Klausmeyer, Alice

    2011-01-01

    Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using

  15. 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.

  16. 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

  17. Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

    PubMed Central

    Mort, Richard L.; Keighren, Margaret; Hay, Leonard; Jackson, Ian J.

    2014-01-01

    Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel. PMID:24894489

  18. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

    PubMed Central

    Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

    2010-01-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

  19. Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system.

    PubMed

    Aflatoonian, Behrouz; Ruban, Ludmila; Shamsuddin, Shamsul; Baker, Duncan; Andrews, Peter; Moore, Harry

    2010-04-01

    The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 microl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs. PMID:20224972

  20. Cultures of human embryonic osteoblasts. A new in vitro model for biocompatibility studies.

    PubMed

    Riccio, V; Della Ragione, F; Marrone, G; Palumbo, R; Guida, G; Oliva, A

    1994-11-01

    Cell populations derived from human embryonic bone were isolated according to the ability of osteoblasts to migrate from bone onto glass fragments. Morphologic and biochemical assays showed (1) osteoblast-like appearance; (2) elevated alkaline phosphatase 1,25(OH)2D3 responsive activity associated with plasma membranes and matrix vesicles; (3) production of a thick extracellular matrix, mainly composed of Type I collagen, which mineralized in the presence of 10 mM beta-glycerophosphate; and (4) higher growth rate and viability when compared with their mature counterpart. Cultures of embryonic cells were challenged with particles of several biomaterials, and their effects on morphology, vitality, and osteogenic capacity of the cultured cells were tested. Stainless steel, titanium alloy, Co-Cr-Mo alloy (vitallium), carbon fiber-reinforced polybutylene terephtalate, ultra-high molecular weight polyethylene, ceramic, calcium phosphate, and hydroxyapatite did not exert any significant deleterious effects on the cultured human osteoblasts. PMID:7955704

  1. Ketamine-induced apoptosis in cultured rat cortical neurons

    SciTech Connect

    Takadera, Tsuneo . E-mail: t-takadera@hokuriku-u.ac.jp; Ishida, Akira; Ohyashiki, Takao

    2006-01-15

    Recent data suggest that anesthetic drugs cause neurodegeneration during development. Ketamine is frequently used in infants and toddlers for elective surgeries. The purpose of this study is to determine whether glycogen synthase kinase-3 (GSK-3) is involved in ketamine-induced apoptosis. Ketamine increased apoptotic cell death with morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation. In addition, insulin growth factor-1 completely blocked the ketamine-induced apoptotic cell death. Ketamine decreased Akt phosphorylation. GSK-3 is known as a downstream target of Akt. The selective inhibitors of GSK-3 prevented the ketamine-induced apoptosis. Moreover, caspase-3 activation was accompanied by the ketamine-induced cell death and inhibited by the GSK-3 inhibitors. These results suggest that activation of GSK-3 is involved in ketamine-induced apoptosis in rat cortical neurons.

  2. Dissection, culture, and analysis of Xenopus laevis embryonic retinal tissue.

    PubMed

    McDonough, Molly J; Allen, Chelsea E; Ng-Sui-Hing, Ng-Kwet-Leok A; Rabe, Brian A; Lewis, Brittany B; Saha, Margaret S

    2012-01-01

    The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation(1-16). The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates (12,14-18). While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells (7,19-23). For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues (8,19-22,24-33). Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level (5,8,21,24,27-30,33-39). Xenopus laevis, a classic model system for the study of early neural development (19,27,29,31-32,40-42), serves as a particularly suitable system for retinal primary cell culture (10,38,43-45). Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction (25,38,43). In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding

  3. Mechanism of soluble beta-amyloid 25-35 neurotoxicity in primary cultured rat cortical neurons.

    PubMed

    Wang, Yong; Liu, Lili; Hu, Weimin; Li, Guanglai

    2016-04-01

    This study aimed to determine the effects of different concentrations of soluble beta-amyloid 25-35 (Aβ25-35) on cell viability, calcium overload, and PI3K-p85 expression in cultured cortical rat neurons. Primary cultured cerebral cortical neurons of newborn rats were divided randomly into six groups. Five groups were treated with soluble Aβ25-35 at concentrations of 10nmol/L, 100nmol/L, 1μmol/L, 10μmol/L, or 30μmol/L. Cell Counting Kit-8 staining was used to measure cell viability, laser-scanning confocal imaging was used to detect changes in intracellular free calcium concentration, and western blot assay was used to measure neuronal PI3K-p85 expression. Soluble Aβ25-35 was found to reduce cell viability and induce calcium overload in primary cultured rat cerebral cortical neurons, in a concentration-dependent manner. At certain concentrations, soluble Aβ25-35 also increased neuronal PI3K-p85 expression. These findings reveal that soluble Aβ25-35 reduces the viability of cultured cerebral cortical rat neurons. The neurotoxicity mechanism may involve calcium overload and disruption of insulin signal transduction pathways. PMID:26940239

  4. Mechanism of soluble beta-amyloid 25-35 neurotoxicity in primary cultured rat cortical neurons.

    PubMed

    Wang, Yong; Liu, Lili; Hu, Weimin; Li, Guanglai

    2016-04-01

    This study aimed to determine the effects of different concentrations of soluble beta-amyloid 25-35 (Aβ25-35) on cell viability, calcium overload, and PI3K-p85 expression in cultured cortical rat neurons. Primary cultured cerebral cortical neurons of newborn rats were divided randomly into six groups. Five groups were treated with soluble Aβ25-35 at concentrations of 10nmol/L, 100nmol/L, 1μmol/L, 10μmol/L, or 30μmol/L. Cell Counting Kit-8 staining was used to measure cell viability, laser-scanning confocal imaging was used to detect changes in intracellular free calcium concentration, and western blot assay was used to measure neuronal PI3K-p85 expression. Soluble Aβ25-35 was found to reduce cell viability and induce calcium overload in primary cultured rat cerebral cortical neurons, in a concentration-dependent manner. At certain concentrations, soluble Aβ25-35 also increased neuronal PI3K-p85 expression. These findings reveal that soluble Aβ25-35 reduces the viability of cultured cerebral cortical rat neurons. The neurotoxicity mechanism may involve calcium overload and disruption of insulin signal transduction pathways.

  5. Comparative analysis of cytosolic and mitochondrial ATP synthesis in embryonic and postnatal hippocampal neuronal cultures

    PubMed Central

    Surin, Alexander M.; Khiroug, Serguei; Gorbacheva, Lubov R.; Khodorov, Boris I.; Pinelis, Vsevolod G.; Khiroug, Leonard

    2013-01-01

    ATP in neurons is commonly believed to be synthesized mostly by mitochondria via oxidative phosphorylation. Neuronal mitochondria have been studied primarily in culture, i.e., in neurons isolated either from embryos or from neonatal pups. Although it is generally assumed that both embryonic and postnatal cultured neurons derive their ATP from mitochondrial oxidative phosphorylation, this has never been tested experimentally. We expressed the FRET-based ATP sensor AT1.03 in cultured hippocampal neurons isolated either from E17 to E18 rat embryos or from P1 to P2 rat pups and monitored [ATP]c simultaneously with mitochondrial membrane potential (ΔΨm; TMRM) and NAD(P)H autofluorescence. In embryonic neurons, transient glucose deprivation induced a near-complete decrease in [ATP]c, which was partially reversible and was accelerated by inhibition of glycolysis with 2-deoxyglucose. In the absence of glucose, pyruvate did not cause any significant increase in [ATP]c in 84% of embryonic neurons, and inhibition of mitochondrial ATP synthase with oligomycin failed to decrease [ATP]c. Moreover, ΔΨm was significantly reduced by oligomycin, indicating that mitochondria acted as consumers rather than producers of ATP in embryonic neurons. In sharp contrast, in postnatal neurons pyruvate added during glucose deprivation significantly increased [ATP]c (by 54 ± 8%), whereas oligomycin induced a sharp decline in [ATP]c and increased ΔΨm. These signs of oxidative phosphorylation were observed in all tested P1–P2 neurons. Measurement of ΔΨm with the potential-sensitive probe JC-1 revealed that neuronal mitochondrial membrane potential was significantly reduced in embryonic cultures compared to the postnatal ones, possibly due to increased proton permeability of inner mitochondrial membrane. We conclude that, in embryonic, but not postnatal neuronal cultures, ATP synthesis is predominantly glycolytic and the oxidative phosphorylation-mediated synthesis of ATP by

  6. Patterns of plasminogen activator production in cultured normal embryonic cells

    PubMed Central

    1977-01-01

    Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor. PMID:21193

  7. Pancreas Development Ex Vivo: Culturing Embryonic Pancreas Explants on Permeable Culture Inserts, with Fibronectin-Coated Glass Microwells, or Embedded in Three-Dimensional Matrigel™

    PubMed Central

    Shih, Hung Ping; Sander, Maike

    2015-01-01

    Pancreas development is a complex and dynamic process orchestrated by cellular and molecular events, including morphogenesis and cell differentiation. As a result of recent explorations into possible cell-therapy-based treatments for diabetes, researchers have made significant progress in deciphering the developmental program of pancreas formation. In vitro pancreas organ culture systems provide a valuable tool for exploring the mechanisms of gene regulation, cellular behaviors, and cell differentiation. In this chapter, we review three common techniques for culturing embryonic pancreas explants. Each technique is suitable for different applications. Specifically, culturing embryonic pancreas on culture inserts provides an excellent platform to test the effects of chemical compounds. Conversely, when the embryonic pancreas is cultured in fibronectin-coated glass microwells, the system provides unique culture conditions to monitor organ growth and cellular dynamic events. Lastly, when the embryonic pancreas is embedded in Matrigel, organogenesis can be studied in a three-dimensional environment instead of limiting the analysis to one plane. PMID:25173172

  8. 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.

  9. 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

  10. Effect of basic fibroblast growth factor in mouse embryonic stem cell culture and osteogenic differentiation.

    PubMed

    Rose, Laura C; Fitzsimmons, Ross; Lee, Poh; Krawetz, Roman; Rancourt, Derrick E; Uludağ, Hasan

    2013-05-01

    Embryonic stem cells are actively explored as a cell source in tissue engineering and regenerative medicine involving bone repair. Basic fibroblast growth factor (bFGF) has been a valuable growth factor to support the culture of human stem cells as well as their osteogenic differentiation, but the influence of bFGF on mouse embryonic stem (mES) cells is not known. Towards this goal, D3 cells were treated with bFGF during maintenance conditions and during spontaneous and osteogenic differentiation. In feeder-free monolayers, up to 40 ng/ml of exogenous bFGF did not support self-renewal of mES without LIF during cell expansion. During spontaneous differentiation in high-density cultures, bFGF stimulated cell proliferation under certain conditions but did not influence differentiation, as judged by stage-specific embryonic antigen-1 expression. The addition of bFGF reduced the alkaline phosphatase (ALP) activity associated with osteoblast activity during differentiation induced by osteogenic supplements, although the extent of mineralization was unaffected by bFGF. The bFGF increased the mesenchymal stem cell marker Sca-1 in an mES cell population and led to an enhanced increase in osteocalcin and runx2 expression in combination with BMP-2. These results suggest that bFGF could be utilized to expand the cell population in high-density cultures in addition to enriching the BMP-2 responsiveness of mES cells. PMID:22674886

  11. Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency.

    PubMed

    Martin Gonzalez, Javier; Morgani, Sophie M; Bone, Robert A; Bonderup, Kasper; Abelchian, Sahar; Brakebusch, Cord; Brickman, Joshua M

    2016-08-01

    Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i) or with knockout serum replacement (KOSR), but not serum, can generate high-level chimeras regardless of how these cells were derived. ESCs cultured in these conditions showed a transcriptional correlation with early pre-implantation embryos (E1.5-E3.5) and contributed to development from the 2-cell stage. Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells derived in one condition can be reprogrammed to another developmental state merely by adaptation to another culture condition. PMID:27509134

  12. Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation.

    PubMed

    Miyasaka, N; Arimatsu, Y; Takiguchihayashi, K

    1999-08-01

    A high level of foreign gene expression in organotypic cultures of the cerebral cortical anlage was achieved by electroporation-mediated gene transfer in vivo. A mammalian expression plasmid for green fluorescent protein (GFP) gene was injected into the lateral ventricle of rat embryos. Immediately after the plasmid DNA injection, the head of the embryo was electroporated between a pair of tweezer-type electrodes. The cortical anlage was isolated and maintained organotypically up to 21 days in vitro (DIV). The GFP-transgene was expressed intensely in neural progenitor cells at 1 DIV. GFP-expressing cells were still detectable and were demonstrated to differentiate into neurons and glia at 21 DIV. This system is expected to be useful for molecular analysis of cerebral cortical development and function.

  13. Distinctive neurophysiological properties of embryonic trigeminal and geniculate neurons in culture.

    PubMed

    Grigaliunas, Arturas; Bradley, Robert M; MacCallum, Donald K; Mistretta, Charlotte M

    2002-10-01

    Neurons in trigeminal and geniculate ganglia extend neurites that share contiguous target tissue fields in the fungiform papillae and taste buds of the mammalian tongue and thereby have principal roles in lingual somatosensation and gustation. Although functional differentiation of these neurons is central to formation of lingual sensory circuits, there is little known about electrophysiological properties of developing trigeminal and geniculate ganglia or the extrinsic factors that might regulate neural development. We used whole cell recordings from embryonic day 16 rat ganglia, maintained in culture as explants for 3-10 days with neurotrophin support to characterize basic properties of trigeminal and geniculate neurons over time in vitro and in comparison to each other. Each ganglion was cultured with the neurotrophin that supports maximal neuron survival and that would be encountered by growing neurites at highest concentration in target fields. Resting membrane potential and time constant did not alter over days in culture, whereas membrane resistance decreased and capacitance increased in association with small increases in trigeminal and geniculate soma size. Small gradual differences in action potential properties were observed for both ganglion types, including an increase in threshold current to elicit an action potential and a decrease in duration and increase in rise and fall slopes so that action potentials became shorter and sharper with time in culture. Using a period of 5-8 days in culture when neural properties are generally stable, we compared trigeminal and geniculate ganglia and revealed major differences between these embryonic ganglia in passive membrane and action potential characteristics. Geniculate neurons had lower resting membrane potential and higher input resistance and smaller, shorter, and sharper action potentials with lower thresholds than trigeminal neurons. Whereas all trigeminal neurons produced a single action potential at

  14. Netrin induces down-regulation of its receptor, Deleted in Colorectal Cancer, through the ubiquitin–proteasome pathway in the embryonic cortical neuron

    PubMed Central

    Kim, Tae-Hong; Lee, Hyun Kyoung; Seo, In Ae; Bae, Hae Rahn; Suh, Duk Joon; Wu, Jane; Rao, Yi; Hwang, Kyu-Geun; Park, Hwan Tae

    2009-01-01

    The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down-regulation of netrin-1 signaling in the embryonic cortical neurons. Netrin-1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a trans-membrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin-1 stimulation. Specific ubiquitin–proteasome inhibitors prevented the netrin-induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand-mediated down-regulation of DCC might participate in the loss of netrin-responsiveness in the developing nervous system. PMID:16181408

  15. Microfluidic isolation of highly pure embryonic stem cells using feeder-separated co-culture system.

    PubMed

    Chen, Qiushui; Wu, Jing; Zhuang, Qichen; Lin, Xuexia; Zhang, Jie; Lin, Jin-Ming

    2013-01-01

    Engineered artificial tissues from stem cells show great potential in regenerative medicine, disease therapies and organ transplantation. To date, stem cells are typically co-cultured with inactivated feeder layers to maintain their undifferentiated state, and to ensure reliable cell purity. Herein, we propose a novel microfabricated approach for feeder-separated coculture of mouse embryonic stem (mES) cells on polydimethylsiloxane (PDMS) porous membrane-assembled 3D-microdevice. Normal mouse embryonic fibroblasts (mEFs) without inactivation were specifically co-cultured with mES cells, resulting in the formation of mES cell colonies on spatially controlled co-culture with feeder layers. An excellent undifferentiated state was confirmed by the expressions of Nanog, octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP) after 5 days culture. As a result, with the significant advantages of efficiency and simplicity, pure mES cell populations (a purity of 89.2%) from mEFs co-cultures were easily collected without any further purification or separation.

  16. Microfluidic isolation of highly pure embryonic stem cells using feeder-separated co-culture system.

    PubMed

    Chen, Qiushui; Wu, Jing; Zhuang, Qichen; Lin, Xuexia; Zhang, Jie; Lin, Jin-Ming

    2013-01-01

    Engineered artificial tissues from stem cells show great potential in regenerative medicine, disease therapies and organ transplantation. To date, stem cells are typically co-cultured with inactivated feeder layers to maintain their undifferentiated state, and to ensure reliable cell purity. Herein, we propose a novel microfabricated approach for feeder-separated coculture of mouse embryonic stem (mES) cells on polydimethylsiloxane (PDMS) porous membrane-assembled 3D-microdevice. Normal mouse embryonic fibroblasts (mEFs) without inactivation were specifically co-cultured with mES cells, resulting in the formation of mES cell colonies on spatially controlled co-culture with feeder layers. An excellent undifferentiated state was confirmed by the expressions of Nanog, octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP) after 5 days culture. As a result, with the significant advantages of efficiency and simplicity, pure mES cell populations (a purity of 89.2%) from mEFs co-cultures were easily collected without any further purification or separation. PMID:23942279

  17. A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells

    PubMed Central

    Zhang, Rong; Mjoseng, Heidi K.; Hoeve, Marieke A.; Bauer, Nina G.; Pells, Steve; Besseling, Rut; Velugotla, Srinivas; Tourniaire, Guilhem; Kishen, Ria E. B.; Tsenkina, Yanina; Armit, Chris; Duffy, Cairnan R. E.; Helfen, Martina; Edenhofer, Frank; de Sousa, Paul A.; Bradley, Mark

    2013-01-01

    Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth, while mechanical, enzymatic or chemical cell dissociation methods are used for cellular passaging. However, these methods are ill defined, thus introducing variability into the system, and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate, which support long-term human embryonic stem cell growth and pluripotency over a period of 2–6 months. The hydrogels permitted gentle, reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used, undefined biological substrates represent a flexible and scalable approach for improving the definition, efficacy and safety of human embryonic stem cell culture systems for research, industrial and clinical applications. PMID:23299885

  18. 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

  19. 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.

  20. Human Immunodeficiency Virus Type 1 Coat Protein Neurotoxicity Mediated by Nitric Oxide in Primary Cortical Cultures

    NASA Astrophysics Data System (ADS)

    Dawson, Valina L.; Dawson, Ted M.; Uhl, George R.; Snyder, Solomon H.

    1993-04-01

    The human immunodeficiency virus type 1 coat protein, gp120, kills neurons in primary cortical cultures at low picomolar concentrations. The toxicity requires external glutamate and calcium and is blocked by glutamate receptor antagonists. Nitric oxide (NO) contributes to gp120 toxicity, since nitroarginine, an inhibitor of NO synthase, prevents toxicity as does deletion of arginine from the incubation medium and hemoglobin, which binds NO. Superoxide dismutase also attenuates toxicity, implying a role for superoxide anions.

  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. 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

  3. 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.

  4. 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.

  5. 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.

  6. BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin.

    PubMed

    Hayashi, Yohei; Furue, Miho Kusuda; Tanaka, Satoshi; Hirose, Michiko; Wakisaka, Noriko; Danno, Hiroki; Ohnuma, Kiyoshi; Oeda, Shiho; Aihara, Yuko; Shiota, Kunio; Ogura, Atsuo; Ishiura, Shoichi; Asashima, Makoto

    2010-05-01

    Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast.

  7. 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.

  8. 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

  9. 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.

  10. Differentiation patterns of embryonic stem cells in two- versus three-dimensional culture.

    PubMed

    Pineda, Emma T; Nerem, Robert M; Ahsan, Tabassum

    2013-01-01

    Pluripotent stem cells are attractive candidates as a cell source for regenerative medicine and tissue engineering therapies. Current methods of differentiation result in low yields and impure populations of target phenotypes, with attempts for improved efficiency often comparing protocols that vary multiple parameters. This basic science study focused on a single variable to understand the effects of two-dimensional (2D) versus three-dimensional (3D) culture on directed differentiation. We compared mouse embryonic stem cells (ESCs) differentiated on collagen type I-coated surfaces (SLIDEs), embedded in collagen type I gels (GELs), and in suspension as embryoid bodies (EBs). For a systematic analysis in these studies, key parameters were kept identical to allow for direct comparison across culture configurations. We determined that all three configurations supported differentiation of ESCs and that the kinetics of differentiation differed greatly for cells cultured in 2D versus 3D. SLIDE cultures induced overall differentiation more quickly than 3D configurations, with earlier expression of cytoskeletal and extracellular matrix proteins. For 3D culture as GELs or EBs, cells clustered similarly, formed complex structures and promoted differentiation towards cardiovascular phenotypes. GEL culture, however, also allowed for contraction of the collagen matrix. For differentiation towards fibroblasts and smooth muscle cells which actively remodel their environment, GEL culture may be particularly beneficial. Overall, this study determined the effects of dimensionality on differentiation and helps in the rational design of protocols to generate phenotypes needed for tissue engineering and regenerative medicine.

  11. 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

  12. 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

  13. Differentiation of cartilaginous anlagen in entire embryonic mouse limbs cultured in a rotating bioreactor

    NASA Astrophysics Data System (ADS)

    Montufar-Solis, D.; Oakley, C. R.; Jefferson, Y.; Duke, P. J.

    2003-10-01

    Mechanisms involved in development of the embryonic limb have remained the same throughout eons of genetic and environmental evolution under Earth gravity (lg). During the spaceflight era it has been of interest to explore the ancient theory that form of the skeleton develops in response to gravity, and that changes in gravitational forces can change the developmental pattern of the limb. This has been shown in vivo and in vitro, allowing the hypergravity of centrifugation and microgravity of space to be used as tools to increase our knowledge of limb development. In recapitulations of spaceflight experiments, premetatarsals were cultured in suspension in a bioreactor, and found to be shorter and less differentiated than those cultured in standard culture dishes. This study only measured length of the metatarsals, and did not account for possible changes due to the skeletal elements having a more in vivo 3D shape while in suspension vs. flattened tissues compressed by their own weight. A culture system with an outcome closer to in vivo and that supports growth of younger limb buds than traditional systems will allow studies of early Hox gene expression, and contribute to the understanding of very early stages of development. The purpose of the current experiment was to determine if entire limb buds could be cultured in the bioreactor, and to compare the growth and differentiation with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were cultured for six days, either in the bioreactor or in center-well organ culture dishes, fixed, and embedded for histology. E13 specimens grown in culture dishes were flat, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections showed excellent cartilage differentiation in both culture systems, with more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Younger limb buds fused together during culture, so an additional set of El 1

  14. 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

  15. 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.

  16. 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.

  17. Automated, scalable culture of human embryonic stem cells in feeder-free conditions.

    PubMed

    Thomas, Rob J; Anderson, David; Chandra, Amit; Smith, Nigel M; Young, Lorraine E; Williams, David; Denning, Chris

    2009-04-15

    Large-scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However, current hESC culture strategies are labor-intensive and employ highly variable processes, presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines, HUES7, and NOTT1, with trypsin in feeder-free conditions, is compatible with complete automation on the CompacT SelecT, a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained, as evidenced by consistent proliferation during serial passage, expression of stem cell markers (OCT4, NANOG, TRA1-81, and SSEA-4), stable karyotype, and multi-germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell-use in clinical, scientific, and industrial applications.

  18. Differential effects of methylmercury on gamma-aminobutyric acid type A receptor currents in rat cerebellar granule and cerebral cortical neurons in culture.

    PubMed

    Herden, Christina J; Pardo, Nicole E; Hajela, Ravindra K; Yuan, Yukun; Atchison, William D

    2008-02-01

    Cerebellar granule cells are particularly sensitive to inhibition by methylmercury (MeHg) on GABA(A) receptor function. This is manifested as a more rapid block of inhibitory postsynaptic currents/inhibitory postsynaptic potentials than for Purkinje cells. The underlying mechanism(s) for differential sensitivity of GABAergic transmission to MeHg in cerebellar neurons is unknown. Differential expression of alpha(6) subunit-containing GABA(A) receptors in cerebellar granule and Purkinje neurons could partially explain this. GABA-evoked currents (I(GABA)) were recorded in response to MeHg in alpha(6) subunit-containing cerebellar granule cells and alpha(6) subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express alpha(6) subunits. They express the same alpha(1)-containing GABA(A) receptor as Purkinje cells but lack characteristics that enhance Purkinje cell resistance to MeHg. I(GABA) were obtained using whole-cell recording and symmetrical [Cl(-)]. MeHg reduced I(GABA) to complete block in both cell types in a time- and concentration-dependent manner. This effect was faster in granule cells than cortical cells. Effects of MeHg on I(GABA) were recorded in granule cells at various developmental stages (days in vitro 4, 6, and 8) to alter the expression level of alpha(6) subunit-containing GABA(A) receptors. Effects of MeHg on I(GABA) were similar in cells at all days. In human embryonic kidney 293 cells expressing either alpha(6) or alpha(1) subunit-containing GABA(A) receptors, time to block of I(GABA) by MeHg was comparable. Thus, the presence of the alpha(6) subunit alone may not underlie the differential effects of MeHg on I(GABA) observed in cerebellar granule and cortical neurons; other factors are likely to be involved as well. PMID:17977981

  19. 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%).

  20. Electrical stimulation promotes the survival of oligodendrocytes in mixed cortical cultures.

    PubMed

    Gary, Devin S; Malone, Misti; Capestany, Paul; Houdayer, Thierry; McDonald, John W

    2012-01-01

    Oligodendrocyte (OLG) death plays a major role in white matter dysfunction and demyelination following injury to the CNS. Axonal contact, communication, and neuronal activity appear to promote OLG survival and function in cell culture and during development. The application of electrical stimulation to mixed neural cultures has been shown to promote OLG differentiation and the formation of myelin in vitro. Here we show that OLG viability can be significantly enhanced in mixed cortical cultures by applying biphasic pulses of electrical stimulation (ESTIM). Enhanced survival via ESTIM requires the presence of neurons and is suppressed by inhibition of voltage-gated sodium channels. Additionally, contact between the axon and OLG is necessary for ESTIM to promote OLG survival. This report suggests that patterned neuronal activity could repress delayed progression of white matter injury and promote CNS repair in neurological conditions that involve white matter damage.

  1. Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces.

    PubMed

    Ahmed, Ijaz; Ponery, Abdul S; Nur-E-Kamal, Alam; Kamal, Jabeen; Meshel, Adam S; Sheetz, Michael P; Schindler, Melvin; Meiners, Sally

    2007-07-01

    Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates. PMID:17294137

  2. Osteogenic cell lineage analysis is facilitated by organ cultures of embryonic chick periosteum.

    PubMed

    Bruder, S P; Caplan, A I

    1990-10-01

    Monoclonal antibodies against the surface of embryonic osteogenic cells (SB-1, SB-2, SB-3, and SB-5) have been used to characterize the sequence of transitions involved in the osteogenic cell lineage. In the present study, immunohistochemical analyses of the expression of osteogenic cell surface antigens in organ cultures of folded chick periosteum were performed. Unlike traditional culture methods using isolated osteoblastic cells, periosteal explants form a mineralized bone tissue in 4 to 6 days which is virtually identical to the in vivo counterpart. Examination of fresh explants confirm that no mature osteoblastic cells were present, although a discontinuous layer of preosteoblasts was evident. As the wave of osteodifferentiation swept through the cultured tissue, antibody SB-1 reacted with the surface of a large family of cells associated with the developing bone. Antibodies SB-3 and SB-2 reacted with progressively smaller subsets of cells, namely those in successively closer physical association with the newly formed and mineralizing bone. Cells recently encased in bone matrix were stained by both SB-2 and SB-5 antibodies, while those cells deep within the matrix reacted only with antibody SB-5. Analysis of this culture model allows dissection of the discrete cellular transition steps of osteogenesis, and reveals that osteogenic precursor cells proceed through the unique lineage stages which have been documented for in vivo osteogenesis. This culture system has furthermore provided evidence which is used to refine our understanding of the osteogenic cell lineage.

  3. Activation of 5-HT2A/2C receptors reduces the excitability of cultured cortical neurons.

    PubMed

    Hu, Lingli; Liu, Chunhua; Dang, Minyan; Luo, Bin; Guo, Yiping; Wang, Haitao

    2016-10-01

    The abundant forebrain serotonergic projections are believed to modulate the activities of cortical neurons. 5-HT2 receptor among multiple subtypes of serotonin receptors contributes to the modulation of excitability, synaptic transmissions and plasticity. In the present study, whole-cell patch-clamp recording was adopted to examine whether activation of 5-HT2A/2C receptors would have any impact on the excitability of cultured cortical neurons. We found that 2,5-Dimethoxy-4-iodoamphetamine (DOI), a selective 5-HT2A/2C receptor agonist, rapidly and reversibly depressed spontaneous action potentials mimicking the effect of serotonin. The decreased excitability was also observed for current-evoked firing. Additionally DOI increased neuronal input resistance. Hyperpolarization-activated cyclic nucleotide-gated cationic channels (HCN) did not account for the inhibition of spontaneous firing. The synaptic contribution was ruled out in that DOI augmented excitation and attenuated inhibition to actually favor an increase in the excitability. Our findings revealed that activation of 5-HT2A/2C receptors reduces neuronal excitability, which would deepen our understanding of serotonergic modulation of cortical activities. PMID:27585751

  4. Regulation of phosphatidylcholine biosynthesis in cultured chick embryonic muscle treated with phospholipase C.

    PubMed

    Sleight, R; Kent, C

    1980-11-25

    Cultures of embryonic chick muscle cells grown in medium containing phospholipase C from Clostridium perfringens incorporated [3H]choline into lipid at a rate 3- to 5-fold higher than control cultures. To determine the mechanism by which stimulation of phosphatidylcholine synthesis occurred in phospholipase C-treated cells, activities of enzymes and levels of intermediates in the biosynthetic pathway for phosphatidylcholine were examined. Activities of choline kinase, choline phosphotransferase, glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, acylglycerol-3-phosphate acyltransferase, and phosphatidic acid phosphatase in phospholipase C-treated cells were the same or only slightly higher than in control cells. CTP:phosphocholine cytidylyltransferase, on the other hand, was 3 times as active in homogenates from phospholipase C-treated cells. Levels of phosphocholine decreased and levels of CDP-choline increased in phospholipase C-treated cells, and a calculation of the disequilibrium ratio indicated that the cytidylyltransferase reaction was not at equilibrium. The cytidylyltransferase was, thus, identified as the regulatory enzyme for choline flux in these cells. The cytidylyltransferase was located in both the cytosolic and particulate fractions from cultured muscle cells and a much larger portion of enzyme activity was associated with the particulate fraction in cells treated with phospholipase C. Sonicated preparations of total chick lipids, phosphatidylethanolamine, and phosphatidylserine greatly stimulated the cytosolic cytidylyltransferase activity but had no effect on the particulate enzyme. Neither stimulation of incorporation of [3H]choline into lipid nor activation of the cytidylyltransferase was dependent on protein synthesis. A model for the mechanism of regulation of phosphatidylcholine synthesis in embryonic chick muscle is presented.

  5. Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells.

    PubMed

    Subramanian, Kartik; Owens, Derek Jason; Raju, Ravali; Firpo, Meri; O'Brien, Timothy D; Verfaillie, Catherine M; Hu, Wei-Shou

    2014-01-15

    Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

  6. Comprehensive Method for Culturing Embryonic Dorsal Root Ganglion Neurons for Seahorse Extracellular Flux XF24 Analysis.

    PubMed

    Lange, Miranda; Zeng, Yan; Knight, Andrew; Windebank, Anthony; Trushina, Eugenia

    2012-01-01

    Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG) neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15) rat DRG neurons suitable for analysis with the Seahorse XF24 platform.

  7. Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

    PubMed Central

    Brown, Aaron C.; Blank, Ulrika; Adams, Derek C.; Karolak, Michele J.; Fetting, Jennifer L.; Hill, Beth L.; Oxburgh, Leif

    2011-01-01

    Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering naïve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated http://www.gudmap.org/, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant

  8. 3D mouse embryonic stem cell culture for generating inner ear organoids.

    PubMed

    Koehler, Karl R; Hashino, Eri

    2014-01-01

    This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16-20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.

  9. 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.

  10. 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

  11. 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

  12. Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures.

    PubMed

    Sikorski, Darek J; Caron, Nicolas J; VanInsberghe, Michael; Zahn, Hans; Eaves, Connie J; Piret, James M; Hansen, Carl L

    2015-10-01

    Heterogeneity in the clonal outputs of individual human embryonic stem cells (hESCs) confounds analysis of their properties in studies of bulk populations and how to manipulate them for clinical applications. To circumvent this problem we developed a microfluidic device that supports the robust generation of colonies derived from single ESCs. This microfluidic system contains 160 individually addressable chambers equipped for perfusion culture of individual hESCs that could be shown to match the growth rates, marker expression and colony morphologies obtained in conventional cultures. Use of this microfluidic device to analyze the clonal growth kinetics of multiple individual hESCs induced to differentiation revealed variable shifts in the growth rate, area per cell and expression of OCT4 in the progeny of individual hESCs. Interestingly, low OCT4 expression, a slower growth rate and low nuclear to cytoplasmic ratios were found to be correlated responses. This study demonstrates how microfluidic systems can be used to enable large scale live-cell imaging of isolated hESCs exposed to changing culture conditions, to examine how different aspects of their variable responses are correlated. PMID:26059045

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

    PubMed

    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

  14. Worldwide survey of published procedures to culture human embryonic stem cells.

    PubMed

    Fernandes, A M; Meletti, T; Guimarães, R; Stelling, M P; Marinho, P A N; Valladão, A S; Rehen, S K

    2010-01-01

    Since their derivation 11 years ago, human embryonic stem (hES) cells have become a powerful tool in both basic biomedical research and developmental biology. Their capacity for self-renewal and differentiation into any tissue type has also brought interest from fields such as cell therapy and drug screening. We conducted an extensive analysis of 750 papers (51% of the total published about hES cells between 1998 and 2008) to present a spectrum of hES cell research including culture protocols developed worldwide. This review may stimulate discussions about the importance of having unvarying methods to culture hES cells, in order to facilitate comparisons among data obtained by research groups elsewhere, especially concerning preclinical studies. Moreover, the description of the most widely used cell lines, reagents, and procedures adopted internationally will help newcomers on deciding the best strategies for starting their own studies. Finally, the results will contribute with the efforts of stem cell researchers on comparing the performance of different aspects related to hES cell culture methods.

  15. Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells.

    PubMed

    Enver, Tariq; Soneji, Shamit; Joshi, Chirag; Brown, John; Iborra, Francisco; Orntoft, Torben; Thykjaer, Thomas; Maltby, Edna; Smith, Kath; Abu Dawud, Raed; Jones, Mark; Matin, Maryam; Gokhale, Paul; Draper, Jonathan; Andrews, Peter W

    2005-11-01

    Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also because they may undergo progressive adaptation upon long-term culture in vitro. Such adaptation may reflect selection of variants with altered propensity for survival and retention of an undifferentiated phenotype. Elucidating the mechanisms involved will be important for understanding normal self-renewal and commitment to differentiation and for validating the safety of HESC-based therapy. We have investigated this process of adaptation at the cellular and molecular levels through a comparison of early passage (normal) and late passage (adapted) sublines of a single HESC line, H7. To account for spontaneous differentiation that occurs in HESC cultures, we sorted cells for SSEA3, which marks undifferentiated HESC. We show that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes. On the basis of growth potential, ability to re-initiate ES cultures and global transcription profiles, we propose a cellular differentiation hierarchy for maintenance cultures of HESC: normal SSEA3+ cells represent pluripotent stem cells. Normal SSEA3- cells have exited this compartment, but retain multilineage differentiation potential. However, adapted SSEA3+ and SSEA3- cells co-segregate within the stem cell territory, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. As this balance is also an essential feature of cancer, the mechanisms of culture adaptation may mirror those of oncogenesis and tumour progression. PMID:16159889

  16. Protective effects of isoatriplicolide tiglate from Paulownia coreana against glutamate-induced neurotoxicity in primary cultured rat cortical cells.

    PubMed

    Chung, Ill-Min; Kim, Eun-Hye; Jeon, Hyun-Seok; Moon, Hyung-In

    2010-06-01

    To examine the neuroprotective effects of Paulownia coreana, we tested its protection against the glutamate-induced neurotoxicity to primary cultured cortical neurons. An aqueous extract of the plants exhibited significant protection against glutamate-induced toxicity in primary cultured rat cortical cells. In order to clarify the neuroprotective mechanism(s) of this observed effect, isolation was performed to seek and identify active fractions and components. By such fractionation, one bioactive sesquiterpene lactone, isoatriplicolide tiglate, was isolated, which exhibited significant neuroprotective activities against glutamate-induced toxicity, exhibiting cell viability of about 50%, at concentrations ranging from 0.1 microM to 10 microM. PMID:20614807

  17. 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.

  18. 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.

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

    PubMed Central

    Mutukula, Naresh; Edri, Reuven; Elkabetz, Yechiel

    2015-01-01

    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. PMID:26473351

  20. Scalable platform for human embryonic stem cell differentiation to cardiomyocytes in suspended microcarrier cultures.

    PubMed

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

    2010-12-01

    A scalable platform for human embryonic stem cell (hESC)-derived cardiomyocyte (CM) production can provide a readily available source of CMs for cell therapy, drug screening, and cardiotoxicity tests. We have designed and optimized a scalable platform using microcarrier cultures in serum-free media supplemented with SB203580 mitogen-activated protein kinase-inhibitor. Different microcarriers (DE-53, Cytodex-1 and 3, FACT, and TOSOH-10) were used to investigate the effects of type, size, shape, and microcarrier concentrations on the differentiation efficiency. hESCs propagated on TOSOH-10 (protamine derivatized 10-μm beads) at the concentration of 0.125 mg/mL produced 80% beating aggregates, threefold cell expansion, and 20% of CMs (determined by fluorescence-activated cell sorting for myosin heavy chain and α-actinin expression). The ratio of CM/hESC seeded in this system was 0.62 compared to 0.22 in the embryoid body control cultures. The platform robustness has been tested with HES-3 and H1 cell lines, and its scalability was demonstrated in suspended spinner cultures. However, spinner culture yields dropped to 0.33 CM/hESC probably due to shear stress causing some cell death. Cells dissociated from differentiated aggregates showed positive staining for cardio-specific markers such as α-actinin, myosin heavy and light chain, troponin I, desmin, and emilin-2. Finally, CM functionality was also shown by QT-prolongation (QTempo) assay with/without Astemizole. This study represents a new scalable bioprocessing system for CM production using reagents that can comply with Good Manufacturing Practice. PMID:20590381

  1. [Propagation of the HTV in primary human embryonic kidney and lung cell culture].

    PubMed

    Liu, B; Dai, J; Wang, X; Wang, X; Shen, G

    1994-08-01

    2 strains of Hantaan virus (HTV, 76-118, Hubei-114) have been propagated successfully in cultured primary human embryonic kidney (HEK) and lung (HEL) cells. Cytopathic effect (CPE) was observed in the two kind of cells on day 5 to 7 postinoculation which showed the cell became round and clustered, then detached. The replicating peak of the Hubei-114 in two kinds of cell cultures appeared on the 11th day and another strain on the 14th or 17th day after infection. The ultrastructure changes were observed with EM and IEM, which stained by ICGT before embedding. It was discovered that the mitochondia atrophied and decreased, and inclusion bodies in the cytoplasma of HEK and KEL cells. A large amount of gold granulae were found in the inclusion bodies and the virions were seen occasionally. Contamination with other agents have been ruled out. Our data suggest that the replicating characters of HTV in these cell systems might be possible for the pathogenicity of HFRS for human. PMID:7801638

  2. 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.

  3. Endogenous cholinergic tone modulates spontaneous network level neuronal activity in primary cortical cultures grown on multi-electrode arrays

    PubMed Central

    2013-01-01

    Background Cortical cultures grown long-term on multi-electrode arrays (MEAs) are frequently and extensively used as models of cortical networks in studies of neuronal firing activity, neuropharmacology, toxicology and mechanisms underlying synaptic plasticity. However, in contrast to the predominantly asynchronous neuronal firing activity exhibited by intact cortex, electrophysiological activity of mature cortical cultures is dominated by spontaneous epileptiform-like global burst events which hinders their effective use in network-level studies, particularly for neurally-controlled animat (‘artificial animal’) applications. Thus, the identification of culture features that can be exploited to produce neuronal activity more representative of that seen in vivo could increase the utility and relevance of studies that employ these preparations. Acetylcholine has a recognised neuromodulatory role affecting excitability, rhythmicity, plasticity and information flow in vivo although its endogenous production by cortical cultures and subsequent functional influence upon neuronal excitability remains unknown. Results Consequently, using MEA electrophysiological recording supported by immunohistochemical and RT-qPCR methods, we demonstrate for the first time, the presence of intrinsic cholinergic neurons and significant, endogenous cholinergic tone in cortical cultures with a characterisation of the muscarinic and nicotinic components that underlie modulation of spontaneous neuronal activity. We found that tonic muscarinic ACh receptor (mAChR) activation affects global excitability and burst event regularity in a culture age-dependent manner whilst, in contrast, tonic nicotinic ACh receptor (nAChR) activation can modulate burst duration and the proportion of spikes occurring within bursts in a spatio-temporal fashion. Conclusions We suggest that the presence of significant endogenous cholinergic tone in cortical cultures and the comparability of its modulatory effects

  4. 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.

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

    PubMed

    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. PMID:12743323

  6. 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.

  7. Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.

    PubMed

    Schulz, Thomas C; Noggle, Scott A; Palmarini, Gail M; Weiler, Deb A; Lyons, Ian G; Pensa, Kate A; Meedeniya, Adrian C B; Davidson, Bruce P; Lambert, Nevin A; Condie, Brian G

    2004-01-01

    The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies. PMID:15579641

  8. Comparison of spike parameters from optically identified GABAergic and glutamatergic neurons in sparse cortical cultures

    PubMed Central

    Weir, Keiko; Blanquie, Oriane; Kilb, Werner; Luhmann, Heiko J.; Sinning, Anne

    2015-01-01

    Primary neuronal cultures share many typical features with the in vivo situation, including similarities in distinct electrical activity patterns and synaptic network interactions. Here, we use multi-electrode array (MEA) recordings from spontaneously active cultures of wildtype and glutamic acid decarboxylase 67 (GAD67)-green fluorescent protein (GFP) transgenic mice to evaluate which spike parameters differ between GABAergic interneurons and principal, putatively glutamatergic neurons. To analyze this question we combine MEA recordings with optical imaging in sparse cortical cultures to assign individual spikes to visually-identified single neurons. In our culture system, excitatory and inhibitory neurons are present at a similar ratio as described in vivo, and spike waveform characteristics and firing patterns are fully developed after 2 weeks in vitro. Spike amplitude, but not other spike waveform parameters, correlated with the distance between the recording electrode and the location of the assigned neuron’s soma. Cluster analysis of spike waveform properties revealed no particular cell population that may be assigned to putative inhibitory or excitatory neurons. Moreover, experiments in primary cultures from transgenic GAD67-GFP mice, which allow optical identification of GABAergic interneurons and thus unambiguous assignment of extracellular signals, did not reveal any significant difference in spike timing and spike waveform parameters between inhibitory and excitatory neurons. Despite of our detailed characterization of spike waveform and temporal spiking properties we could not identify an unequivocal electrical parameter to discriminate between individual excitatory and inhibitory neurons in vitro. Our data suggest that under in vitro conditions cellular classifications of single neurons on the basis of their extracellular firing properties should be treated with caution. PMID:25642167

  9. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension.

    PubMed

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E; Qvortrup, Klaus; Baar, Keith; Svensson, René B; Magnusson, S Peter; Krogsgaard, Michael; Koch, Manuel; Kjaer, Michael

    2010-06-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned along the axis of tension. The fibrils had a homogeneous narrow diameter that was similar to collagen fibrils occurring in embryonic tendon. Immunostaining showed colocalization of collagen type I with collagen III, XII and XIV. A fibronectin network was formed in parallel with the collagen, and fibroblasts stained positive for integrin alpha(5). Finally, the presence of cell extensions into the extracellular space with membrane-enclosed fibrils in fibripositors indicated characteristics of embryonic tendon. We conclude that mature human tendon fibroblasts retain an intrinsic capability to perform collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon.

  10. Assessment of developmental cardiotoxic effects of some commonly used phytochemicals in mouse embryonic D3 stem cell differentiation and chick embryonic cardiomyocyte micromass culture models.

    PubMed

    Mohammed, Omar J; McAlpine, Roseanna; Chiewhatpong, Phasawee; Latif, Muhammad Liaque; Pratten, Margaret K

    2016-09-01

    Pregnant women often use herbal medicines to alleviate symptoms of pregnancy. The active phytochemicals eugenol (from holy basil) and α-bisabolol (from chamomile) are recommended to promote calmness and reduce stress. There is evidence that both eugenol and α-bisabolol possess pro-apoptotic and anti-proliferative effects and induce reactive oxygen species. The potential effect was examined by monitoring cardiomyocyte contractile activity (differentiation), cell activity, protein content and ROS production for mouse D3 embryonic stem cell and ‎chick embryonic micromass culture. The results showed that eugenol (0.01-80μM) demonstrated effects on cell activity (both systems) and ROS production (stem cell system only), as well as decreasing the contractile activity and protein content at high concentrations in both systems. Additionally, α-bisabolol (0.01-80μM) at high concentrations decreased the contractile activity and cell activity and in the stem cell system induced ROS production and decreased protein content. The results suggest only low concentrations should be ingested in pregnancy.‎. PMID:27105832

  11. Generation, culture, and differentiation of human embryonic stem cells for therapeutic applications.

    PubMed

    Moon, Shin Yong; Park, Yong Bin; Kim, Dae-Sung; Oh, Sun Kyung; Kim, Dong-Wook

    2006-01-01

    Embryonic stem (ES) cells, derived from the inner cell mass of the mammalian blastocyst, can continuously proliferate in an undifferentiated state and can also be induced to differentiate into a desired cell lineage. These abilities make ES cells an appealing source for cell replacement therapies, the study of developmental biology, and drug/toxin screening studies. As compared to mouse ES cells, human ES cells have only recently been derived and studied. Although there are many differences in properties between mouse and human ES cells, the study of mouse ES cells has provided important insights into human ES cell research. In this review, we describe the advantages and disadvantages of methods used for human ES cell derivation, the expansion of human ES cells, and the current status of human ES cell differentiation research. In addition, we discuss the endeavor that scientists have undertaken toward the therapeutic application of these cells, which includes therapeutic cloning and the improvement of human ES cell culture conditions. PMID:16242999

  12. AXONAL AGRANULAR RETICULUM AND SYNAPTIC VESICLES IN CULTURED EMBRYONIC CHICK SYMPATHETIC NEURONS

    PubMed Central

    Teichberg, Saul; Holtzman, Eric

    1973-01-01

    Cultured chick embryonic sympathetic neurons contain an extensive axonal network of sacs and tubules of agranular reticulum. The reticulum is also seen branching into networks in axon terminals and varicosities. The axonal reticulum and perikaryal endoplasmic reticulum resemble one another in their content of cytochemically demonstrable enzyme activities (G6Pase and IDPase) and in their characteristic membrane thicknesses (narrower than plasma membrane or some Golgi membranes). From the reticulum, both along the axon and at terminals, there appear to form dense-cored vesicles ranging in size from 400 to 1,000 Å in diameter. These vesicles behave pharmacologically and cytochemically like the classes of large and small catecholamine storage vesicles found in several adrenergic systems; for example, they can accumulate exogenous 5-hydroxydopamine. In addition, dense-cored vesicles at the larger (1,000 Å) end of the size spectrum appear to arise within perikaryal membrane systems associated with the Golgi apparatus; this is true also of very large (800–3,500 Å) dense-cored vesicles found in some perikarya. PMID:4347980

  13. Extracellular glutathione promotes migration of hydrogen peroxide-stressed cultured chick embryonic skin cells.

    PubMed

    Denunzio, Mia; Gomez, George

    2014-04-01

    The ability of glutathione to affect melanocyte survival has fostered its use in a variety of applications related to epithelial cells. Our study focused on fibroblast migration and the effects of oxidative stress. We used scratch assays to measure cell migration: fibroblasts were harvested from embryonic chicks, grown to confluence in a monolayer, and the layer was scratched to initiate migration. Migration rates were measured over 8 h using photomicrographs, and vinculin expression as an indicator focal adhesion formation was measured using immunofluorescence. Addition of 200 μM glutathione to the culture media in which the cells grew resulted in a significantly increased rate of scratch closure. When the scratch assays were performed in the presence of 100 μM H2O2 (to simulate oxidative stress), the cells ceased to migrate. Addition of 200 μM glutathione to the H2O2-treated scratched layers resulted in a restoration of the scratch closure capabilities. At the subcellular level, addition of extracellular glutathione resulted in a redistribution of vinculin into fewer but larger aggregates. In cells at the edge of scratched monolayers that were treated with H2O2, vinculin particles were distributed throughout the cell in smaller aggregates; addition of glutathione resulted in vinculin aggregates that were larger and closer to the edges of the cell, indicating that these cells were more migratory. Our results suggest that glutathione promotes fibroblast migration, possibly via a mechanism that promotes the formation of focal adhesions.

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

    PubMed

    Isomura, Takuya; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2015-12-01

    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

  15. Substance P receptors in primary cultures of cortical astrocytes from the mouse.

    PubMed Central

    Torrens, Y; Beaujouan, J C; Saffroy, M; Daguet de Montety, M C; Bergström, L; Glowinski, J

    1986-01-01

    Binding sites for substance P were labeled on intact cortical glial cells from newborn mice in primary culture using 125I-labeled Bolton-Hunter-labeled substance P. Maximal specific binding (95% of total binding) was reached after 2-3 weeks in culture. The binding was saturable, reversible, and temperature dependent. Scatchard and Hill analysis revealed a single population of noninteracting high-affinity binding sites (Kd, 0.33 nM; Bmax, 14.4 fmol per dish). Competition studies made with tachykinins and substance P analogues indicated that the characteristics of the 125I-labeled Bolton-Hunter labeled substance P binding sites on glial cells were identical to those on rat brain synaptosomes. 125I-labeled Bolton-Hunter labeled substance P binding sites were visualized by autoradiography, and differences in the intensity of labeling were seen among astrocytes. Substance P was found to stimulate phosphatidylinositol turnover; the EC50 value (0.36 nM) was identical to the IC50 value (0.38 nM) determined in binding studies. 125I-labeled Bolton-Hunter labeled substance P binding sites were also found on astrocytes derived from other brain structures and from the spinal cord of mice. Images PMID:2431412

  16. Assessment of general anaesthetic cytotoxicity in murine cortical neurones in dissociated culture.

    PubMed

    Campbell, Laura L; Tyson, Jennifer A; Stackpole, Emily E; Hokenson, Kristen E; Sherrill, Hanna; McKeon, Jeanne E; Kim, Sarah A; Edmands, Scott D; Suarez, Cristina; Hall, Adam C

    2011-04-28

    General anaesthetics are proposed to cause unconsciousness by modulating neuronal excitability in the mammalian brain through mechanisms that include enhancement of inhibitory GABA(A) receptor currents and suppression of excitatory glutamate receptor responses. Both intravenous and volatile agents may produce neurotoxic effects during early postnatal rodent brain development through similar mechanisms. In the following study, we investigated anaesthetic cytotoxicity in primary cortical neurones and glia from postnatal day 2-8 mice. Cultures at 4-20 days in vitro were exposed to combinations of ketamine (100 μM to 3 mM), nitrous oxide (75%, v/v) and/or isoflurane (1.5-5%, v/v) for 6-12 h. Neuronal survival and cell death were measured via microtubule associated protein 2 immunoassay and lactate dehydrogenase release assays, respectively. Clinically relevant anaesthetic concentrations of ketamine, nitrous oxide and isoflurane had no significant neurotoxic effects individually or when given as anaesthetic cocktails, even with up to 12 h exposure. This lack of neurotoxicity was observed regardless of whether cultures were prepared from postnatal day 0-2 or day 8 mice, and was also unaffected by number of days in vitro (DIV 4-20). Significant neurotoxic effects were only observed at supraclinical concentrations (e.g. 1-3 mM ketamine). Our study suggests that neurotoxicity previously reported in vivo is not due to direct cytotoxicity of anaesthetic agents, but results from other impacts of the anaesthetised state during early brain development. PMID:21277931

  17. 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

  18. Effects of gentiopicroside, sweroside and swertiamarine, secoiridoids from gentian (Gentiana lutea ssp. symphyandra), on cultured chicken embryonic fibroblasts.

    PubMed

    Oztürk, Nilgün; Korkmaz, Seval; Oztürk, Yusuf; Başer, K Hüsnü Can

    2006-03-01

    Wound healing properties of Gentian (Gentiana lutea ssp. symphyandra) extract and its main constituents, gentiopicroside, sweroside and swertiamarine (compounds 1-3, respectively) were evaluated by comparison with dexpanthenol on cultured chicken embryonic fibroblasts. The extract was also analyzed by HPLC to quantify its constituents. Chicken embryonic fibroblasts from fertilized eggs were incubated with the plant extract and its constituents, compounds 1-3. Using microscopy, mitotic ability, morphological changes and collagen production in the cultured fibroblasts were evaluated as parameters. Wound healing activity of Gentian seems to be mainly due to the increase in the stimulation of collagen production and the mitotic activity by compounds 2 and 3, respectively (p < 0.005 in all cases). All three compounds also exhibited cytoprotective effects, which may cause a synergism in terms of wound healing activity of Gentian. The findings demonstrated the wound healing activity of Gentian, which has previously been based only on ethnomedical data.

  19. 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.

  20. Exogenous nitric oxide enhances calcification in embryonic stem cell-derived osteogenic cultures.

    PubMed

    Ehnes, D D; Geransar, R M; Rancourt, D E; Zur Nieden, N I

    2015-01-01

    While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N(5)-(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stage-specific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases.

  1. 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.

  2. The accumulation of nontargeted quantum dots in cultured human embryonic kidney cells

    NASA Astrophysics Data System (ADS)

    Knight, V. Bleu; Serrano, Elba E.

    2006-02-01

    Quantum dots (QDs) could offer significant advantages in clinical settings due to their high photostability and quantum yield. We are investigating the uptake and compartmentalization of QDs by cells because these processes are not fully characterized and there is potential for heavy metal toxicity when semiconductor nanocrystals are sequestered. Here we demonstrate the intracellular accumulation of QDs in human embryonic kidney cells (HEK-293; ATCC) exposed to nontargeted (Qtracker 565nm; QDot Corp.) or targeted (Qtracker 565 Cell Labeling Kit; QDot Corp.) QDs. As expected, 10 nM targeted QDs (Lagerholm et al., 2004, Nano Letters, 4:2019-2022) accumulated in HEK-293 cells and normal human astrocytes (NHA; Cambrex Biosciences) after 1 hr, while nontargeted QDs (200 nM) could be detected after 24 hr in HEK-293 but not NHA. The uptake of 10 nM targeted QDs was greater than the uptake of 200 nM nontargeted QDs as confirmed by the number and intensity of puncta visible in HEK-293 cells imaged with confocal microscopy. QD uptake was not detected in two Xenopus kidney cell lines (XLK-WG and A6; ATCC) exposed to nontargeted QDs (10-500 nM) for 18 hours. Co-labeling of HEK-293 cultures with CellTracker Red CMTPX (Invitrogen) following QD uptake verified that QD accumulation does not affect cell viability. Differences in QD uptake between cell lines could be species-specific or due to different growth conditions. The unexpected accumulation of nontargeted QDs raises questions about the uptake mechanism and the intracellular location that are being investigated with TEM. Supported by NIH-NIDCD (DC003292) and NMSU-ADVANCE (NSF0123690) to EES.

  3. 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

  4. Toxicity evaluation of new agricultural fungicides in primary cultured cortical neurons.

    PubMed

    Regueiro, Jorge; Olguín, Nair; Simal-Gándara, Jesús; Suñol, Cristina

    2015-07-01

    Fungicides are crucial for food protection as well as for the production of crops of suitable quality and quantity to provide a viable economic return. Like other pesticides, fungicides are widely sprayed on agricultural land, especially in wine-growing areas, from where they can move-off after application. Furthermore, residues of these agrochemicals can remain on crops after harvest and even after some food processing operations, being a major exposure pathway. Although a relatively low toxicity has been claimed for this kind of compounds, information about their neurotoxicity is still scarce. In the present study, nine fungicides recently approved for agricultural uses in the EU - ametoctradin, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone and pyraclostrobin - have been evaluated for their toxicity in primary cultured mouse cortical neurons. Exposure to 0.1-100µM for 7 days in vitro resulted in a dose-dependent toxicity in the MTT cell viability assay. Strobilurin fungicides kresoxim-methyl (KR) and pyraclostrobin (PY) were the most neurotoxic compounds (lethal concentration 50 were in the low micromolar and nanomolar levels, respectively) causing a rapid raise in intracellular calcium [Ca(2+)]i and strong depolarization of mitochondrial membrane potential. KR- and PY-induced cell death was reversed by the calcium channels blockers MK-801 and verapamil, suggesting that calcium entry through NMDA receptors and voltage-operated calcium channels are involved in KR- and PY-induced neurotoxicity. These results highlight the need for further evaluation of their neurotoxic effects in vivo.

  5. 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.

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

    PubMed

    Beggs, John M; Plenz, Dietmar

    2004-06-01

    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.

  7. 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

  8. Scale-up of human embryonic stem cell culture using a hollow fibre bioreactor.

    PubMed

    Roberts, Iwan; Baila, Stefano; Rice, R Brent; Janssens, Michiel Etienne; Nguyen, Kim; Moens, Nathalie; Ruban, Ludmila; Hernandez, Diana; Coffey, Pete; Mason, Chris

    2012-12-01

    The commercialisation of human embryonic stem cell derived cell therapies for large patient populations is reliant on both minimising expensive and variable manual-handling methods whilst realising economies of scale. The Quantum Cell Expansion System, a hollow fibre bioreactor (Terumo BCT), was used in a pilot study to expand 60 million human embryonic stem cells to 708 million cells. Further improvements can be expected with optimisation of media flow rates throughout the run to better control the cellular microenvironment. High levels of pluripotency marker expression were maintained on the bioreactor, with 97.7 % of cells expressing SSEA-4 when harvested.

  9. 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

  10. 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

    2016-04-01

    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. PMID:27068738

  11. Isolation of swine influenza virus in cell cultures and embryonated chicken eggs.

    PubMed

    Zhang, Jianqiang; Gauger, Phillip C

    2014-01-01

    Influenza virus isolation is a procedure to obtain a live and infectious virus that can be used for antigenic characterization, pathogenesis investigation, and vaccine production. Embryonated chicken egg inoculation is traditionally considered the "gold standard" method for influenza virus isolation and propagation. However, many primary cells and continuous cell lines have also been examined or developed for influenza virus isolation and replication. Specifically, swine influenza virus (SIV) isolation and propagation have been attempted and compared in embryonated chicken eggs, some primary porcine cells, and a number of continuous cell lines. Currently Madin-Darby canine kidney (MDCK) cells remain the most commonly used cell line for isolation, propagation, and titration of SIV. Virus isolation in embryonated chicken eggs or in different cell lines offers alternative approaches when SIV isolation in MDCK cells is unsuccessful. Nasal swabs, lung tissues, and oral fluids are three major specimen types for SIV isolation. In this chapter, we describe the procedures of sample processing, SIV isolation in MDCK cells and in embryonated chicken eggs, as well as methods used for confirming the virus isolation results.

  12. 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.

  13. 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.

  14. Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.

    PubMed

    Zhu, Yu; Carido, Madalena; Meinhardt, Andrea; Kurth, Thomas; Karl, Mike O; Ader, Marius; Tanaka, Elly M

    2013-01-01

    A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia. PMID:23358448

  15. Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.

    PubMed

    Zhu, Yu; Carido, Madalena; Meinhardt, Andrea; Kurth, Thomas; Karl, Mike O; Ader, Marius; Tanaka, Elly M

    2013-01-01

    A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

  16. Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons

    PubMed Central

    1995-01-01

    The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n- acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more

  17. 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

  18. 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. PMID:26074427

  19. 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

  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. Direct Conversion of Pluripotent Human Embryonic Stem Cells Under Defined Culture Conditions into Human Neuronal or Cardiomyocyte Cell Therapy Derivatives.

    PubMed

    Parsons, Xuejun H

    2016-01-01

    Developing novel strategies for well-controlled efficiently directing pluripotent human embryonic stem cells (hESCs) exclusively and uniformly towards clinically relevant cell types in a lineage-specific manner is not only crucial for unveiling the molecular and cellular cues that direct human embryogenesis but also vital to harnessing the power of hESC biology for tissue engineering and cell-based therapies. Conventional hESC differentiation methods require uncontrollable simultaneous multi-lineage differentiation of pluripotent cells, which yield embryoid bodies (EB) or aggregates consisting of a mixed population of cell types of three embryonic germ layers, among which only a very small fraction of cells display targeted differentiation, impractical for commercial and clinical applications. Here, a protocol for lineage-specific differentiation of hESCs, maintained under defined culture systems, direct from the pluripotent stage using small-molecule induction exclusively and uniformly to a neural or a cardiac lineage is described. Lineage-specific differentiation of pluripotent hESCs by small-molecule induction enables well-controlled highly efficient direct conversion of nonfunctional pluripotent hESCs into a large supply of high-purity functional human neuronal or cardiomyocyte cell therapy derivatives for commercial and therapeutic uses.

  2. Fabrication of uniform-sized poly-ɛ-caprolactone microspheres and their applications in human embryonic stem cell culture.

    PubMed

    Li, Jian; Lam, Alan Tin-Lun; Toh, Jessica Pei Wen; Reuveny, Shaul; Oh, Steve Kah-Weng; Birch, William R

    2015-12-01

    The generation of liquefied poly-ɛ-caprolactone (PCL) droplets by means of a microfluidic device results in uniform-sized microspheres, which are validated as microcarriers for human embryonic stem cell culture. Formed droplet size and size distribution, as well as the resulting PCL microsphere size, are correlated with the viscosity and flow rate ratio of the dispersed (Q d) and continuous (Q c) phases. PCL in dichloromethane increases its viscosity with concentration and molecular weight. Higher viscosity and Q d/Q c lead to the formation of larger droplets, within two observed formation modes: dripping and jetting. At low viscosity of dispersed phase and Q d/Q c, the microfluidic device is operated in dripping mode, which generates droplets and microspheres with greater size uniformity. Solutions with lower molecular weight PCL have lower viscosity, resulting in a wider concentration range for the dripping mode. When coated with extracellular matrix (ECM) proteins, the fabricated PCL microspheres are demonstrated capable of supporting the expansion of human embryonic stem cells.

  3. Three-dimensional culture of human embryonic stem cell derived hepatic endoderm and its role in bioartificial liver construction.

    PubMed

    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.

  4. 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

  5. IL-10 Promotes Neurite Outgrowth and Synapse Formation in Cultured Cortical Neurons after the Oxygen-Glucose Deprivation via JAK1/STAT3 Pathway

    PubMed Central

    Chen, Hongbin; Lin, Wei; Zhang, Yixian; Lin, Longzai; Chen, Jianhao; Zeng, Yongping; Zheng, Mouwei; Zhuang, Zezhong; Du, Houwei; Chen, Ronghua; Liu, Nan

    2016-01-01

    As a classic immunoregulatory and anti-inflammatory cytokine, interleukin-10 (IL-10) provides neuroprotection in cerebral ischemia in vivo or oxygen-glucose deprivation (OGD)-induced injury in vitro. However, it remains blurred whether IL-10 promotes neurite outgrowth and synapse formation in cultured primary cortical neurons after OGD injury. In order to evaluate its effect on neuronal apoptosis, neurite outgrowth and synapse formation, we administered IL-10 or IL-10 neutralizing antibody (IL-10NA) to cultured rat primary cortical neurons after OGD injury. We found that IL-10 treatment activated the Janus kinase 1 (JAK1)/signal transducers and activators of transcription 3 (STAT3) signaling pathway. Moreover, IL-10 attenuated OGD-induced neuronal apoptosis by down-regulating the Bax expression and up-regulating the Bcl-2 expression, facilitated neurite outgrowth by increasing the expression of Netrin-1, and promoted synapse formation in cultured primary cortical neurons after OGD injury. These effects were partly abolished by JAK1 inhibitor GLPG0634. Contrarily, IL-10NA produced opposite effects on the cultured cortical neurons after OGD injury. Taken together, our findings suggest that IL-10 not only attenuates neuronal apoptosis, but also promotes neurite outgrowth and synapse formation via the JAK1/STAT3 signaling pathway in cultured primary cortical neurons after OGD injury. PMID:27456198

  6. Pharmacological profiling of native group II metabotropic glutamate receptors in primary cortical neuronal cultures using a FLIPR.

    PubMed

    Sanger, Helen; Hanna, Lydia; Colvin, Ellen M; Grubisha, Olivera; Ursu, Daniel; Heinz, Beverly A; Findlay, Jeremy D; Vivier, Richard G; Sher, Emanuele; Lodge, David; Monn, James A; Broad, Lisa M

    2013-03-01

    The group II metabotropic glutamate (mGlu) receptors comprised of the mGlu2 and mGlu3 receptor subtypes have gained recognition in recent years as potential targets for psychiatric disorders, including anxiety and schizophrenia. In addition to studies already indicating which subtype mediates the anxiolytic and anti-psychotic effects observed in disease models, studies to help further define the preferred properties of selective group II mGlu receptor ligands will be essential. Comparison of the in vitro properties of these ligands to their in vivo efficacy and tolerance profiles may help provide these additional insights. We have developed a relatively high-throughput native group II mGlu receptor functional assay to aid this characterisation. We have utilised dissociated primary cortical neuronal cultures, which after 7 days in vitro have formed functional synaptic connections and display periodic and spontaneous synchronised calcium (Ca(2+)) oscillations in response to intrinsic action potential bursts. We herein demonstrate that in addition to non-selective group II mGlu receptor agonists, (2R,4R)-APDC, LY379268 and DCG-IV, a selective mGlu2 agonist, LY541850, and mGlu2 positive allosteric modulators, BINA and CBiPES, inhibit the frequency of synchronised Ca(2+) oscillations in primary cultures of rat and mouse cortical neurons. Use of cultures from wild-type, mGlu2(-/-), mGlu3(-/-) and mGlu2/3(-/-) mice allowed us to further probe the contribution of mGlu2 and mGlu3, and revealed LY541850 to be a partial mGlu2 agonist and a full mGlu3 antagonist. Overnight pre-treatment of cultures with these ligands revealed a preferred desensitisation profile after treatment with a positive allosteric modulator. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22659090

  7. Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells

    PubMed Central

    Ozone, Chikafumi; Suga, Hidetaka; Eiraku, Mototsugu; Kadoshima, Taisuke; Yonemura, Shigenobu; Takata, Nozomu; Oiso, Yutaka; Tsuji, Takashi; Sasai, Yoshiki

    2016-01-01

    Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine. PMID:26762480

  8. Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells.

    PubMed

    Ozone, Chikafumi; Suga, Hidetaka; Eiraku, Mototsugu; Kadoshima, Taisuke; Yonemura, Shigenobu; Takata, Nozomu; Oiso, Yutaka; Tsuji, Takashi; Sasai, Yoshiki

    2016-01-01

    Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine. PMID:26762480

  9. 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...

  10. 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.

  11. Pigmented epithelium induces complete retinal reconstitution from dispersed embryonic chick retinae in reaggregation culture.

    PubMed Central

    Rothermel, A; Willbold, E; Degrip, W J; Layer, P G

    1997-01-01

    Reaggregation of dispersed retinal cells of the chick embryo leads to histotypic retinospheroids in which the laminar organization remains incomplete: photoreceptors form rosettes which are surrounded by constituents of the other retinal layers. Here, for the first time, a complete arrangement of layers is achieved in cellular spheres (stratoids), provided that fully dispersed retinal cells are younger than embryonic day E6, and are reaggregated in the presence of a monolayer of retinal pigmented epithelium (RPE). A remarkable mechanism of stratoid formation from 1 to 15 days in vitro is revealed by the establishment of a radial Müller glia scaffold and of photoreceptors. During the first two days of reaggregation on RPE, rosettes are still observed. At this stage immunostaining with vimentin and F11 antibodies for radial Müller glia reveal a disorganized pattern. Subsequently, radial glia processes organize into long parallel fibre bundles which are arranged like spokes to stabilize the surface and centre of the stratoid. The opsin-specific antibody CERN 901 detects photoreceptors as they gradually build up an outer nuclear layer at the surface. These findings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina. PMID:9332014

  12. Continuous Hypoxic Culturing of Human Embryonic Stem Cells Enhances SSEA-3 and MYC Levels

    PubMed Central

    Laiho, Asta; Rahkonen, Nelly; Emani, Maheswara Reddy; Viitala, Miro; Laurila, Kirsti; Sahla, Roosa; Lund, Riikka; Lähdesmäki, Harri; Jaakkola, Panu; Lahesmaa, Riitta

    2013-01-01

    Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However, it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9, HS401 and HS360) on short (2 hours), intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore, the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs, were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover, transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further, MYC protein expression in hypoxia was affected by silencing HIF2α, but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state. PMID:24236059

  13. Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation*

    PubMed Central

    Meade, Kate A.; White, Kathryn J.; Pickford, Claire E.; Holley, Rebecca J.; Marson, Andrew; Tillotson, Donna; van Kuppevelt, Toin H.; Whittle, Jason D.; Day, Anthony J.; Merry, Catherine L. R.

    2013-01-01

    As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells. PMID:23235146

  14. GLUT3 and PKM2 regulate OCT4 expression and support the hypoxic culture of human embryonic stem cells.

    PubMed

    Christensen, David R; Calder, Philip C; Houghton, Franchesca D

    2015-12-07

    Human embryonic stem cells (hESCs) have the capacity to differentiate into all cell types and thus have great potential for regenerative medicine. hESCs cultured at low oxygen tensions are more pluripotent and display an increased glycolytic rate but how this is regulated is unknown. This study therefore aimed to investigate the regulation of glucose metabolism in hESCs and whether this might impact OCT4 expression. In contrast to the glucose transporter GLUT1, GLUT3 was regulated by environmental oxygen and localised to hESC membranes. Silencing GLUT3 caused a reduction in glucose uptake and lactate production as well as OCT4 expression. GLUT3 and OCT4 expression were correlated suggesting that hESC self-renewal is regulated by the rate of glucose uptake. Surprisingly, PKM2, a rate limiting enzyme of glycolysis displayed a nuclear localisation in hESCs and silencing PKM2 did not alter glucose metabolism suggesting a role other than as a glycolytic enzyme. PKM2 expression was increased in hESCs cultured at 5% oxygen compared to 20% oxygen and silencing PKM2 reduced OCT4 expression highlighting a transcriptional role for PKM2 in hESCs. Together, these data demonstrate two separate mechanisms by which genes regulating glucose uptake and metabolism are involved in the hypoxic support of pluripotency in hESCs.

  15. Chitosan scaffolds for in vitro buffalo embryonic stem-like cell culture: an approach to tissue engineering.

    PubMed

    Thein-Han, Wah W; Kitiyanant, Yindee

    2007-01-01

    Three-dimensional (3D) porous chitosan scaffolds are attractive candidates for tissue engineering applications. Chitosan scaffolds of 70, 88, and 95% degree of deacetylation (% DD) with the same molecular weight were developed and their properties with buffalo embryonic stem-like (ES-like) cells were investigated in vitro. Scaffolds were fabricated by freezing and lyophilization. They showed open pore structure with interconnecting pores under scanning electron microscopy (SEM). Higher % DD chitosan scaffolds had greater mechanical strength, slower degradation rate, lower water uptake ability, but similar water retention ability, when compared to lower % DD chitosan. As a strategy to tissue engineering, buffalo ES-like cells were cultured on scaffolds for 28 days. It appeared that chitosan was cytocompatible and cells proliferated well on 88 and 95% DD scaffolds. In addition, the buffalo ES-like cells maintained their pluripotency during the culture period. Furthermore, the SEM and histological study showed that the polygonal buffalo ES-like cells proliferated well and attached to the pores. This study proved that 3D biodegradable highly deacetylated chitosan scaffolds are promising candidates for ES-like cell based tissue engineering and this chitosan scaffold and ES cell based system can be used as in vitro model for subsequent clinical applications.

  16. Three diterpenes from Leonurus japonicus Houtt protect primary cultured rat cortical cells from glutamate-induced toxicity.

    PubMed

    Moon, Hyung-In

    2010-08-01

    To examine the cytoprotective effects of Leonurus japonicus Houtt, its protection against the glutamate-induced toxicity in primary cortical cultured neurons was tested. In order to clarify the cytoprotective mechanism(s) of this observed effect, isolation was performed to seek and identify active fractions and components. By such fractionation, three known diterpenes--prehispanolone (1), leojaponin (2) and iso-preleoheterin (3)--were isolated from the methanol extracts from the air-dried L. japonicus. Among these three compounds, leojaponin (2) exhibited significant cytoprotective activities against glutamate-induced toxicity, exhibiting cell viability of about 50%, at concentrations ranging from 0.1 microm to 10 microm. Therefore, the cytoprotective effect of L. japonicus might be due to the inhibition of glutamate-induced toxicity by the diterpenes it contains.

  17. Micro-electrode array recordings reveal reductions in both excitation and inhibition in cultured cortical neuron networks lacking Shank3.

    PubMed

    Lu, C; Chen, Q; Zhou, T; Bozic, D; Fu, Z; Pan, J Q; Feng, G

    2016-02-01

    Numerous risk genes have recently been implicated in susceptibility to autism and schizophrenia. Translating such genetic findings into disease-relevant neurobiological mechanisms is challenging due to the lack of throughput assays that can be used to assess their functions on an appropriate scale. To address this issue, we explored the feasibility of using a micro-electrode array (MEA) as a potentially scalable assay to identify the electrical network phenotypes associated with risk genes. We first characterized local and global network firing in cortical neurons with MEAs, and then developed methods to analyze the alternation between the network active period (NAP) and the network inactive period (NIP), each of which lasts tens of seconds. We then evaluated the electric phenotypes of neurons derived from Shank3 knockout (KO) mice. Cortical neurons cultured on MEAs displayed a rich repertoire of spontaneous firing, and Shank3 deletion led to reduced firing activity. Enhancing excitation with CX546 rescued the deficit in the spike rate in the Shank3 KO network. In addition, the Shank3 KO network produced a shorter NIP, and this altered network firing pattern was normalized by clonazepam, a positive modulator of the GABAA receptor. MEA recordings revealed electric phenotypes that displayed altered excitation and inhibition in the network lacking Shank3. Thus, our study highlights MEAs as an experimental framework for measuring multiple robust neurobiological end points in dynamic networks and as an assay system that could be used to identify electric phenotypes in cultured neuronal networks and to analyze additional risk genes identified in psychiatric genetics. PMID:26598066

  18. 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.

  19. 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.

  20. [Effect of ascorbic acid, epidermal growth factor and follicle stimulating hormone on in vitro culture of sheep ovarian cortical tissue].

    PubMed

    Peng, Xiayu; Wang, Liqin; Yang, Mei; Chen, Tong; Guo, Zhiqin

    2010-06-01

    In this study, we evaluated the effects of ascorbic acid (VC), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) on in vitro culture of sheep ovarian cortical tissue. Using 2 x 2 x 2 factor experimental design, we cultured sheep ovarian cortex fragments in 8 media with MEM (control), MEM+VC (50 microg/mL), MEM +EGF (100 ng/mL), MEM+FSH (50 ng/mL), MEM+VC+EGF, MEM+VC+FSH, MEM+EGF+FSH, MEM+VC+EGF+FSH. After 0 (non-cultured control), 2, 6, 12 days of culture, the pieces of ovarian cortex were proceed to histological and proliferating cell nuclear antigen (PCNA) examination, or observed by transmission electron microscopy (TEM). The percentages of developing follicles were increased (P < 0.05) and the percentages of healthy follicles were reduced (P < 0.05). When compared to the MEM group, the addition of FSH with VC or EGF promoted a significant increase of follicles diameter and follicles survival rate (P < 0.05), and stimulated the proliferation of granulosa cells. After 12 days of culture, medium supplemented with MEM+VC+EGF resulted the lowest proportion of developing follicles (49.3% +/- 3.2%), follicles diameter((32.3 +/- 2.3) microm), follicles survival rate (41.6% +/- 3.1%) and the proportion of PCNA stained follicles (26.4% +/- 1.2%, P < 0.05). In contrast, MEM+VC+EGF+FSH resulted the highest follicles diameter ((42.5 +/- 5.1) microm), follicles survival rate (59.7% +/- 6.1%) and proportion of PCNA stained follicles (43.5% +/- 4.1%, P < 0.05). Ultrastructural analysis confirmed the integrity of follicles cultured in VC+EGF+FSH group, while follicles cultured in MEM+VC+EGF groups showed more degeneration characters. In conclusion, the addition of VC and EGF to culture medium inhibited follicular development, VC+EGF+FSH was the most effective treatment to maintain follicular integrity and promote sheep primordial follicular activation and growth during in vitro culture.

  1. 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

  2. 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...

  3. 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.

  4. Rat embryonic hippocampus and induced pluripotent stem cell derived cultured neurons recover from laser-induced subaxotomy

    PubMed Central

    Selfridge, Aaron; Chiang, Chai-Chun; Reyna, Sol M.; Weissmiller, April M.; Shi, Linda Z.; Preece, Daryl; Mobley, William C.; Berns, Michael W.

    2015-01-01

    Abstract. Axonal injury and stress have long been thought to play a pathogenic role in a variety of neurodegenerative diseases. However, a model for studying single-cell axonal injury in mammalian cells and the processes of repair has not been established. The purpose of this study was to examine the response of neuronal growth cones to laser-induced axonal damage in cultures of embryonic rat hippocampal neurons and induced pluripotent stem cell (iPSC) derived human neurons. A 532-nm pulsed Nd:YVO4 picosecond laser was focused to a diffraction limited spot at a precise location on an axon using a laser energy/power that did not rupture the cell membrane (subaxotomy). Subsequent time series images were taken to follow axonal recovery and growth cone dynamics. After laser subaxotomy, axons thinned at the damage site and initiated a dynamic cytoskeletal remodeling process to restore axonal thickness. The growth cone was observed to play a role in the repair process in both hippocampal and iPSC-derived neurons. Immunofluorescence staining confirmed structural tubulin damage and revealed initial phases of actin-based cytoskeletal remodeling at the damage site. The results of this study indicate that there is a repeatable and cross-species repair response of axons and growth cones after laser-induced damage. PMID:26157985

  5. 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. PMID:21354615

  6. Silver nanoparticles (AgNPs) cause degeneration of cytoskeleton and disrupt synaptic machinery of cultured cortical neurons

    PubMed Central

    2013-01-01

    Background Silver nanoparticles (AgNPs), owing to their effective antimicrobial properties, are being widely used in a broad range of applications. These include, but are not limited to, antibacterial materials, the textile industry, cosmetics, coatings of various household appliances and medical devices. Despite their extensive use, little is known about AgNP safety and toxicity vis-à-vis human and animal health. Recent studies have drawn attention towards potential neurotoxic effects of AgNPs, however, the primary cellular and molecular targets of AgNP action/s remain to be defined. Results Here we examine the effects of ultra fine scales (20 nm) of AgNPs at various concentrations (1, 5, 10 and 50 μg/ml) on primary rat cortical cell cultures. We found that AgNPs (at 1-50 μg/ml) not only inhibited neurite outgrowth and reduced cell viability of premature neurons and glial cells, but also induced degeneration of neuronal processes of mature neurons. Our immunocytochemistry and confocal microscopy studies further demonstrated that AgNPs induced the loss of cytoskeleton components such as the β-tubulin and filamentous actin (F-actin). AgNPs also dramatically reduced the number of synaptic clusters of the presynaptic vesicle protein synaptophysin, and the postsynaptic receptor density protein PSD-95. Finally, AgNP exposure also resulted in mitochondria dysfunction in rat cortical cells. Conclusions Taken together, our data show that AgNPs induce toxicity in neurons, which involves degradation of cytoskeleton components, perturbations of pre- and postsynaptic proteins, and mitochondrial dysfunction leading to cell death. Our study clearly demonstrates the potential detrimental effects of AgNPs on neuronal development and physiological functions and warns against its prolific usage. PMID:23782671

  7. 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…

  8. Synergistic Effects of Hypoxia and Morphogenetic Factors on Early Chondrogenic Commitment of Human Embryonic Stem Cells in Embryoid Body Culture

    PubMed Central

    Yodmuang, Supansa; Marolt, Darja; Marcos-Campos, Ivan; Gadjanski, Ivana

    2015-01-01

    Derivation of articular chondrocytes from human stem cells would advance our current understanding of chondrogenesis, and accelerate development of new stem cell therapies for cartilage repair. Chondrogenic differentiation of human embryonic stem cells (hESCs) has been studied using supplemental and cell-secreted morphogenetic factors. The use of bioreactors enabled insights into the effects of physical forces and controlled oxygen tension. In this study, we investigated the interactive effects of controlled variation of oxygen tension and chondrocyte-secreted morphogenetic factors on chondrogenic differentiation of hESCs in the embryoid body format (hESC-EB). Transient hypoxic culture (2 weeks at 5 % O2 followed by 1 week at 21 % O2) of hESC-EBs in medium conditioned with primary chondrocytes up-regulated the expression of SOX9 and suppressed pluripotent markers OCT4 and NANOG. Pellets derived from these cells showed significant up-regulation of chondrogenic genes (SOX9, COL2A1, ACAN) and enhanced production of cartilaginous matrix (collagen type II and proteoglycan) as compared to the pellets from hESC-EBs cultured under normoxic conditions. Gene expression profiles corresponded to those associated with native cartilage development, with early expression of N-cadherin (indicator of cell condensation) and late expression of aggrecan (ACAN, indicator of proteoglycan production). When implanted into highly vascularized subcutaneous area in immunocompromised mice for 4 weeks, pellets remained phenotypically stable and consisted of cartilaginous extracellular matrix (ECM), without evidence of dedifferentiation or teratoma formation. Based on these results, we propose that chondrogenesis in hESC can be synergistically enhanced by a control of oxygen tension and morphogenetic factors secreted by chondrocytes. PMID:25618295

  9. Distinct activation profiles in microglia of different ages: a systematic study in isolated embryonic to aged microglial cultures.

    PubMed

    Lai, A Y; Dibal, C D; Armitage, G A; Winship, I R; Todd, K G

    2013-12-19

    Microglia have been implicated in disease progression for several age-related brain disorders. However, while microglia's contribution to the progression of these disorders is accepted, the effect of aging on their endogenous cellular characteristics has received limited attention. In fact, a comprehensive study of how the structure and function of microglia changes as a function of developmental age has yet to be performed. Here, we describe the functional response characteristics of primary microglial cultures prepared from embryonic, neonatal (Neo), 2-3month-old, 6-8month-old, 9-11month-old, and 13-15month-old rats. Microglial morphology, glutamate (GLU) uptake, and release of trophic and inflammatory factors were assessed under basal conditions and in microglia activated with adenosine 5'-triphosphate (ATP) or lipopolysaccharide. We found that microglia from different age groups were both morphologically and functionally distinct. Upon activation by ATP, Neo microglia were the most reactive, upregulating nitric oxide, tumor necrosis factor-α, and brain-derived neurotrophic factor release as well as GLU uptake. This upregulation translated into neurotoxicity in microglia-neuron co-cultures that were not observed with microglia of different developmental ages. Interestingly, 13-15month-old microglia exhibited similar activation profiles to Neo microglia, whereas microglia from younger adults and embryos were activated less by ATP. Our data also identify age-dependent differences in purinergic receptor subtype expression that contribute to the regulation of neuronal survival. Combined, our data demonstrate that microglial activation and purinergic receptor profiles vary non-linearly with developmental age, a potentially important finding for studies examining the role of microglia in neurodegenerative disorders.

  10. Synergistic effects of hypoxia and morphogenetic factors on early chondrogenic commitment of human embryonic stem cells in embryoid body culture.

    PubMed

    Yodmuang, Supansa; Marolt, Darja; Marcos-Campos, Ivan; Gadjanski, Ivana; Vunjak-Novakovic, Gordana

    2015-04-01

    Derivation of articular chondrocytes from human stem cells would advance our current understanding of chondrogenesis, and accelerate development of new stem cell therapies for cartilage repair. Chondrogenic differentiation of human embryonic stem cells (hESCs) has been studied using supplemental and cell-secreted morphogenetic factors. The use of bioreactors enabled insights into the effects of physical forces and controlled oxygen tension. In this study, we investigated the interactive effects of controlled variation of oxygen tension and chondrocyte-secreted morphogenetic factors on chondrogenic differentiation of hESCs in the embryoid body format (hESC-EB). Transient hypoxic culture (2 weeks at 5 % O2 followed by 1 week at 21 % O2) of hESC-EBs in medium conditioned with primary chondrocytes up-regulated the expression of SOX9 and suppressed pluripotent markers OCT4 and NANOG. Pellets derived from these cells showed significant up-regulation of chondrogenic genes (SOX9, COL2A1, ACAN) and enhanced production of cartilaginous matrix (collagen type II and proteoglycan) as compared to the pellets from hESC-EBs cultured under normoxic conditions. Gene expression profiles corresponded to those associated with native cartilage development, with early expression of N-cadherin (indicator of cell condensation) and late expression of aggrecan (ACAN, indicator of proteoglycan production). When implanted into highly vascularized subcutaneous area in immunocompromised mice for 4 weeks, pellets remained phenotypically stable and consisted of cartilaginous extracellular matrix (ECM), without evidence of dedifferentiation or teratoma formation. Based on these results, we propose that chondrogenesis in hESC can be synergistically enhanced by a control of oxygen tension and morphogenetic factors secreted by chondrocytes.

  11. Uptake and release of [3H]gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture

    PubMed Central

    1979-01-01

    We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio. PMID:457763

  12. 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

  13. Electrochemical lactate biosensor based upon chitosan/carbon nanotubes modified screen-printed graphite electrodes for the determination of lactate in embryonic cell cultures.

    PubMed

    Hernández-Ibáñez, Naiara; García-Cruz, Leticia; Montiel, Vicente; Foster, Christopher W; Banks, Craig E; Iniesta, Jesús

    2016-03-15

    l-lactate is an essential metabolite present in embryonic cell culture. Changes of this important metabolite during the growth of human embryo reflect the quality and viability of the embryo. In this study, we report a sensitive, stable, and easily manufactured electrochemical biosensor for the detection of lactate within embryonic cell cultures media. Screen-printed disposable electrodes are used as electrochemical sensing platforms for the miniaturization of the lactate biosensor. Chitosan/multi walled carbon nanotubes composite have been employed for the enzymatic immobilization of the lactate oxidase enzyme. This novel electrochemical lactate biosensor analytical efficacy is explored towards the sensing of lactate in model (buffer) solutions and is found to exhibit a linear response towards lactate over the concentration range of 30.4 and 243.9 µM in phosphate buffer solution, with a corresponding limit of detection (based on 3-sigma) of 22.6 µM and exhibits a sensitivity of 3417 ± 131 µAM(-1) according to the reproducibility study. These novel electrochemical lactate biosensors exhibit a high reproducibility, with a relative standard deviation of less than 3.8% and an enzymatic response over 82% after 5 months stored at 4 °C. Furthermore, high performance liquid chromatography technique has been utilized to independently validate the electrochemical lactate biosensor for the determination of lactate in a commercial embryonic cell culture medium providing excellent agreement between the two analytical protocols. PMID:26579934

  14. Electrochemical lactate biosensor based upon chitosan/carbon nanotubes modified screen-printed graphite electrodes for the determination of lactate in embryonic cell cultures.

    PubMed

    Hernández-Ibáñez, Naiara; García-Cruz, Leticia; Montiel, Vicente; Foster, Christopher W; Banks, Craig E; Iniesta, Jesús

    2016-03-15

    l-lactate is an essential metabolite present in embryonic cell culture. Changes of this important metabolite during the growth of human embryo reflect the quality and viability of the embryo. In this study, we report a sensitive, stable, and easily manufactured electrochemical biosensor for the detection of lactate within embryonic cell cultures media. Screen-printed disposable electrodes are used as electrochemical sensing platforms for the miniaturization of the lactate biosensor. Chitosan/multi walled carbon nanotubes composite have been employed for the enzymatic immobilization of the lactate oxidase enzyme. This novel electrochemical lactate biosensor analytical efficacy is explored towards the sensing of lactate in model (buffer) solutions and is found to exhibit a linear response towards lactate over the concentration range of 30.4 and 243.9 µM in phosphate buffer solution, with a corresponding limit of detection (based on 3-sigma) of 22.6 µM and exhibits a sensitivity of 3417 ± 131 µAM(-1) according to the reproducibility study. These novel electrochemical lactate biosensors exhibit a high reproducibility, with a relative standard deviation of less than 3.8% and an enzymatic response over 82% after 5 months stored at 4 °C. Furthermore, high performance liquid chromatography technique has been utilized to independently validate the electrochemical lactate biosensor for the determination of lactate in a commercial embryonic cell culture medium providing excellent agreement between the two analytical protocols.

  15. 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

  16. Evaluation of Chang's culture medium for mouse in vitro fertilization and embryonic development.

    PubMed

    Ariff, B; Ng, S C; Mok, H; Lim, M N; Wong, P C; Shan, R

    1988-04-01

    Chang's medium [with and without human serum (HS)] was compared with T6 medium [with and without bovine serum albumin (BSA)] for in vitro fertilization (IVF) and development of two-cell mouse embryos to the blastocyst stage. Chang's medium without any supplementation gave significantly better fertilization rates (83.3%) than Chang's with 10% HS (76.4%) or T6 and BSA (76.6%) (P less than 0.01). In a separate experiment 87.7% of the two-cell mouse embryos developed to the blastocyst stage in Chang's medium, compared to 90.6% for T6 with BSA and 93.6% without BSA, respectively (P greater than 0.01). In Chang's medium supplemented with 10% HS, 76.6% of the embryos developed to the blastocyst stage and 17.2% stopped development after the morula stage. After 72 hr in vitro hatched trophoblast and inner-cell-mass cells from 26.5 and 30.8% of the embryos grown in Chang's medium (with and without HS) attached to the plastic culture dishes and grew to form a mixed monolayer of epithelioid and fibroblastic cells. Chang's medium can thus be successfully used for IVF and growth of mammalian embryos. Further, inner cell mass and trophoblast cell lines could be established for various reproductive studies using this medium.

  17. Development of electrophysiological and biochemical membrane properties during differentiation of embryonic skeletal muscle in culture.

    PubMed Central

    Spector, I; Prives, J M

    1977-01-01

    Newly fused chick myotubes undergo simultaneous and rapid changes in cell membrane properties during synchronous differentiation in culture. These changes are coordinately regulated and include increases in acetylcholine receptor, acetylcholinesterase, and resting potential, as well as the appearance of action potentials in discrete membrane areas upon stimulation. Subsequently, the acetylcholine receptor reaches maximal levels, whereas the development of electrical properties is marked by a further increase in resting potential, changes in the characteristics of the elicited action potential, and the recruitment of additional membrane areas for action potential generation. Maturation of electrical excitability, marked by the acquisition of the ability to fire repetitively and to conduct action potentials along the membrane, occurs well after resting potential has reached a maximum. During post-maturational development, myotubes exhibit spontaneous electrical and contractile activity, and levels of acetylcholine receptor accessible to externally applied 125I-labeled alpha-bungarotoxin decrease markedly. It is suggested that electrophysiological membrane maturation is autonomously regulated with no requirement for neuronal intervention and involves the coordinated biosynthesis of discrete membrane components and their subsequent organization in the myotube membrane. Images PMID:270755

  18. 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

  19. Neuroprotective effects of triterpene glycosides from glycine max against glutamate induced toxicity in primary cultured rat cortical cells.

    PubMed

    Moon, Hyung-In; Lee, Jai-Heon

    2012-01-01

    To examine the neuroprotective effects of Glycine max, we tested its protection against the glutamate-induced toxicity in primary cortical cultured neurons. In order to clarify the neuroprotective mechanism(s) of this observed effect, isolation was performed to seek and identify active fractions and components. From such fractionation, two triterpene glycosides, 3-O-[α-l-rhamnopyranosyl(1-2)-β-d-glucopyranosyl(1-2)-β-d-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (1) and 3-O-[β-d-glucopyranosyl(1-2)-β-d-galactopyranosyl(1-2)-β-d-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (2) were isolated with the methanol extracts with of air-dried Glycine max. Among these compounds, compound 2 exhibited significant neuroprotective activities against glutamate-induced toxicity, exhibiting cell viability of about 50% at concentrations ranging from 0.1 μM to 10 μM. Therefore, the neuroprotective effect of Glycine max might be due to the inhibition of glutamate-induced toxicity by triterpene glycosides.

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

    PubMed

    Yuan, Shi-Ying; Liu, Jue; Zhou, Jun; 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

  1. NMDA-mediated and self-induced bdnf exon IV transcriptions are differentially regulated in cultured cortical neurons.

    PubMed

    Zheng, Fei; Wang, Hongbing

    2009-01-01

    Activity-dependent transcriptional up-regulation of bdnf (brain-derived neurotrophic factor) is involved in regulating many aspects of neuronal functions. The NMDA (N-methyl-D-aspartic acid)-mediated and BDNF-mediated exon IV transcription may represent mechanistically different responses, and relevant to activity-dependent changes in neurons. We found that the activities of ERK (extracellular signal regulated kinase), CaM KII/IV (calmodulin-dependent protein kinase II and IV), PI3K (phosphoinositide 3-kinase), and PLC (phospholipase C) are required for NMDA receptor-mediated bdnf exon IV transcription in cultured cortical neurons. In contrast, the BDNF-induced and TrkB-dependent exon IV transcription was regulated by ERK and CaM KII/IV, but not by PI3K and PLC. While ERK and CaM KII/IV are separate signaling pathways in BDNF-stimulated neurons, CaM KII/IV appeared to regulate exon IV transcription through ERK in NMDA-stimulated neurons. Similarly, the PI3K and PLC signaling pathways converged on ERK in NMDA- but not BDNF-stimulated neurons. Our results implicate that the NMDA-induced and the self-maintenance of bdnf transcription are differentially regulated.

  2. 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

  3. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells.

    PubMed

    Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T; Henningsen, Jeanette; Kratchmarova, Irina; Kassem, Moustapha; Blagoev, Blagoy

    2009-05-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.

  4. Effects of estradiol and IGF-1 on the sodium calcium exchanger in rat cultured cortical neurons.

    PubMed

    Sánchez, Julio C; López-Zapata, Diego F; Francis, Liliana; De Los Reyes, Lina

    2011-05-01

    The Na(+)/Ca(2+) exchanger (NCX) is an important bidirectional transporter of calcium in neurons and has been shown to be involved in neuroprotection. Calcium can activate a number of cascades that can result in apoptosis and cell death, and NCX is a key factor in regulating the cytoplasmic concentration of this ion. 17-β-estradiol and insulin-like growth factor 1 (IGF-1) are known neuroprotective hormones with interacting mechanisms and effects on intracellular calcium; however, their relationship with the NCX has not been explored. In this article, the effects of these two hormones on neuronal NCX were tested using the whole-cell patch clamp technique on rat primary culture neurons. Both 17-β-estradiol and IGF-1 produced an increase in the NCX-mediated inward current and a decrease in the NCX-mediated outward current. However, the IGF-1 effect was lower than that of 17-β-estradiol, and the effect of both agents together was greater than the sum of each agent alone. Neither of the agents affected the pattern of regulation by extracellular or intrapipette calcium. Inhibitors of the IGF-1 and 17-β-estradiol receptors and inhibitors of the main signaling pathways failed to change the observed effects, indicating that these actions were not mediated by the classical receptors of these hormones. These effects on the NCX could be a mechanism explaining the neuroprotective actions of 17-β-estradiol and IGF-1, and these findings could help researchers to understand the role of the NCX in neuroprotection.

  5. Effects of transforming growth factor beta1 (TGFbeta-1) and dentin non-collagenous proteins (DNCP) on human embryonic ectomesenchymal cells in a three-dimensional culture system.

    PubMed

    Deng, Manjing; Shi, Junnan; Smith, Anthony J; Jin, Yan

    2005-11-01

    Cranial neural crest-derived ectomesenchymal cells represent a population of pluripotent stem cells giving rise to many of the various oro-facial and dental tissues. The factors determining the terminal fate of these cells are still unclear. The potentiality of human embryonic ectomesenchymal cells from the first branchial arch have been investigated when isolated and grown in a three-dimensional (3D)-collagen gel culture system in the presence of dentin matrix-derived non-collagenous proteins (DNCP) and TGFbeta-1. Functional differentiation of cells showing some characteristics of odontoblast-like cells could be observed when the cells were cultured with DNCP+TGFbeta-1 or DNCP, however, only cytological differentiation was observed during culture with TGFbeta-1 alone. The characteristics of these cells was assessed by morphological appearance, expression of the odontoblast phenotype marker dentin sialophosphoprotein (DSPP), increased alkaline phosphatase levels and formation of mineralised nodules in vitro. The results indicate that these embryonic cells from the first branchial arch are capable of responding to the inductive stimulus of DNCP or DNCP+TGFbeta-1 when isolated and grown in the 3D collagen gel culture system. The capacity of the isolated cells to differentiate into mineralizing cells showing some characteristics of odontoblast-like cells under these growth conditions highlights the potential of such approaches for tissue engineering strategies for hard-tissue regeneration after injury. PMID:15871903

  6. alpha-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with alpha 7 subunits and up-regulation by chronic nicotine treatment.

    PubMed

    Barrantes, G E; Rogers, A T; Lindstrom, J; Wonnacott, S

    1995-02-20

    High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [3H]nicotine or [3H]cytisine to live cells in situ was detected although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [3H]QNB were present at a density of 0.75 pmol/mg protein. [125I]alpha-Bungarotoxin (alpha Bgt) bound to hippocampal cultures with a Bmax of 128 fmol/mg protein and a Kd of 0.6 nM; cortical cultures expressed five times fewer [125I]alpha-Bgt binding sites. Fluorescence cytochemistry with rhodamine-alpha-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 x 10(4) and 2 x 10(4) binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat alpha 7 nicotinic receptor subunit) and rhodamine-alpha-Bgt gave coincident labelling patterns, supporting the correlation between the alpha 7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 microM nicotine for 14 days elicited a 40% increase in the numbers of [125I]alpha-Bgt binding sites, mimicking the up-regulation observed in in vivo studies. Primary cultures offer a useful in vitro system for investigating the expression and regulation of brain alpha-Bgt-sensitive receptors.

  7. 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.

  8. Autophagy Activation Is Involved in 3,4-Methylenedioxymethamphetamine (‘Ecstasy’)—Induced Neurotoxicity in Cultured Cortical Neurons

    PubMed Central

    Li, I-Hsun; Ma, Kuo-Hsing; Weng, Shao-Ju; Huang, Shiang-Suo; Liang, Chang-Min; Huang, Yuahn-Sieh

    2014-01-01

    Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation. PMID:25551657

  9. 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.

  10. Chronic Alcohol Exposure Decreases 53BP1 Protein Levels Leading to a Defective DNA Repair in Cultured Primary Cortical Neurons.

    PubMed

    Romero, Ana M; Palanca, Ana; Ruiz-Soto, Maria; Llorca, Javier; Marín, María P; Renau-Piqueras, Jaime; Berciano, Maria T; Lafarga, Miguel

    2016-01-01

    Chronic alcohol consumption may cause neurodevelopmental and neurodegenerative disorders. Alcohol neurotoxicity is associated with the production of acetaldehyde and reactive oxygen species that induce oxidative DNA damage. However, the molecular mechanisms by which ethanol disturbs the DNA damage response (DDR), resulting in a defective DNA repair, remain unknown. Here, we have used cultured primary cortical neurons exposed to 50 or 100 mM ethanol for 7 days to analyze the ethanol-induced DDR. Ethanol exposure produced a dose-dependent generation of double strand breaks and the formation of DNA damage foci immunoreactive for the histone γH2AX, a DNA damage marker, and for the ubiquitylated H2A, which is involved in chromatin remodeling at DNA damage sites. Importantly, these DNA damage foci failed to recruit the protein 53BP1, a crucial DNA repair factor. This effect was associated with a drop in 53BP1 mRNA and protein levels and with an inhibition of global transcription. Moreover, ethanol-exposed neurons treated with ionizing radiation (2 Gy) also failed to recruit 53BP1 at DNA damage foci and exhibited a greater vulnerability to DNA lesions than irradiated control neurons. Our results support that defective DNA repair, mediated by the deficient expression and recruitment of 53BP1 to DNA damage sites, represents a novel mechanism involved in ethanol neurotoxicity. The design of therapeutic strategies that increase or stabilize 53BP1 levels might potentially promote DNA repair and partially compensate alcohol neurotoxicity.

  11. Stimulation of α7 nicotinic acetylcholine receptor regulates glutamate transporter GLAST via basic fibroblast growth factor production in cultured cortical microglia.

    PubMed

    Morioka, Norimitsu; Harano, Sakura; Tokuhara, Masato; Idenoshita, Yuko; Zhang, Fang Fang; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2015-11-01

    The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18 h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and (14)C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and (14)C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

  12. 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...

  13. 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.

  14. Application of three-dimensional culture conditions to human embryonic stem cell-derived definitive endoderm cells enhances hepatocyte differentiation and functionality.

    PubMed

    Ramasamy, Thamil Selvee; Yu, Jason S L; Selden, Clare; Hodgson, Humphery; Cui, Wei

    2013-02-01

    Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of human hepatocytes, owing to their indefinite self-renewal and pluripotent properties. Both hESC-/iPSC-derived hepatocytes hold great promise in treating liver diseases as potential candidates for cell replacement therapies or as an in vitro platform to conduct new drug trials. It has been previously demonstrated that the initiation of hESC differentiation in monolayer cultures increases the generation of definitive endoderm (DE) and subsequently of hepatocyte differentiation. However, monolayer culture may hinder the maturation of hESC-derived hepatocytes, since such two-dimensional (2D) conditions do not accurately reflect the complex nature of three-dimensional (3D) hepatocyte specification in vivo. Here, we report the sequential application of 2D and 3D culture systems to differentiate hESCs to hepatocytes. Human ESCs were initially differentiated in a monolayer culture to DE cells, which were then inoculated into Algimatrix scaffolds. Treatments of hESC-DE cells with a ROCK inhibitor before and after inoculation dramatically enhanced their survival and the formation of spheroids, which are distinct from HepG2 carcinoma cells. In comparison with monolayer culture alone, sequential 2D and 3D cultures significantly improved hepatocyte differentiation and function. Our results demonstrate that hESC-DE cells can be incorporated into Algimatrix 3D culture systems to enhance hepatocyte differentiation and function.

  15. 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...

  16. 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…

  17. Group II mGlu receptor agonists fail to protect against various neurotoxic insults induced in murine cortical, striatal and cerebellar granular pure neuronal cultures.

    PubMed

    Moldrich, R X; Giardina, S F; Beart, P M

    2001-07-01

    Since group II metabotropic glutamate (mGlu) receptors are a potential target for the amelioration of neuronal injury, we evaluated the ability of group II mGlu receptor agonists to attenuate toxicity induced by various insults in cortical, striatal and cerebellar granular (CGCs) pure neuronal cultures. The three cultures, when maintained under serum-free, anti-oxidant rich conditions for up to 13 days in vitro (div) were shown by immunocytochemistry to contain a maximum of 2-7% glia. At 6, 9 and 13 div a graded pattern of injury to cortical and striatal cultures was achieved with either hydrogen peroxide (60-110 microM), staurosporine (1 microM), N-methyl-D-aspartate (NMDA, 70 microM), alpha-amino-3-hydroxy-methylisoxazole-4-propionate (AMPA, 100 microM) or kainate (100 microM) over either 4, 24 or 48 h. CGCs were similarly exposed to low K(+) (5.4 mM KCl). Cell viability was examined via phase-contrast microscopy and assessed by a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. Treatment with group II mGlu receptor agonists (1-300 microM), 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC), (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) and N-acetylaspartylglutamate (NAAG) failed to attenuate the toxicity. Pretreatment of cultures with the agonists and treatment following acute insult also failed to attenuate toxicity. Further investigations demonstrated the presence of second messenger activation whereby (2R,4R)-APDC reduced forskolin-stimulated production of cAMP in each culture. Thus, despite receptor coupling to intracellular signaling cascades, and regardless of culture development, agonist concentration, extent and mode of injury, group II mGlu receptor agonists were unable to protect against injury induced in cortical, striatal and cerebellar granular pure neuronal cultures. This result is in contrast to mixed cultures of neurones and glia and implies an

  18. Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters.

    PubMed

    Hunt, Megan M; Meng, Guoliang; Rancourt, Derrick E; Gates, Ian D; Kallos, Michael S

    2014-01-01

    Traditional optimization of culture parameters for the large-scale culture of human embryonic stem cells (ESCs) as aggregates is carried out in a stepwise manner whereby the effect of varying each culture parameter is investigated individually. However, as evidenced by the wide range of published protocols and culture performance indicators (growth rates, pluripotency marker expression, etc.), there is a lack of systematic investigation into the true effect of varying culture parameters especially with respect to potential interactions between culture variables. Here we describe the design and execution of a two-parameter, three-level (3(2)) factorial experiment resulting in nine conditions that were run in duplicate 125-mL stirred suspension bioreactors. The two parameters investigated here were inoculation density and agitation rate, which are easily controlled, but currently, poorly characterized. Cell readouts analyzed included fold expansion, maximum density, and exponential growth rate. Our results reveal that the choice of best case culture parameters was dependent on which cell property was chosen as the primary output variable. Subsequent statistical analyses via two-way analysis of variance indicated significant interaction effects between inoculation density and agitation rate specifically in the case of exponential growth rates. Results indicate that stepwise optimization has the potential to miss out on the true optimal case. In addition, choosing an optimum condition for a culture output of interest from the factorial design yielded similar results when repeated with the same cell line indicating reproducibility. We finally validated that human ESCs remain pluripotent in suspension culture as aggregates under our optimal conditions and maintain their differentiation capabilities as well as a stable karyotype and strong expression levels of specific human ESC markers over several passages in suspension bioreactors.

  19. Endothelial cells in co-culture enhance embryonic stem cell differentiation to pancreatic progenitors and insulin-producing cells through BMP signaling.

    PubMed

    Talavera-Adame, Dodanim; Wu, Gordon; He, Yao; Ng, Tina T; Gupta, Ankur; Kurtovic, Silvia; Hwang, Jae Y; Farkas, Daniel L; Dafoe, Donald C

    2011-09-01

    Endothelial cells (ECs) represent the major component of the embryonic pancreatic niche and play a key role in the differentiation of insulin-producing β cells in vivo. However, it is unknown if ECs promote such differentiation in vitro. We investigated whether interaction of ECs with mouse embryoid bodies (EBs) in culture promotes differentiation of pancreatic progenitors and insulin-producing cells and the mechanisms involved. We developed a co-culture system of mouse EBs and human microvascular ECs (HMECs). An increase in the expression of the pancreatic markers PDX-1, Ngn3, Nkx6.1, proinsulin, GLUT-2, and Ptf1a was observed at the interface between EBs and ECs (EB-EC). No expression of these markers was found at the periphery of EBs cultured without ECs or those co-cultured with mouse embryonic fibroblasts (MEFs). At EB-EC interface, proinsulin and Nkx6.1 positive cells co-expressed phospho-Smad1/5/8 (pSmad1/5/8). Therefore, EBs were treated with HMEC conditioned media (HMEC-CM) suspecting soluble factors involved in bone morphogenetic protein (BMP) pathway activation. Upregulation of PDX-1, Ngn3, Nkx6.1, insulin-1, insulin-2, amylin, SUR1, GKS, and amylase as well as down-regulation of SST were detected in treated EBs. In addition, higher expression of BMP-2/-4 and their receptor (BMPR1A) were also found in these EBs. Recombinant human BMP-2 (rhBMP-2) mimicked the effects of the HMEC-CM on EBs. Noggin (NOG), a BMP antagonist, partially inhibited these effects. These results indicate that the differentiation of EBs to pancreatic progenitors and insulin-producing cells can be enhanced by ECs in vitro and that BMP pathway activation is central to this process.

  20. The embryonic stem cell test.

    PubMed

    Schulpen, Sjors H W; Piersma, Aldert H

    2013-01-01

    The embryonic stem cell test is an animal-free alternative test method for developmental toxicity. Mouse embryonic stem cells are cultured in a hanging drop method to form embryoid bodies. These embryoid bodies, when plated on tissue culture dishes, differentiate to form contracting myocardial cell foci within 10 days. Inhibition of cardiomyocyte differentiation by test compounds serves as the end point of the assay, as monitored by counting contracting muscle foci under the microscope.

  1. Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation

    PubMed Central

    Hue, Isabelle; Evain-Brion, Danièle; Fournier, Thierry; Degrelle, Séverine A.

    2015-01-01

    In addition to nourishing the embryo, extra-embryonic tissues (EETs) contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC), endoderm (bXEC), and mesoderm (bXMC) cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression) of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify “cores” of cell-type-specific (and substrate-independent) genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21–D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED) at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in

  2. Isoflurane post-conditioning protects primary cultures of cortical neurons against oxygen and glucose deprivation injury via upregulation of Slit2/Robo1.

    PubMed

    Zhao, Xiao-Chun; Zhang, Li-Min; Li, Qiang; Tong, Dong-Yi; Fan, Long-Chang; An, Ping; Wu, Xiu-Ying; Chen, Wei-Min; Zhao, Ping; Wang, Jian

    2013-11-01

    Different mechanisms have been suggested to contribute to isoflurane-mediated neuroprotection. Previous studies have suggested that the protein Slit can abrogate neuronal death in mixed neuronal-glial cultures exposed to oxygen-glucose deprivation (OGD) and reperfusion (OGD/R). We hypothesized that isoflurane increases the expression of Slit and its receptor Robo when cortical neurons are exposed to OGD/R. To test this hypothesis, we exposed primary cortical neurons to OGD for 90 min and reperfusion for 24h and investigated how isoflurane post-conditioning affected cell survival and expression of Slit2 and receptors Robo1 and Robo4. Cell survival increased after administration of isoflurane, as assessed by the lactate dehydrogenase assay, trypan blue analysis, and propidium iodide staining. Western blot analysis showed that cleaved caspase-3 was increased after OGD/R(P<0.01) but reduced by isoflurane post-conditioning. Real-time PCR and Western blot analysis showed that the expression levels of Slit2 and Robo1, but not Robo4, were increased after OGD/R (P<0.5) and increased even further by isoflurane post-conditioning (P<0.01). Our results suggest that isoflurane post-conditioning markedly attenuates apoptosis and necrosis of cortical neurons exposed to OGD/R possibly in part via elevation of Slit2 and Robo1 expression. These findings provide a novel explanation for the pleiotropic effects of isoflurane that could benefit the central nervous system.

  3. 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

  4. 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.

  5. 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.

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

    PubMed Central

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

    2015-01-01

    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 in 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. PMID:25913222

  7. Translating human embryonic stem cells from 2-dimensional to 3-dimensional cultures in a defined medium on laminin- and vitronectin-coated surfaces.

    PubMed

    Heng, Boon Chin; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul; Cool, Simon M; Birch, William R; Oh, Steve Kah-Weng

    2012-07-01

    While defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3-dimensional (3D) system, thus enabling better control, automation, and volumetric scale-up in bioreactors. The current study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESCs and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and laminin (LN) were chosen as matrices for the long-term propagation of hESCs in a defined culture medium (STEMPRO(®)) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm(2) for VN and LN, respectively, was attained above 20 μg/mL deposition solution concentration. Adsorption of these proteins onto spherical (97±10 μm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm(2) for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESCs was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, MAB-84, and TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold increase in cell numbers on VN-coated MC per serial passage was 8.5±1.0, which was similar to LN-coated MC (8.5±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESCs retained the ability to differentiate into lineages of all 3 germ layers, thus demonstrating the first translation to a fully defined MC-based environment for the expansion of hESCs.

  8. Long-term culture and cryopreservation does not affect the stability and functionality of human embryonic stem cell-derived hepatocyte-like cells.

    PubMed

    Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

    2016-02-01

    Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs, obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology.

  9. 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

  10. The effect of Portuguese Man-of-war (Physalia physalis) venom on calcium, sodium and potassium fluxes of cultured embryonic chick heart cells.

    PubMed

    Edwards, L; Luo, E; Hall, R; Gonzalez, R R; Hessinger, D A

    2000-03-01

    Portuguese Man-of-war venom markedly increases calcium (45Ca2+) influx into primary, cultured, embryonic chick heart cells. This action is dose-dependent, but is unaffected by organic calcium blockers (diltiazem, verapamil, nifedipine, nimodipine and mibefradil). On the other hand, certain trivalent (La3+, Gd3+) and divalent (Zn2+, Ni2+, Cu2+, Mn2+) metals inhibit venom-induced calcium influx. Sodium (22Na+) influx into chick heart cells is also significantly increased by Man-of-war venom. Flecainide does not block venom-induced sodium influx. The efflux of the potassium analogue, 86Rb+, from heart cells is also significantly increased by the venom. The venom, however, has little or no effect on rubidium (86Rb+) or 2-deoxy-D-[2-3H] glucose influx. PMID:10669022

  11. [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

  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. Glycine receptors influence radial migration in the embryonic mouse neocortex.

    PubMed

    Nimmervoll, Birgit; Denter, Denise G; Sava, Irina; Kilb, Werner; Luhmann, Heiko J

    2011-07-13

    To investigate whether glycine receptors influence radial migration in the neocortex, we analyzed the effect of glycine and the glycinergic antagonist strychnine, on the distribution of 5-bromo-2'deoxyuridine-labeled neurons in organotypic slice cultures from embryonic mice cortices. Application of glycine impeded radial migration only in the presence of the glycine-transport blockers, ALX-5407 and ALX-1393. This effect was blocked by the specific glycine receptor antagonist strychnine, whereas application of strychnine in the absence of glycine was without effect. We conclude from these observations that an activation of glycine receptors can impede radial migration, but that the glycinergic system is not directly implicated in the regulation of radial migration in organotypic slice cultures.

  14. Early Exposure of Murine Embryonic Stem Cells to Hematopoietic Cytokines Differentially Directs Definitive Erythropoiesis and Cardiomyogenesis in Alginate Hydrogel Three-Dimensional Cultures

    PubMed Central

    Fauzi, Iliana

    2014-01-01

    HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and β-major globin), and proteins (Gata-1 and β-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development. PMID:24926614

  15. 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.

  16. 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. PMID:25593208

  17. 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

  18. Amitriptyline up-regulates connexin43-gap junction in rat cultured cortical astrocytes via activation of the p38 and c-Fos/AP-1 signalling pathway

    PubMed Central

    Morioka, N; Suekama, K; Zhang, F F; Kajitani, N; Hisaoka-Nakashima, K; Takebayashi, M; Nakata, Y

    2014-01-01

    Background and Purpose Intercellular communication via gap junctions, comprised of connexin (Cx) proteins, allow for communication between astrocytes, which in turn is crucial for maintaining CNS homeostasis. The expression of Cx43 is decreased in post-mortem brains from patients with major depression. A potentially novel mechanism of tricyclic antidepressants is to increase the expression and functioning of gap junctions in astrocytes. Experimental Approach The effect of amitriptyline on the expression of Cx43 and gap junction intercellular communication (GJIC) in rat primary cultured cortical astrocytes was investigated. We also investigated the role of p38 MAPK intracellular signalling pathway in the amitriptyline-induced expression of Cx43 and GJIC. Key Results Treatment with amitriptyline for 48 h significantly up-regulated Cx43 mRNA, protein and GJIC. The up-regulation of Cx43 was not monoamine-related since noradrenaline, 5-HT and dopamine did not induce Cx43 expression and pretreatment with α- and β-adrenoceptor antagonists had no effect. Intracellular signalling involved p38 MAPK, as amitriptyline significantly increased p38 MAPK phosphorylation and Cx43 expression and GJIC were significantly blocked by the p38 inhibitor SB 202190. Furthermore, amitriptyline-induced Cx43 expression and GJIC were markedly reduced by transcription factor AP-1 inhibitors (curcumin and tanshinone IIA). The translocation of c-Fos from the cytosol and the nucleus of cortical astrocytes was increased by amitriptyline, and this response was dependent on p38 activity. Conclusion and Implication These findings indicate a novel mechanism of action of amitriptyline through cortical astrocytes, and further suggest that targeting this mechanism could lead to the development of a new class of antidepressants. PMID:24641259

  19. STC1 induction by PACAP is mediated through cAMP and ERK1/2 but not PKA in cultured cortical neurons

    PubMed Central

    Holighaus, Yvonne; Weihe, Eberhard; Eiden, Lee E.

    2011-01-01

    The neuroprotective actions of PACAP (pituitary adenylate cyclase-activating polypeptide) in vitro and in vivo suggest that activation of its cognate G protein-coupled receptor PAC1 or downstream signaling molecules, and thus activation of PACAP target genes, could be of therapeutic benefit. Here we show, that cultured rat cortical neurons predominantly expressed the PAC1hop and null variants, activation of which resulted in elevation of the two second messengers cAMP and Ca2+ and expression of the putative neuroprotectant stanniocalcin 1 (STC1). PACAP signaling to the STC1 gene proceeded through the extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not through the cAMP dependent protein kinase (PKA), and was mimicked by the adenylate cyclase activator forskolin. PACAP- and forskolin-mediated activation of ERK1/2 occurred through cAMP, but not PKA. These results suggest that STC1 gene induction proceeds through cAMP and ERK1/2, independently of PKA, the canonical cAMP effector. In contrast, PACAP signaling to the BDNF gene proceeded through PKA, suggesting that two different neuroprotective cAMP pathways co-exist in differentiated cortical neurons. The selective activation of a potentially neuroprotective cAMP dependent pathway different from the canonical cAMP pathway used in many physiological processes, such as memory storage, has implications for pharmacological activation of neuroprotection in vivo. PMID:21975601

  20. Synthesis and secretion of plasma proteins by embryonic chick hepatocytes: changing patterns during the first three days of culture

    PubMed Central

    1978-01-01

    A simple model system is described for studying synthesis of plasma proteins. The system is based on chick embryo hepatocytes in primary monolayer culture which synthesize a broad spectrum of plasma proteins and secrete them into the culture medium. The secreted proteins are stable and consist almost exclusively of plasma proteins. The cultured cells are nonproliferating hepatic parenchymal cells whose cell mass remains constant in culture. By a modification of Laurell's rocket immunoelectrophoresis, the secreted plasma proteins can be detected in nanogram amounts in 3 microliter of unconcentrated culture medium. Kinetics of secretion are obtained by sequential assay of proteins accumulating in the medium. In this system it is demonstrated that: (a) intracellular plasma protein levels are equivalent to less than 5% of the daily secretion; (b) synthesis and secretion are continuous; and (c) the overall half-time for plasma protein movement along the secretory pathway is less than 10 min. From these results, it follows that the rate at which the plasma proteins are secreted gives a valid estimate of their rate of synthesis. This feature of the culture and the sensitivity of the assay allow routine measurements of plasma protein synthesis without disruption of the cells and without the use of radioisotopes. It is shown, furthermore, that the overall rate of plasma protein synthesis in cultured hepatocytes is constant over a 3- day period and is similar to that of the intact liver. 3,000,000 cells, containing 1 mg cell protein, synthesize 0.2 mg of plasma proteins daily, amounting to one-fifth of hepatocellular protein synthesis. Under the conditions used, albumin synthesis steadily decreases with culture time whereas the synthesis of many other plasma proteins increases. The observed phenotypic changes and reorganization of plasma protein synthesis illustrate how the system may be exploited for studying the regulatory processes governing plasma protein synthesis. PMID

  1. Comparison of in vitro mineralization by murine embryonic and adult stem cells cultured in an osteogenic medium.

    PubMed

    Shimko, Daniel A; Burks, Chris A; Dee, Kay C; Nauman, Eric A

    2004-01-01

    Nearly half a million bone-grafting procedures occurred in the United States in the year 2000. Tissue-engineered bone substitutes may mitigate difficulties associated with current grafting options. Embryonic stem cells (ESCs) could be a potential cell source for bone substitutes; however, direct comparisons between ESCs and other cell sources are lacking. Here we provide a direct, long-term, in vitro comparison of mineralization processes in adult, marrow-derived, mesenchymal stem cells (MSCs) and ESCs from the 129/Sv+c/+p mouse strain. MSCs were observed to grow at a slower rate than ESCs. MSCs expressed seven times more alkaline phosphatase (AP) per cell than did ESCs and immediately showed type I collagen and osteocalcin production. ESCs also produced type I collagen and osteocalcin, but production was delayed. Mineral deposition by ESCs was nearly 50 times higher than by MSCs. Spectroscopic analysis showed the calcium-to-phosphorus ratio (Ca:P) of the ESC mineral (1.26:1) to be significantly higher than that of the MSCs (0.29:1), but still 25% lower than hydroxyapatite (1.67:1). Addition of basic fibroblast growth factor significantly inhibited AP expression, mineral deposition, and Ca:P ratios in MSCs and had little effect on ESCs. These functional characteristics may assist with cell selection for purposes of bone tissue engineering.

  2. Development of retrovirus vectors useful for expressing genes in cultured murine embryonal cells and hematopoietic cells in vivo.

    PubMed Central

    Guild, B C; Finer, M H; Housman, D E; Mulligan, R C

    1988-01-01

    A series of retrovirus vectors were constructed in which cellular promoter elements derived from the chicken beta-actin and human histone H4 genes were introduced within the proviral transcriptional unit of Moloney murine leukemia virus in order to promote expression of inserted sequences. Each of these vectors gave rise to high titer of virus capable of transferring the expected proviral structure to cells. Inclusion of normal 5' splice sequences or a portion of viral gag sequences in these constructions resulted in significant increases in virus titer. Each construction was transcriptionally active in NIH 3T3 cells and in undifferentiated F9 cells. One of the vectors, HSG-neo, which contained the human histone H4 promoter, was shown to be transcriptionally active in hematopoietic cells derived from long-term reconstituted bone marrow transplant recipients engrafted with transduced stem cells. These vectors should be of general use for obtaining efficient gene expression in embryonal and hematopoietic cells. Images PMID:3418785

  3. The Effect of Topography on Differentiation Fates of Matrigel-Coated Mouse Embryonic Stem Cells Cultured on PLGA Nanofibrous Scaffolds

    PubMed Central

    Abasi, Mozhgan; Babaloo, Hamideh; Terraf, Panieh; Safi, Mojtaba; Saeed, Mahdi; Barzin, Jalal; Zandi, Mojgan; Soleimani, Masoud

    2012-01-01

    Due to pluripotency of embryonic stem (ES) cells, these cells are an invaluable in vitro model that investigates the influence of different physical and chemical cues on differentiation/development pathway of specialized cells. We sought the effect of roughness and alignment, as topomorpholocial properties of scaffolds on differentiation of green fluorescent protein-expressing ES (GFP-ES) cells into three germ layers derivates simultaneously. Furthermore, the effect of Matrigel as a natural extracellular matrix in combination with poly(lactic-co-glycolic acid) (PLGA) nanofibrous scaffolds on differentiation of mouse ES cells has been investigated. The PLGA nanofibrous scaffolds with different height and distribution of roughness and alignments were fabricated. Then, the different cell differentiation fats of GFP-ES cells plated on PLGA and PLGA/Matrigel scaffolds were analyzed by gene expression profiling. The findings demonstrated that distinct ranges of roughness, height, and distribution can support/promote a specific cell differentiation fate on scaffolds. Coating of scaffolds with Matrigel has a synergistic effect in differentiation of mesoderm-derived cells and germ cells from ES cells, whereas it inhibits the derivation of endodermal cell lineages. It was concluded that the topomorpholocial cues such as roughness and alignment should be considered in addition to other scaffolds properties to design an efficient electrospun scaffold for specific tissue engineering. PMID:21981309

  4. Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture.

    PubMed

    Sakakura, Eriko; Eiraku, Mototsugu; Takata, Nozomu

    2016-08-01

    The eyes are subdivided from the rostral diencephalon in early development. How the neuroectoderm regulates this subdivision, however, is largely unknown. Taking advantage of embryonic stem cell (ESC) culture using a Rax reporter line to monitor rostral diencephalon formation, we found that ESC-derived tissues at day 7 grown in Glasgow Minimum Expression Media (GMEM) containing knockout serum replacement (KSR) exhibited higher levels of expression of axin2, a Wnt target gene, than those grown in chemically defined medium (CDM). Surprisingly, Wnt agonist facilitated eye field-like tissue specification in CDM. In contrast, the addition of Wnt antagonist diminished eye field tissue formation in GMEM+KSR. Furthermore, the morphological formation of the eye tissue anlage, including the optic vesicle, was accompanied by Wnt signaling activation. Additionally, using CDM culture, we developed an efficient method for generating Rax+/Chx10+ retinal progenitors, which could become fully stratified retina. Here we provide a new avenue for exploring the mechanisms of eye field specification in vitro.

  5. 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. PMID:20683844

  6. Structures of 1,4-benzodioxane derivatives from the seeds of Phytolacca americana and their neuritogenic activity in primary cultured rat cortical neurons.

    PubMed

    Takahasi, Hironobu; Yanagi, Kazue; Ueda, Masumi; Nakade, Kousuke; Fukuyama, Yoshiyasu

    2003-12-01

    The methanol extract of the seeds of Phytolacca americana was reinvestigated to yield three new 1,4-benzodioxane-type compounds, americanoic acid methyl ester (1), isoamericanoic acid A methyl ester (2), and 9'-O-methylamericanol A (3) along with the previously isolated neolignans 6-9. The structures of 1-3 were characterized by 2D NMR and long-range selective proton-decoupling (LSPD) techniques. The neuritogenic effects of compounds 1-3, and dicarboxilic acids 4 and 5, which had been previously synthesized with horseradish peroxidase-catalyzed oxidative coupling of caffeic acid, were examined in primary cultured rat cortical neurons. Americanoic acid A methyl ester (1) exhibited neurite outgrowth-promoting activity at concentration of 0.01-1.0 microM, whereas dicarboxilic acids 4 and 5 were found to induce neuritogenesis dose dependently at the concentrations from 0.1 microM to 10 microM.

  7. Synthesis of a growth-associated protein by embryonic rat cerebrocortical neurons in vitro.

    PubMed

    Perrone-Bizzozero, N I; Finklestein, S P; Benowitz, L I

    1986-12-01

    Proteins synthesized by embryonic rat cortical cultures were studied under conditions that were either permissive or nonpermissive to neurite outgrowth. Freshly dissected cortex from embryonic day 17 rat pups was mechanically dissociated and plated on poly(L-lysine) substrate in the presence of (1) serum-free media, which allowed neuronal survival but no outgrowth; (2) serum, which allowed survival of both neurons and glia as well as neurite outgrowth; or (3) a hormone-supplemented defined media, which allowed preferential survival and outgrowth of neurons. In addition, postnatal tissue was cultured as a source of glia. Cultures were pulse-labeled with 35S-methionine 48 hr after plating and the protein synthesis patterns examined by 2-dimensional gel electrophoresis followed by fluorography. The expression of an acidic 50 kDa protein, associated with the particulate fraction of cells, was found to be a prominent correlate of neurite outgrowth. This protein was synthesized in serum- or hormone-treated embryonic cultures showing neurite outgrowth but was undetectable in embryonic cultures without outgrowth or in postnatal glial cultures. By virtue of its migration position on 2-dimensional gels, its presence in a light membrane fraction, and its cleavage products after Staphylococcus aureus protease treatment, the 50 kDa protein appears to be identical to an acidic 43-49 kDa protein that has been identified in several developing and regenerating neural pathways, as well as to the B-50 phosphoprotein. These findings lend support for a critical role of this protein in neural development and demonstrate the feasibility of using primary CNS cell cultures to study its biosynthesis and function. PMID:2947982

  8. Development and characterization of a primary culture of chicken embryonic tracheal epithelial cells and their use in avian studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major route of infection of avian influenza is through cells of the airway epithelium. To study the molecular mechanism of infection and early host responses we created a primary chicken tracheal cell culture. Epithelial cells were isolated from the trachea of 18 day old chicken embryos and cult...

  9. Embryonic development in culture of two dasyurid marsupials, Sminthopsis crassicaudata (Gould) and Sminthopsis macroura (Spencer), during cleavage and blastocyst formation.

    PubMed

    Selwood, L

    1987-04-01

    Embryos of Sminthopsis crassicaudata and Sminthopsis macroura were cultured for up to 96 hours during cleavage and early expansion of the blastocyst in Dulbecco's modified Eagle's medium (DMEG), DMEG containing 2.76 gm/liter sodium lactate (DMEGL), DMEG containing 3.5 gm/liter galactose (DMEGAL), DMEG containing 15 ng/ml progesterone (DMEGP) or 150 ng/ml progesterone (DMEGP10), and DMEGL containing 15 ng/ml progesterone (DMEGLP). The disappearance of sperm was used to indicate the time of ovulation (day 0). Fertilized eggs were found in the uterus at the end of day 1, four-cell stages at the end of day 2, and embryos completing the fourth division by the end of day 3 in S. macroura and day 4 in S. crassicaudata. Estimated developmental times in culture were similar to those obtained in vivo. In both species, the first two divisions take about 24 hours, cleavage is arrested for 24 hours or longer at the rounded four-cell stage, and the third and fourth divisions take a further 24 hours. The blastocyst expands during the next 24 hours in which time the fifth and sixth divisions occur. It was possible to culture embryos from S. macroura but not S. crassicaudata over the four-cell stage to early expanding blastocysts. DMEGAL did not support cleavage in culture. DMEG, DMEGL, DMEGP, DMEGP10, and DMEGLP all supported culture during cleavage and early blastocyst expansion. Blastocyst expansion was slightly enhanced using media containing sodium lactate. More embryos completed the fifth division and formed expanding blastocysts in DMEG, DMEGL, and DMEGLP.

  10. 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

  11. Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition.

    PubMed

    Ho, Sing Yee; Goh, Crystal Wei Pin; Gan, Jen Yang; Lee, Youn Sing; Lam, Millie Kuen Kuen; Hong, Ni; Hong, Yunhan; Chan, Woon Khiong; Shu-Chien, Alexander Chong

    2014-10-01

    Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.

  12. The Ketone Body, β-Hydroxybutyrate Stimulates the Autophagic Flux and Prevents Neuronal Death Induced by Glucose Deprivation in Cortical Cultured Neurons.

    PubMed

    Camberos-Luna, Lucy; Gerónimo-Olvera, Cristian; Montiel, Teresa; Rincon-Heredia, Ruth; Massieu, Lourdes

    2016-03-01

    Glucose is the major energy substrate in brain, however, during ketogenesis induced by starvation or prolonged hypoglycemia, the ketone bodies (KB), acetoacetate and β-hydroxybutyrate (BHB) can substitute for glucose. KB improve neuronal survival in diverse injury models, but the mechanisms by which KB prevent neuronal damage are still not well understood. In the present study we have investigated whether protection by the D isomer of BHB (D-BHB) against neuronal death induced by glucose deprivation (GD), is related to autophagy. Autophagy is a lysosomal-dependent degradation process activated during nutritional stress, which leads to the digestion of damaged proteins and organelles providing energy for cell survival. Results show that autophagy is activated in cortical cultured neurons during GD, as indicated by the increase in the levels of the lipidated form of the microtubule associated protein light chain 3 (LC3-II), and the number of autophagic vesicles. At early phases of glucose reintroduction (GR), the levels of p62 declined suggesting that the degradation of the autophagolysosomal content takes place at this time. In cultures exposed to GD and GR in the presence of D-BHB, the levels of LC3-II and p62 rapidly declined and remained low during GR, suggesting that the KB stimulates the autophagic flux preventing autophagosome accumulation and improving neuronal survival.

  13. Effects of amyloid-beta on cholinergic and acetylcholinesterase-positive cells in cultured basal forebrain neurons of embryonic rat brain.

    PubMed

    Kasa, Peter; Papp, Henrietta; Kasa, Peter; Pakaski, Magdolna; Balaspiri, Lajos

    2004-02-13

    The neurotoxic effects of amyloid-beta(1-42) and amyloid-beta(25-35) (A beta) on cholinergic and acetylcholinesterase-positive neurons were investigated in primary cultures derived from embryonic 18-day-old rat basal forebrain. After various time intervals, the cultures were treated with 1, 5, 10 or 20 microM A beta for different time periods. The cholinergic neurons and their axon terminals were revealed by vesicular acetylcholine transporter immunohistochemistry and the cholinoceptive cells by acetylcholinesterase histochemical staining. To assess the toxic effects of these A beta peptides on the cholinergic neurons, image analysis was applied for quantitative determination of the numbers of axon varicosities/terminals and cells. The results demonstrate that, following treatment with 1 or 5 microM A beta for 5, 10, 30, 60 or 120 min, no changes in vesicular acetylcholine transporter immunohistochemical staining were observed. However, after treatment for 30 min with 10 or 20 microM A beta, the number of stained axon varicosities was reduced, and treatment for 2 h they had disappeared. In contrast, vesicular acetylcholine transporter-positivity could be seen in some of the neuronal perikarya even after 3 days after treatment. The acetylcholinesterase staining was homogeneously distributed in the control neurons. After A beta treatment, the histochemical reaction end-product was detected in some of the neuronal perikarya or in the dendritic processes near to the soma. It is concluded that the neurotoxic effects of A beta appear more rapidly in the cholinergic axon terminals than in the cholinergic and acetylcholinesterase-positive neuronal perikarya. PMID:14725970

  14. 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

  15. The Effects of 1α, 25-dihydroxyvitamin D3 and Transforming Growth Factor-β3 on Bone Development in an Ex Vivo Organotypic Culture System of Embryonic Chick Femora

    PubMed Central

    Smith, Emma L.; Rashidi, Hassan; Kanczler, Janos M.; Shakesheff, Kevin M.; Oreffo, Richard O. C.

    2015-01-01

    Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life. PMID:25835745

  16. The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

    PubMed

    Smith, Emma L; Rashidi, Hassan; Kanczler, Janos M; Shakesheff, Kevin M; Oreffo, Richard O C

    2015-01-01

    Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

  17. 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

  18. Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format.

    PubMed

    Medda, X; Mertens, L; Versweyveld, S; Diels, A; Barnham, L; Bretteville, A; Buist, A; Verheyen, A; Royaux, I; Ebneth, A; Cabrera-Socorro, A

    2016-09-01

    Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer's disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting β-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD. PMID:26984927

  19. Down-regulation of ASICs current and the calcium transients by disrupting PICK1 protects primary cultured mouse cortical neurons from OGD-Rep insults.

    PubMed

    Cheng, Jin; Chen, Yu; Xing, Hui; Jiang, Hua; Ye, Xihong

    2015-01-01

    Acid sensing ion channels (ASICs), activated by lowering extracellular pH, play an important role in normal synaptic transmission in brain and in the pathology of brain ischemia. ASICs activation involving in glutamate receptor-independent ischemic brain injury has been generally accepted, and PICK1 is recently shown to be one of partner proteins interacting with ASICs through its PDZ domain. Here we showed that ASICs and PICK1 played key roles in OGD-Rep process. In wild-type cultured cortical neurons, not only the amplitude of ASICs current and the calcium transients induced by acidosis were both increased after OGD-Rep, but also the total protein levels of ASIC1 and ASIC2a were up-regulated progressively after ischemia insults, indicating that ASICs play a vital role in neuronal ischemia. However, these activities were reversed with PICK1-knockout after OGD-Rep, accompanied with the dramatically down-regulating the protein abundances of ASIC1 and ASIC2a, which suggested the neuroprotection activity in brain ischemia by PICK1-knockout. These results indicate that knocking-out PICK1 gene casts the neuroprotection effect by reducing ASICs current and the calcium transients in OGD-Rep neuronal cells, which will offer a promising strategy in the therapy of brain ischemia.

  20. Rare cell proteomic reactor applied to stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics study of human embryonic stem cell differentiation.

    PubMed

    Tian, Ruijun; Wang, Shuai; Elisma, Fred; Li, Li; Zhou, Hu; Wang, Lisheng; Figeys, Daniel

    2011-02-01

    The molecular basis governing the differentiation of human embryonic stem cells (hESCs) remains largely unknown. Systems-level analysis by proteomics provides a unique approach to tackle this question. However, the requirement of a large number of cells for proteomics analysis (i.e. 10(6)-10(7) cells) makes this assay challenging, especially for the study of rare events during hESCs lineage specification. Here, a fully integrated proteomics sample processing and analysis platform, termed rare cell proteomic reactor (RCPR), was developed for large scale quantitative proteomics analysis of hESCs with ∼50,000 cells. hESCs were completely extracted by a defined lysis buffer, and all of the proteomics sample processing procedures, including protein preconcentration, reduction, alkylation, and digestion, were integrated into one single capillary column with a strong cation exchange monolith matrix. Furthermore, on-line two-dimensional LC-MS/MS analysis was performed directly using RCPR as the first dimension strong cation exchange column. 2,281 unique proteins were identified on this system using only 50,000 hESCs. For stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative study, a ready-to-use and chemically defined medium and an in situ differentiation procedure were developed for complete SILAC labeling of hESCs with well characterized self-renewal and differentiation properties. Mesoderm-enriched differentiation was studied by RCPR using 50,000 hESCs, and 1,086 proteins were quantified with a minimum of two peptides per protein. Of these, 56 proteins exhibited significant changes during mesoderm-enriched differentiation, and eight proteins were demonstrated for the first time to be overexpressed during early mesoderm development. This work provides a new platform for the study of rare cells and in particular for further elucidating proteins that govern the mesoderm lineage specification of human pluripotent stem cells.

  1. 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

    2015-12-01

    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.

  2. Large-Scale, High-Resolution Multielectrode-Array Recording Depicts Functional Network Differences of Cortical and Hippocampal Cultures

    PubMed Central

    Ito, Shinya; Yeh, Fang-Chin; Hiolski, Emma; Rydygier, Przemyslaw; Gunning, Deborah E.; Hottowy, Pawel; Timme, Nicholas; Litke, Alan M.; Beggs, John M.

    2014-01-01

    Understanding the detailed circuitry of functioning neuronal networks is one of the major goals of neuroscience. Recent improvements in neuronal recording techniques have made it possible to record the spiking activity from hundreds of neurons simultaneously with sub-millisecond temporal resolution. Here we used a 512-channel multielectrode array system to record the activity from hundreds of neurons in organotypic cultures of cortico-hippocampal brain slices from mice. To probe the network structure, we employed a wavelet transform of the cross-correlogram to categorize the functional connectivity in different frequency ranges. With this method we directly compare, for the first time, in any preparation, the neuronal network structures of cortex and hippocampus, on the scale of hundreds of neurons, with sub-millisecond time resolution. Among the three frequency ranges that we investigated, the lower two frequency ranges (gamma (30–80 Hz) and beta (12–30 Hz) range) showed similar network structure between cortex and hippocampus, but there were many significant differences between these structures in the high frequency range (100–1000 Hz). The high frequency networks in cortex showed short tailed degree-distributions, shorter decay length of connectivity density, smaller clustering coefficients, and positive assortativity. Our results suggest that our method can characterize frequency dependent differences of network architecture from different brain regions. Crucially, because these differences between brain regions require millisecond temporal scales to be observed and characterized, these results underscore the importance of high temporal resolution recordings for the understanding of functional networks in neuronal systems. PMID:25126851

  3. 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

  4. 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

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

    PubMed

    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

  6. 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

  7. Characterizing HSF1 Binding and Post-Translational Modifications of hsp70 Promoter in Cultured Cortical Neurons: Implications in the Heat-Shock Response

    PubMed Central

    Gómez, Andrea V.; Córdova, Gonzalo; Munita, Roberto; Parada, Guillermo E.; Barrios, Álvaro P.; Cancino, Gonzalo I.; Álvarez, Alejandra R.; Andrés, María E.

    2015-01-01

    Causes of lower induction of Hsp70 in neurons during heat shock are still a matter of debate. To further inquire into the mechanisms regulating Hsp70 expression in neurons, we studied the activity of Heat Shock Factor 1 (HSF1) and histone posttranslational modifications (PTMs) at the hsp70 promoter in rat cortical neurons. Heat shock induced a transient and efficient translocation of HSF1 to neuronal nuclei. However, no binding of HSF1 at the hsp70 promoter was detected while it bound to the hsp25 promoter in cortical neurons during heat shock. Histone PTMs analysis showed that the hsp70 promoter harbors lower levels of histone H3 and H4 acetylation in cortical neurons compared to PC12 cells under basal conditions. Transcriptomic profiling data analysis showed a predominant usage of cryptic transcriptional start sites at hsp70 gene in the rat cerebral cortex, compared with the whole brain. These data support a weaker activation of hsp70 canonical promoter. Heat shock increased H3Ac at the hsp70 promoter in PC12 cells, which correlated with increased Hsp70 expression while no modifications occurred at the hsp70 promoter in cortical neurons. Increased histone H3 acetylation by Trichostatin A led to hsp70 mRNA and protein induction in cortical neurons. In conclusion, we found that two independent mechanisms maintain a lower induction of Hsp70 in cortical neurons. First, HSF1 fails to bind specifically to the hsp70 promoter in cortical neurons during heat shock and, second, the hsp70 promoter is less accessible in neurons compared to non-neuronal cells due to histone deacetylases repression. PMID:26053851

  8. 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.

  9. Conventional protein kinase Cβ-mediated phosphorylation inhibits collapsin response-mediated protein 2 proteolysis and alleviates ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.

    PubMed

    Yang, Xuan; Zhang, Xinxin; Li, Yun; Han, Song; Howells, David W; Li, Shujuan; Li, Junfa

    2016-05-01

    We previously reported that conventional protein kinase C (cPKC)β participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 (CRMP2) was identified as a cPKCβ interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by cPKCβ, and their role in ischemic injury of oxygen-glucose deprivation (OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that cPKCβ-mediated CRMP2 phosphorylation via the cPKCβ-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 (TAT-CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT-CRMP2 in cortical neurons. In addition, both cPKCβ activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT-CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that cPKCβ modulates CRMP2 phosphorylation and proteolysis, and cPKCβ activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. Focal cerebral ischemia induces a large flux of Ca(2+) to activate calpain which cleaves collapsin response mediator (CRMP) 2 into breakdown product (BDP). Inhibition of CRMP2 cleavage by calpeptin and TAT-CRMP2 alleviates ischemic injury. Conventional protein kinase C (c

  10. Decision by division: making cortical maps.

    PubMed

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

    2009-05-01

    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.

  11. Ginsenoside Rg1 exerts a protective effect against Aβ₂₅₋₃₅-induced toxicity in primary cultured rat cortical neurons through the NF-κB/NO pathway.

    PubMed

    Wu, Jiaying; Yang, Hongyu; Zhao, Qingwei; Zhang, Xingguo; Lou, Yijia

    2016-03-01

    Ginsenoside Rg1 (Rg1) is a multipotent triterpene saponin extracted from ginseng, and has been proven to act as a nootropic agent against various types of neurological damage. The present study was designed to investigate the neuroprotective effect and the underlying mechanisms of Rg1 on apoptosis induced by β-amyloid peptide 25-35 (Aβ25-35) in primary cultured cortical neurons. The primary neurons were preincubated with 20 µM Rg1 for 24 h and exposed to 10 µM Aβ25-35 for 72 h. In the present study, we found that Rg1 prevented nuclear factor κ-light-chain‑enhancer of activated B cells (NF-κB) nuclear translocation and IκB-α phosphorylation in primary cultured cortical neurons after Aβ25-35 exposure by scavenging excess reactive oxygen species (ROS); ROS was measured using DCFDA and examined using a fluorescence microscope. In addition, Rg1 successfully suppressed Aβ25‑35-inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in a NF-κB-dependent manner; the suppression of NO was clearly illustrated by the NO production assay. Pretreatment of the cells with Rg1 elevated the proportion of Bcl-2/Bax, lessened the release of cytochrome c from mitochondria into cytoplasm and then blocked mitochondrial apoptotic cascades after Aβ25-35 insult by lowering NO generation. Taken together, our data demonstrate that Rg1 rescues primary cultured cortical neurons from Aβ25-35-induced cell apoptosis through the downregulation of the NF-κB/NO signaling pathway. PMID:26865401

  12. 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.

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

    PubMed

    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

  14. 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

  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. Radial glial cell transformation to astrocytes is bidirectional: regulation by a diffusible factor in embryonic forebrain.

    PubMed Central

    Hunter, K E; Hatten, M E

    1995-01-01

    During development of mammalian cerebral cortex, two classes of glial cells are thought to underlie the establishment of cell patterning. In the embryonic period, migration of young neurons is supported by a system of radial glial cells spanning the thickness of the cortical wall. In the neonatal period, neuronal function is assisted by the physiological support of a second class of astroglial cell, the astrocyte. Here, we show that expression of embryonic radial glial identity requires extrinsic soluble signals present in embryonic forebrain. Moreover, astrocytes reexpress features of radial glia in vitro in the presence of the embryonic cortical signals and in vivo after transplantation into embryonic neocortex. These findings suggest that the transformation of radial glia cells into astrocytes is regulated by availability of inducing signals rather than by changes in cell potential. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7892225

  17. Programming embryonic stem cells to neuronal subtypes

    PubMed Central

    Peljto, Mirza; Wichterle, Hynek

    2010-01-01

    Richness of neural circuits and specificity of neuronal connectivity depends on the diversification of nerve cells into functionally and molecularly distinct subtypes. While efficient methods for directed differentiation of embryonic stem cells (ESCs) into multiple principal neuronal classes have been established, only a few studies systematically examined the subtype diversity of in vitro derived nerve cells. Here we review evidence based on molecular and in vivo transplantation studies that ESC-derived spinal motor neurons and cortical layer V pyramidal neurons acquire subtype specific functional properties. We discuss similarities and differences in the role of cell intrinsic transcriptional programs, extrinsic signals and cell-cell interactions during subtype diversification of the two classes of nerve cells. We conclude that the high degree of fidelity with which differentiating ESCs recapitulate normal embryonic development provides a unique opportunity to explore developmental processes underlying specification of mammalian neuronal diversity in a simplified and experimentally accessible system. PMID:20970319

  18. Freshly frozen E18 rat cortical cells can generate functional neural networks after standard cryopreservation and thawing procedures.

    PubMed

    Quasthoff, Kim; Ferrea, Stefano; Fleischer, Wiebke; Theiss, Stephan; Schnitzler, Alfons; Dihné, Marcel; Walter, Janine

    2015-05-01

    Primary dissociated brain tissue from rodents is widely used in a variety of different scientific methods to investigate cellular processes in vitro. Often, for this purpose cell cultures need to be generated just on time, requiring extensive animal lab infrastructure. We show here that cryopreservation and thawing of dissociated tissue from rat cerebral cortex at embryonic day 18 is feasible without affecting its ability to form functional neuronal networks in vitro. Vitality of fresh and re-thawed cortical cells was comparable, assessed by CellTiter-Blue-assay, CytoTox-ONE assay, immunocytochemical characterization and in vitro neuronal network activity recordings on microelectrode arrays. These findings suggest that planning and execution of experiments might be considerably facilitated by using cryo-preserved neurons instead of acutely dissociated neural cultures due to fewer logistical issues with regard to animal breeding and pregnancy timed preparations.

  19. 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

  20. 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.

  1. Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

    PubMed Central

    Pilaz, Louis-Jan; Silver, Debra L.

    2014-01-01

    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. PMID:24961595

  2. 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

  3. 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

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A; Harris, William A

    2013-04-01

    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

  4. Human embryonic stem cell-derived mesodermal progenitors display substantially increased tissue formation compared to human mesenchymal stem cells under dynamic culture conditions in a packed bed/column bioreactor.

    PubMed

    de Peppo, Giuseppe Maria; Sladkova, Martina; Sjövall, Peter; Palmquist, Anders; Oudina, Karim; Hyllner, Johan; Thomsen, Peter; Petite, Hervé; Karlsson, Camilla

    2013-01-01

    Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.

  5. Regulation of Extrasynaptic GABAA α4 Receptors by Ethanol-Induced Protein Kinase A, but Not Protein Kinase C Activation in Cultured Rat Cerebral Cortical Neurons.

    PubMed

    Carlson, Stephen L; Bohnsack, J Peyton; Patel, Vraj; Morrow, A Leslie

    2016-01-01

    Ethanol produces changes in GABAA receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic γ-aminobutyric acid A receptors (GABAA-R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABAA α4δ receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABAA α4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic α4δ receptors. In contrast, prolonged ethanol exposure (4 hours) reduced α4δ receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic α4δ subunit abundance or activity. We conclude that ethanol alters extrasynaptic α4δ receptor function and expression in cortical neurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.

  6. 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

  7. Regulation of embryonic development and apoptotic-related gene expression by brain-derived neurotrophic factor in two different culture conditions in ovine.

    PubMed

    Abazari-Kia, Amir Hossein; Dehghani-Mohammadabadi, Maryam; Mohammadi-Sangcheshmeh, Abdollah; Zhandi, Mahdi; Salehi, Mohammad

    2015-07-01

    In the present study, we aimed to evaluate effects of brain-derived neurotrophic factor (BDNF) which is a member of neurotrophic factor family on developmental competence of oocytes in sheep. In vitro maturation was performed in presence of various concentrations (0, 10, and 100 ng/mL) of BDNF. Meiotic maturation, levels of intracellular glutathione, embryonic developmental potential after parthenogenetic activation, number of total and apoptotic cells in blastocysts, and expression of Bax and Bcl-2 genes in blastocyst cells were determined. Under unstressed condition, while at 100 ng/mL concentration, BDNF increased the IVM rate; an increase of glutathione level was observed at 10 ng/mL concentration. Moreover, when BDNF-treated oocytes were used for parthenogenetic activation, more blastocyst at both 10 and 100 ng/mL levels was obtained in comparison with the untreated group. Under heat stress (HS), the blastocyst rate was dramatically reduced in untreated oocytes compared to that obtained from 10 ng/mL BDNF groups. Total cell number in blastocysts was not affected by the treatment groups. The mean of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in blastocysts was not influenced by addition of BDNF in medium and that presence or absence of thermal stress during IVM than the control group. Moreover, our data revealed that the expression of Bax and Bcl-2 genes in blastocysts was affected by both BDNF concentration and HS. Conclusively, supplementation of IVM medium with 10 ng/mL BDNF had a beneficial effect on sheep oocyte competence by increasing the rate of blastocyst especially when HS exists.

  8. 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.

  9. An optogenetic approach for assessing formation of neuronal connections in a co-culture system.

    PubMed

    Su, Colin T E; Yoon, Su-In; Marcy, Guillaume; Chin, Eunice W M; Augustine, George J; Goh, Eyleen L K

    2015-01-01

    Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. PMID:25742527

  10. 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. PMID:27401343

  11. 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...

  12. Relative embryotoxic potency of p-substituted phenols in the embryonic stem cell test (EST) and comparison to their toxic potency in vivo and in the whole embryo culture (WEC) assay.

    PubMed

    Strikwold, Marije; Woutersen, Ruud A; Spenkelink, Bert; Punt, Ans; Rietjens, Ivonne M C M

    2012-09-01

    The applicability of the embryonic stem cell test (EST) as an alternative for in vivo embryotoxicity testing was evaluated for a series of five p-substituted phenols. To this purpose, the potency ranking for this class of compounds derived from the inhibition of cardiomyocyte differentiation in the EST was compared to in vivo embryotoxic potency data obtained from literature and to the potency ranking defined in the in vitro whole embryo culture (WEC) assay. From the results obtained it appears that the EST was able to identify the embryotoxic potential for p-substituted phenols, providing an identical potency ranking compared to the WEC assay. However, the EST was not able to predict an accurate ranking for the phenols compared to their potency observed in vivo. Only phenol, the least potent compound within this series, was correctly ranked. Furthermore, p-mercaptophenol was correctly identified as a relative potent congener of the phenols tested, but its ranking was distorted by p-heptyloxyphenol, of which the toxicity was overestimated in the EST. It is concluded that when attempting to explain the observed disparity in potency rankings between in vitro and in vivo embryotoxicity, the in vitro models should be combined with a kinetic model describing in vivo absorption, distribution, metabolism and excretion processes of the compounds.

  13. 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.

  14. 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.

  15. Hyperpolarization-activated cation current contributes to spontaneous network activity in developing neocortical cultures.

    PubMed

    Klueva, Julia; Lima, Ana D de; Meis, Susanne; Voigt, Thomas; Munsch, Thomas

    2012-01-01

    The mechanisms underlying spontaneous burst activity (SBA), appearing in networks of embryonic cortical neurons at the end of the first week in vitro, remain elusive. Here we investigated the contribution of the hyperpolarization-activated cation current (I(h)) to SBA in cortical cultures of GAD67-GFP mice. I(h) current could be detected in GFP-positive large GABAergic interneurons (L-INs) and glutamatergic principal neurons (PNs) as early as DIV 5. Under current-clamp conditions, blockers of I(h) current, ZD7288 and Cs⁺, abolished the voltage sag and rebound depolarization. ZD7288 induced a hyperpolarization concomitant with an increase in the membrane input resistance in L-INs and PNs. Voltage-clamp recordings revealed I(h) as slowly activating inward current with a reversal potential close to -50 mV and a mid-activation point around -90 mV. Both, ZD7288 (1-10 μM) and Cs⁺ (1-2 mM) reduced SBA, spontaneous activity-driven Ca²⁺ transients, and frequency as well as amplitude of miniature GABAergic postsynaptic currents. Immunocytochemistry and Western blot demonstrated that HCN1 and HCN2 were the prevalent isoforms of HCN channels expressed in L-INs and PNs. These results suggest an important contribution of HCN channels to the maintenance of SBA in embryonic cortical cultures. PMID:22094222

  16. Infiltrating cells from host brain restore the microglial population in grafted cortical tissue.

    PubMed

    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.

  17. Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture

    PubMed Central

    Odawara, A.; Katoh, H.; Matsuda, N.; Suzuki, I.

    2016-01-01

    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. PMID:27188845

  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. Effect of long-term culturing on the potential of mouse embryonic stem cells for in vitro and in vivo development

    SciTech Connect

    Mitalipov, Sh.M.; Mitalipova, M.M.; Ivanov, V.I.

    1994-11-01

    We performed comparative analysis of in vitro and in vivo pluripotency for two clones of ES-D3 cells subjected to different number of passages after the beginning of subcloning. Both clones of ES cells produced characteristically shaped colonies and embryoid bodies during culturing in suspension. High activity of alkaline phosphatase was demonstrated in ES cells by cytochemical staining. The proportion of aneuploid ES cells increased with the increase in the number of passages, as shown by karyotyping. Experiments on producing chimeric mice using ES cells have shown that clone D3W (passage 17) exceeds clone D3M (passage 42) both in terms of chimera proportion among the offspring and in terms of the extent of coat chimerism (proportion of agouti coat color (ES component) in the coat of chimeras). 26 refs., 4 figs., 3 tabs.

  20. Fabrication of microengineered templates and their applications into micropatterned cell culture.

    PubMed

    Choi, Jin Ho; Lee, Hyun; Jin, Hee Kyung; Bae, Jae-Sung; Kim, Gyu Man

    2013-03-01

    We present a fabrication method of PDMS microtemplates and their applications into a localized culture of primary mammalian cells. Three types of microtemplates of microwell, microstencil and microcontact printing stamp (mCP) were fabricated, and their feasibilities into cell culture were studied. All of microtemplates were fabricated by PDMS casting from SU-8 mold prepared by photolithography. The pattern used in the test was 500 microm dots. In the culture test of microstencil, a stencil was placed on a glass disk coated with Poly-D-Lysine (PDL) layer. In the other test of microcontact printing, PDL layer was patterned by microcontact printing. The mammalian cells of mouse embryonic fibroblasts (MEF) and cortical neuron were successfully cultured into micropatterns by using the microtemplates. The results showed that cells could be cultured into micropatterns with precisely controlled manner at any shapes and specific size for bioscience study and bioengineering applications. PMID:23620992

  1. 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.

  2. Sex stratified neuronal cultures to study ischemic cell death pathways.

    PubMed

    Fairbanks, Stacy L; Vest, Rebekah; 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

  3. The histamine H3 receptor antagonist clobenpropit enhances GABA release to protect against NMDA-induced excitotoxicity through the cAMP/protein kinase A pathway in cultured cortical neurons.

    PubMed

    Dai, Haibin; Fu, Qiuli; Shen, Yao; Hu, Weiwei; Zhang, Zhongmiao; Timmerman, Henk; Leurs, Rob; Chen, Zhong

    2007-06-01

    Using the histamine H3 receptor antagonist clobenpropit, the roles of histamine H3 receptors in NMDA-induced necrosis were investigated in rat cultured cortical neurons. Clobenpropit reversed the neurotoxicity in a concentration-dependent manner, and showed peak protection at a concentration of 10(-7) M. This protection was antagonized by the histamine H3 receptor agonist (R)-alpha-methylhistamine, but not by the histamine H1 receptor antagonist pyrilamine or the histamine H2 receptor antagonist cimetidine. In addition, the protection by clobenpropit was inhibited by the GABAA receptor antagonists picrotoxin and bicuculline. Further study demonstrated that the protection by clobenpropit was due to increased GABA release. The inducible GABA release was also inhibited by (R)-alpha-methylhistamine, but not by pyrilamine or cimetidine. Furthermore, both the adenylyl cyclase inhibitor SQ-22536 and the protein kinase A (PKA) inhibitor H-89 reversed the protection and the GABA release by clobenpropit. In addition, clobenpropit reversed the NMDA-induced increase in intracellular calcium level, which was antagonized by (R)-alpha-methylhistamine. These results indicate that clobenpropit enhanced GABA release to protect against NMDA-induced excitotoxicity, which was induced through the cAMP/PKA pathway, and reduction of intracellular calcium level may also be involved.

  4. 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).

  5. Retinoic acid from the meninges regulates cortical neuron generation.

    PubMed

    Siegenthaler, Julie A; Ashique, Amir M; Zarbalis, Konstantinos; Patterson, Katelin P; Hecht, Jonathan H; Kane, Maureen A; Folias, Alexandra E; Choe, Youngshik; May, Scott R; Kume, Tsutomu; Napoli, Joseph L; Peterson, Andrew S; Pleasure, Samuel J

    2009-10-30

    Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.

  6. The PB1 segment of an influenza A virus H1N1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs.

    PubMed

    Mostafa, Ahmed; Kanrai, Pumaree; Ziebuhr, John; Pleschka, Stephan

    2016-03-01

    Influenza vaccine strains (IVSs) contain the haemagglutinin (HA) and neuraminidase (NA) genome segments of relevant circulating strains in the genetic background of influenza A/PR/8/1934 virus (PR8). Previous work has shown that the nature of the PB1 segment may be a limiting factor for the efficient production of IVSs. Here, we showed that the PB1 segment (PB1Gi) from the 2009 pandemic influenza A virus (IAV) A/Giessen/06/2009 (Gi wt, H1N1pdm) may help to resolve (some of) these limitations. We produced a set of recombinant PR8-derived viruses that contained (i) the HA and NA segments from representative IAV strains (H3N2, H5N1, H7N9, H9N2); (ii) the PB1 segment from PR8 or Gi wt, respectively; and (iii) the remaining five genome segments from PR8. Viruses containing the PB1Gi segment, together with the heterologous HA/NA segments and five PR8 segments (5+2+1), replicated to higher titres compared with their 6+2 counterparts containing six PR8 segments and the equivalent heterologous HA/NA segments. Compared with PB1PR8-containing IVSs, viruses with the PB1Gi segment replicated to higher or similar titres in both cell culture and embryonated eggs, most profoundly IVSs of the H5N1 and H7N9 subtype, which are known to grow poorly in these systems. IVSs containing either the PB1Gi or the cognate PB1 segment of the respective specific HA/NA donor strain showed enhanced or similar virus replication levels. This study suggests that substitution of PB1PR8 with the PB1Gi segment may greatly improve the large-scale production of PR8-derived IVSs, especially of those known to replicate poorly in vitro.

  7. The PB1 segment of an influenza A virus H1N1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs.

    PubMed

    Mostafa, Ahmed; Kanrai, Pumaree; Ziebuhr, John; Pleschka, Stephan

    2016-03-01

    Influenza vaccine strains (IVSs) contain the haemagglutinin (HA) and neuraminidase (NA) genome segments of relevant circulating strains in the genetic background of influenza A/PR/8/1934 virus (PR8). Previous work has shown that the nature of the PB1 segment may be a limiting factor for the efficient production of IVSs. Here, we showed that the PB1 segment (PB1Gi) from the 2009 pandemic influenza A virus (IAV) A/Giessen/06/2009 (Gi wt, H1N1pdm) may help to resolve (some of) these limitations. We produced a set of recombinant PR8-derived viruses that contained (i) the HA and NA segments from representative IAV strains (H3N2, H5N1, H7N9, H9N2); (ii) the PB1 segment from PR8 or Gi wt, respectively; and (iii) the remaining five genome segments from PR8. Viruses containing the PB1Gi segment, together with the heterologous HA/NA segments and five PR8 segments (5+2+1), replicated to higher titres compared with their 6+2 counterparts containing six PR8 segments and the equivalent heterologous HA/NA segments. Compared with PB1PR8-containing IVSs, viruses with the PB1Gi segment replicated to higher or similar titres in both cell culture and embryonated eggs, most profoundly IVSs of the H5N1 and H7N9 subtype, which are known to grow poorly in these systems. IVSs containing either the PB1Gi or the cognate PB1 segment of the respective specific HA/NA donor strain showed enhanced or similar virus replication levels. This study suggests that substitution of PB1PR8 with the PB1Gi segment may greatly improve the large-scale production of PR8-derived IVSs, especially of those known to replicate poorly in vitro. PMID:26743314

  8. Zic deficiency in the cortical marginal zone and meninges results in cortical lamination defects resembling those in type II lissencephaly.

    PubMed

    Inoue, Takashi; Ogawa, Masaharu; Mikoshiba, Katsuhiko; Aruga, Jun

    2008-04-30

    The formation of the highly organized cortical structure depends on the production and correct placement of the appropriate number and types of neurons. The Zic family of zinc-finger transcription factors plays essential roles in regulating the proliferation and differentiation of neuronal progenitors in the medial forebrain and the cerebellum. Examination of the expression of Zic genes demonstrated that Zic1, Zic2, and Zic3 were expressed by the progenitor cells in the septum and cortical hem, the sites of generation of the Cajal-Retzius (CR) cells. Immunohistochemical studies have revealed that Zic proteins were abundantly expressed in the meningeal cells and that the majority of the CR cells distributed in the medial and dorsal cortex also expressed Zic proteins in the mid-late embryonic and postnatal cortical marginal zones. During embryonic cortical development, Zic1/Zic3 double-mutant and hypomorphic Zic2 mutant mice showed a reduction in the number of CR cells in the rostral cortex, whereas the cell number remained unaffected in the caudal cortex. These mutants also showed mislocalization of the CR cells and cortical lamination defects, resembling the changes noted in type II (cobblestone) lissencephaly, throughout the brain. In the Zic1/3 mutant, reduced proliferation of the meningeal cells was observed before the thinner and disrupted organization of the pial basement membrane (BM) with reduced expression of the BM components and the meningeal cell-derived secretory factor. These defects correlated with the changes in the end feet morphology of the radial glial cells. These findings indicate that the Zic genes play critical roles in cortical development through regulating the proliferation of meningeal cells and the pial BM assembly.

  9. 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.

  10. Expression of microRNAs miR21, miR146a, and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA-derived cell cultures.

    PubMed

    van Scheppingen, J; Iyer, A M; Prabowo, A S; Mühlebner, A; Anink, J J; Scholl, T; Feucht, M; Jansen, F E; Spliet, W G; Krsek, P; Zamecnik, J; Buccoliero, A M; Giordano, F; Genitori, L; Kotulska, K; Jozwiak, S; Jaworski, J; Liszewska, E; van Vliet, E A; Aronica, E

    2016-06-01

    Tuberous sclerosis complex (TSC) is a genetic disease presenting with multiple neurological symptoms including epilepsy, mental retardation, and autism. Abnormal activation of various inflammatory pathways has been observed in astrocytes in brain lesions associated with TSC. Increasing evidence supports the involvement of microRNAs in the regulation of astrocyte-mediated inflammatory response. To study the role of inflammation-related microRNAs in TSC, we employed real-time PCR and in situ hybridization to characterize the expression of miR21, miR146a, and miR155 in TSC lesions (cortical tubers and subependymal giant cell astrocytomas, SEGAs). We observed an increased expression of miR21, miR146a, and miR155 in TSC tubers compared with control and perituberal brain tissue. Expression was localized in dysmorphic neurons, giant cells, and reactive astrocytes and positively correlated with IL-1β expression. In addition, cultured human astrocytes and SEGA-derived cell cultures were used to study the regulation of the expression of these miRNAs in response to the proinflammatory cytokine IL-1β and to evaluate the effects of overexpression or knockdown of miR21, miR146a, and miR155 on inflammatory signaling. IL-1β stimulation of cultured glial cells strongly induced intracellular miR21, miR146a, and miR155 expression, as well as miR146a extracellular release. IL-1β signaling was differentially modulated by overexpression of miR155 or miR146a, which resulted in pro- or anti-inflammatory effects, respectively. This study provides supportive evidence that inflammation-related microRNAs play a role in TSC. In particular, miR146a and miR155 appear to be key players in the regulation of astrocyte-mediated inflammatory response, with miR146a as most interesting anti-inflammatory therapeutic candidate. PMID:27014996

  11. Cortical State and Attention

    PubMed Central

    Harris, Kenneth D.; Thiele, Alexander

    2012-01-01

    Preface The brain continuously adapts its processing machinery to behavioural demands. To achieve this it rapidly modulates the operating mode of cortical circuits, controlling the way information is transformed and routed. This article will focus on two experimental approaches by which the control of cortical information processing has been investigated: the study of state-dependent cortical processing in rodents, and attention in the primate visual system. Both processes involve a modulation of low-frequency activity fluctuations and spiking correlation, and are mediated by common receptor systems. We suggest that selective attention involves processes similar to state change, operating at a local columnar level to enhance the representation of otherwise nonsalient features while suppressing internally generated activity patterns. PMID:21829219

  12. 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.

  13. 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 USA" (Alisa Woodring);…

  14. 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

  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. Bidirectional radial Ca2+ activity regulates neurogenesis and migration during early cortical column formation

    PubMed Central

    Rash, Brian G.; Ackman, James B.; Rakic, Pasko

    2016-01-01

    Cortical columns are basic cellular and functional units of the cerebral cortex that are malformed in many brain disorders, but how they initially develop is not well understood. Using an optogenetic sensor in the mouse embryonic forebrain, we demonstrate that Ca2+ fluxes propagate bidirectionally within the elongated fibers of radial glial cells (RGCs), providing a novel communication mechanism linking the proliferative and postmitotic zones before the onset of synaptogenesis. Our results indicate that Ca2+ activity along RGC fibers provides feedback information along the radial migratory pathway, influencing neurogenesis and migration during early column development. Furthermore, we find that this columnar Ca2+ propagation is induced by Notch and fibroblast growth factor activities classically implicated in cortical expansion and patterning. Thus, cortical morphogens and growth factors may influence cortical column assembly in part by regulating long-distance Ca2+ communication along the radial axis of cortical development. PMID:26933693

  17. 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

  18. Cortical dynamics revisited.

    PubMed

    Singer, Wolf

    2013-12-01

    Recent discoveries on the organisation of the cortical connectome together with novel data on the dynamics of neuronal interactions require an extension of classical concepts on information processing in the cerebral cortex. These new insights justify considering the brain as a complex, self-organised system with nonlinear dynamics in which principles of distributed, parallel processing coexist with serial operations within highly interconnected networks. The observed dynamics suggest that cortical networks are capable of providing an extremely high-dimensional state space in which a large amount of evolutionary and ontogenetically acquired information can coexist and be accessible to rapid parallel search.

  19. Human Pluripotent Stem Cell-Derived Radial Glia Recapitulate Developmental Events and Provide Real-Time Access to Cortical Neurons and Astrocytes

    PubMed Central

    Peng, Chian-Yu; Pan, Liuliu; Kessler, John A.

    2015-01-01

    Studies of human cerebral cortex development are limited by difficulties in accessing and manipulating human neural tissue at specific development stages. We have derived human radial glia (hRG), which are responsible for most cerebral cortex neurogenesis, from human pluripotent stem cells. These hRG display the hallmark morphological, cellular, and molecular features of radial glia in vitro. They can be passaged and generate layer-specific subtypes of cortical neurons in a temporal and passage-dependent fashion. In later passages, they adopt a distinct progenitor phenotype that gives rise to cortical astrocytes and GABAergic interneurons. These hRG are also capable of following developmental cues to engraft, differentiate, migrate, and integrate into the embryonic mouse cortex when injected into E14 lateral ventricles. Moreover, hRG-derived cells can be cryopreserved at specific stages and retain their stage-specific phenotypes and competence when revived. Our study demonstrates that cultured hRG maintain a cell-intrinsic clock that regulates the progressive generation of stage-specific neuronal and glial subtypes. It also describes an easily accessible cell source for studying hRG lineage specification and progression and an on-demand supply of specific cortical neuron subtypes and astrocytes. PMID:25834120

  20. 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.

  1. Improved Oocyte Isolation and Embryonic Development of Outbred Deer Mice.

    PubMed

    Choi, Jung Kyu; He, Xiaoming

    2015-01-01

    In this study, we improved the protocol for isolating cumulus-oocyte complexes (COCs) from the outbred deer mice by using only one hormone (instead of the widely used combination of two hormones) with reduced dose. Moreover, we identified that significantly more metaphase II (MII) oocytes could be obtained by supplementing epidermal growth factor (EGF) and leukemia inhibition factor (LIF) into the previously established medium for in vitro maturation (IVM) of the COCs. Furthermore, we overcame the major challenge of two-cell block during embryonic development of deer mice after either in vitro fertilization (IVF) or parthenogenetic activation (PA) of the MII oocytes, by culturing the two-cell stage embryos on the feeder layer of inactivated mouse embryonic fibroblasts (MEFs) in the medium of mouse embryonic stem cells. Collectively, this work represents a major step forward in using deer mice as an outbred animal model for biomedical research on reproduction and early embryonic development. PMID:26184014

  2. Mouse embryonic fibroblasts exhibit extensive developmental and phenotypic diversity.

    PubMed

    Singhal, Prabhat K; Sassi, Slim; Lan, Lan; Au, Patrick; Halvorsen, Stefan C; Fukumura, Dai; Jain, Rakesh K; Seed, Brian

    2016-01-01

    Analysis of embryonic fibroblasts from GFP reporter mice indicates that the fibroblast cell type harbors a large collection of developmentally and phenotypically heterogeneous subtypes. Some of these cells exhibit multipotency, whereas others do not. Multiparameter flow cytometry analysis shows that a large number of distinct populations of fibroblast-like cells can be found in cultures initiated from different embryonic organs, and cells sorted according to their surface phenotype typically retain their characteristics on continued propagation in culture. Similarly, surface phenotypes of individual cloned fibroblast-like cells exhibit significant variation. The fibroblast cell class appears to contain a very large number of denumerable subtypes. PMID:26699463

  3. Visualization of Cortical Dynamics

    NASA Astrophysics Data System (ADS)

    Grinvald, Amiram

    2003-03-01

    Recent progress in studies of cortical dynamics will be reviewed including the combination of real time optical imaging based on voltage sensitive dyes, single and multi- unit recordings, LFP, intracellular recordings and microstimulation. To image the flow of neuronal activity from one cortical site to the next, in real time, we have used optical imaging based on newly designed voltage sensitive dyes and a Fuji 128x 128 fast camera which we modified. A factor of 20-40 fold improvement in the signal to noise ratio was obtained with the new dye during in vivo imaging experiments. This improvements has facilitates the exploration of cortical dynamics without signal averaging in the millisecond time domain. We confirmed that the voltage sensitive dye signal indeed reflects membrane potential changes in populations of neurons by showing that the time course of the intracellular activity recorded intracellularly from a single neuron was highly correlated in many cases with the optical signal from a small patch of cortex recorded nearby. We showed that the firing of single cortical neurons is not a random process but occurs when the on-going pattern of million of neurons is similar to the functional architecture map which correspond to the tuning properties of that neuron. Chronic optical imaging, combined with electrical recordings and microstimulation, over a long period of times of more than a year, was successfully applied also to the study of higher brain functions in the behaving macaque monkey.

  4. Cortical thinning in psychopathy

    PubMed Central

    Ly, Martina; Motzkin, Julian C.; Philippi, Carissa L.; Kirk, Gregory R.; Newman, Joseph P.; Kiehl, Kent A.; Koenigs, Michael

    2013-01-01

    Objective Psychopathy is a personality disorder associated with severely antisocial behavior and a host of cognitive and affective deficits. The neuropathological basis of the disorder has not been clearly established. Cortical thickness is a sensitive measure of brain structure that has been used to identify neurobiological abnormalities in a number of psychiatric disorders. The purpose of this study is to evaluate cortical thickness and corresponding functional connectivity in criminal psychopaths. Method Using T1 MRI data, we computed cortical thickness maps in a sample of adult male prison inmates selected based on psychopathy diagnosis (n=21 psychopathic inmates, n=31 non-psychopathic inmates). Using rest-fMRI data from a subset of these inmates (n=20 psychopathic inmates, n=20 non-psychopathic inmates), we then computed functional connectivity within networks exhibiting significant thinning among psychopaths. Results Relative to non-psychopaths, psychopaths exhibited significantly thinner cortex in a number of regions, including left insula and dorsal anterior cingulate cortex, bilateral precentral gyrus, bilateral anterior temporal cortex, and right inferior frontal gyrus. These neurostructural differences were not due to differences in age, IQ, or substance abuse. Psychopaths also exhibited a corresponding reduction in functional connectivity between left insula and left dorsal anterior cingulate cortex. Conclusions Psychopathy is associated with a distinct pattern of cortical thinning and reduced functional connectivity. PMID:22581200

  5. 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].

  6. 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

  7. Peptides regulate cortical thymocytes differentiation, proliferation, and apoptosis.

    PubMed

    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.

  8. 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

  9. Evaluation of different embryonating bird eggs and cell cultures for isolation efficiency of avian influenza A virus and avian paramyxovirus serotype 1 from real-time reverse transcription polymerase chain reaction--positive

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two hundred samples collected from Anseriformes, Charadriiformes, Gruiformes, and Galliformes were assayed using real-time reverse transcriptase polymerase chain reaction (RRT-PCR) for presence of avian influenza virus and avian paramyxovirus-1. Virus isolation using embryonating chicken eggs, embr...

  10. Embryonal rhabdomyosarcoma of foot with expansive growth between metatarsals.

    PubMed

    Suzuki, Y; Ehara, S; Shiraishi, H; Nishida, J; Murooka, G; Tamakawa, Y

    1997-02-01

    The case of a 14-year-old girl with rhabdomyosarcoma of the right foot is reported. Plain radiography showed a large nonspecific soft tissue tumor between the metatarsals with bowing of the metatarsals away from the mass. MR imaging showed a large soft tissue mass involving the metatarsals. The findings were conflicting, because the tumor had an infiltrative soft tissue mass and bowing of the metatarsals more suggestive of slow expansive growth. Bowing of short tubular bones may be a process similar to cortical saucerization, which is typically seen in Ewing's sarcoma, and it can be one of the findings of high grade neoplasms, such as embryonal rhabdomyosarcoma. PMID:9060106

  11. Mechanical signaling coordinates the embryonic heartbeat.

    PubMed

    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

    2016-08-01

    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

  12. [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.

  13. Purely Cortical Anaplastic Ependymoma

    PubMed Central

    Romero, Flávio Ramalho; Zanini, Marco Antônio; Ducati, Luis Gustavo; Vital, Roberto Bezerra; de Lima Neto, Newton Moreira; Gabarra, Roberto Colichio

    2012-01-01

    Ependymomas are glial tumors derived from ependymal cells lining the ventricles and the central canal of the spinal cord. It may occur outside the ventricular structures, representing the extraventicular form, or without any relationship of ventricular system, called ectopic ependymona. Less than fifteen cases of ectopic ependymomas were reported and less than five were anaplastic. We report a rare case of pure cortical ectopic anaplastic ependymoma. PMID:23119204

  14. Purely cortical anaplastic ependymoma.

    PubMed

    Romero, Flávio Ramalho; Zanini, Marco Antônio; Ducati, Luis Gustavo; Vital, Roberto Bezerra; de Lima Neto, Newton Moreira; Gabarra, Roberto Colichio

    2012-01-01

    Ependymomas are glial tumors derived from ependymal cells lining the ventricles and the central canal of the spinal cord. It may occur outside the ventricular structures, representing the extraventicular form, or without any relationship of ventricular system, called ectopic ependymona. Less than fifteen cases of ectopic ependymomas were reported and less than five were anaplastic. We report a rare case of pure cortical ectopic anaplastic ependymoma.

  15. 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

  16. Potential of embryonic and adult stem cells in vitro.

    PubMed

    Czyz, Jaroslaw; Wiese, Cornelia; Rolletschek, Alexandra; Blyszczuk, Przemyslaw; Cross, Michael; Wobus, Anna M

    2003-01-01

    Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissue-specific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.

  17. 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

  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. 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.

  20. 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

  1. Cortical Clefts and Cortical Bumps: A Continuous Spectrum.

    PubMed

    Biswas, Asthik; Furruqh, Farha; Thirunavukarasu, Suresh; Vivekandan, Ravichandran

    2016-07-01

    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

  2. Derivation of human embryonic stem cells in defined conditions.

    PubMed

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  3. The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone

    PubMed Central

    Arnold, Sebastian J.; Huang, Guo-Jen; Cheung, Amanda F.P.; Era, Takumi; Nishikawa, Shin-Ichi; Bikoff, Elizabeth K.; Molnár, Zoltán; Robertson, Elizabeth J.; Groszer, Matthias

    2008-01-01

    The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Here we demonstrate that conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ. PMID:18794345

  4. Cortical Basal Ganglionic Degeneration

    PubMed Central

    Scarmeas, Nikolaos; Chin, Steven S.; Marder, Karen

    2011-01-01

    In this case study, we describe the symptoms, neuropsychological testing, and brain pathology of a retired mason's assistant with cortical basal ganglionic degeneration (CBGD). CBGD is an extremely rare neurodegenerative disease that is categorized under both Parkinsonian syndromes and frontal lobe dementias. It affects men and women nearly equally, and the age of onset is usually in the sixth decade of life. CBGD is characterized by Parkinson's-like motor symptoms and by deficits of movement and cognition, indicating focal brain pathology. Neuronal cell loss is ultimately responsible for the neurological symptoms. PMID:14602941

  5. Pluripotent states of human embryonic stem cells.

    PubMed

    Chen, Yifei; Lai, Dongmei

    2015-02-01

    Since human embryonic stem cells (hESCs) were first isolated and successfully cultured in vitro, the pluripotent potential of hESCs has been underestimated. The pluripotency of mouse embryonic stem cells (mESCs) can be categorized as naïve and primed, depending on their corresponding in vivo developing phases. mESC morphology differs at distinct pluripotent states, which differ in signaling dependence, gene expression, epigenetic features, and developmental potential. hESCs resemble mouse stem cells at primed pluripotency, and consequently are believed to correspond to a later developmental stage in vivo than mESCs. Nevertheless, recent studies indicate that a naïve state of pluripotency may exist in hESCs, and the pluripotency of hESCs also can be enhanced by genetic modification or optimized culture systems. These findings provide novel insight into the properties and differentiation potential of hESCs. Here, we review the recent advances in characterization of ESC states and investigate the mechanisms regulating hESC pluripotency. PMID:25393391

  6. Time in Cortical Circuits

    PubMed Central

    Shadlen, Michael N.; Jazayeri, Mehrdad; Nobre, Anna C.; Buonomano, Dean V.

    2015-01-01

    Time is central to cognition. However, the neural basis for time-dependent cognition remains poorly understood. We explore how the temporal features of neural activity in cortical circuits and their capacity for plasticity can contribute to time-dependent cognition over short time scales. This neural activity is linked to cognition that operates in the present or anticipates events or stimuli in the near future. We focus on deliberation and planning in the context of decision making as a cognitive process that integrates information across time. We progress to consider how temporal expectations of the future modulate perception. We propose that understanding the neural basis for how the brain tells time and operates in time will be necessary to develop general models of cognition. SIGNIFICANCE STATEMENT Time is central to cognition. However, the neural basis for time-dependent cognition remains poorly understood. We explore how the temporal features of neural activity in cortical circuits and their capacity for plasticity can contribute to time-dependent cognition over short time scales. We propose that understanding the neural basis for how the brain tells time and operates in time will be necessary to develop general models of cognition. PMID:26468192

  7. In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

    PubMed Central

    Grapin-Botton, Anne

    2014-01-01

    The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity. PMID:25079453

  8. In vitro pancreas organogenesis from dispersed mouse embryonic progenitors.

    PubMed

    Greggio, Chiara; De Franceschi, Filippo; Figueiredo-Larsen, Manuel; Grapin-Botton, Anne

    2014-07-19

    The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells (1). The whole embryonic organ can be cultured at multiple stages of development (2-4). These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.

  9. In vitro pancreas organogenesis from dispersed mouse embryonic progenitors.

    PubMed

    Greggio, Chiara; De Franceschi, Filippo; Figueiredo-Larsen, Manuel; Grapin-Botton, Anne

    2014-01-01

    The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells (1). The whole embryonic organ can be cultured at multiple stages of development (2-4). These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity. PMID:25079453

  10. 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

  11. 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.

  12. Cortical plasticity and rehabilitation.

    PubMed

    Moucha, Raluca; Kilgard, Michael P

    2006-01-01

    The brain is constantly adapting to environmental and endogenous changes (including injury) that occur at every stage of life. The mechanisms that regulate neural plasticity have been refined over millions of years. Motivation and sensory experience directly shape the rewiring that makes learning and neurological recovery possible. Guiding neural reorganization in a manner that facilitates recovery of function is a primary goal of neurological rehabilitation. As the rules that govern neural plasticity become better understood, it will be possible to manipulate the sensory and motor experience of patients to induce specific forms of plasticity. This review summarizes our current knowledge regarding factors that regulate cortical plasticity, illustrates specific forms of reorganization induced by control of each factor, and suggests how to exploit these factors for clinical benefit.

  13. Nicotinic acetylcholine receptors containing alpha 7 subunits on rat cortical neurons do not undergo long-lasting inactivation even when up-regulated by chronic nicotine exposure.

    PubMed

    Kawai, H; Berg, D K

    2001-09-01

    Chronic exposure to (-)nicotine has been widely reported to up-regulate nicotinic acetylcholine receptors on neurons and induce long-term inactivation as a possible cause. Nicotinic receptors containing alpha 7 subunits are among the most abundant in brain and influence diverse cellular events. Whole-cell patch clamp recording from embryonic rat cortical neurons in culture was used to identify responses from alpha 7-containing receptors. Immunochemical staining for glutamic acid decarboxylase (GAD) indicated that both GABAergic and non-GABAergic neurons expressed the receptors. Exposure to micromolar concentrations of nicotine for 1-4 days caused up-regulation of the receptors as measured by [alpha-(125)I]-bungarotoxin binding. Carbachol produced the same up-regulation, and cell counts demonstrated that neuronal survival was unchanged. The up-regulation was accompanied by an increased whole-cell response; no evidence was found for long-lasting inactivation. Autonomic alpha 7-containing receptors also avoided long-lasting inactivation, even though the receptors were down-regulated by nicotine. Blocking protein synthesis or protein glycosylation prevented receptor up-regulation on cortical neurons, suggesting that new synthesis was required. No evidence was found for a pre-existing intracellular pool that supplied receptors to the surface. The results indicate that alpha 7-containing receptors differ from other receptor subtypes in their regulation by nicotine and demonstrate further that long-lasting inactivation is not an obligatory requirement for up-regulation in this case.

  14. 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.

  15. 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

  16. Chromosomal Aneuploidies and Early Embryonic Developmental Arrest

    PubMed Central

    Maurer, Maria; Ebner, Thomas; Puchner, Manuela; Mayer, Richard Bernhard; Shebl, Omar; Oppelt, Peter; Duba, Hans-Christoph

    2015-01-01

    Background Selecting the best embryo for transfer, with the highest chance of achieving a vital pregnancy, is a major goal in current in vitro fertilization (IVF) technology. The high rate of embryonic developmental arrest during IVF treatment is one of the limitations in achieving this goal. Chromosomal abnormalities are possibly linked with chromosomal arrest and selection against abnormal fertilization products. The objective of this study was to evaluate the frequency and type of chromosomal abnormalities in preimplantation embryos with developmental arrest. Materials and Methods This cohort study included blastomeres of embryos with early developmental arrest that were biopsied and analyzed by fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 16, 18, 21 and 22. Forty-five couples undergoing IVF treatment were included, and 119 arrested embryos were biopsied. All probes were obtained from the Kinderwunsch Zentrum, Linz, Austria, between August 2009 and August 2011. Results Of these embryos, 31.6% were normal for all chromosomes tested, and 68.4% were abnormal. Eleven embryos were uniformly aneuploid, 20 were polyploid, 3 were haploid, 11 displayed mosaicism and 22 embryos exhibited chaotic chromosomal complement. Conclusion Nearly 70% of arrested embryos exhibit chromosomal errors, making chromosomal abnormalities a major cause of embryonic arrest and may be a further explanation for the high developmental failure rates during culture of the embryos in the IVF setting. PMID:26644858

  17. 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.

  18. Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

    PubMed

    Hertsenberg, Andrew J; Funderburgh, James L

    2016-01-01

    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.

  19. Compatibility of embryonic stem cells with biomaterials.

    PubMed

    Handschel, Jörg; Berr, Karin; Depprich, Rita; Naujoks, Christian; Kübler, Norbert R; Meyer, Ulrich; Ommerborn, Michelle; Lammers, Lydia

    2009-05-01

    Periodontal bone defects and atrophy of the jaws in an aging population are of special concern. Tissue engineering using embryonic stem cells (ESCs) and biomaterials may offer new therapeutic options. The purpose of this study is to evaluate the compatibility of ESCs with biomaterials and the influence of biomaterials on the osteogenic gene expression profile.Therefore, ESCs are cultured with various biomaterials. The cytocompatibility of murine ESCs is measured regarding the proliferation of the cells on the materials by CyQUANT assay, the morphology by scanning electron microscopy, and the influence on the gene expression by real time PCR.The results show that insoluble collagenous bone matrix, followed by beta-tricalciumphosphate, is most suitable for bone tissue engineering regarding cell proliferation, and phenotype. The gene expression analysis indicates that biomaterials do influence the gene expression of ESCs.Our results provide new insight into the cytocompatibility of ESCs on different scaffolds.

  20. 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

  1. Spatial integration and cortical dynamics.

    PubMed Central

    Gilbert, C D; Das, A; Ito, M; Kapadia, M; Westheimer, G

    1996-01-01

    Cells in adult primary visual cortex are capable of integrating information over much larger portions of the visual field than was originally thought. Moreover, their receptive field properties can be altered by the context within which local features are presented and by changes in visual experience. The substrate for both spatial integration and cortical plasticity is likely to be found in a plexus of long-range horizontal connections, formed by cortical pyramidal cells, which link cells within each cortical area over distances of 6-8 mm. The relationship between horizontal connections and cortical functional architecture suggests a role in visual segmentation and spatial integration. The distribution of lateral interactions within striate cortex was visualized with optical recording, and their functional consequences were explored by using comparable stimuli in human psychophysical experiments and in recordings from alert monkeys. They may represent the substrate for perceptual phenomena such as illusory contours, surface fill-in, and contour saliency. The dynamic nature of receptive field properties and cortical architecture has been seen over time scales ranging from seconds to months. One can induce a remapping of the topography of visual cortex by making focal binocular retinal lesions. Shorter-term plasticity of cortical receptive fields was observed following brief periods of visual stimulation. The mechanisms involved entailed, for the short-term changes, altering the effectiveness of existing cortical connections, and for the long-term changes, sprouting of axon collaterals and synaptogenesis. The mutability of cortical function implies a continual process of calibration and normalization of the perception of visual attributes that is dependent on sensory experience throughout adulthood and might further represent the mechanism of perceptual learning. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8570604

  2. Establishment and Characterization of Naïve Pluripotency in Human Embryonic Stem Cells.

    PubMed

    Warrier, Sharat; Popovic, Mina; Van der Jeught, Margot; Heindryckx, Björn

    2016-01-01

    Mouse embryonic stem cells are known to represent the naïve state of pluripotency, while human embryonic stem cells typically represented the primed state of pluripotency, characterized by a higher drift toward differentiation and some other disadvantages. Here we describe an efficient method for rapid, transgene free induction of the naïve pluripotent state in human by applying a novel combination of small molecules and growth factors in the culture medium (2i, LIF, basic fibroblast growth factor, ascorbic acid, and forskolin). Conversion of primed human embryonic stem cells towards the naive pluripotent state should be confirmed by a detailed characterization of the cells, as described in this chapter.

  3. 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

  4. Infiltrating cells from host brain restore the microglial population in grafted cortical tissue.

    PubMed

    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

  5. 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).

  6. Nitric oxide synthase-3 promotes embryonic development of atrioventricular valves.

    PubMed

    Liu, Yin; Lu, Xiangru; Xiang, Fu-Li; Lu, Man; Feng, Qingping

    2013-01-01

    Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3(-/-) mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3(-/-) compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3(-/-) mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1(+) cells in the AV cushion were decreased in NOS3(-/-) compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ), bone morphogenetic protein (BMP2) and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3(-/-) compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.

  7. 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, ...

  8. 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. PMID:16180222

  9. Cell cycle synchronization of embryonic stem cells: Effect of serum deprivation on the differentiation of embryonic bodies in vitro

    SciTech Connect

    Zhang Enming; Li Xiaolong; Zhang Shufang; Chen Liangqiang; Zheng Xiaoxiang . E-mail: zxx@mail.bme.zju.edu.cn

    2005-08-12

    Research on stem-cell transplantation has indicated that the success of transplantation largely depends on synchronizing donor cells into the G0/G1 phase. In this study, we investigated the profile of embryonic stem (ES) cell synchronization and its effect on the formation of embryonic bodies (EBs) using cell culture with serum deprivation. The D3 cell line of ES cells was used, and parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher than that in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully after serum supply. EBs formed from synchronized ES cells have higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells. Our study provides a method for ES treatment before cell transplantation that possibly helps to decrease the rate of cell death after transplantation.

  10. Synaptogenesis in purified cortical subplate neurons.

    PubMed

    McKellar, Claire E; Shatz, Carla J

    2009-08-01

    An ideal preparation for investigating events during synaptogenesis would be one in which synapses are sparse, but can be induced at will using a rapid, exogenous trigger. We describe a culture system of immunopurified subplate neurons in which synaptogenesis can be triggered, providing the first homogeneous culture of neocortical neurons for the investigation of synapse development. Synapses in immunopurified rat subplate neurons are sparse, and can be induced by a 48-h exposure to feeder layers of neurons and glia, an induction more rapid than any previously reported. Induced synapses are electrophysiologically functional and ultrastructurally normal. Microarray and real-time PCR experiments reveal a new program of gene expression accompanying synaptogenesis. Surprisingly few known synaptic genes are upregulated during the first 24 h of synaptogenesis; Gene Ontology annotation reveals a preferential upregulation of synaptic genes only at a later time. In situ hybridization confirms that some of the genes regulated in cultures are also expressed in the developing cortex. This culture system provides both a means of studying synapse formation in a homogeneous population of cortical neurons, and better synchronization of synaptogenesis, permitting the investigation of neuron-wide events following the triggering of synapse formation.

  11. Synaptogenesis in Purified Cortical Subplate Neurons

    PubMed Central

    Shatz, Carla J.

    2009-01-01

    An ideal preparation for investigating events during synaptogenesis would be one in which synapses are sparse, but can be induced at will using a rapid, exogenous trigger. We describe a culture system of immunopurified subplate neurons in which synaptogenesis can be triggered, providing the first homogeneous culture of neocortical neurons for the investigation of synapse development. Synapses in immunopurified rat subplate neurons are sparse, and can be induced by a 48-h exposure to feeder layers of neurons and glia, an induction more rapid than any previously reported. Induced synapses are electrophysiologically functional and ultrastructurally normal. Microarray and real-time PCR experiments reveal a new program of gene expression accompanying synaptogenesis. Surprisingly few known synaptic genes are upregulated during the first 24 h of synaptogenesis; Gene Ontology annotation reveals a preferential upregulation of synaptic genes only at a later time. In situ hybridization confirms that some of the genes regulated in cultures are also expressed in the developing cortex. This culture system provides both a means of studying synapse formation in a homogeneous population of cortical neurons, and better synchronization of synaptogenesis, permitting the investigation of neuron-wide events following the triggering of synapse formation. PMID:19029062

  12. 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.

  13. 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.

  14. 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

    2015-01-01

    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. PMID:26133243

  15. Directed Differentiation of Human Embryonic Stem Cells into Neural Progenitors.

    PubMed

    Banda, Erin; Grabel, Laura

    2016-01-01

    A variety of protocols have been used to produce neural progenitors from human embryonic stem cells. We have focused on a monolayer culture approach that generates neural rosettes. To initiate differentiation, cells are plated in a serum-free nutrient-poor medium in the presence of a BMP inhibitor. Depending on the cell line used, additional growth factor inhibitors may be required to promote neural differentiation. Long-term culture and addition of the Notch inhibitor DAPT can promote terminal neuronal differentiation. Extent of differentiation is monitored using immunocytochemistry for cell type-specific markers.

  16. A circuit for motor cortical modulation of auditory cortical activity.

    PubMed

    Nelson, Anders; Schneider, David M; Takatoh, Jun; Sakurai, Katsuyasu; Wang, Fan; Mooney, Richard

    2013-09-01

    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

  17. A proapoptotic effect of valproic acid on progenitors of embryonic stem cell-derived glutamatergic neurons

    PubMed Central

    Fujiki, R; Sato, A; Fujitani, M; Yamashita, T

    2013-01-01

    Valproic acid (VPA) is a branched-chain saturated fatty acid with a long history of clinical use as an antiepileptic drug (AED). VPA is also known to inhibit histone deacetylases (HDACs) and to cause diverse effects on neural progenitor cells (NPCs) and neurons. Although the neuroprotective or neurodestructive effects of VPA have been investigated in heterogeneous cell populations, in this study, we used homogeneous populations of NPCs and glutamatergic cortical pyramidal neurons, which were differentiated from embryonic stem (ES) cells. At therapeutic concentrations, VPA had a proapoptotic effect on ES cell-derived NPCs of glutamatergic neurons, but not on their progeny. This effect of VPA most likely occurred through the inhibition of HDACs, because similar phenotypes were observed following treatment with other HDAC inhibitors (HDACis) such as trichostatin A and sodium butyrate. The proapoptotic phenotype was not observed when cells were exposed to a structural analog of VPA, valpromide (VPM), which has the same antiepileptic effect as VPA, but does not inhibit HDACs. Western blotting confirmed that treatment with HDACis, but not VPM, significantly increased the levels of histone H3 acetylation in NPCs. HDACi treatments did not affect the survival of neurons, although the acetylation levels were increased to a limited extent. These results, which are based on a homogeneous culture system, suggest that VPA inhibits HDAC activity and induces the apoptosis of NPCs that are fated to differentiate into glutamatergic neurons. The dose-dependent effects of VPA both on apoptosis and hyperacetylation of histone H3 in NPCs supported this notion. These cell type- and differentiation stage-specific effects of VPA imply that dysfunction of HDACs during pregnancy significantly increase the risk of congenital malformations associated with VPA administration. PMID:23788034

  18. In vitro thermal effects on embryonic cells of endangered hawksbill turtle Eretmochelys imbricata.

    PubMed

    Takeshita, Satoshi; Matsuda, Naoki; Kodama, Seiji; Suzuki, Keiji; Watanabe, Masami

    2013-12-01

    The hawksbill turtle is an ectotherm, whose sex is determined by temperature during embryonic development. This study aimed to determine whether embryonic hawksbill turtle cells respond differently to temperature than mammalian cells. Embryonic hawksbill turtle cells were established in culture, and thermal effects on these cells were investigated in vitro. Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with non-essential amino acids, vitamin solution, sodium pyruvate, and 10% fetal bovine serum at 33°C and cell proliferation occurred at 25-33°C. When cells were incubated at 37°C (the temperature of mammalian cell culture) for 24 h, cell growth was completely inhibited. This growth inhibition was evidently recovered by changing the incubation temperature back to 33°C. Expression of heat shock protein was found to increase with elevating culture temperature from 25 to 33°C.

  19. In vitro thermal effects on embryonic cells of endangered hawksbill turtle Eretmochelys imbricata.

    PubMed

    Takeshita, Satoshi; Matsuda, Naoki; Kodama, Seiji; Suzuki, Keiji; Watanabe, Masami

    2013-12-01

    The hawksbill turtle is an ectotherm, whose sex is determined by temperature during embryonic development. This study aimed to determine whether embryonic hawksbill turtle cells respond differently to temperature than mammalian cells. Embryonic hawksbill turtle cells were established in culture, and thermal effects on these cells were investigated in vitro. Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with non-essential amino acids, vitamin solution, sodium pyruvate, and 10% fetal bovine serum at 33°C and cell proliferation occurred at 25-33°C. When cells were incubated at 37°C (the temperature of mammalian cell culture) for 24 h, cell growth was completely inhibited. This growth inhibition was evidently recovered by changing the incubation temperature back to 33°C. Expression of heat shock protein was found to increase with elevating culture temperature from 25 to 33°C. PMID:24320182

  20. Imprinting and recalling cortical ensembles.

    PubMed

    Carrillo-Reid, Luis; Yang, Weijian; Bando, Yuki; Peterka, Darcy S; Yuste, Rafael

    2016-08-12

    Neuronal ensembles are coactive groups of neurons that may represent building blocks of cortical circuits. These ensembles could be formed by Hebbian plasticity, whereby synapses between coactive neurons are strengthened. Here we report that repetitive activation with two-photon optogenetics of neuronal populations from ensembles in the visual cortex of awake mice builds neuronal ensembles that recur spontaneously after being imprinted and do not disrupt preexisting ones. Moreover, imprinted ensembles can be recalled by single- cell stimulation and remain coactive on consecutive days. Our results demonstrate the persistent reconfiguration of cortical circuits by two-photon optogenetics into neuronal ensembles that can perform pattern completion. PMID:27516599

  1. Grid cells and cortical representation.

    PubMed

    Moser, Edvard I; Roudi, Yasser; Witter, Menno P; Kentros, Clifford; Bonhoeffer, Tobias; Moser, May-Britt

    2014-07-01

    One of the grand challenges in neuroscience is to comprehend neural computation in the association cortices, the parts of the cortex that have shown the largest expansion and differentiation during mammalian evolution and that are thought to contribute profoundly to the emergence of advanced cognition in humans. In this Review, we use grid cells in the medial entorhinal cortex as a gateway to understand network computation at a stage of cortical processing in which firing patterns are shaped not primarily by incoming sensory signals but to a large extent by the intrinsic properties of the local circuit.

  2. Horizontal integration and cortical dynamics.

    PubMed

    Gilbert, C D

    1992-07-01

    We have discussed several results that lead to a view that cells in the visual system are endowed with dynamic properties, influenced by context, expectation, and long-term modifications of the cortical network. These observations will be important for understanding how neuronal ensembles produce a system that perceives, remembers, and adapts to injury. The advantage to being able to observe changes at early stages in a sensory pathway is that one may be able to understand the way in which neuronal ensembles encode and represent images at the level of their receptive field properties, of cortical topographies, and of the patterns of connections between cells participating in a network.

  3. Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics

    PubMed Central

    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

    2016-01-01

    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. PMID:26740550

  4. The myokine irisin increases cortical bone mass

    PubMed Central

    Colaianni, Graziana; Cuscito, Concetta; Mongelli, Teresa; Pignataro, Paolo; Buccoliero, Cinzia; Liu, Peng; Lu, Ping; Sartini, Loris; Di Comite, Mariasevera; Mori, Giorgio; Di Benedetto, Adriana; Brunetti, Giacomina; Yuen, Tony; Sun, Li; Reseland, Janne E.; Colucci, Silvia; New, Maria I.; Zaidi, Mone; Cinti, Saverio; Grano, Maria

    2015-01-01

    It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg−1. We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg−1 per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, β-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin–injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle–bone connectivity. PMID:26374841

  5. The myokine irisin increases cortical bone mass.

    PubMed

    Colaianni, Graziana; Cuscito, Concetta; Mongelli, Teresa; Pignataro, Paolo; Buccoliero, Cinzia; Liu, Peng; Lu, Ping; Sartini, Loris; Di Comite, Mariasevera; Mori, Giorgio; Di Benedetto, Adriana; Brunetti, Giacomina; Yuen, Tony; Sun, Li; Reseland, Janne E; Colucci, Silvia; New, Maria I; Zaidi, Mone; Cinti, Saverio; Grano, Maria

    2015-09-29

    It is unclear how physical activity stimulates new bone synthesis. We explored whether irisin, a newly discovered myokine released upon physical activity, displays anabolic actions on the skeleton. Young male mice were injected with vehicle or recombinant irisin (r-irisin) at a low cumulative weekly dose of 100 µg kg(-1). We observed significant increases in cortical bone mass and strength, notably in cortical tissue mineral density, periosteal circumference, polar moment of inertia, and bending strength. This anabolic action was mediated primarily through the stimulation of bone formation, but with parallel notable reductions in osteoclast numbers. The trabecular compartment of the same bones was spared, as were vertebrae from the same mice. Higher irisin doses (3,500 µg kg(-1) per week) cause browning of adipose tissue; this was not seen with low-dose r-irisin. Expectedly, low-dose r-irisin modulated the skeletal genes, Opn and Sost, but not Ucp1 or Pparγ expression in white adipose tissue. In bone marrow stromal cell cultures, r-irisin rapidly phosphorylated Erk, and up-regulated Atf4, Runx2, Osx, Lrp5, β-catenin, Alp, and Col1a1; this is consistent with a direct receptor-mediated action to stimulate osteogenesis. We also noted that, although the irisin precursor Fndc5 was expressed abundantly in skeletal muscle, other sites, such as bone and brain, also expressed Fndc5, albeit at low levels. Furthermore, muscle fibers from r-irisin-injected mice displayed enhanced Fndc5 positivity, and irisin induced Fdnc5 mRNA expression in cultured myoblasts. Our data therefore highlight a previously unknown action of the myokine irisin, which may be the molecular entity responsible for muscle-bone connectivity. PMID:26374841

  6. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells

    PubMed Central

    Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice. PMID:26091287

  7. Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

    PubMed

    Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

    2015-01-01

    Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

  8. Hbp1 regulates the timing of neuronal differentiation during cortical development by controlling cell cycle progression.

    PubMed

    Watanabe, Naoki; Kageyama, Ryoichiro; Ohtsuka, Toshiyuki

    2015-07-01

    In the developing mammalian brain, neural stem cells (NSCs) initially expand the progenitor pool by symmetric divisions. NSCs then shift from symmetric to asymmetric division and commence neurogenesis. Although the precise mechanisms regulating the developmental timing of this transition have not been fully elucidated, gradual elongation in the length of the cell cycle and coinciding accumulation of determinants that promote neuronal differentiation might function as a biological clock that regulates the onset of asymmetric division and neurogenesis. We conducted gene expression profiling of embryonic NSCs in the cortical regions and found that expression of high mobility group box transcription factor 1 (Hbp1) was upregulated during neurogenic stages. Induced conditional knockout mice of Hbp1, generated by crossing with Nestin-CreER(T2) mice, exhibited a remarkable dilatation of the telencephalic vesicles with a tangentially expanded ventricular zone and a thinner cortical plate containing reduced numbers of neurons. In these Hbp1-deficient mouse embryos, neural stem/progenitor cells continued to divide with a shorter cell cycle length. Moreover, downstream target genes of the Wnt signaling, such as cyclin D1 (Ccnd1) and c-jun (Jun), were upregulated in the germinal zone of the cortical regions. These results indicate that Hbp1 plays a crucial role in regulating the timing of cortical neurogenesis by elongating the cell cycle and that it is essential for normal cortical development.

  9. 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

  10. 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.

  11. 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. PMID:27514653

  12. 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.

  13. [Epigenetic influence on embryonic development].

    PubMed

    Donkin, Ida; Barrès, Romain; Pinborg, Anja

    2016-09-12

    The epigenome is sensitive to environmental changes and can sustainably alter gene expression, notably during embryonic development. New research indicates that epigenetic factors are heritable, which is why paternal lifestyle may affect fetal development and risk of disease. Children conceived by assisted reproduction technology (ART) have an increased risk of peri- and postnatal complications, and as specific ART protocols associate with specific risk profiles, the procedures themselves may cause epigenetic changes contributing to the altered outcomes of the 5,000 Danish children annually conceived by ART. PMID:27649584

  14. 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.

  15. 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.

  16. Abnormalities in structural covariance of cortical gyrification in schizophrenia.

    PubMed

    Palaniyappan, Lena; Park, Bert; Balain, Vijender; Dangi, Raj; Liddle, Peter

    2015-07-01

    The highly convoluted shape of the adult human brain results from several well-coordinated maturational events that start from embryonic development and extend through the adult life span. Disturbances in these maturational events can result in various neurological and psychiatric disorders, resulting in abnormal patterns of morphological relationship among cortical structures (structural covariance). Structural covariance can be studied using graph theory-based approaches that evaluate topological properties of brain networks. Covariance-based graph metrics allow cross-sectional study of coordinated maturational relationship among brain regions. Disrupted gyrification of focal brain regions is a consistent feature of schizophrenia. However, it is unclear if these localized disturbances result from a failure of coordinated development of brain regions in schizophrenia. We studied the structural covariance of gyrification in a sample of 41 patients with schizophrenia and 40 healthy controls by constructing gyrification-based networks using a 3-dimensional index. We found that several key regions including anterior insula and dorsolateral prefrontal cortex show increased segregation in schizophrenia, alongside reduced segregation in somato-sensory and occipital regions. Patients also showed a lack of prominence of the distributed covariance (hubness) of cingulate cortex. The abnormal segregated folding pattern in the right peri-sylvian regions (insula and fronto-temporal cortex) was associated with greater severity of illness. The study of structural covariance in cortical folding supports the presence of subtle deviation in the coordinated development of cortical convolutions in schizophrenia. The heterogeneity in the severity of schizophrenia could be explained in part by aberrant trajectories of neurodevelopment.

  17. 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

  18. 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.

  19. Functional Calcium Imaging in Developing Cortical Networks

    PubMed Central

    Dawitz, Julia; Kroon, Tim; Hjorth, J.J. Johannes; Meredith, Rhiannon M.

    2011-01-01

    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

  20. Reconstitution of cortical Dynein function.

    PubMed

    Roth, Sophie; Laan, Liedewij; Dogterom, Marileen

    2014-01-01

    Cytoplasmic dynein is a major microtubule (MT)-associated motor in nearly all eukaryotic cells. A subpopulation of dyneins associates with the cell cortex and the interaction of this cortical dynein with MTs helps to drive processes such as nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during wound healing. In this chapter, we describe three types of assays in which interactions between cortical dynein and MTs are reconstituted in vitro at increasing levels of complexity. In the first 1D assay, MTs, nucleated from a centrosome attached to a surface, grow against dynein-coated gold barriers. In this assay configuration, the interactions between MTs and dynein attached to a barrier can be studied in great detail. In the second and third assays, a freely moving dynamic aster is placed in either a 2D microfabricated chamber or a 3D water-in-oil emulsion droplet, with dynein-coated boundaries. These assays can be used to study how cortical dynein positions centrosomes. Finally, we discuss future possibilities for increasing the complexity of these reconstituted systems.

  1. Cortical Control of Affective Networks

    PubMed Central

    Kumar, Sunil; Black, Sherilynn J.; Hultman, Rainbo; Szabo, Steven T.; DeMaio, Kristine D.; Du, Jeanette; Katz, Brittany M.; Feng, Guoping; Covington, Herbert E.; Dzirasa, Kafui

    2013-01-01

    Transcranial magnetic stimulation and deep brain stimulation have emerged as therapeutic modalities for treatment refractory depression; however, little remains known regarding the circuitry that mediates the therapeutic effect of these approaches. Here we show that direct optogenetic stimulation of prefrontal cortex (PFC) descending projection neurons in mice engineered to express Chr2 in layer V pyramidal neurons (Thy1–Chr2 mice) models an antidepressant-like effect in mice subjected to a forced-swim test. Furthermore, we show that this PFC stimulation induces a long-lasting suppression of anxiety-like behavior (but not conditioned social avoidance) in socially stressed Thy1–Chr2 mice: an effect that is observed >10 d after the last stimulation. Finally, we use optogenetic stimulation and multicircuit recording techniques concurrently in Thy1–Chr2 mice to demonstrate that activation of cortical projection neurons entrains neural oscillatory activity and drives synchrony across limbic brain areas that regulate affect. Importantly, these neural oscillatory changes directly correlate with the temporally precise activation and suppression of limbic unit activity. Together, our findings show that the direct activation of cortical projection systems is sufficient to modulate activity across networks underlying affective regulation. They also suggest that optogenetic stimulation of cortical projection systems may serve as a viable therapeutic strategy for treating affective disorders. PMID:23325249

  2. Genetic Manipulation of Human Embryonic Stem Cells.

    PubMed

    Eiges, Rachel

    2016-01-01

    One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent, and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods proved to be extremely useful in mouse ES cells, for monitoring and directing differentiation, discovering unknown genes, and studying their function, and are now being extensively implemented in human ES cells (HESCs). This chapter describes the different approaches and methodologies that have been applied for the genetic manipulation of HESCs and their applications. Detailed protocols for generating clones of genetically modified HESCs by transfection, electroporation, and infection will be described, with special emphasis on the important technical details that are required for this purpose. All protocols are equally effective in human-induced pluripotent stem (iPS) cells.

  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. ETS transcription factors in embryonic vascular development.

    PubMed

    Craig, Michael P; Sumanas, Saulius

    2016-07-01

    At least thirteen ETS-domain transcription factors are expressed during embryonic hematopoietic or vascular development and potentially function in the formation and maintenance of the embryonic vasculature or blood lineages. This review summarizes our current understanding of the specific roles played by ETS factors in vasculogenesis and angiogenesis and the implications of functional redundancies between them.

  5. ERK5 MAP Kinase Regulates Neurogenin1 during Cortical Neurogenesis

    PubMed Central

    Cundiff, Paige; Liu, Lidong; Wang, Yupeng; Zou, Junhui; Pan, Yung-Wei; Abel, Glen; Duan, Xin; Ming, Guo-li; Englund, Chris; Hevner, Robert; Xia, Zhengui

    2009-01-01

    The commitment of multi-potent cortical progenitors to a neuronal fate depends on the transient induction of the basic-helix-loop-helix (bHLH) family of transcription factors including Neurogenin 1 (Neurog1). Previous studies have focused on mechanisms that control the expression of these proteins while little is known about whether their pro-neural activities can be regulated by kinase signaling pathways. Using primary cultures and ex vivo slice cultures, here we report that both the transcriptional and pro-neural activities of Neurog1 are regulated by extracellular signal-regulated kinase (ERK) 5 signaling in cortical progenitors. Activation of ERK5 potentiated, while blocking ERK5 inhibited Neurog1-induced neurogenesis. Furthermore, endogenous ERK5 activity was required for Neurog1-initiated transcription. Interestingly, ERK5 activation was sufficient to induce Neurog1 phosphorylation and ERK5 directly phosphorylated Neurog1 in vitro. We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1. Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1. Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors. PMID:19365559

  6. [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. PMID:19160841

  7. [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.

  8. 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.

  9. Scalable production of embryonic stem cell-derived cells.

    PubMed

    Dang, Stephen M; Zandstra, Peter W

    2005-01-01

    Embryonic stem (ES) cells have the ability to self-renew as well as differentiate into any cell type in the body. These traits make ES cells an attractive "raw material" for a variety of cell-based technologies. However, uncontrolled cell aggregation in ES cell differentiation culture inhibits cell proliferation and differentiation and thwarts the use of stirred suspension bioreactors. Encapsulation of ES cells in agarose microdrops prevents physical interaction between developing embryoid bodies (EBs) that, in turn, prevents EB agglomeration. This enables use of stirred suspension bioreactors that can generate large numbers of ES-derived cells under controlled conditions.

  10. 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.

  11. 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.

  12. Spontaneous embryonic motility: an enduring legacy.

    PubMed

    Bekoff, A

    2001-04-01

    This chapter addresses the influential contributions Viktor Hamburger has made to our understanding of embryonic motor behavior. With his classic review, published in 1963, Viktor Hamburger opened up the field of embryonic motor behavior, which had lain almost completely dormant for many years. He focused his observations and experimental studies on the spontaneously generated embryonic movements rather than on reflex responses. As a result, he and his colleagues firmly established the central generation of embryonic motility as a basic component of embryonic behavior in chicks. These studies were also extended to rat fetuses, showing that similar principles apply to mammalian fetuses. All of us who have followed after him owe Viktor Hamburger an enormous debt of gratitude for his pioneering work. PMID:11255029

  13. 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.

  14. 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...

  15. 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.

  16. 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 governing their tangential migration have begun to be elucidated. To date, the mechanisms of SPs and CPTs tangential migration remain unknown. We therefore review the known signaling pathways, which regulate parameters of CRs migration including their motility, contact-redistribution and adhesion to the pial surface, and discuss this in the context of how CR migration may regulate their signaling activity in a spatial and temporal manner. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 847-881, 2016.

  17. On the dynamics of cortical development: synchrony and synaptic self-organization

    PubMed Central

    Wright, James Joseph; Bourke, Paul David

    2013-01-01

    We describe a model for cortical development that resolves long-standing difficulties of earlier models. It is proposed that, during embryonic development, synchronous firing of neurons and their competition for limited metabolic resources leads to selection of an array of neurons with ultra-small-world characteristics. Consequently, in the visual cortex, macrocolumns linked by superficial patchy connections emerge in anatomically realistic patterns, with an ante-natal arrangement which projects signals from the surrounding cortex onto each macrocolumn in a form analogous to the projection of a Euclidean plane onto a Möbius strip. This configuration reproduces typical cortical response maps, and simulations of signal flow explain cortical responses to moving lines as functions of stimulus velocity, length, and orientation. With the introduction of direct visual inputs, under the operation of Hebbian learning, development of mature selective response “tuning” to stimuli of given orientation, spatial frequency, and temporal frequency would then take place, overwriting the earlier ante-natal configuration. The model is provisionally extended to hierarchical interactions of the visual cortex with higher centers, and a general principle for cortical processing of spatio-temporal images is sketched. PMID:23596410

  18. Cortical microtubule rearrangements and cell wall patterning

    PubMed Central

    Oda, Yoshihisa

    2015-01-01

    Plant cortical microtubules, which form a highly ordered array beneath the plasma membrane, play essential roles in determining cell shape and function by directing the arrangement of cellulosic and non-cellulosic compounds on the cell surface. Interphase transverse arrays of cortical microtubules self-organize through their dynamic instability and inter-microtubule interactions, and by branch-form microtubule nucleation and severing. Recent studies revealed that distinct spatial signals including ROP GTPase, cellular geometry, and mechanical stress regulate the behavior of cortical microtubules at the subcellular and supercellular levels, giving rise to dramatic rearrangements in the cortical microtubule array in response to internal and external cues. Increasing evidence indicates that negative regulators of microtubules also contribute to the rearrangement of the cortical microtubule array. In this review, I summarize recent insights into how the rearrangement of the cortical microtubule array leads to proper, flexible cell wall patterning. PMID:25904930

  19. Circadian regulation of human cortical excitability

    PubMed Central

    Ly, Julien Q. M.; Gaggioni, Giulia; Chellappa, Sarah L.; Papachilleos, Soterios; Brzozowski, Alexandre; Borsu, Chloé; Rosanova, Mario; Sarasso, Simone; Middleton, Benita; Luxen, André; Archer, Simon N.; Phillips, Christophe; Dijk, Derk-Jan; Maquet, Pierre; Massimini, Marcello; Vandewalle, Gilles

    2016-01-01

    Prolonged wakefulness alters cortical excitability, which is essential for proper brain function and cognition. However, besides prior wakefulness, brain function and cognition are also affected by circadian rhythmicity. Whether the regulation of cognition involves a circadian impact on cortical excitability is unknown. Here, we assessed cortical excitability from scalp electroencephalography (EEG) responses to transcranial magnetic stimulation in 22 participants during 29 h of wakefulness under constant conditions. Data reveal robust circadian dynamics of cortical excitability that are strongest in those individuals with highest endocrine markers of circadian amplitude. In addition, the time course of cortical excitability correlates with changes in EEG synchronization and cognitive performance. These results demonstrate that the crucial factor for cortical excitability, and basic brain function in general, is the balance between circadian rhythmicity and sleep need, rather than sleep homoeostasis alone. These findings have implications for clinical applications such as non-invasive brain stimulation in neurorehabilitation. PMID:27339884

  20. Inhibitory Circuits in Cortical Layer 5

    PubMed Central

    Naka, Alexander; Adesnik, Hillel

    2016-01-01

    Inhibitory neurons play a fundamental role in cortical computation and behavior. Recent technological advances, such as two photon imaging, targeted in vivo recording, and molecular profiling, have improved our understanding of the function and diversity of cortical interneurons, but for technical reasons most work has been directed towards inhibitory neurons in the superficial cortical layers. Here we review current knowledge specifically on layer 5 (L5) inhibitory microcircuits, which play a critical role in controlling cortical output. We focus on recent work from the well-studied rodent barrel cortex, but also draw on evidence from studies in primary visual cortex and other cortical areas. The diversity of both deep inhibitory neurons and their pyramidal cell targets make this a challenging but essential area of study in cortical computation and sensory processing. PMID:27199675

  1. Hamilton-Jacobi skeleton on cortical surfaces.

    PubMed

    Shi, Y; Thompson, P M; Dinov, I; Toga, A W

    2008-05-01

    In this paper, we propose a new method to construct graphical representations of cortical folding patterns by computing skeletons on triangulated cortical surfaces. In our approach, a cortical surface is first partitioned into sulcal and gyral regions via the solution of a variational problem using graph cuts, which can guarantee global optimality. After that, we extend the method of Hamilton-Jacobi skeleton [1] to subsets of triangulated surfaces, together with a geometrically intuitive pruning process that can trade off between skeleton complexity and the completeness of representing folding patterns. Compared with previous work that uses skeletons of 3-D volumes to represent sulcal patterns, the skeletons on cortical surfaces can be easily decomposed into branches and provide a simpler way to construct graphical representations of cortical morphometry. In our experiments, we demonstrate our method on two different cortical surface models, its ability of capturing major sulcal patterns and its application to compute skeletons of gyral regions. PMID:18450539

  2. Circadian regulation of human cortical excitability.

    PubMed

    Ly, Julien Q M; Gaggioni, Giulia; Chellappa, Sarah L; Papachilleos, Soterios; Brzozowski, Alexandre; Borsu, Chloé; Rosanova, Mario; Sarasso, Simone; Middleton, Benita; Luxen, André; Archer, Simon N; Phillips, Christophe; Dijk, Derk-Jan; Maquet, Pierre; Massimini, Marcello; Vandewalle, Gilles

    2016-06-24

    Prolonged wakefulness alters cortical excitability, which is essential for proper brain function and cognition. However, besides prior wakefulness, brain function and cognition are also affected by circadian rhythmicity. Whether the regulation of cognition involves a circadian impact on cortical excitability is unknown. Here, we assessed cortical excitability from scalp electroencephalography (EEG) responses to transcranial magnetic stimulation in 22 participants during 29 h of wakefulness under constant conditions. Data reveal robust circadian dynamics of cortical excitability that are strongest in those individuals with highest endocrine markers of circadian amplitude. In addition, the time course of cortical excitability correlates with changes in EEG synchronization and cognitive performance. These results demonstrate that the crucial factor for cortical excitability, and basic brain function in general, is the balance between circadian rhythmicity and sleep need, rather than sleep homoeostasis alone. These findings have implications for clinical applications such as non-invasive brain stimulation in neurorehabilitation.

  3. A Rare Hydrocephalus Complication: Cortical Blindness.

    PubMed

    Ünal, Emre; Göçmen, Rahşan; Işıkay, Ayşe İlksen; Tekşam, Özlem

    2015-01-01

    Cortical blindness related to bilateral occipital lobe infarction is an extremely rare complication of hydrocephalus. Compression of the posterior cerebral artery, secondary to tentorial herniation, is the cause of occipital infarction. Particularly in children and mentally ill patients, cortical blindness may be missed. Therefore, early diagnosis and treatment of hydrocephalus is important. We present herein a child of ventricular shunt malfunction complicated by cortical blindness. PMID:27411424

  4. Communication and wiring in the cortical connectome

    PubMed Central

    Budd, Julian M. L.; Kisvárday, Zoltán F.

    2012-01-01

    In cerebral cortex, the huge mass of axonal wiring that carries information between near and distant neurons is thought to provide the neural substrate for cognitive and perceptual function. The goal of mapping the connectivity of cortical axons at different spatial scales, the cortical connectome, is to trace the paths of information flow in cerebral cortex. To appreciate the relationship between the connectome and cortical function, we need to discover the nature and purpose of the wiring principles underlying cortical connectivity. A popular explanation has been that axonal length is strictly minimized both within and between cortical regions. In contrast, we have hypothesized the existence of a multi-scale principle of cortical wiring where to optimize communication there is a trade-off between spatial (construction) and temporal (routing) costs. Here, using recent evidence concerning cortical spatial networks we critically evaluate this hypothesis at neuron, local circuit, and pathway scales. We report three main conclusions. First, the axonal and dendritic arbor morphology of single neocortical neurons may be governed by a similar wiring principle, one that balances the conservation of cellular material and conduction delay. Second, the same principle may be observed for fiber tracts connecting cortical regions. Third, the absence of sufficient local circuit data currently prohibits any meaningful assessment of the hypothesis at this scale of cortical organization. To avoid neglecting neuron and microcircuit levels of cortical organization, the connectome framework should incorporate more morphological description. In addition, structural analyses of temporal cost for cortical circuits should take account of both axonal conduction and neuronal integration delays, which appear mostly of the same order of magnitude. We conclude the hypothesized trade-off between spatial and temporal costs may potentially offer a powerful explanation for cortical wiring patterns

  5. Cortical Tremor (CT) with coincident orthostatic movements.

    PubMed

    Termsarasab, Pichet; Frucht, Steven J

    2015-01-01

    Cortical tremor (CT) is a form of cortical reflex myoclonus that can mimic essential tremor (ET). Clinical features that are helpful in distinguishing CT from ET are the irregular and jerky appearance of the movements. We report two patients with CT with coexisting orthostatic movements, either orthostatic tremor (OT) or myoclonus, who experienced functional improvement in both cortical myoclonus and orthostatic movements when treated with levetiracetam. PMID:26788343

  6. A Rare Hydrocephalus Complication: Cortical Blindness.

    PubMed

    Ünal, Emre; Göçmen, Rahşan; Işıkay, Ayşe İlksen; Tekşam, Özlem

    2015-01-01

    Cortical blindness related to bilateral occipital lobe infarction is an extremely rare complication of hydrocephalus. Compression of the posterior cerebral artery, secondary to tentorial herniation, is the cause of occipital infarction. Particularly in children and mentally ill patients, cortical blindness may be missed. Therefore, early diagnosis and treatment of hydrocephalus is important. We present herein a child of ventricular shunt malfunction complicated by cortical blindness.

  7. Avian influenza virus isolation, propagation, and titration in embryonated chicken eggs.

    PubMed

    Spackman, Erica; Killian, Mary Lea

    2014-01-01

    Avian influenza virus and some mammalian influenza A viruses are usually isolated, propagated, and titrated in embryonated chicken 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, for example, swine influenza in turkey specimens. Culture is highly sensitive, but is not specific for influenza A, which may be an advantage because a sample may be screened for several agents at once. Once an agent is isolated in culture, the presence of influenza viruses is confirmed with any of several assays. The methods for propagating virus isolates in ECE are described.

  8. 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.

  9. Cortical auditory disorders: clinical and psychoacoustic features.

    PubMed Central

    Mendez, M F; Geehan, G R

    1988-01-01

    The symptoms of two patients with bilateral cortical auditory lesions evolved from cortical deafness to other auditory syndromes: generalised auditory agnosia, amusia and/or pure word deafness, and a residual impairment of temporal sequencing. On investigation, both had dysacusis, absent middle latency evoked responses, acoustic errors in sound recognition and matching, inconsistent auditory behaviours, and similarly disturbed psychoacoustic discrimination tasks. These findings indicate that the different clinical syndromes caused by cortical auditory lesions form a spectrum of related auditory processing disorders. Differences between syndromes may depend on the degree of involvement of a primary cortical processing system, the more diffuse accessory system, and possibly the efferent auditory system. Images PMID:2450968

  10. 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

  11. Role of microglia in embryonic neurogenesis.

    PubMed

    Tong, Chih Kong; Vidyadaran, Sharmili

    2016-09-01

    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

  12. Retinoic acid-induced neural differentiation of embryonal carcinoma cells.

    PubMed Central

    Jones-Villeneuve, E M; Rudnicki, M A; Harris, J F; McBurney, M W

    1983-01-01

    We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs. Images PMID:6656766

  13. 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.

  14. Cortical Specializations Underlying Fast Computations

    PubMed Central

    Volgushev, Maxim

    2016-01-01

    The time course of behaviorally relevant environmental events sets temporal constraints on neuronal processing. How does the mammalian brain make use of the increasingly complex networks of the neocortex, while making decisions and executing behavioral reactions within a reasonable time? The key parameter determining the speed of computations in neuronal networks is a time interval that neuronal ensembles need to process changes at their input and communicate results of this processing to downstream neurons. Theoretical analysis identified basic requirements for fast processing: use of neuronal populations for encoding, background activity, and fast onset dynamics of action potentials in neurons. Experimental evidence shows that populations of neocortical neurons fulfil these requirements. Indeed, they can change firing rate in response to input perturbations very quickly, within 1 to 3 ms, and encode high-frequency components of the input by phase-locking their spiking to frequencies up to 300 to 1000 Hz. This implies that time unit of computations by cortical ensembles is only few, 1 to 3 ms, which is considerably faster than the membrane time constant of individual neurons. The ability of cortical neuronal ensembles to communicate on a millisecond time scale allows for complex, multiple-step processing and precise coordination of neuronal activity in parallel processing streams, while keeping the speed of behavioral reactions within environmentally set temporal constraints. PMID:25689988

  15. Cortical control of facial expression.

    PubMed

    Müri, René M

    2016-06-01

    The present Review deals with the motor control of facial expressions in humans. Facial expressions are a central part of human communication. Emotional face expressions have a crucial role in human nonverbal behavior, allowing a rapid transfer of information between individuals. Facial expressions can be either voluntarily or emotionally controlled. Recent studies in nonhuman primates and humans have revealed that the motor control of facial expressions has a distributed neural representation. At least five cortical regions on the medial and lateral aspects of each hemisphere are involved: the primary motor cortex, the ventral lateral premotor cortex, the supplementary motor area on the medial wall, and the rostral and caudal cingulate cortex. The results of studies in humans and nonhuman primates suggest that the innervation of the face is bilaterally controlled for the upper part and mainly contralaterally controlled for the lower part. Furthermore, the primary motor cortex, the ventral lateral premotor cortex, and the supplementary motor area are essential for the voluntary control of facial expressions. In contrast, the cingulate cortical areas are important for emotional expression, because they receive input from different structures of the limbic system. PMID:26418049

  16. Gyrification from constrained cortical expansion

    PubMed Central

    Tallinen, Tuomas; Chung, Jun Young; Biggins, John S.; Mahadevan, L.

    2014-01-01

    The exterior of the mammalian brain—the cerebral cortex—has a conserved layered structure whose thickness varies little across species. However, selection pressures over evolutionary time scales have led to cortices that have a large surface area to volume ratio in some organisms, with the result that the brain is strongly convoluted into sulci and gyri. Here we show that the gyrification can arise as a nonlinear consequence of a simple mechanical instability driven by tangential expansion of the gray matter constrained by the white matter. A physical mimic of the process using a layered swelling gel captures the essence of the mechanism, and numerical simulations of the brain treated as a soft solid lead to the formation of cusped sulci and smooth gyri similar to those in the brain. The resulting gyrification patterns are a function of relative cortical expansion and relative thickness (compared with brain size), and are consistent with observations of a wide range of brains, ranging from smooth to highly convoluted. Furthermore, this dependence on two simple geometric parameters that characterize the brain also allows us to qualitatively explain how variations in these parameters lead to anatomical anomalies in such situations as polymicrogyria, pachygyria, and lissencephalia. PMID:25136099

  17. Sleep and olfactory cortical plasticity

    PubMed Central

    Barnes, Dylan C.; Wilson, Donald A.

    2014-01-01

    In many systems, sleep plays a vital role in memory consolidation and synaptic homeostasis. These processes together help store information of biological significance and reset synaptic circuits to facilitate acquisition of information in the future. In this review, we describe recent evidence of sleep-dependent changes in olfactory system structure and function which contribute to odor memory and perception. During slow-wave sleep, the piriform cortex becomes hypo-responsive to odor stimulation and instead displays sharp-wave activity similar to that observed within the hippocampal formation. Furthermore, the functional connectivity between the piriform cortex and other cortical and limbic regions is enhanced during slow-wave sleep compared to waking. This combination of conditions may allow odor memory consolidation to occur during a state of reduced external interference and facilitate association of odor memories with stored hedonic and contextual cues. Evidence consistent with sleep-dependent odor replay within olfactory cortical circuits is presented. These data suggest that both the strength and precision of odor memories is sleep-dependent. The work further emphasizes the critical role of synaptic plasticity and memory in not only odor memory but also basic odor perception. The work also suggests a possible link between sleep disturbances that are frequently co-morbid with a wide range of pathologies including Alzheimer’s disease, schizophrenia and depression and the known olfactory impairments associated with those disorders. PMID:24795585

  18. Differential expression of gamma-aminobutyric acid type B receptor subunit mRNAs in the developing nervous system and receptor coupling to adenylyl cyclase in embryonic neurons.

    PubMed

    Martin, Stella C; Steiger, Janine L; Gravielle, María Clara; Lyons, Helen R; Russek, Shelley J; Farb, David H

    2004-05-17

    gamma-Aminobutyric acid type B receptors (GABA(B)Rs) mediate both slow inhibitory synaptic activity in the adult nervous system and motility signals for migrating embryonic cortical cells. Previous papers have described the expression of GABA(B)Rs in the adult brain, but the expression and functional significance of these gene products in the embryo are largely unknown. Here we examine GABA(B)R expression from rat embryonic day 10 (E10) to E18 compared with adult and ask whether embryonic cortical neurons contain functional GABA(B)R. GABA(B)R1 transcript levels greatly exceed GABA(B)R2 levels in the developing neural tube at E11, and olfactory bulb and striatum at E17 but equalize in most regions of adult nervous tissue, except for the glomerular and granule cell layers of the main olfactory bulb and the striatum. Consistent with expression differences, the binding affinity of GABA for GABA(B)Rs is significantly lower in adult striatum compared with cerebellum. Multiple lines of evidence from in situ hybridization, RNase protection, and real-time PCR demonstrate that GABA(B)R1a, GABA(B)R1b, GABA(B)R1h (a subunit subtype, lacking a sushi domain, that we have identified in embryonic rat brain), GABA(B)R2, and GABA(B)L transcript levels are not coordinately regulated. Despite the functional requirement for a heterodimer of GABA(B)R subunits, the expression of each subunit mRNA is under independent control during embryonic development, and, by E18, GABA(B)Rs are negatively coupled to adenylyl cyclase in neocortical neurons. The presence of embryonic GABA(B)R transcripts and protein and functional receptor coupling indicates potentially important roles for GABA(B)Rs in modulation of synaptic transmission in the developing embryonic nervous system.

  19. Epigenetic stability of embryonic stem cells and developmental potential.

    PubMed

    Pannetier, Maëlle; Feil, Robert

    2007-12-01

    Recent studies highlight the tremendous potential of human embryonic stem (ES) cells and their derivatives as therapeutic tools for degenerative diseases. However, derivation and culture of ES cells can induce epigenetic alterations, which can have long lasting effects on gene expression and phenotype. Research on human and mouse stem cells indicates that developmental, cancer-related genes, and genes regulated by genomic imprinting are particularly susceptible to changes in DNA methylation. Together with the occurrence of genetic alterations, epigenetic instability needs to be monitored when considering human stem cells for therapeutic and technological purposes. Here, we discuss the maintenance of epigenetic information in cultured stem cells and embryos and how this influences their developmental potential.

  20. Serotonin homeostasis and serotonin receptors as actors of cortical construction: special attention to the 5-HT3A and 5-HT6 receptor subtypes

    PubMed Central

    Vitalis, Tania; Ansorge, Mark S.; Dayer, Alexandre G.

    2013-01-01

    Cortical circuits control higher-order cognitive processes and their function is highly dependent on their structure that emerges during development. The construction of cortical circuits involves the coordinated interplay between different types of cellular processes such as proliferation, migration, and differentiation of neural and glial cell subtypes. Among the multiple factors that regulate the assembly of cortical circuits, 5-HT is an important developmental signal that impacts on a broad diversity of cellular processes. 5-HT is detected at the onset of embryonic telencephalic formation and a variety of serotonergic receptors are dynamically expressed in the embryonic developing cortex in a region and cell-type specific manner. Among these receptors, the ionotropic 5-HT3A receptor and the metabotropic 5-HT6 receptor have recently been identified as novel serotonergic targets regulating different aspects of cortical construction including neuronal migration and dendritic differentiation. In this review, we focus on the developmental impact of serotonergic systems on the construction of cortical circuits and discuss their potential role in programming risk for human psychiatric disorders. PMID:23801939

  1. 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

  2. Assessment of Bisphenol A (BPA) neurotoxicity in vitro with mouse embryonic stem cells.

    PubMed

    Yin, Nuoya; Yao, Xinglei; Qin, Zhanfen; Wang, Yuan-Liang; Faiola, Francesco

    2015-10-01

    The adverse effects of environmental pollution on our well-being have been intensively studied with many in vitro and in vivo systems. In our group, we focus on stem cell toxicology due to the multitude of embryonic stem cell (ESC) properties which can be exerted in toxicity assays. In fact, ESCs can differentiate in culture to mimic embryonic development in vivo, or specifically to virtually any kind of somatic cells. Here, we used the toxicant Bisphenol A (BPA), a chemical known as a hazard to infants and children, and showed that our stem cell toxicology system was able to efficiently recapitulate most of the toxic effects of BPA previously detected by in vitro system or animal tests. More precisely, we demonstrated that BPA affected the proper specification of germ layers during our in vitro mimicking of the embryonic development, as well as the establishment of neural ectoderm and neural progenitor cells. PMID:26456621

  3. Assessment of Bisphenol A (BPA) neurotoxicity in vitro with mouse embryonic stem cells.

    PubMed

    Yin, Nuoya; Yao, Xinglei; Qin, Zhanfen; Wang, Yuan-Liang; Faiola, Francesco

    2015-10-01

    The adverse effects of environmental pollution on our well-being have been intensively studied with many in vitro and in vivo systems. In our group, we focus on stem cell toxicology due to the multitude of embryonic stem cell (ESC) properties which can be exerted in toxicity assays. In fact, ESCs can differentiate in culture to mimic embryonic development in vivo, or specifically to virtually any kind of somatic cells. Here, we used the toxicant Bisphenol A (BPA), a chemical known as a hazard to infants and children, and showed that our stem cell toxicology system was able to efficiently recapitulate most of the toxic effects of BPA previously detected by in vitro system or animal tests. More precisely, we demonstrated that BPA affected the proper specification of germ layers during our in vitro mimicking of the embryonic development, as well as the establishment of neural ectoderm and neural progenitor cells.

  4. Live 4D optical coherence tomography for early embryonic mouse cardiac phenotyping

    NASA Astrophysics Data System (ADS)

    Lopez, Andrew L.; Wang, Shang; Larin, Kirill V.; Overbeek, Paul A.; Larina, Irina V.

    2016-03-01

    Studying embryonic mouse development is important for our understanding of normal human embryogenesis and the underlying causes of congenital defects. Our research focuses on imaging early development in the mouse embryo to specifically understand cardiovascular development using optical coherence tomography (OCT). We have previously developed imaging approaches that combine static embryo culture, OCT imaging and advanced image processing to visualize the whole live mouse embryos and obtain 4D (3D+time) cardiodynamic datasets with cellular resolution. Here, we present the study of using 4D OCT for dynamic imaging of early embryonic heart in live mouse embryos to assess mutant cardiac phenotypes during development, including a cardiac looping defect. Our results indicate that the live 4D OCT imaging approach is an efficient phenotyping tool that can reveal structural and functional cardiac defects at very early stages. Further studies integrating live embryonic cardiodynamic phenotyping with molecular and genetic approaches in mouse mutants will help to elucidate the underlying signaling defects.

  5. Establishment and Characterization of an Embryonic Cell Line from Sarconesiopsis magellanica

    PubMed Central

    Cruz, Mónica; Bello, Felio J.

    2013-01-01

    Sarconesiopsis magellanica (Le Guillou) (Diptera: Calliphoridae) is a necrophagous fly that is important in both human and veterinary medicines. This insect has been registered in Colombia as a biological indicator in estimating post-mortem interval. Insect cell cultures are an important biotechnological tool for basic and applied studies, and cell cultures derived from S. magellanica embryonic tissues are described in this study. S. magellanica embryonated eggs were taken for tissue explants. These were seeded in L-15, Grace/L-15, Eagle MEM, MM, VP12, MM/VP12, and Schneider culture media. The morphological, cytogenetic, biochemical, and molecular characteristics of the cell cultures were examined. Cell growth was achieved in the L15, Grace/L15, and Schneider culture media, and the confluent monolayers were obtained 8, 10, and 19 days after the embryonated eggs were explanted. However, the Schneider medium was the most efficient to develop the subcultures, and 21 passages have been maintained. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer and in the subcultures there was greater cell morphology uniformity, fibroblastoid types being predominant. Cultured cells had a chromosomal number of 12, and the karyotypic complement consisted of five pairs of somatic chromosomes and one sexual pair. The cell culture isozyme patterns of S. magellanica coincided with adult samples from the same species. The molecular analysis, using RAPD-PCR, demonstrated the authentication of the cell cultures of this fly and their differentiation from other cultures derived from two sand flies species. This cell line is a new in vitro model that will be used in biomedical and biotechnological studies. PMID:24766352

  6. Ultra-slow oscillations in cortical networks in vitro.

    PubMed

    Mok, S Y; Nadasdy, Z; Lim, Y M; Goh, S Y

    2012-03-29

    An ultra-slow oscillation (<0.01 Hz) in the network-wide activity of dissociated cortical networks is described in this article. This slow rhythm is characterized by the recurrence of clusters of large synchronized bursts of activity lasting approximately 1-3 min, separated by an almost equivalent interval of relatively smaller bursts. Such rhythmic activity was detected in cultures starting from the fourth week in vitro. Our analysis revealed that the propagation motifs of constituent bursts were strongly conserved across multiple oscillation cycles, and these motifs were more consistent at the electrode level compared with the neuronal level.

  7. In ovo electroporation in embryonic chick retina.

    PubMed

    Islam, Mohammed M; Doh, Sung Tae; Cai, Li

    2012-02-05

    Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons(1), which has been difficult with the conventional method of injection and electroporation at E1.5(2).

  8. Cortical thickness gradients in structural hierarchies

    PubMed Central

    Wagstyl, Konrad; Ronan, Lisa; Goodyer, Ian M.; Fletcher, Paul C.

    2015-01-01

    MRI, enabling in vivo analysis of cortical morphology, offers a powerful tool in the assessment of brain development and pathology. One of the most ubiquitous measures used—the thickness of the cortex—shows abnormalities in a number of diseases and conditions, but the functional and biological correlates of such alterations are unclear. If the functional connotations of structural MRI measures are to be understood, we must strive to clarify the relationship between measures such as cortical thickness and their cytoarchitectural determinants. We therefore sought to determine whether patterns of cortical thickness mirror a key motif of the cortex, specifically its structural hierarchical organisation. We delineated three sensory hierarchies (visual, somatosensory and auditory) in two species—macaque and human—and explored whether cortical thickness was correlated with specific cytoarchitectural characteristics. Importantly, we controlled for cortical folding which impacts upon thickness and may obscure regional differences. Our results suggest that an easily measurable macroscopic brain parameter, namely, cortical thickness, is systematically related to cytoarchitecture and to the structural hierarchical organisation of the cortex. We argue that the measurement of cortical thickness gradients may become an important way to develop our understanding of brain structure–function relationships. The identification of alterations in such gradients may complement the observation of regionally localised cortical thickness changes in our understanding of normal development and neuropsychiatric illnesses. PMID:25725468

  9. Cortical Correspondence with Probabilistic Fiber Connectivity

    PubMed Central

    Oguz, Ipek; Niethammer, Marc; Cates, Josh; Whitaker, Ross; Fletcher, Thomas; Vachet, Clement; Styner, Martin

    2009-01-01

    This paper presents a novel method of optimizing point-based correspondence among populations of human cortical surfaces by combining structural cues with probabilistic connectivity maps. The proposed method establishes a tradeoff between an even sampling of the cortical surfaces (a low surface entropy) and the similarity of corresponding points across the population (a low ensemble entropy). The similarity metric, however, isn’t constrained to be just spatial proximity, but uses local sulcal depth measurements as well as probabilistic connectivity maps, computed from DWI scans via a stochastic tractography algorithm, to enhance the correspondence definition. We propose a novel method for projecting this fiber connectivity information on the cortical surface, using a surface evolution technique. Our cortical correspondence method does not require a spherical parameterization. Experimental results are presented, showing improved correspondence quality demonstrated by a cortical thickness analysis, as compared to correspondence methods using spatial metrics as the sole correspondence criterion. PMID:19694301

  10. Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

    PubMed

    Shaker, Mohammed R; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

    2015-05-15

    Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

  11. Cadherins in cerebellar development: translation of embryonic patterning into mature functional compartmentalization.

    PubMed

    Redies, Christoph; Neudert, Franziska; Lin, Juntang

    2011-09-01

    Cadherins are cell adhesion molecules with multiple morphogenic functions in brain development, for example, in neuroblast migration and aggregation, axon navigation, neural circuit formation, and synaptogenesis. More than 100 members of the cadherin superfamily are expressed in the developing and mature brain. Most of the cadherins investigated, in particular classic cadherins and δ-protocadherins, are expressed in the cerebellum. For several cadherin subtypes, expression begins at early embryonic stages and persists until mature stages of cerebellar development. At intermediate stages, distinct Purkinje cell clusters exhibit unique rostrocaudal and mediolateral expression profiles for each cadherin. In the chicken, mouse, and other species, the Purkinje cell clusters are separated by intervening raphes of migrating granule cells. This pattern of Purkinje cell clusters/raphes is, at least in part, continuous with the parasagittal striping pattern that is apparent in the mature cerebellar cortex, for example, for zebrin II/aldolase C. Moreover, subregions of the deep cerebellar nuclei, vestibular nuclei and the olivary complex also express cadherins differentially. Neuroanatomical evidence suggests that the nuclear subregions and cortical domains that express the same cadherin subtype are connected to each other, to form neural subcircuits of the cerebellar system. Cadherins thus provide a molecular code that specifies not only embryonic structures but also functional cerebellar compartmentalization. By following the implementation of this code, it can be revealed how mature functional architecture emerges from embryonic patterning during cerebellar development. Dysfunction of some cadherins is associated with psychiatric diseases and developmental impairments and may also affect cerebellar function.

  12. Expression of neuronal nitric oxide synthase during embryonic development of the rat optic vesicle.

    PubMed

    Nobakht, M; Majidzadeh, S; Fattahi, M; Samadi, M; Tabatabaeei, P

    2007-04-01

    The expression of neuronal nitric oxide synthase during the development of rat optic vesicle from embryonic day E14 to E18 was analyzed by histochemical procedures. The samples were frozen and cut on a cryostat and then studied by using the light microscope. Expression of nNOS was first seen on E14 in cells of Cajal-Retzius located in the marginal zone of optic vesicle. NADPH-d persisted in this layer throughout the embryonic period and began to decrease on E20. At E16, the optic vesicle has four NADPH-d positive layers. At E18, NADPH-d reactivity observed at low magnification showed five clearly defined layers. In the late stages, the most notable feature was a decrease in histochemical reaction of the marginal zone and at these stages, the layer IV showed less staining than the rest of the cortical plate. The observations suggest that nitric oxide is synthesized during embryonic life processes and this is related to maturational processes.

  13. Cortical Cartography and Caret Software

    PubMed Central

    Van Essen, David C.

    2011-01-01

    Caret software is widely used for analyzing and visualizing many types of fMRI data, often in conjunction with experimental data from other modalities. This article places Caret’s development in a historical context that spans three decades of brain mapping – from the early days of manually generated flat maps to the nascent field of human connectomics. It also highlights some of Caret’s distinctive capabilities. This includes the ease of visualizing data on surfaces and/or volumes and on atlases as well as individual subjects. Caret can display many types of experimental data using various combinations of overlays (e.g., fMRI activation maps, cortical parcellations, areal boundaries), and it has other features that facilitate the analysis and visualization of complex neuroimaging datasets. PMID:22062192

  14. Gyrification from constrained cortical expansion

    NASA Astrophysics Data System (ADS)

    Tallinen, Tuomas

    The convolutions of the human brain are a symbol of its functional complexity. But how does the outer surface of the brain, the layered cortex of neuronal gray matter get its folds? In this talk, we ask to which extent folding of the brain can be explained as a purely mechanical consequence of unpatterned growth of the cortical layer relative to the sublayers. Modeling the growing brain as a soft layered solid leads to elastic instabilities and the formation of cusped sulci and smooth gyri consistent with observations across species in both normal and pathological situations. Furthermore, we apply initial geometries obtained from fetal brain MRI to address the question of how the brain geometry and folding patterns may be coupled via mechanics.

  15. Cortical cartography and Caret software.

    PubMed

    Van Essen, David C

    2012-08-15

    Caret software is widely used for analyzing and visualizing many types of fMRI data, often in conjunction with experimental data from other modalities. This article places Caret's development in a historical context that spans three decades of brain mapping--from the early days of manually generated flat maps to the nascent field of human connectomics. It also highlights some of Caret's distinctive capabilities. This includes the ease of visualizing data on surfaces and/or volumes and on atlases as well as individual subjects. Caret can display many types of experimental data using various combinations of overlays (e.g., fMRI activation maps, cortical parcellations, areal boundaries), and it has other features that facilitate the analysis and visualization of complex neuroimaging datasets.

  16. Nicotinic modulation of cortical circuits

    PubMed Central

    Arroyo, Sergio; Bennett, Corbett; Hestrin, Shaul

    2014-01-01

    The ascending cholinergic neuromodulatory system sends projections throughout cortex and has been shown to play an important role in a number of cognitive functions including arousal, working memory, and attention. However, despite a wealth of behavioral and anatomical data, understanding how cholinergic synapses modulate cortical function has been limited by the inability to selectively activate cholinergic axons. Now, with the development of optogenetic tools and cell-type specific Cre-driver mouse lines, it has become possible to stimulate cholinergic axons from the basal forebrain (BF) and probe cholinergic synapses in the cortex for the first time. Here we review recent work studying the cell-type specificity of nicotinic signaling in the cortex, synaptic mechanisms mediating cholinergic transmission, and the potential functional role of nicotinic modulation. PMID:24734005

  17. Unsupervised fetal cortical surface parcellation

    NASA Astrophysics Data System (ADS)

    Dahdouh, Sonia; Limperopoulos, Catherine

    2016-03-01

    At the core of many neuro-imaging studies, atlas-based brain parcellations are used for example to study normal brain evolution across the lifespan. These atlases rely on the assumption that the same anatomical features are present on all subjects to be studied and that these features are stable enough to allow meaningful comparisons between different brain surfaces and structures These methods, however, often fail when applied to fetal MRI data, due to the lack of consistent anatomical features present across gestation. This paper presents a novel surface-based fetal cortical parcellation framework which attempts to circumvent the lack of consistent anatomical features by proposing a brain parcellation scheme that is based solely on learned geometrical features. A mesh signature incorporating both extrinsic and intrinsic geometrical features is proposed and used in a clustering scheme to define a parcellation of the fetal brain. This parcellation is then learned using a Random Forest (RF) based learning approach and then further refined in an alpha-expansion graph-cut scheme. Based on the votes obtained by the RF inference procedure, a probability map is computed and used as a data term in the graph-cut procedure. The smoothness term is defined by learning a transition matrix based on the dihedral angles of the faces. Qualitative and quantitative results on a cohort of both healthy and high-risk fetuses are presented. Both visual and quantitative assessments show good results demonstrating a reliable method for fetal brain data and the possibility of obtaining a parcellation of the fetal cortical surfaces using only geometrical features.

  18. Cortical spreading depression: An enigma

    NASA Astrophysics Data System (ADS)

    Miura, R. M.; Huang, H.; Wylie, J. J.

    2007-08-01

    The brain is a complex organ with active components composed largely of neurons, glial cells, and blood vessels. There exists an enormous experimental and theoretical literature on the mechanisms involved in the functioning of the brain, but we still do not have a good understanding of how it works on a gross mechanistic level. In general, the brain maintains a homeostatic state with relatively small ion concentration changes, the major ions being sodium, potassium, and chloride. Calcium ions are present in smaller quantities but still play an important role in many phenomena. Cortical spreading depression (CSD for short) was discovered over 60 years ago by A.A.P. Leão, a Brazilian physiologist doing his doctoral research on epilepsy at Harvard University, “Spreading depression of activity in the cerebral cortex," J. Neurophysiol., 7 (1944), pp. 359-390. Cortical spreading depression is characterized by massive changes in ionic concentrations and slow nonlinear chemical waves, with speeds on the order of mm/min, in the cortex of different brain structures in various experimental animals. In humans, CSD is associated with migraine with aura, where a light scintillation in the visual field propagates, then disappears, and is followed by a sustained headache. To date, CSD remains an enigma, and further detailed experimental and theoretical investigations are needed to develop a comprehensive picture of the diverse mechanisms involved in producing CSD. A number of mechanisms have been hypothesized to be important for CSD wave propagation. In this paper, we briefly describe several characteristics of CSD wave propagation, and examine some of the mechanisms that are believed to be important, including ion diffusion, membrane ionic currents, osmotic effects, spatial buffering, neurotransmitter substances, gap junctions, metabolic pumps, and synaptic connections. Continuum models of CSD, consisting of coupled nonlinear diffusion equations for the ion concentrations, and

  19. N-Cadherin Sustains Motility and Polarity of Future Cortical Interneurons during Tangential Migration

    PubMed Central

    Luccardini, Camilla; Hennekinne, Laetitia; Viou, Lucie; Yanagida, Mitsutoshi; Murakami, Fujio; Kessaris, Nicoletta; Ma, Xufei; Adelstein, Robert S.; Mège, René-Marc

    2013-01-01

    In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell–cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation. N-cadherin is also expressed in embryonic territories crossed by MGE cells during their migration. In this study, we demonstrate that N-cadherin is a key player in the long-distance migration of future cortical interneurons. Using N-cadherin-coated substrate, we show that N-cadherin-dependent adhesion promotes the migration of mouse MGE cells in vitro. Conversely, mouse MGE cells electroporated with a construct interfering with cadherin function show reduced cell motility, leading process instability, and impaired polarization associated with abnormal myosin IIB dynamics. In vivo, the capability of electroporated MGE cells to invade the developing cortical plate is altered. Using genetic ablation of N-cadherin in mouse embryos, we show that N-cadherin-depleted MGEs are severely disorganized. MGE cells hardly exit the disorganized proliferative area. N-cadherin ablation at the postmitotic stage, which does not affect MGE morphogenesis, alters MGE cell motility and directionality. The tangential migration to the cortex of N-cadherin ablated MGE cells is delayed, and their radial migration within the cortical plate is perturbed. Altogether, these results identify N-cadherin as a pivotal adhesion substrate that activates cell motility in future cortical interneurons and maintains cell polarity over their long-distance migration to the developing cortex. PMID:24227724

  20. Prenatal Exposure to Autism-Specific Maternal Autoantibodies Alters Proliferation of Cortical Neural Precursor Cells, Enlarges Brain, and Increases Neuronal Size in Adult Animals.

    PubMed

    Martínez-Cerdeño, Verónica; Camacho, Jasmin; Fox, Elizabeth; Miller, Elaine; Ariza, Jeanelle; Kienzle, Devon; Plank, Kaela; Noctor, Stephen C; Van de Water, Judy

    2016-01-01

    Autism spectrum disorders (ASDs) affect up to 1 in 68 children. Autism-specific autoantibodies directed against fetal brain proteins have been found exclusively in a subpopulation of mothers whose children were diagnosed with ASD or maternal autoantibody-related autism. We tested the impact of autoantibodies on brain development in mice by transferring human antigen-specific IgG directly into the cerebral ventricles of embryonic mice during cortical neurogenesis. We show that autoantibodies recognize radial glial cells during development. We also show that prenatal exposure to autism-specific maternal autoantibodies increased stem cell proliferation in the subventricular zone (SVZ) of the embryonic neocortex, increased adult brain size and weight, and increased the size of adult cortical neurons. We propose that prenatal exposure to autism-specific maternal autoantibodies directly affects radial glial cell development and presents a viable pathologic mechanism for the maternal autoantibody-related prenatal ASD risk factor.

  1. N-Methyl D-Aspartate Receptor Antagonist Kynurenic Acid Affects Human Cortical Development

    PubMed Central

    Bagasrawala, Inseyah; Zecevic, Nada; Radonjić, Nevena V.

    2016-01-01

    Kynurenic acid (KYNA), a neuroactive metabolite of tryptophan degradation, acts as an endogenous N-methyl-D-aspartate receptor (NMDAR) antagonist. Elevated levels of KYNA have been observed in pregnant women after viral infections and are considered to play a role in neurodevelopmental disorders. However, the consequences of KYNA-induced NMDAR blockade in human cortical development still remain elusive. To study the potential impact of KYNA on human neurodevelopment, we used an in vitro system of multipotent cortical progenitors, i.e., radial glia cells (RGCs), enriched from human cerebral cortex at mid-gestation (16–19 gestational weeks). KYNA treatment significantly decreased RGCs proliferation and survival by antagonizing NMDAR. This alteration resulted in a reduced number of cortical progenitors and neurons while number and activation of astrocytes increased. KYNA treatment reduced differentiation of RGCs into GABAergic neurons, while differentiation into glutamatergic neurons was relatively spared. Furthermore, in mixed cortical cultures KYNA triggered an inflammatory response as evidenced by increased levels of the pro-inflammatory cytokine IL-6. In conclusion, elevated levels of KYNA play a significant role in human RGC fate determination by antagonizing NMDARs and by activating an inflammatory response. The altered cell composition observed in cell culture following exposure to elevated KYNA levels suggests a mechanism for impairment of cortical circuitry formation in the fetal brain after viral infection, as seen in neurodevelopmental disorders such as schizophrenia. PMID:27746712

  2. Caloric restriction stimulates autophagy in rat cortical neurons through neuropeptide Y and ghrelin receptors activation

    PubMed Central

    Carmo-Silva, Sara; Botelho, Mariana; de Almeida, Luís Pereira; Cavadas, Cláudia

    2016-01-01

    Caloric restriction is an anti-aging intervention known to extend lifespan in several experimental models, at least in part, by stimulating autophagy. Caloric restriction increases neuropeptide Y (NPY) in the hypothalamus and plasma ghrelin, a peripheral gut hormone that acts in hypothalamus to modulate energy homeostasis. NPY and ghrelin have been shown to be neuroprotective in different brain areas and to induce several physiological modifications similar to those induced by caloric restriction. However, the effect of NPY and ghrelin in autophagy in cortical neurons is currently not known. Using a cell culture of rat cortical neurons we investigate the involvement of NPY and ghrelin in caloric restriction-induced autophagy. We observed that a caloric restriction mimetic cell culture medium stimulates autophagy in rat cortical neurons and NPY or ghrelin receptor antagonists blocked this effect. On the other hand, exogenous NPY or ghrelin stimulate autophagy in rat cortical neurons. Moreover, NPY mediates the stimulatory effect of ghrelin on autophagy in rat cortical neurons. Since autophagy impairment occurs in aging and age-related neurodegenerative diseases, NPY and ghrelin synergistic effect on autophagy stimulation may suggest a new strategy to delay aging process. PMID:27441412

  3. Low level laser therapy reduces oxidative stress in cortical neurons in vitro

    NASA Astrophysics Data System (ADS)

    Huang, Ying-Ying; Tedford, Clark E.; McCarthy, Thomas; Hamblin, Michael R.

    2012-03-01

    It is accepted that the mechanisms of low level laser therapy (LLLT) involves photons that are absorbed in the mitochondria of cells and lead to increase of mitochondrial metabolism resulting in more electron transport, increase of mitochondrial membrane potential, and more ATP production. Intracellular calcium changes are seen that correlate with mitochondrial stimulation. The situation with two other intermediates is more complex however: reactive oxygen species (ROS) and nitric oxide (NO). Evidence exists that low levels of ROS are produced by LLLT in normal cells that can be beneficial by (for instance) activating NF-kB. However high fluences of light can produce large amounts of ROS that can damage the cells. In oxidatively stressed cells the situation may be different. We exposed primary cultured cortical neurons to hydrogen peroxide (H2O2) or cobalt chloride (CoCl2) oxidative insults in the presence or absence of LLLT (810-nm laser at 0.3 or 3 J/cm2). Cell viability of cortical neurons was determined by lactate dehydrogenase assay. ROS in neurons was detected using an ROS probe, MitoRox with confocal microscopy. Results showed that LLLT dose-dependently reversed ROS production and protected cortical neurons against H2O2 or CoCl2 induced oxidative injury in cultured cortical neurons. Conclusion: LLLT can protect cortical neurons against oxidative stress by reversing the levels of ROS.

  4. Dynamic expression of calretinin in embryonic and early fetal human cortex

    PubMed Central

    González-Gómez, Miriam; Meyer, Gundela

    2014-01-01

    Calretinin (CR) is one of the earliest neurochemical markers in human corticogenesis. In embryos from Carnegie stages (CS) 17 to 23, calbindin (CB) and CR stain opposite poles of the incipient cortex suggesting early regionalization: CB marks the neuroepithelium of the medial boundary of the cortex with the choroid plexus (cortical hem). By contrast, CR is confined to the subventricular zone (SVZ) of the lateral and caudal ganglionic eminences at the pallial-subpallial boundary (PSB, or antihem), from where CR+/Tbr1- neurons migrate toward piriform cortex and amygdala as a component of the lateral cortical stream. At CS 19, columns of CR+ cells arise in the rostral cortex, and contribute at CS 20 to the “monolayer” of horizontal Tbr1+/CR+ and GAD+ cells in the preplate. At CS 21, the “pioneer cortical plate” appears as a radial aggregation of CR+/Tbr1+ neurons, which cover the entire future neocortex and extend the first corticofugal axons. CR expression in early human corticogenesis is thus not restricted to interneurons, but is also present in the first excitatory projection neurons of the cortex. At CS 21/22, the cortical plate is established following a lateral to medial gradient, when Tbr1+/CR- neurons settle within the pioneer cortical plate, and thus separate superficial and deep pioneer neurons. CR+ pioneer neurons disappear shortly after the formation of the cortical plate. Reelin+ Cajal-Retzius cells begin to express CR around CS21 (7/8 PCW). At CS 21–23, the CR+ SVZ at the PSB is the source of CR+ interneurons migrating into the cortical SVZ. In turn, CB+ interneurons migrate from the subpallium into the intermediate zone following the fibers of the internal capsule. Early CR+ and CB+ interneurons thus have different origins and migratory routes. CR+ cell populations in the embryonic telencephalon take part in a complex sequence of events not analyzed so far in other mammalian species, which may represent a distinctive trait of the initial

  5. Spatiotemporal SERT expression in cortical map development.

    PubMed

    Chen, Xiaoning; Petit, Emilie I; Dobrenis, Kostantin; Sze, Ji Ying

    2016-09-01

    The cerebral cortex is organized into morphologically distinct areas that provide biological frameworks underlying perception, cognition, and behavior. Profiling mouse and human cortical transcriptomes have revealed temporal-specific differential gene expression modules in distinct neocortical areas during cortical map establishment. However, the biological roles of spatiotemporal gene expression in cortical patterning and how cortical topographic gene expression is regulated are largely unknown. Here, we characterize temporal- and spatial-defined expression of serotonin (5-HT) transporter (SERT) in glutamatergic neurons during sensory map development in mice. SERT is transiently expressed in glutamatergic thalamic neurons projecting to sensory cortices and in pyramidal neurons in the prefrontal cortex (PFC) and hippocampus (HPC) during the period that lays down the basic functional neural circuits. We previously identified that knockout of SERT in the thalamic neurons blocks 5-HT uptake by their thalamocortical axons, resulting in excessive 5-HT signaling that impairs sensory map architecture. In contrast, here we show that selective SERT knockout in the PFC and HPC neurons does not perturb sensory map patterning. These data suggest that transient SERT expression in specific glutamatergic neurons provides area-specific instructions for cortical map patterning. Hence, genetic and pharmacological manipulations of this SERT function could illuminate the fundamental genetic programming of cortex-specific maps and biological roles of temporal-specific cortical topographic gene expression in normal development and mental disorders. PMID:27282696

  6. Perilipin+ embryonic preadipocytes actively proliferate along growing vasculatures for adipose expansion.

    PubMed

    Hong, Ki Yong; Bae, Hosung; Park, Intae; Park, Dae-Young; Kim, Kyun Hoo; Kubota, Yoshiaki; Cho, Eui-Sic; Kim, Hail; Adams, Ralf H; Yoo, Ook-Joon; Koh, Gou Young

    2015-08-01

    Despite the growing interest in adipose tissue as a therapeutic target of metabolic diseases, the identity of adipocyte precursor cells (preadipocytes) and the formation of adipose tissue during embryonic development are still poorly understood. Here, we clarified the identity and dynamic processes of preadipocytes in mouse white adipose tissue during embryogenesis through direct examination, lineage tracing and culture systems. Surprisingly, we found that lipid-lacking but perilipin(+) or adiponectin(+) proliferating preadipocytes started to emerge at embryonic day 16.5, and these cells underwent active proliferation until birth. Moreover, these preadipocytes resided as clusters and were distributed along growing adipose vasculatures. Importantly, the embryonic preadipocytes exhibited considerable coexpression of stem cell markers, such as CD24, CD29 and PDGFRα, and a small portion of preadipocytes were derived from PDGFRβ(+) mural cells, in contrast to the adult preadipocytes present in the stromal vascular fraction. Further analyses with in vitro and ex vivo culture systems revealed a stepwise but dynamic regulation of preadipocyte formation and differentiation during prenatal adipogenesis. To conclude, we unraveled the identity and characteristics of embryonic preadipocytes, which are crucial for the formation and expansion of adipose tissue during embryogenesis.

  7. A Turing Reaction-Diffusion Model for Human Cortical Folding Patterns and Cortical Pattern Malformations

    NASA Astrophysics Data System (ADS)

    Hurdal, Monica K.; Striegel, Deborah A.

    2011-11-01

    Modeling and understanding cortical folding pattern formation is important for quantifying cortical development. We present a biomathematical model for cortical folding pattern formation in the human brain and apply this model to study diseases involving cortical pattern malformations associated with neural migration disorders. Polymicrogyria is a cortical malformation disease resulting in an excessive number of small gyri. Our mathematical model uses a Turing reaction-diffusion system to model cortical folding. The lateral ventricle (LV) and ventricular zone (VZ) of the brain are critical components in the formation of cortical patterning. In early cortical development the shape of the LV can be modeled with a prolate spheroid and the VZ with a prolate spheroid surface. We use our model to study how global cortex characteristics, such as size and shape of the LV, affect cortical pattern formation. We demonstrate increasing domain scale can increase the number of gyri and sulci formed. Changes in LV shape can account for sulcus directionality. By incorporating LV size and shape, our model is able to elucidate which parameters can lead to excessive cortical folding.

  8. Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain.

    PubMed

    Kim, Sun Kwang; Hayashi, Hideaki; Ishikawa, Tatsuya; Shibata, Keisuke; Shigetomi, Eiji; Shinozaki, Youichi; Inada, Hiroyuki; Roh, Seung Eon; Kim, Sang Jeong; Lee, Gihyun; Bae, Hyunsu; Moorhouse, Andrew J; Mikoshiba, Katsuhiko; Fukazawa, Yugo; Koizumi, Schuichi; Nabekura, Junichi

    2016-05-01

    Long-term treatments to ameliorate peripheral neuropathic pain that includes mechanical allodynia are limited. While glial activation and altered nociceptive transmission within the spinal cord are associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompany peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following nerve injury; however, the underlying cellular mechanisms of this plasticity and whether it has a causal relationship to allodynia remain unsolved. Furthermore, it is not known whether glial activation occurs within the S1 cortex following injury or whether it contributes to this S1 synaptic plasticity. Using in vivo 2-photon imaging with genetic and pharmacological manipulations of murine models, we have shown that sciatic nerve ligation induces a re-emergence of immature metabotropic glutamate receptor 5 (mGluR5) signaling in S1 astroglia, which elicits spontaneous somatic Ca2+ transients, synaptogenic thrombospondin 1 (TSP-1) release, and synapse formation. This S1 astrocyte reactivation was evident only during the first week after injury and correlated with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking the astrocytic mGluR5-signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (>1 month) allodynia. We conclude that reawakened astrocytes are a key trigger for S1 circuit rewiring and that this contributes to neuropathic mechanical allodynia. PMID:27064281

  9. Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain

    PubMed Central

    Hayashi, Hideaki; Ishikawa, Tatsuya; Shibata, Keisuke; Inada, Hiroyuki; Roh, Seung Eon; Kim, Sang Jeong; Moorhouse, Andrew J.

    2016-01-01

    Long-term treatments to ameliorate peripheral neuropathic pain that includes mechanical allodynia are limited. While glial activation and altered nociceptive transmission within the spinal cord are associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompany peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following nerve injury; however, the underlying cellular mechanisms of this plasticity and whether it has a causal relationship to allodynia remain unsolved. Furthermore, it is not known whether glial activation occurs within the S1 cortex following injury or whether it contributes to this S1 synaptic plasticity. Using in vivo 2-photon imaging with genetic and pharmacological manipulations of murine models, we have shown that sciatic nerve ligation induces a re-emergence of immature metabotropic glutamate receptor 5 (mGluR5) signaling in S1 astroglia, which elicits spontaneous somatic Ca2+ transients, synaptogenic thrombospondin 1 (TSP-1) release, and synapse formation. This S1 astrocyte reactivation was evident only during the first week after injury and correlated with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking the astrocytic mGluR5-signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (>1 month) allodynia. We conclude that reawakened astrocytes are a key trigger for S1 circuit rewiring and that this contributes to neuropathic mechanical allodynia. PMID:27064281

  10. Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

    PubMed Central

    Degrelle, Séverine A.; Jaffrezic, Florence; Campion, Evelyne; Lê Cao, Kim-Anh; Le Bourhis, Daniel; Richard, Christophe; Rodde, Nathalie; Fleurot, Renaud; Everts, Robin E.; Lecardonnel, Jérôme; Heyman, Yvan; Vignon, Xavier; Tian, Xiuchun C.; Lewin, Harris A.; Renard, Jean-Paul; Hue, Isabelle

    2012-01-01

    Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters

  11. Cortical hypoplasia and ventriculomegaly of p73-deficient mice: Developmental and adult analysis.

    PubMed

    Medina-Bolívar, Carolina; González-Arnay, Emilio; Talos, Flaminia; González-Gómez, Miriam; Moll, Ute M; Meyer, Gundela

    2014-08-01

    Trp73, a member of the p53 gene family, plays a crucial role in neural development. We describe two main phenotypic variants of p73 deficiency in the brain, a severe one characterized by massive apoptosis in the cortex leading to early postnatal death and a milder, non-/low-apoptosis one in which 50% of pups may reach adulthood using an intensive-care breeding protocol. Both variants display the core triad of p73 deficiency: cortical hypoplasia, hippocampal malformations, and ventriculomegaly. We studied the development of the neocortex in p73 KO mice from early embryonic life into advanced age (25 months). Already at E14.5, the incipient cortical plate of the p73 KO brains showed a reduced width. Examination of adult neocortex revealed a generalized, nonprogressive reduction by 10-20%. Area-specific architectonic landmarks and lamination were preserved in all cortical areas. The surviving adult animals had moderate ventricular distension, whereas pups of the early lethal phenotypic variant showed severe ventriculomegaly. Ependymal cells of wild-type ventricles strongly express p73 and are particularly vulnerable to p73 deficiency. Ependymal denudation by apoptosis and reduction of ependymal cilia were already evident in young mice, with complete absence of cilia in older animals. Loss of p73 function in the ependyma may thus be one determining factor for chronic hydrocephalus, which leads to atrophy of subcortical structures (striatum, septum, amygdala). p73 Is thus involved in a variety of CNS activities ranging from embryonic regulation of brain size to the control of cerebrospinal fluid homeostasis in the adult brain via maintenance of the ependyma.

  12. 2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size

    PubMed Central

    Otani, Tomoki; Marchetto, Maria C.; Gage, Fred H.; Simons, Benjamin D.; Livesey, Frederick J.

    2016-01-01

    Summary Variation in cerebral cortex size and complexity is thought to contribute to differences in cognitive ability between humans and other animals. Here we compare cortical progenitor cell output in humans and three nonhuman primates using directed differentiation of pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems. Clonal lineage analysis showed that primate cortical progenitors proliferate for a protracted period of time, during which they generate early-born neurons, in contrast to rodents, where this expansion phase largely ceases before neurogenesis begins. The extent of this additional cortical progenitor expansion differs among primates, leading to differences in the number of neurons generated by each progenitor cell. We found that this mechanism for controlling cortical size is regulated cell autonomously in culture, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output. PMID:27049876

  13. Measuring the micromechanical properties of embryonic tissues.

    PubMed

    Chevalier, Nicolas R; Gazguez, Elodie; Dufour, Sylvie; Fleury, Vincent

    2016-02-01

    Local mechanical properties play an important role in directing embryogenesis, both at the cell (differentiation, migration) and tissue level (force transmission, organ formation, morphogenesis). Measuring them is a challenge as embryonic tissues are small (μm to mm) and soft (0.1-10 kPa). We describe here how glass fiber cantilevers can be fabricated, calibrated and used to apply small forces (0.1-10 μN), measure contractile activity and assess the bulk tensile elasticity of embryonic tissue. We outline how pressure (hydrostatic or osmotic) can be applied to embryonic tissue to quantify stiffness anisotropy. These techniques can be assembled at low cost and with a minimal amount of equipment. We then present a protocol to prepare tissue sections for local elasticity and adhesion measurements using the atomic force microscope (AFM). We compare AFM nanoindentation maps of native and formaldehyde fixed embryonic tissue sections and discuss how the local elastic modulus obtained by AFM compares to that obtained with other bulk measurement methods. We illustrate all of the techniques presented on the specific example of the chick embryonic digestive tract, emphasizing technical issues and common pitfalls. The main purpose of this report is to make these micromechanical measurement techniques accessible to a wide community of biologists and biophysicists.

  14. Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

    PubMed

    Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-02-25

    Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process.

  15. Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model.

    PubMed

    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E

    2014-01-01

    Neurodevelopmental diseases such as the Rett syndrome (RTT) have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2) gene (MeCP2-/y) and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind RTT pathophysiology.

  16. Linking cortical network synchrony and excitability

    PubMed Central

    Meisel, Christian

    2016-01-01

    ABSTRACT Theoretical approaches based on dynamical systems theory can provide useful frameworks to guide experiments and analysis techniques when investigating cortical network activity. The notion of phase transitions between qualitatively different kinds of network dynamics has been such a framework inspiring novel approaches to neurophysiological data analysis over the recent years. One particular intriguing hypothesis has been that cortical networks reside in the vicinity of a phase transition. Although the final verdict on this hypothesis is still out, trying to understand cortex dynamics from this viewpoint has recently led to interesting insights on cortical network function with relevance for clinical practice. PMID:27065159

  17. Focal Cortical Dysplasia in Childhood Epilepsy.

    PubMed

    Shaker, Tarek; Bernier, Anne; Carmant, Lionel

    2016-05-01

    Focal cortical dysplasia is a common cause of medication resistant epilepsy. A better understanding of its presentation, pathophysiology and consequences have helped us improved its treatment and outcome. This paper reviews the most recent classification, pathophysiology and imaging findings in clinical research as well as the knowledge gained from studying genetic and lesional animal models of focal cortical dysplasia. This review of this recently gained knowledge will most likely help develop new research models and new therapeutic targets for patients with epilepsy associated with focal cortical dysplasia. PMID:27544467

  18. In vitro bioengineered model of cortical brain tissue.

    PubMed

    Chwalek, Karolina; Tang-Schomer, Min D; Omenetto, Fiorenzo G; Kaplan, David L

    2015-09-01

    A bioengineered model of 3D brain-like tissue was developed using silk-collagen protein scaffolds seeded with primary cortical neurons. The scaffold design provides compartmentalized control for spatial separation of neuronal cell bodies and neural projections, which resembles the layered structure of the brain (cerebral cortex). Neurons seeded in a donut-shaped porous silk sponge grow robust neuronal projections within a collagen-filled central region, generating 3D neural networks with structural and functional connectivity. The silk scaffold preserves the mechanical stability of the engineered tissues, allowing for ease of handling, long-term culture in vitro and anchoring of the central collagen gel to avoid shrinkage, and enabling neural network maturation. This protocol describes the preparation and manipulation of silk-collagen constructs, including the isolation and seeding of primary rat cortical neurons. This 3D technique is useful for mechanical injury studies and as a drug screening tool, and it could serve as a foundation for brain-related disease models. The protocol of construct assembly takes 2 d, and the resulting tissues can be maintained in culture for several weeks.

  19. Cortical dynamics as a therapeutic mechanism for touch healing.

    PubMed

    Kerr, Catherine E; Wasserman, Rachel H; Moore, Christopher I

    2007-01-01

    Touch Healing (TH) therapies, defined here as treatments whose primary route of administration is tactile contact and/or active guiding of somatic attention, are ubiquitous across cultures. Despite increasing integration of TH into mainstream medicine through therapies such as Reiki, Therapeutic Touch,(TM) and somatically focused meditation practices such as Mindfulness-Based Stress Reduction, relatively little is known about potential underlying mechanisms. Here, we present a neuroscientific explanation for the prevalence and effectiveness of TH therapies for relieving chronic pain. We begin with a cross-cultural review of several different types of TH treatments and identify common characteristics, including: light tactile contact and/or a somatosensory attention directed toward the body, a behaviorally relevant context, a relaxed context and repeated treatment sessions. These cardinal features are also key elements of established mechanisms of neural plasticity in somatosensory cortical maps, suggesting that sensory reorganization is a mechanism for the healing observed. Consideration of the potential health benefits of meditation practice specifically suggests that these practices provide training in the regulation of neural and perceptual dynamics that provide ongoing resistance to the development of maladaptive somatic representations. This model provides several direct predictions for investigating ways that TH may induce cortical plasticity and dynamics in pain remediation.

  20. In vitro bioengineered model of cortical brain tissue

    PubMed Central

    Chwalek, Karolina; Tang-Schomer, Min D.; Omenetto, Fiorenzo G.; Kaplan, David L.

    2016-01-01

    A bioengineered model of three-dimensional (3D) brain-like tissue was developed using silk-collagen protein scaffolds seeded with primary cortical neurons. The scaffold design provides compartmentalized control for spatial separation of neuronal cell bodies and neural projections, resembling the layered structure of the brain (cerebral cortex). Neurons seeded in a donut-shaped porous silk sponge grow robust neuronal projections within a collagen-filled central region, generating 3D neural networks with structural and functional connectivity. The silk scaffold preserves the mechanical stability of the engineered tissues, allowing for ease of handling, long-term culture in vitro, anchoring of the central collagen gel to avoid shrinkage, and neural network maturation. This protocol describes the preparation and manipulation of silk-collagen constructs, including the isolation and seeding of primary rat cortical neurons. This 3D technique is useful for mechanical injury studies, as a drug screening tool and could serve as a foundation for brain-related disease models. The protocol of construct assembly takes 2 days and the resulting tissues can be maintained in culture for several weeks. PMID:26270395

  1. EDA-containing fibronectin increases proliferation of embryonic stem cells.

    PubMed

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  2. Embryonic development of circadian clocks in the mammalian suprachiasmatic nuclei

    PubMed Central

    Landgraf, Dominic; Koch, Christiane E.; Oster, Henrik

    2014-01-01

    In most species, self-sustained molecular clocks regulate 24-h rhythms of behavior and physiology. In mammals, a circadian pacemaker residing in the hypothalamic suprachiasmatic nucleus (SCN) receives photic signals from the retina and synchronizes subordinate clocks in non-SCN tissues. The emergence of circadian rhythmicity during development has been extensively studied for many years. In mice, neuronal development in the presumptive SCN region of the embryonic hypothalamus occurs on days 12–15 of gestation. Intra-SCN circuits differentiate during the following days and retinal projections reach the SCN, and thus mediate photic entrainment, only after birth. In contrast the genetic components of the clock gene machinery are expressed much earlier and during midgestation SCN explants and isolated neurons are capable of generating molecular oscillations in culture. In vivo metabolic rhythms in the SCN, however, are observed not earlier than the 19th day of rat gestation, and rhythmic expression of clock genes is hardly detectable until after birth. Together these data indicate that cellular coupling and, thus, tissue-wide synchronization of single-cell rhythms, may only develop very late during embryogenesis. In this mini-review we describe the developmental origin of the SCN structure and summarize our current knowledge about the functional initiation and entrainment of the circadian pacemaker during embryonic development. PMID:25520627

  3. EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

    PubMed Central

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  4. EDA-containing fibronectin increases proliferation of embryonic stem cells.

    PubMed

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

  5. High-Degree Neurons Feed Cortical Computations

    PubMed Central

    Timme, Nicholas M.; Ito, Shinya; Shimono, Masanori; Yeh, Fang-Chin; Litke, Alan M.; Beggs, John M.

    2016-01-01

    Recent work has shown that functional connectivity among cortical neurons is highly varied, with a small percentage of neurons having many more connections than others. Also, recent theoretical developments now make it possible to quantify how neurons modify information from the connections they receive. Therefore, it is now possible to investigate how information modification, or computation, depends on the number of connections a neuron receives (in-degree) or sends out (out-degree). To do this, we recorded the simultaneous spiking activity of hundreds of neurons in cortico-hippocampal slice cultures using a high-density 512-electrode array. This preparation and recording method combination produced large numbers of neurons recorded at temporal and spatial resolutions that are not currently available in any in vivo recording system. We utilized transfer entropy (a well-established method for detecting linear and nonlinear interactions in time series) and the partial information decomposition (a powerful, recently developed tool for dissecting multivariate information processing into distinct parts) to quantify computation between neurons where information flows converged. We found that computations did not occur equally in all neurons throughout the networks. Surprisingly, neurons that computed large amounts of information tended to receive connections from high out-degree neurons. However, the in-degree of a neuron was not related to the amount of information it computed. To gain insight into these findings, we developed a simple feedforward network model. We found that a degree-modified Hebbian wiring rule best reproduced the pattern of computation and degree correlation results seen in the real data. Interestingly, this rule also maximized signal propagation in the presence of network-wide correlations, suggesting a mechanism by which cortex could deal with common random background input. These are the first results to show that the extent to which a neuron

  6. APC/C-Cdh1 coordinates neurogenesis and cortical size during development

    NASA Astrophysics Data System (ADS)

    Delgado-Esteban, Maria; García-Higuera, Irene; Maestre, Carolina; Moreno, Sergio; Almeida, Angeles

    2013-12-01

    The morphology of the adult brain is the result of a delicate balance between neural progenitor proliferation and the initiation of neurogenesis in the embryonic period. Here we assessed whether the anaphase-promoting complex/cyclosome (APC/C) cofactor, Cdh1—which regulates mitosis exit and G1-phase length in dividing cells—regulates neurogenesis in vivo. We use an embryo-restricted Cdh1 knockout mouse model and show that functional APC/C-Cdh1 ubiquitin ligase activity is required for both terminal differentiation of cortical neurons in vitro and neurogenesis in vivo. Further, genetic ablation of Cdh1 impairs the ability of APC/C to promote neurogenesis by delaying the exit of the progenitor cells from the cell cycle. This causes replicative stress and p53-mediated apoptotic death resulting in decreased number of cortical neurons and cortex size. These results demonstrate that APC/C-Cdh1 coordinates cortical neurogenesis and size, thus posing Cdh1 in the molecular pathogenesis of congenital neurodevelopmental disorders, such as microcephaly.

  7. MEF2C regulates cortical inhibitory and excitatory synapses and behaviors relevant to neurodevelopmental disorders

    PubMed Central

    Harrington, Adam J; Raissi, Aram; Rajkovich, Kacey; Berto, Stefano; Kumar, Jaswinder; Molinaro, Gemma; Raduazzo, Jonathan; Guo, Yuhong; Loerwald, Kris; Konopka, Genevieve; Huber, Kimberly M; Cowan, Christopher W

    2016-01-01

    Numerous genetic variants associated with MEF2C are linked to autism, intellectual disability (ID) and schizophrenia (SCZ) – a heterogeneous collection of neurodevelopmental disorders with unclear pathophysiology. MEF2C is highly expressed in developing cortical excitatory neurons, but its role in their development remains unclear. We show here that conditional embryonic deletion of Mef2c in cortical and hippocampal excitatory neurons (Emx1-lineage) produces a dramatic reduction in cortical network activity in vivo, due in part to a dramatic increase in inhibitory and a decrease in excitatory synaptic transmission. In addition, we find that MEF2C regulates E/I synapse density predominantly as a cell-autonomous, transcriptional repressor. Analysis of differential gene expression in Mef2c mutant cortex identified a significant overlap with numerous synapse- and autism-linked genes, and the Mef2c mutant mice displayed numerous behaviors reminiscent of autism, ID and SCZ, suggesting that perturbing MEF2C function in neocortex can produce autistic- and ID-like behaviors in mice. DOI: http://dx.doi.org/10.7554/eLife.20059.001 PMID:27779093

  8. Cortical circuits for perceptual inference.

    PubMed

    Friston, Karl; Kiebel, Stefan

    2009-10-01

    This paper assumes that cortical circuits have evolved to enable inference about the causes of sensory input received by the brain. This provides a principled specification of what neural circuits have to achieve. Here, we attempt to address how the brain makes inferences by casting inference as an optimisation problem. We look at how the ensuing recognition dynamics could be supported by directed connections and message-passing among neuronal populations, given our knowledge of intrinsic and extrinsic neuronal connections. We assume that the brain models the world as a dynamic system, which imposes causal structure on the sensorium. Perception is equated with the optimisation or inversion of this internal model, to explain sensory input. Given a model of how sensory data are generated, we use a generic variational approach to model inversion to furnish equations that prescribe recognition; i.e., the dynamics of neuronal activity that represents the causes of sensory input. Here, we focus on a model whose hierarchical and dynamical structure enables simulated brains to recognise and predict sequences of sensory states. We first review these models and their inversion under a variational free-energy formulation. We then show that the brain has the necessary infrastructure to implement this inversion and present stimulations using synthetic birds that generate and recognise birdsongs.

  9. Cortical circuits for perceptual inference.

    PubMed

    Friston, Karl; Kiebel, Stefan

    2009-10-01

    This paper assumes that cortical circuits have evolved to enable inference about the causes of sensory input received by the brain. This provides a principled specification of what neural circuits have to achieve. Here, we attempt to address how the brain makes inferences by casting inference as an optimisation problem. We look at how the ensuing recognition dynamics could be supported by directed connections and message-passing among neuronal populations, given our knowledge of intrinsic and extrinsic neuronal connections. We assume that the brain models the world as a dynamic system, which imposes causal structure on the sensorium. Perception is equated with the optimisation or inversion of this internal model, to explain sensory input. Given a model of how sensory data are generated, we use a generic variational approach to model inversion to furnish equations that prescribe recognition; i.e., the dynamics of neuronal activity that represents the causes of sensory input. Here, we focus on a model whose hierarchical and dynamical structure enables simulated brains to recognise and predict seq