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  • Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains. 20520777

    New mechanisms that regulate neural stem cell (NSC) expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers.Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen.Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Two cell circuits of oriented adult hippocampal neurons on self-assembled monolayers for use in the study of neuronal communication in a defined system. 23611164

    In this study, we demonstrate the directed formation of small circuits of electrically active, synaptically connected neurons derived from the hippocampus of adult rats through the use of engineered chemically modified culture surfaces that orient the polarity of the neuronal processes. Although synaptogenesis, synaptic communication, synaptic plasticity, and brain disease pathophysiology can be studied using brain slice or dissociated embryonic neuronal culture systems, the complex elements found in neuronal synapses makes specific studies difficult in these random cultures. The study of synaptic transmission in mature adult neurons and factors affecting synaptic transmission are generally studied in organotypic cultures, in brain slices, or in vivo. However, engineered neuronal networks would allow these studies to be performed instead on simple functional neuronal circuits derived from adult brain tissue. Photolithographic patterned self-assembled monolayers (SAMs) were used to create the two-cell "bidirectional polarity" circuit patterns. This pattern consisted of a cell permissive SAM, N-1[3-(trimethoxysilyl)propyl] diethylenetriamine (DETA), and was composed of two 25 μm somal adhesion sites connected with 5 μm lines acting as surface cues for guided axonal and dendritic regeneration. Surrounding the DETA pattern was a background of a non-cell-permissive poly(ethylene glycol) (PEG) SAM. Adult hippocampal neurons were first cultured on coverslips coated with DETA monolayers and were later passaged onto the PEG-DETA bidirectional polarity patterns in serum-free medium. These neurons followed surface cues, attaching and regenerating only along the DETA substrate to form small engineered neuronal circuits. These circuits were stable for more than 21 days in vitro (DIV), during which synaptic connectivity was evaluated using basic electrophysiological methods.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Derivation, characterization, and in vitro differentiation of canine embryonic stem cells. 18065395

    Canine embryonic stem (cES) cell lines were generated to establish a large-animal preclinical model for testing the safety and efficacy of embryonic stem (ES) cell-derived tissue replacement therapy. Putative cES cell lines were initiated from canine blastocysts harvested from natural matings. Times of harvest were estimated as 12-16 days after the presumed surge in circulating levels of luteinizing hormone. Four lines established from blastocysts harvested at days 13-14 postsurge satisfied most of the criteria for embryonic stem cells, whereas lines established after day 14 did not. One line, Fred Hutchinson dog (FHDO)-7, has been maintained through 34 passages and is presented here. FHDO-7 cells are alkaline phosphatase-positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and telomerase but do not express message for Cdx2, which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miRs) (miR-302b, miR-302c, and miR-367) characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders, the cells form embryoid bodies (EBs). Under various culture conditions, the EBs give rise to ectoderm-derived neuronal cells expressing gamma-enolase and beta 3-tubulin; mesoderm-derived cells producing collagen IIA1, cartilage, and bone; and endoderm-derived cells expressing alpha-fetoprotein or Clara cell-specific protein.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4381
    Nombre del producto:
    Anti-TRA-1-81 Antibody, clone TRA-1-81

  • Procedures for derivation and characterisation of human embryonic stem cells from Odense, Denmark. 22528347

    In 1998, a development occurred in stem cell biology with the first report of the derivation of a human embryonic stem cell (hESC) line. Since then a number of techniques have been used to derive and characterise hESCs. Here, we describe the derivation methods used by our laboratory for isolation of the ICM by immunosurgery and outgrowth of the whole blastocyst. We have added protocols for routine culture, passaging and cryopreservation of our hESC lines as well as the methods we have used for characterisation (flow cytometry, karyotyping, immunocytochemistry, in vitro and in vivo differentiation). Additionally, we have included gene sequences for PCR and an antibody list for immunocytochemistry.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4381
    Nombre del producto:
    Anti-TRA-1-81 Antibody, clone TRA-1-81

  • Rac1 plays an essential role in axon growth and guidance and in neuronal survival in the central and peripheral nervous systems. 26395878

    Rac1 is a critical regulator of cytoskeletal dynamics in multiple cell types. In the nervous system, it has been implicated in the control of cell proliferation, neuronal migration, and axon development.To systematically investigate the role of Rac1 in axon growth and guidance in the developing nervous system, we have examined the phenotypes associated with deleting Rac1 in the embryonic mouse forebrain, in cranial and spinal motor neurons, in cranial sensory and dorsal root ganglion neurons, and in the retina. We observe a widespread requirement for Rac1 in axon growth and guidance and a cell-autonomous defect in axon growth in Rac1 (-/-) motor neurons in culture. Neuronal death, presumably a secondary consequence of the axon growth and/or guidance defects, was observed in multiple locations. Following deletion of Rac1 in the forebrain, thalamocortical axons were misrouted inferiorly, with the majority projecting to the contralateral thalamus and a minority projecting ipsilaterally to the ventral cortex, a pattern of misrouting that is indistinguishable from the pattern previously observed in Frizzled3 (-/-) and Celsr3 (-/-) forebrains. In the limbs, motor-neuron-specific deletion of Rac1 produced a distinctive stalling of axons within the dorsal nerve of the hindlimb but a much milder loss of axons in the ventral hindlimb and forelimb nerves, a pattern that is virtually identical to the one previously observed in Frizzled3 (-/-) limbs.The similarities in axon growth and guidance phenotypes caused by Rac1, Frizzled3, and Celsr3 loss-of-function mutations suggest a mechanistic connection between tissue polarity/planar cell polarity signaling and Rac1-dependent cytoskeletal regulation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Manipulation of human pluripotent embryonal carcinoma stem cells and the development of neural subtypes. 12743319

    There are few reliable cell systems available to study the process of human neural development. Embryonal carcinoma (EC) cells are pluripotent stem cells derived from teratocarcinomas and offer a robust culture system to research cell differentiation in a manner pertinent to embryogenesis. Here, we describe the recent development of a series of culture procedures that together can be used to induce the differentiation of human EC stem cells, resulting in the formation of either pure populations of differentiated neurons, populations of differentiated astrocytes, or populations of immature neuronal cell types. Cell-type-specific markers were used to examine the induction of EC stem cell differentiation by retinoic acid. In direct response to manipulation of the culture environment, the expression of cell type markers correlated with the differentiation and appearance of distinct neural cell types, including neurons and astrocytes. These experiments demonstrate that cultured human EC stem cells provide a robust model cell system capable of reproducibly forming neural subtypes for research purposes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB324
    Nombre del producto:
    Anti-Neuron Specific Enolase Antibody, clone 5E2

  • Disruption of intermolecular disulfide bonds in PDGF-BB dimers by N-acetyl-L-cysteine does not prevent PDGF signaling in cultured hepatic stellate cells. 16289037

    Oxidative stress is important in the pathogenesis of liver fibrosis through its induction of hepatic stellate cell (HSC) proliferation and enhancement of collagen synthesis. Reactive oxygen species have been found to be essential second messengers in the signaling of both major fibrotic growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), in cultured HSC and liver fibrosis. The non-toxic aminothiol N-acetyl-L-cysteine (NAC) inhibits cellular activation and attenuates experimental fibrosis in liver. Prior reports show that NAC is capable of reducing the effects of TGF-beta in biological systems, in cultured endothelial cells, and HSC through its direct reducing activity upon TGF-beta molecules. We here analyzed the effects of NAC on PDGF integrity, receptor binding, and downstream signaling in culture-activated HSC. We found that NAC dose-dependently induces disintegration of PDGF in vitro. However, even high doses (>20mM) were not sufficient to prevent the phosphorylation of the PDGF receptor type beta, extracellular signal-regulated kinase, or protein kinase B (PKB/Akt). Therefore, we conclude that the PDGF monomer is still active. The described antifibrotic effects are therefore mainly attributable to the structural impairment of TGF-beta signaling components reported previously.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-321
    Nombre del producto:
    Anti-Phosphotyrosine Antibody, clone 4G10®

  • Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture. 10602491

    The proliferation of most normal cells depends on the synergistic interaction of several growth factors and hormones, but the cell cycle basis for this combined requirement remains largely uncharacterized. We have addressed the question of the requirement for insulin/IGF-1 also observed in many cell culture systems in the physiologically relevant system of primary cultures of dog thyroid epithelial cells stimulated by TSH, which exerts its mitogenic activity only via cAMP. The induction of cyclin A and cdc2, the phosphorylation of cdk2, the nuclear translocation of cdk4 and the assembly of cyclin D3-cdk4 complexes required the synergy of TSH and insulin. Cyclin D3 (the most abundant cyclin D) was necessary for the proliferation stimulated by TSH in the presence of insulin as shown by microinjection of a neutralizing antibody. Cyclin D3 accumulation and activity were differentially regulated by insulin and TSH, which points out this cyclin as an integrator that ranks these comitogenic pathways as supportive and activatory, respectively. Paradoxically TSH alone strongly repressed cyclin D3 accumulation. This inhibition was overridden by insulin, which markedly stimulated cyclin D3 mRNA and protein accumulation, but failed to assemble cyclin D3-cdk4 complexes in the absence of TSH. TSH unmasked the DCS-22 epitope of cyclin D3 and assembled cyclin D3-cdk4 in the presence of insulin. These data demonstrate that cyclin D synthesis and cyclin D-cdk assembly can be dissociated and complementarily regulated by different agents and signalling pathways.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Roles of cell adhesion molecules in tumor angiogenesis induced by cotransplantation of cancer and endothelial cells to nude rats. 12414659

    Roles of cell adhesion molecules mediating the interaction of cancer and endothelial cells in tumor angiogenesis were investigated using new in vitro and in vivo model systems with a cultured murine endothelial cell line (F-2) and human cultured epidermoid cancer cells (A431). The A431 cells exhibited typical in vitro cell adhesion to the endothelial F-2 cells. The initial step of adhesion was mediated by sialyl Lewis(x) (Le(x)) and sialyl Le(a), the carbohydrate determinants expressed on the cancer cells, and E-selectin expressed constitutively on F-2 cells. Prolonged culture led to the implantation of cancer cells into the monolayer of the F-2 cells, which was mediated mainly by alpha(3)beta(1)-integrin. F-2 cells cultured on Matrigel showed evident tube formation, and coculture of F-2 cells with A431 cells led to the formation of A431 cell nests constantly surrounded by tube-like networks consisting of F-2 cells. This in vitro morphogenesis was inhibited by the addition of anti-sialyl Le(x)/Le(a) or anti-beta(1)-integrin antibodies, which led to the formation of cancer cell aggregates that were independent from the F-2 cell networks. This in vitro morphological appearance was exactly reproduced in the in vivo tumors, which were formed when the mixture of A431 and F-2 cells at the ratio of 10:1 were cotransplanted s.c. into the back of nude rats. The tumors of A431 supplemented with F-2 cells were profoundly vascularized throughout by the tubular structures formed by F-2 cells, the lumen of which contained the host rat blood cells. The tumor mass thus formed was an average 5.8-fold as large as control A431 tumors that were grown without F-2 cells. The co-injection of anti-Le(x)/Le(a) or anti-beta(1)-integrin antibodies produced a marked reduction in the size of A431 tumors, which were not vascularized and accompanied an independent tiny remnant clump of F-2 cells. The size of these A431 tumors did not differ significantly from those of control A431 tumors raised without F-2 cells. These results indicate that the interaction of tumor cells and endothelial cells in orderly tumor angiomorphogenesis is highly dependent on the action of cell adhesion molecules mediating the adhesion of cancer cells to endothelial cells, inhibition of which remarkably retards tumor growth and angiogenesis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1980

  • Optimization of flowrate for expansion of human embryonic stem cells in perfusion microbioreactors. 21732331

    Microfluidic systems create significant opportunities to establish highly controlled microenvironmental conditions for screening pluripotent stem cell fate. However, since cell fate is crucially dependent on this microenvironment, it remains unclear as to whether continual perfusion of culture medium supports pluripotent stem cell maintenance in feeder-free, chemically defined conditions, and further, whether optimum perfusion conditions exist for subsequent use of human embryonic stem cell (hESCs) in other microfludic systems. To investigate this, we designed microbioreactors based on resistive flow to screen hESCs under a linear range of flowrates. We report that at low rates (conditions where glucose transport is convection-limited with Péclet number <1), cells are affected by apparent nutrient depletion and waste accumulation, evidenced by reduced cell expansion and altered morphology. At higher rates, cells are spontaneously washed out, and display morphological changes which may be indicative of early-stage differentiation. However, between these thresholds exists a narrow range of flowrates in which hESCs expand comparably to the equivalent static culture system, with regular morphology and maintenance of the pluripotency marker TG30 in >95% of cells over 7 days. For MEL1 hESCs the optimum flowrate also coincided with the time-averaged medium exchange rate in static cultures, which may therefore provide a good first estimate of appropriate perfusion rates. Overall, we demonstrate hESCs can be maintained in microbioreactors under continual flow for up to 7 days, a critical outcome for the future development of microbioreactor-based screening systems and assays for hESC culture.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4427