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  • Long-term dynamic loading improves the mechanical properties of chondrogenic mesenchymal stem cell-laden hydrogel. 20186667

    Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering given their ability to undergo chondrogenesis in 3D culture systems. Mechanical forces play an important role in regulating both cartilage development and MSC chondrogenic gene expression, however, mechanical stimulation has yet to enhance the mechanical properties of engineered constructs. In this study, we applied long-term dynamic compression to MSC-seeded constructs and assessed whether varying pre-culture duration, loading regimens and inclusion of TGF-beta3 during loading would influence functional outcomes and these phenotypic transitions. Loading initiated before chondrogenesis decreased functional maturation, although chondrogenic gene expression increased. In contrast, loading initiated after chondrogenesis and matrix elaboration further improved the mechanical properties of MSC-based constructs, but only when TGF-beta3 levels were maintained and under specific loading parameters. Although matrix quantity was not affected by dynamic compression, matrix distribution, assessed histologically and by FT-IRIS analysis, was significantly improved on the micro- (pericellular) and macro- (construct expanse) scales. Further, whole genome expression profiling revealed marked shifts in the molecular topography with dynamic loading. These results demonstrate, for the first time, that dynamic compressive loading initiated after a sufficient period of chondro-induction and with sustained TGF-beta exposure enhances matrix distribution and the mechanical properties of MSC-seeded constructs.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3391
    Nombre del producto:
    Anti-Collagen Type I Antibody, clone 5D8-G9

  • Feedback regulation of the ?2(1) collagen gene via the Mek-Erk signaling pathway. 22131293

    The extracellular matrix (ECM) provides the microenvironment that is pivotal for cell growth, motility, attachment, and differentiation. Advances in cell culture techniques have led to the development of cell-derived ECM model systems that are more reflective of the in vivo architecture of the ECM in tissue. In this study, a fibroblast-derived ECM (fd-ECM) was used to study the feedback regulation of type I collagen synthesis in fibroblasts. Fibroblasts plated on a preformed fd-ECM showed a significant decrease in the production of type I collagen and pro-?2(1) collagen mRNA compared to cells grown in the absence of a matrix. Function-blocking antibodies showed that this downregulation of type I collagen gene expression is mediated via ?2?1 integrin. The use of several kinase inhibitors and a dominant negative ras construct (N17Ras) showed that the matrix-mediated downregulation of COL1A2 occurs via Ras-dependent activation of the MEK/ERK signaling pathway. Deletion analysis of the COL1A2 promoter implicated the region between -375 and -107 as containing a potential matrix responsive element. The use of Sp1 siRNA demonstrated that Sp1 is an important mediator of this feedback inhibition. This study provides some new insights into the feedback regulation of COL1A2 gene expression.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1998
    Nombre del producto:
    Anti-Integrin α2β1 Antibody, clone BHA2.1

  • 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

  • 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®

  • Differentiation of rhesus monkey embryonic stem cells in three-dimensional collagen matrix. 16846041

    During normal embryogenesis, embryonic stem cells (ESCs) reside in the context of complex three-dimensional tissue structures, in particular of extracellular matrices (ECMs), which determine cell migration, proliferation, and differentiation. Therefore, to study ESC differentiation in an in vivo-like microenvironment, three-dimensional culture systems are necessary. Here, we developed protocols for ESC cultures in three-dimensional systems consisting of collagen matrices (collagen gels and porous collagen sponges) to investigate the mechanisms of ESC differentiation as well as the formation of tissue-like structures. In collagen matrices, ESCs differentiate into neural, epithelial, and endothelial lineages. In this system, ESCs form various tissue-like structures. The abilities of ESCs to form such structures in two chemically similar but topologically different matrices are different. In particular, in collagen gels ESCs form gland-like circular structures, whereas in collagen sponges ESCs are scattered through the matrix and form aggregates. To mimic the in vivo situation further, we developed a protocol for co-cultures of ESCs with human dermal fibroblasts or keratinocytes in collagen matrixes. Co-culture with fibroblasts in collagen gel facilitates ESC differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule (NCAM), and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ESC differentiation into cells of an endothelial lineage expressing factor VIII. Thus, the developed protocols promote ESC differentiation into a particular lineage, accompanied by the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for directing ESC differentiation and the formation of organs and tissues.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1637
    Nombre del producto:
    Anti-Tubulin Antibody, beta III isoform, CT, clone TU-20 (Similar to TUJ1)

  • 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