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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

  • 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

  • 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®

  • Lead exposure during synaptogenesis alters vesicular proteins and impairs vesicular release: potential role of NMDA receptor-dependent BDNF signaling. 20375082

    Lead (Pb(2+)) exposure is known to affect presynaptic neurotransmitter release in both in vivo and cell culture models. However, the precise mechanism by which Pb(2+) impairs neurotransmitter release remains unknown. In the current study, we show that Pb(2+) exposure during synaptogenesis in cultured hippocampal neurons produces the loss of synaptophysin (Syn) and synaptobrevin (Syb), two proteins involved in vesicular release. Pb(2+) exposure also increased the number of presynaptic contact sites. However, many of these putative presynaptic contact sites lack Soluble NSF attachment protein receptor complex proteins involved in vesicular exocytosis. Analysis of vesicular release using FM 1-43 dye confirmed that Pb(2+) exposure impaired vesicular release and reduced the number of fast-releasing sites. Because Pb(2+) is a potent N-methyl-D-aspartate receptor (NMDAR) antagonist, we tested the hypothesis that NMDAR inhibition may be producing the presynaptic effects. We show that NMDAR inhibition by aminophosphonovaleric acid mimics the presynaptic effects of Pb(2+) exposure. NMDAR activity has been linked to the signaling of the transsynaptic neurotrophin brain-derived neurotrophic factor (BDNF), and we observed that both the cellular expression of proBDNF and release of BDNF were decreased during the same period of Pb(2+) exposure. Furthermore, exogenous addition of BDNF rescued the presynaptic effects of Pb(2+). We suggest that the presynaptic deficits resulting from Pb(2+) exposure during synaptogenesis are mediated by disruption of NMDAR-dependent BDNF signaling.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Neurogenic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum-free suspension and adherent cultures. 17152062

    The great potential of human embryonic stem (hES) cells offers the opportunity both for studying basic developmental processes in vitro as well as for drug screening, modeling diseases, or future cell therapy. Defining protocols for the generation of human neural progenies represents a most important prerequisite. Here, we have used six hES cell lines to evaluate defined conditions for neural differentiation in suspension and adherent culture systems. Our protocol does not require fetal serum, feeder cells, or retinoic acid at any step, to induce neural fate decisions in hES cells. We monitored neurogenesis in differentiating cultures using morphological (including on-line follow up), immunocytochemical, and RT-PCR assays. For each hES cell line, in suspension or adherent culture, the same longitudinal progression of neural differentiation occurs. We showed the dynamic transitions from hES cells to neuroepithelial (NE) cells, to radial glial (RG) cells, and to neurons. Thus, 7 days after neural induction the majority of cells were NE, expressing nestin, Sox1, and Pax6. During neural proliferation and differentiation, NE cells transformed in RG cells, which acquired vimentin, BLBP, GLAST, and GFAP, proliferated and formed radial scaffolds. gamma-Aminobutyric acid (GABA)-positive and glutamate positive neurons, few oligodendrocyte progenitors and astrocytes were formed in our conditions and timing. Our system successfully generates human RG cells and could be an effective source for neuronal replacement, since RG cells predominantly generate neurons and provide them with support and guidance.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Congestive heart failure effects on atrial fibroblast phenotype: differences between freshly-isolated and cultured cells. 23251678

    Fibroblasts are important in the atrial fibrillation (AF) substrate resulting from congestive heart failure (CHF). We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks). Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM) gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+)-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold), collagen-3 (5-fold), and fibronectin-1 (3-fold) vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm) and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02), consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA)-sensitive K(+)-currents that were strongly downregulated in CHF. The TEA-sensitive K(+)-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3242
    Nombre del producto:
    Anti-Smoothelin Antibody, clone R4A

  • Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression. 12933809

    Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-beta (TGF-beta) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-beta in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-beta on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-beta was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-beta during the initial culture period (days 1-7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-beta was present during the entire culture period (days 1-21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-beta was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-beta, we examined RANK expression and p38 MAPK activation. We found the TGF-beta strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-beta to monocytes down-regulated RANK expression. The current results suggest that TGF-beta promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-beta abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo