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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

  • 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

  • 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®

  • Skeletal muscle tissue engineering: a maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus a ... 19625080

    The use of defined in vitro systems to study the developmental and physiological characteristics of a variety of cell types is increasing, due in large part to their ease of integration with tissue engineering, regenerative medicine, and high-throughput screening applications. In this study, myotubes derived from fetal rat hind limbs were induced to develop several aspects of mature muscle including: sarcomere assembly, development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching. Utilizing immunocytochemical analysis, anisotropic and isotropic band formation (striations) within the myotubes was established, indicative of sarcomere formation. In addition, clusters of ryanodine receptors were colocalized with dihydropyridine complex proteins which signaled development of the excitation-contraction coupling apparatus and transverse tubule biogenesis. The myotubes also exhibited MHC class switching from embryonic to neonatal MHC. Lastly, the myotubes survived significantly longer in culture (70-90 days) than myotubes from our previously developed system (20-25 days). These results were achieved by modifying the culture timeline as well as the development of a new medium formulation. This defined model system for skeletal muscle maturation supports the goal of developing physiologically relevant muscle constructs for use in tissue engineering and regenerative medicine as well as for high-throughput screening applications.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB9078
    Nombre del producto:
    Anti-Ryanodine Receptor 1 Antibody

  • Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells. 24465853

    Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4304
    Nombre del producto:
    Anti-Stage-Specific Embryonic Antigen-4 Antibody, clone MC-813-70

  • 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