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  • Increased expression of cyclo-oxygenase 2 and vascular endothelial growth factor in lesioned spinal cord by transplanted olfactory ensheathing cells. 15319002

    Olfactory ensheathing cells (OECs) were transplanted in adult rats after photochemical injury of the spinal cord. Rats received either 180,000 OECs suspended in DMEM or DMEM alone. Locomotor ability scored by the BBB-scale, pain sensibility, and motor and somatosensory evoked potentials were evaluated during the first 14 days post-surgery. At 3, 7, and 14 days, 5 rats per day of both groups were perfused and transverse sections from proximal, lesioned and distal spinal cord blocks were stained for COX-2, VEGF, GFAP and lectin. The BBB-score and the amplitude of motor and somatosensory evoked potentials were significantly higher in OEC- than in DMEM-injected animals throughout follow-up, whereas the withdrawal latency to heat noxious stimulus was lower in OEC- than in DMEM-injected rats. The area of preserved spinal cord and the levels of COX-2 and VEGF staining were significantly higher in OEC- than in DMEM-injected rats. GFAP- but no LEC-positive cells expressed COX-2 staining in OEC-transplanted rats. The density of blood vessels was also significantly increased in OEC- with respect to DMEM-injected rats. Our results show that OECs promote functional and morphological preservation of the spinal cord after photochemical injury, increasing neoangiogenesis and up-regulation of COX-2 and VEGF expression in astrocytes.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    MAB365
    Produktbezeichnung:
    Anti-Nerve Growth Factor Receptor Antibody, extracellular, clone 192-IgG

  • Novel functional hepatocyte cell line derived from spontaneous dwarf rat: model of growth hormone function in vitro. 21166888

    Currently there is no good hepatocyte model for studying growth hormone (GH) function that reflects its normal physiological roles. Here we report the establishment of a functional hepatocyte cell line, SDRL-1, from the liver of young male spontaneous dwarf rats (SDR), with isolated GH deficiency. This line has been maintained in Dulbecco's Modified Eagle Medium (DMEM)/F12 medium supplemented with 10% fetal bovine serum (FBS) with retention of a near diploid karyotype for extended periods of time. When grown as a monolayer sheet, it displayed a pavement-like appearance and contact inhibition. These cells have a poorly developed rough endoplasmic reticulum (r-ER), few mitochondria and glycogen granules, and produce a small amount of albumin and α-fetoprotein, that is enhanced when grown on a collagen gel sponge. Human recombinant GH stimulated JAK2 and STAT5b tyrosine phosphorylation and IGF-I production in a concentration-dependent manner. When the cells were cultured with GH-supplemented medium, the number of mitochondria and glycogen granules increased together with the r-ER and Golgi apparatus. A number of microvilli were observed on the surface of the cultured cells, further suggesting that this cell line is composed of normally functioning hepatocytes. In summary, we established a novel hepatocyte cell line (SDRL-1), that appears to display normal function, which we propose can serve as a good in vitro model for studying GH-target organ interactions.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    06-255
    Produktbezeichnung:
    Anti-JAK2 Antibody

  • An in vitro culture system for long-term expansion of epithelial and mesenchymal salivary gland cells: role of TGF-β1 in salivary gland epithelial and mesenchymal differe ... 23841093

    Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere

  • Strategies for repair of white matter: influence of osmolarity and microglia on proliferation and apoptosis of oligodendrocyte precursor cells in different basal culture ... 24421756

    The aim of the present study has been to obtain high yields of oligodendrocyte precursor cells (OPCs) in culture. This is a first step in facilitation of myelin repair. We show that, in addition to factors, known to promote proliferation, such as basic fibroblast growth factor (FGF-2) and platelet derived growth factor (PDGF) the choice of the basal medium exerts a significant influence on the yield of OPCs in cultures from newborn rats. During a culture period of up to 9 days we observed larger numbers of surviving cells in Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute Medium (RPMI) compared with Neurobasal Medium (NB). A larger number of A2B5-positive OPCs was found after 6 days in RPMI based media compared with NB. The percentage of bromodeoxyuridine (BrdU)-positive cells was largest in cultures maintained in DMEM and RPMI. The percentage of caspase-3 positive cells was largest in NB, suggesting that this medium inhibits OPC proliferation and favors apoptosis. A difference between NB and DMEM as well as RPMI is the reduced Na(+)-content. The addition of equiosmolar supplements of mannitol or NaCl to NB medium rescued the BrdU-incorporation rate. This suggested that the osmolarity influences the proliferation of OPCs. Plating density as well as residual microglia influence OPC survival, BrdU incorporation, and caspase-3 expression. We found, that high density cultures secrete factors that inhibit BrdU incorporation whereas the presence of additional microglia induces an increase in caspase-3 positive cells, indicative of enhanced apoptosis. An enhanced number of microglia could thus also explain the stronger inhibition of OPC differentiation observed in high density cultures in response to treatment with the cytokines TNF-α and IFN-γ. We conclude that a maximal yield of OPCs is obtained in a medium of an osmolarity higher than 280 mOsm plated at a relatively low density in the presence of as little microglia as technically achievable.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Differentiation of human breast-milk stem cells to neural stem cells and neurons. 25506428

    Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC) toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12) containing fibroblast growth factor (bFGF). The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7-10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial growth factor (EGF) 20 µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing growth factor in the presence of 5% FBS (fetal bovine serum). The immunofluorescence was done for β-tubulin III, O4, and GFAP (glial fibrillary acidic protein). Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC) markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Optimization and integration of expansion and neural commitment of mouse embryonic stem cells. 17596123

    To harness the potential of ES (embryonic stem) cells for human therapy, technology to develop the large-scale expansion and differentiation of these cells is required. In the present study, we tested various conditions for the expansion and neural commitment of mouse ES cells, using a cell line with a fluorescent reporter, which allows the monitoring of these processes by flow cytometry. The expansion of the 46C ES cell line in the presence of two different media [serum-free ESGRO Completetrade mark and DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) fetal bovine serum] was compared. Both media yielded similar cell fold increases at two different initial cell densities and were able to maintain neural commitment potential during expansion. The influence of inocula concentration in the presence of two different media on cell proliferation and efficiency of neural commitment was evaluated. Two different chemically defined serum-free media were tested: the more conventional N2B27 and the second-generation medium RHB-A (Stem Cell Sciences, Edinburgh, Scotland, U.K.). The kinetics of neural commitment was followed during 8 days in the presence of both media. Our results show that inocula concentrations between 5x10(3) and 10(4) cells/cm(2) are the most appropriate to achieve a better cell growth and more efficient neural commitment. We also show that cell culture in RHB-A medium results in higher rates of cell proliferation and neural commitment of ES cells, when compared with N2B27.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    SCM012
    Produktbezeichnung:
    NDiff Neuro-2 Medium Supplement (200x)

  • Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning. 25680105

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P less than 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P greater than 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P less than 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Multiphasic collagen fibre-PLA composites seeded with human mesenchymal stem cells for osteochondral defect repair: an in vitro study. 19434664

    A promising approach for the repair of osteochondral defects is the use of a scaffold with a well-defined cartilage-bone interface. In this study, we used a multiphasic composite scaffold with an upper collagen I fibre layer for articular cartilage repair, separated by a hydrophobic interface from a lower polylactic acid (PLA) part for bone repair. Focusing initially on the engineering of cartilage, the upper layer was seeded with human mesenchymal stem cells (hMSCs) suspended in a collagen I hydrogel for homogeneous cell distribution. The constructs were cultured in a defined chondrogenic differentiation medium supplemented with 10 ng/ml transforming growth factor-beta1 (TGFbeta1) or in DMEM with 10% fetal bovine serum as a control. After 3 weeks a slight contraction of the collagen I fibre layer was seen in the TGFbeta1-treated group. Furthermore, a homogeneous cell distribution and chondrogenic differentiation was achieved in the upper third of the collagen I fibre layer. In the TGFbeta1-treated group cells showed a chondrocyte-like appearance and were surrounded by a proteoglycan and collagen type II-rich extracellular matrix. Also, a high deposition of glycosaminoglycans could be measured in this group and RT-PCR analyses confirmed the induction of chondrogenesis, with the expression of cartilage-specific marker genes, such as aggrecan and collagen types II and X. This multiphasic composite scaffold with the cartilage layer on top might be a promising construct for the repair of osteochondral defects.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    AB3209
    Produktbezeichnung:
    Anti-Oct-4 Antibody