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  • High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighbori ... 25919140

    We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the "cytokine fingerprints" identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    06-182

  • Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools. 25889980

    Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity.SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation.SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons.The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture. 18513158

    Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    SCR004
    Produktbezeichnung:
    Alkaline Phosphatase Detection Kit

  • Serum inhibitors for human mast cell growth: possible role of retinol. 14510724

    BACKGROUND: In vitro culture systems have been used to study the physiological and pathological characteristics of human mast cells. However, there are some differences in proliferation and maturation of mast cells between fetal bovine serum (FBS)-containing and serum-deprived cultures. Accordingly, we attempted to identify circulating factor(s) affecting the development of human mast cells. METHODS: We measured the serum levels of retinol and several cytokines. To elucidate the antiproliferative effects of the serum, a retinoic acid receptor (RARalpha) antagonist and neutralizing antibodies against cytokines were used. RESULTS: Similar to FBS, human serum dose-dependently suppressed the growth of tryptase+ cells from CD34+ cord blood cells or 20-week cultured mast cells under stimulation with stem cell factor (SCF). The serum-mediated inhibition might be based on a decline in proliferation rate. Among inhibitors for mast cell growth, retinol and transforming growth factor (TGF)-beta1 were present at high levels in human serum. In contrast with anti-TGF-beta1 antibody, an RARalpha antagonist counteracted the serum-induced suppression of human mast cell proliferation. CONCLUSIONS: Our results suggest that retinol and its derivatives act as a circulating regulator for human mast cell growth. The RARalpha antagonist may be a useful tool to obtain higher numbers of mast cells in FBS-containing cultures.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    MAB1222
    Produktbezeichnung:
    Anti-Tryptase Antibody, Mast Cell, clone G3

  • 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

  • Early acquisition of typical metabolic features upon differentiation of mouse neural stem cells into astrocytes. 14999809

    Specific metabolic features, such as glutamate reuptake, have been associated with normal functions of mature astrocytes. In this study, we examined whether these characteristics are acquired together with classical phenotypic markers of differentiated astrocytes. Differentiation of E14 mouse neurospheres into astrocytes was induced by the addition of fetal bovine serum (FBS). Degree of differentiation was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence for both GFAP and nestin. Neural stem cells expressed nestin but not GFAP, while differentiated astrocytes were immunopositive for GFAP but displayed low levels of nestin expression. A strong increase in the expression of the glutamate transporter GLAST and the monocarboxylate transporter MCT1 accompanied phenotypic changes. In addition, active glutamate transport appeared in differentiated astrocytes, as well as their capacity to increase aerobic glycolysis in response to glutamate. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, but not interleukin-6, triggered the expression of phenotypic and morphological characteristics of astrocytes. In addition, exposure to LIF led to the appearance of metabolic features typically associated with astrocytes. Altogether, our results show that acquisition of some specific metabolic features by astrocytes occurs early in their differentiation process and that LIF represents a candidate signal to induce their expression.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    AB5922

  • Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells. 10446906

    The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    06-411

  • 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