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  • FLOW-CYTOMETRY  (3)
  • 1
    Keywords: IN-VIVO ; BONE-MARROW ; PROGENITOR CELLS ; VASCULATURE ; FLOW-CYTOMETRY ; PERIPHERAL-BLOOD ; GENE-THERAPY ; PRECURSORS ; PROTOCOL ; MESENCHYMAL STEM-CELLS
    Abstract: BACKGROUND AIMS: Endothelial progenitor cells (EPCs) specifically home to sites of malignant growth, rendering them attractive for anti-cancer therapies. Data are conflicting on the phenotype and quantitative contribution toward tumor angiogenesis based on differing culture assays to outgrow EPCs. To evaluate the origin and early phenotype of EPCs and to define a population with enhanced tumor-targeting capacity, we evaluated a hierarchy of cord blood-derived EPCs modeling the multi-step nature of tumor homing. METHODS: CD34(+) mononuclear cells were isolated from fresh cord blood and cultured to derive endothelial colony-forming cells (ECFCs). Human umbilical vein endothelial cells (HUVECs) served as control. Using intra-vital microscopy, the recruitment was analyzed in mice bearing C6 xenografts. Adhesion, migration, transmigration and differentiation were further addressed. RESULTS: Within the primary passage, ECFCs underwent a rapid maturation from a CD45(+) and CD31(+) phenotype to a CD45(-) and endothelial marker positive phenotype. Assessing in vivo tumor recruitment, ECFCs had the highest activity in all steps analyzed. In vitro, ECFCs demonstrated significantly higher adhesion under static and flow conditions. Similarly, ECFCs exhibited highest migratory and trans-migratory activity toward tumor-conditioned medium. On subcutaneous implantation, only ECFCs formed blood vessels covered with perivascular cells, similar to HUVECs. CONCLUSIONS: Our study indicates that ECFCs emerge from a CD45(+) and CD31(+) progenitor and rapidly mature in culture. ECFCs have a significantly higher potential for tumor targeting than non-cultured CD34(+) cells and HUVECs. They are ideal candidates for future cell-based anti-cancer therapies.
    Type of Publication: Journal article published
    PubMed ID: 23491253
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  • 2
    Keywords: CELLS ; EXPRESSION ; tumor ; BLOOD ; CELL ; Germany ; human ; IN-VIVO ; THERAPY ; VIVO ; FOLLOW-UP ; POPULATION ; GENE ; DRUG ; gene therapy ; MICE ; TRANSDUCTION ; gene transfer ; GENE-TRANSFER ; MARKER ; FLOW ; BIOLOGY ; MATURATION ; TARGET ; HUMANS ; resistance ; VECTOR ; MARKERS ; STEM-CELLS ; FLOW-CYTOMETRY ; HEMATOPOIETIC PROGENITOR CELLS ; HEMATOPOIETIC-CELLS ; PERIPHERAL-BLOOD ; MULTIDRUG-RESISTANCE ; MULTIDRUG-RESISTANCE-1 GENE ; P-GLYCOPROTEIN ; EX-VIVO ; LINEAGE ; RESISTANCE 1 GENE ; SEVERE COMBINED IMMUNODEFICIENCY ; DRUGS ; REPOPULATING CELLS ; in vivo ; progenitor cell ; VARIETIES ; MDR1 gene therapy ; myeloid differentiation ; P-GLYCOPROTEIN EXPRESSION ; peripheral blood ; TRANSDUCED CELLS
    Abstract: Background The objective of multidrug resistance-1 (MDR1) gene therapy is protection of the myeloid cell lineage. It is therefore important to examine the effect of retroviral transduction on myeloid maturation. Transfer of the human MDR1 gene can confer resistance to a variety of cytostatic drugs. For a safe application in humans it is paramount to follow-up the development of transduced cells. Methods We transduced human mobilized peripheral blood progenitor cells (PBPC) with a viral vector containing the human MDR1 cDNA and transplanted the transduced cells into non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. The progeny of the transduced cells was analyzed in detail by flow cytometry. Results A detailed analysis by four-color flow cytometry showed that MDR1 transgene-expressing CD33(+) myeloid cells were preferentially negative for the maturation-associated myeloid markers CD11b and CD10, while the untransduced CD33(+) myeloid cells expressed significantly higher proportions of these Ag (PB 〈 0.01 each). There was no difference in the expression of B- or T-lymphoid Ag among the MDR1-transduced and untransduced lymphoid cells. Discussion These data indicate that retroviral MDR1 gene transfer results in preferential P-glycoprotein expression in myeloid progenitor cells, which is the target cell population for myelotoxicity of cytostatic drugs
    Type of Publication: Journal article published
    PubMed ID: 17148033
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  • 3
    Keywords: CANCER ; CELLS ; EXPRESSION ; IN-VITRO ; proliferation ; tumor ; BLOOD ; CELL ; Germany ; DIFFERENTIATION ; MOLECULES ; RELEASE ; ACTIVATION ; DENDRITIC CELLS ; T cell ; T cells ; T-CELL ; T-CELLS ; FLOW ; BIOLOGY ; MOLECULE ; culture ; cytokines ; MATURATION ; STIMULATION ; ASSAY ; NUMBER ; METASTATIC MELANOMA ; PHENOTYPE ; VACCINE ; IMMUNOTHERAPY ; ADAPTIVE IMMUNITY ; FLOW-CYTOMETRY ; INTERFERON-GAMMA ; COLONY-STIMULATING FACTOR ; SURFACE EXPRESSION ; T-cell response ; CYTOKINE ; ELISA ; cancer vaccine ; LEVEL ; methods ; dendritic cell ; microbiology ; coagulation activation ; IL-6 ; MEDICINE ; MONOCYTES ; biotechnology ; E2 ; response ; discussion ; NORWAY ; CELL BIOLOGY ; LIGATION ; red ; CD86 ; DC maturation ; dendritic cell differentiation ; dendritic cell function ; platelet contamination ; T-HELPER-CELLS ; VITRO LIPOPOLYSACCHARIDE-CHALLENGE
    Abstract: Background Monocytapheresis has been established to collect a sufficient number of monocytes (MO) for differentiation to dendritic cells (DC) as a cancer vaccine. Platelets (Plt) are invariably found as a contaminant in the final monocytapheresis product. The aim of this study was to investigate DC differentiation under the influence of Plt with regard to their function and phenotype. Methods MO were isolated and co-cultured with autologous Plt at different MO:Plt ratios (1:1.7, 1:5, 1:15, 1:45 and 1:135) in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-12p70 release after ligation of CD40L was determined in the supernatant by enzyme-linked immunosorbent assay (ELISA). For T-cell stimulation, tetanus toxoid was added to immature DC and maturation was induced by adding cytokines (IL-1, IL-6, tumor necrosis factor- and prostaglandin E2). Stimulated T cells were analyzed for activation and proliferation as well as for intracellular cytokines by flow cytometry. Results All DC cultures were strongly positive for CD83. At a contaminating concentration of 5 Plt/MO, matured DC showed the highest expression of HLA-DR, CD80 and CD86, inducing a strong T-cell proliferation with high production of IL-4 and interferon-. The highest level of IL-12p70 production was observed by the same DC group. Discussion Plt did not negatively influence DC maturation but enhanced the expression of co-stimulatory molecules and the release of IL-12. Functionally this was reflected by a strong T-cell response that involved T-helper 1 (Th1)- as well as Th2-biased T cells. Our findings show that controlling the Plt concentration may provide important advantages for the generation of DC for use in immunotherapy
    Type of Publication: Journal article published
    PubMed ID: 18985478
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