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  • 1
    Keywords: CELLS ; BLOOD ; CELL ; Germany ; THERAPY ; VIVO ; GENERATION ; SYSTEM ; DISEASE ; POPULATION ; TISSUE ; MARKER ; BIOLOGY ; DISCOVERY ; CELL THERAPY ; MARKERS ; STEM-CELLS ; cord blood ; EXPANSION ; STEM ; FETAL CALF SERUM ; CELL BIOLOGY ; mesenchymal stromal cells ; Genetic ; ANIMAL SERUM ; AUTOLOGOUS SERUM ; BOVINE SERUM ; clinical-scale expansion ; closed process ; CULTURE-CONDITIONS ; good manufacturing practice ; IN-VITRO DIFFERENTIATION ; mononuclear cell separation ; regenerative medicine ; Sepax ; unrestricted somatic stem cell
    Abstract: Background aims. The discovery of unrestricted somatic stem cells (USSC), a non-hematopoietic stem cell population, brought cord blood (CB) to the attention of regenerative medicine for defining more protocols for non-hematopoietic indications. We demonstrate that a reliable and reproducible method for good manufacturing practice (GMP)-conforming generation of USSC is possible that fulfils safety requirements as well as criteria for clinical applications, such as adherence of strict regulations on cell isolation and expansion. Methods. In order to maintain GMP conformity, the automated cell processing system Sepax (Biosafe) was implemented for mononucleated cell (MNC) separation from fresh CB. After USSC generation, clinical-scale expansion was achieved by multi-layered CellSTACKs (Costar/Corning). Infectious disease markers, pyrogen and endotoxin levels, immunophenotype, potency, genetic stability and sterility of the cell product were evaluated. Results. The MNC isolation and cell cultivation methods used led to safe and reproducible GMP-conforming USSC production while maintaining somatic stem cell character. Conclusions. Together with implemented in-process controls guaranteeing contamination-free products with adult stem cell character, USSC produced as suggested here may serve as a universal allogeneic stem cell source for future cell treatment and clinical settings
    Type of Publication: Journal article published
    PubMed ID: 20370349
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  • 2
    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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  • 3
    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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  • 4
  • 5
    Keywords: CELLS ; EXPRESSION ; IN-VITRO ; proliferation ; tumor ; IN-VIVO ; MODEL ; THERAPY ; DISEASE ; DISEASES ; GENE ; MICE ; TRANSPLANTATION ; BONE-MARROW ; NOD/Scid mice ; MOUSE ; MOUSE MODEL ; cord blood ; ONCOLOGY ; interaction ; BLOOD PROGENITOR CELLS ; SDF-1 ; FATE ; REPOPULATION ; mesenchymal stromal cells ; ENGRAFTMENT ; NOD/SCID/B2M(NULL) MICE
    Abstract: Abstract Background aims. Transplantation of allogeneic hematopoietic stem cells (HSC) within the framework of hematologic oncology or inherited diseases may be associated with complications such as engraftment failure and long-term pancytopenia. HSC engraftment can be improved, for example by co-transplantation with mesenchymal stem cells (MSC). Recently, a new multipotent MSC line from umbilical cord blood, unrestricted somatic stem cells (USSC), has been described. It was demonstrated that USSC significantly support proliferation of HSC in an in vitro feeder layer assay. Methods. A NOD/SCID mouse model was used to assess the effect of USSC on co-transplanted CD34(+) cells and look for the fate of transplanted USSC. The migration potential of USSC was studied in a Boyden chamber migration assay and in vivo. Quantitative real-time polymerase chain reaction (qRT-PCR) for CXCR4, CD44, LFA1, CD62L, VLA4, RAC2, VLA5A and RAC1 were performed. NMR1 nu/nu mice were used for a tumorigenicity test. Results. After 4 weeks, homing of human cells (CD45(+)) to the bone marrow of NOD/SCID mice was significantly increased in mice co-transplanted with CD34(+) cells and USSC (median 30.9%, range 7-50%) compared with the CD34(+) cell-only control group (median 5.9%, range 3-10%; P = 0.004). Homing of USSC could not be shown in the bone marrow. A cell-cell contact was not required for the graft enhancing effect of USSC. An in vivo tumorigenicity assay showed no tumorigenic potential of USSC. Conclusions. This pre-clinical study clearly shows that USSC have an enhancing effect on engraftment of human CD34(+) cells. USSC are a safe graft adjunct.
    Type of Publication: Journal article published
    PubMed ID: 20950214
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  • 6
    Keywords: THERAPY ; TRANSPLANTATION ; T-CELLS ; NK cells ; CANCER-PATIENTS ; GENE-EXPRESSION ANALYSIS ; PHASE-II ; ACUTE MYELOID-LEUKEMIA ; EX-VIVO EXPANSION ; LARGE-SCALE
    Abstract: BACKGROUND AIMS: Ex vivo expansion of natural killer (NK) cells is a strategy to produce large numbers of these effector cells for immunotherapy. However, the transfer of bench-top expansion protocols to clinically applicable methods is challenging for NK cell-based therapy because of regulatory aspects and scale-up issues. Therefore, we developed an automated, large-scale NK cell expansion process. METHODS: Enriched NK cells were expanded with interleukin-2 and irradiated clinical-grade Epstein-Barr virus-transformed lymphoblastoid feeder cells with the use of an automated system in comparison to manual expansion, and the cells were investigated for their functionality, phenotype and gene expression. RESULTS: Automated expansion resulted in a mean 850-fold expansion of NK cells by day 14, yielding 1.3 (+/-0.9) x 10(9) activated NK cells. Automatically and manually produced NK cells were comparable in target cell lysis, degranulation and production of interferon-gamma and tumor necrosis factor-alpha and had similar high levels of antibody-dependent cellular cytotoxicity against rituximab-treated leukemic cells. NK cells after automated or manual expansion showed similar gene expression and marker profiles. However, expanded NK cells differed significantly from primary NK cells including upregulation of the functional relevant molecules TRAIL and FasL and NK cell-activating receptors NKp30, NKG2D and DNAM-1. Neither automatically nor manually expanded NK cells showed reduced telomere length indicative of a conserved proliferative potential. CONCLUSIONS: We established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.
    Type of Publication: Journal article published
    PubMed ID: 25881519
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  • 7
    Abstract: BACKGROUND AIMS: Natural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications. METHODS: To enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3zeta (CAR 63.z), composite CD28-CD3zeta or CD137-CD3zeta signaling domains (CARs 63.28.z and 63.137.z). RESULTS: Exposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R gammanull mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo. CONCLUSIONS: Our data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent.
    Type of Publication: Journal article published
    PubMed ID: 27887866
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  • 8
    Keywords: EXPRESSION ; IN-VITRO ; THERAPY ; DIFFERENTIATION ; DOWN-REGULATION ; BONE-MARROW ; culture ; STEM-CELLS ; SAFETY ; LONG-TERM CULTURE ; senescence ; POTENCY ; EXPANSION ; fetal bovine serum ; HUMAN SERUM ; adipose tissue-derived mesenchymal stromal cells ; CALF SERUM ; HUMAN PLATELET LYSATE ; replicative aging
    Abstract: BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are promising candidates for innovative cell therapeutic applications. For clinical-scale manufacturing, different supplements have been evaluated as alternatives for the commonly used fetal bovine serum (FBS). We have reported previously that pooled human AB serum (HS) accelerates the proliferation of adipose tissue-derived MSC (ASC) while maintaining key functions of MSC biology such as differentiation, immune suppression and growth factor secretion. ASC expanded in FBS-supplemented culture media undergo replicative aging that is associated with a progressive loss of differentiation capacity but without indications of cellular transformation. The effects of HS media on ASC long-term culture, however, remain poorly characterized. METHODS: Long-term cultures of ASC in FBS and HS media were analyzed with respect to proliferation, marker expression, differentiation and immune suppression. RESULTS: Despite signs of an accelerated proliferation, extended life span and clonogenic capacity of ASC cultivated in HS-supplemented media, HS and FBS cultures revealed no significant differences with respect to differentiation potential and expression of senescence markers. Anchorage-independent growth, which is indicative of tumorigenic properties, was not observed in either culture conditions. Similarly, immune suppressive activities were maintained. Donor variation regarding differentiation potential and marker expression became apparent in this study independent of the culture supplement or culture duration. CONCLUSIONS: We have demonstrated that the use of pooled allogeneic HS maintains the characteristics of ASC even after long-term expansion, further demonstrating that the use of HS is an alternative to FBS.
    Type of Publication: Journal article published
    PubMed ID: 22300364
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  • 9
    Keywords: ADHERENS JUNCTIONS, ADIPOSE-TISSUE, ADULT, adult stem cells, ADULT STEM-CELLS, animal, BIOLOGY, biot
    Abstract: As an archetype of human adult stem cells that can readily be harvested, enriched and expanded in vitro, mesenchymal stromal cells (MSC) have been reported to be of significance for regenerative medicine. The literature is replete with reports on their developmental potentials in pre-clinical model systems. Different preparative protocols have been shown to yield MSC-like cell cultures or even cell lines, from starting materials as diverse as bone marrow, fat tissue, fetal cord blood and peripheral blood. However, MSC are still ill-defined by physical, phenotypic and functional properties. The quality of preparations from different laboratories varies tremendously and the cell products are notoriously heterogeneous. The source and freshness of the starting material, culture media used, presence of animal sera, cytokines, cell density, number of passages upon culture, etc., all have a significant impact on the (1) cell type components and heterogeneity of the initial population, (2) differential expansion of specific subsets, with different potentials of the end products, and (3) long-term functional fate of MSC as well as other types of progenitor cells that are co-cultivated with them. Consequently, there is an urgent need for the development of reliable reagents, common guidelines and standards for MSC preparations and of precise molecular and cellular markers to define subpopulations with diverse pathways of differentiation and divergent potentials
    Type of Publication: Journal article published
    PubMed ID: 18574765
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  • 10
    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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