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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: APOPTOSIS ; CELLS ; EXPRESSION ; IONIZING-RADIATION ; IRRADIATION ; radiotherapy ; tumor ; CELL ; Germany ; human ; IN-VIVO ; imaging ; SUPPORT ; SYSTEM ; GENE ; PROTEIN ; PROTEINS ; DIFFERENTIATION ; NUCLEAR-MEDICINE ; TRANSDUCTION ; gene transfer ; GENE-TRANSFER ; MARKER ; INDEX ; BIOLOGY ; culture ; ASSAY ; VECTOR ; IMMUNODEFICIENT MICE ; STEM-CELLS ; OVEREXPRESSION ; MULTIDRUG-RESISTANCE ; nuclear medicine ; EFFLUX ; radiology ; RE ; INCREASE ; P-GLYCOPROTEIN ; RESISTANCE 1 GENE ; NUCLEAR ; USA ; STEM ; MEDICINE ; NOD/SCID-REPOPULATING CELLS ; MARROW-CELLS ; RETROVIRAL TRANSFER
    Abstract: Tumor radiotherapy with large-field irradiation results in an increase in apoptosis of the radiosensitive hematopoietic stem cells (CD34(+)). The aim of this study was to demonstrate the radioprotective potential of MDR1 overexpression in human CD34+ cells using a lentiviral self-inactivating vector. Transduced human undifferentiated CD34(+) cells were irradiated with 0-8 Gy and held in liquid culture under myeloid-specific maturation conditions. After 12 days, MDR1 expression was determined by the rhodamine efflux assay. The proportion of MDR1-positive cells in cells from four human donors increased with increasing radiation dose (up to a 14-fold increase at 8 Gy). Determination of expression of myeloid-specific surface marker proteins revealed that myeloid differentiation was not affected by transduction and MDR1 over-expression. Irradiation after myeloid differentiation also led to an increase of MDR1-positive cells with escalating radiation doses (e.g. 12.5-16% from 0-8 Gy). Most importantly, fractionated irradiation (3 x 2 Gy; 24-h intervals) of MDR1-transduced CD34(+) cells resulted in an increase in MDR1-positive cells (e.g. 3-8% from 0-3 x 2 Gy). Our results clearly support a radioprotective effect of lentiviral MDR1 overexpression in human CD34(+) cells. Thus enhancing repopulation by surviving stem cells may increase the radiation tolerance of the hematopoietic system, which will contribute to widening the therapeutic index in radiotherapy. (C) 2008 by Radiation Research Society
    Type of Publication: Journal article published
    PubMed ID: 18302483
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  • 3
    Keywords: CELLS ; EXPRESSION ; CELL ; COMBINATION ; Germany ; human ; MODEL ; THERAPY ; FOLLOW-UP ; QUANTIFICATION ; SITE ; GENE ; SAMPLE ; SAMPLES ; EFFICIENCY ; gene therapy ; TIME ; DNA ; primary ; VECTORS ; IMMUNODEFICIENT MICE ; leukemia ; PCR ; PROGENITOR CELLS ; sensitivity ; GENE-THERAPY ; MARROW-REPOPULATING CELLS ; MULTIDRUG-RESISTANCE ; RE ; THERAPIES ; INCREASE ; analysis ; USA ; microbiology ; quantitative ; INSERTION ; WHOLE-GENOME AMPLIFICATION
    Abstract: Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material
    Type of Publication: Journal article published
    PubMed ID: 18077708
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  • 4
    Keywords: OVEREXPRESSION ; nuclear medicine ; ONCOLOGY ; radiology ; Jun ; Germany ; human ; NUCLEAR-MEDICINE ; imaging ; NUCLEAR
    Type of Publication: Meeting abstract published
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  • 5
    Keywords: Radioprotection ; Germany ; imaging ; OVEREXPRESSION ; radiology
    Type of Publication: Meeting abstract published
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  • 6
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; BLOOD ; Germany ; human ; THERAPY ; GENE ; GENE-EXPRESSION ; GENES ; GENOME ; microarray ; RNA ; RELEASE ; PATIENT ; FLOW ; cell cycle ; CYCLE ; bone marrow ; BONE-MARROW ; TRANSPORT ; LYMPHOMA ; gene expression ; NUMBER ; REPAIR ; HUMAN GENOME ; POLYMERASE-CHAIN-REACTION ; MICROARRAY ANALYSIS ; CD34(+) CELLS ; PERIPHERAL-BLOOD ; HEMATOPOIETIC STEM-CELLS ; non-hodgkin's lymphoma ; OXYGEN ; COLONY-STIMULATING FACTOR ; MULTIPLE-MYELOMA ; INCREASED EXPRESSION ; multiple myeloma ; SUBSET ; INCREASE ; function ; MOBILIZATION ; BONE ; clinical studies ; non-Hodgkin's ; MOTILITY ; peripheral blood ; CXCR4 ; CORD
    Abstract: Objective. AMD3100 is a new CXCR4 antagonist that induces a rapid release of hematopoiefic progenitors from the bone marrow to the peripheral blood. We conducted a clinical study where patients with multiple myeloma and non-Hodgkin's lymphoma were treated with AMD3100 (A) to increase the number of peripheral blood progenitor cells (PBPCs) when given a mobilization regimen of granulocyte colony-stimulating factor (G-CSF, G). Because experimental data suggest that A+G-mobilized PBPCs are functionally different from G-mobilized PBPCs, we were interested in an intraindividual comparison of the gene expression profile of CD34(+) cells in the two different settings. Methods. To this end peripheral blood CD34(+) cells of three patients (three G, three A+G samples) were isolated by immunomagnetic followed by flow cytometric sorting to a purity of 〉 99%. Total RNA was purified. Differentially expressed genes were analyzed by using the Affymetrix GeneChip Human Genome U133 Plus2.0 and the software package Micro Array Solutions 1.3 (SAS Institute Inc.). Results. We found a pattern of unanimously higher (81 genes, log2 ratio 〉 0.5; p 〈 0.0001) or lower (29 genes, log2 ratio 〈 -0.4; p 〈 0.0001) expressed genes in the A+G-mobilized vs G-mobilized CD34+ PBPCs. Significant changes of four selected genes noted in the microarray analysis were validated by quantitative real-time polymerase chain reaction. Genes were grouped according to gene function. Only increased expression was found in the categories antiapoptosis (e.g., MPO, HSPA1B), cell cycle (e.g., MS4A3, RRM2), replication/DNA repair (e.g. MPO, HSPA1B), cell motility (e.g., TNFSF4, HMMR), and oxygen transport. Decreased expression occurred in the proapoptosis gene group (e.g., MDA5, BCL10). CXCR4 receptor gene expression itself was significantly 1.5-fold higher in the A+G vs G group. Conclusion. We conclude that A+G-mobilized CD34(+) PBPCs express significantly higher amounts of genes that potentially promote superior engraftment after myeloablative therapy than G-mobilized CD34(+) PBPCs. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc
    Type of Publication: Journal article published
    PubMed ID: 16863911
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  • 7
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; IONIZING-RADIATION ; IRRADIATION ; radiotherapy ; tumor ; Germany ; human ; THERAPY ; GENE ; GENE-EXPRESSION ; GENES ; GENOME ; PROTEIN ; PROTEINS ; TISSUE ; TUMORS ; gene therapy ; radiation ; MECHANISM ; TISSUES ; mechanisms ; treatment ; virus ; gene expression ; ASSAY ; resistance ; VECTOR ; RATES ; IMMUNODEFICIENT MICE ; CELL-LINE ; chemotherapy ; LINE ; DAMAGE ; HUMAN GENOME ; PHENOTYPE ; SAFETY ; OVEREXPRESSION ; HEMATOPOIETIC STEM-CELLS ; MULTIDRUG-RESISTANCE ; DIFFERENTIAL EXPRESSION ; INCREASED EXPRESSION ; signaling ; INCREASE ; P-GLYCOPROTEIN ; RESISTANCE 1 GENE ; CASPASE ACTIVATION ; LEVEL ; ASSAYS ; RESISTANT ; LYMPHOBLAST CELL-LINES ; MEDIATED TRANSFER ; ORAL MUCOSITIS
    Abstract: Overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, might complement chemotherapy and radiotherapy in the treatment of tumors. However, for safety and mechanistic reasons, it is important to know whether MDR1 overexpression influences the expression of other genes. Therefore, we analyzed differential gene expression in cells of the human lymphoblast cell line TK6 retrovirally transduced with MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). Sixty-one annotated genes showed a significant change in expression (P 〈 10(-4)) in MDR1-overexpressing cells compared to untransduced cells and cells transduced with a control virus expressing the neomycin phosphotransferase gene. Several genes coding for proteins involved in detoxification and exocytosis showed similar to 1.4-4-fold increases in transcript levels (e.g. ALDHIA, UNC13). Additionally, pro-apoptosis genes were down-regulated (e.g. twofold for CASP1, 2.5-fold for NALP7) with concomitant increased expression of the potential anti-apoptosis gene AKT3. In functional assays the influence of MDR1 overexpression on apoptosis signaling was further corroborated by showing reduced rates of apoptosis in response to irradiation in TK6 cells transduced with MDR1. In conclusion, the resistant phenotype of MDR1-mediated P-gp-overexpressing cells is associated with differential expression of genes coding for metabolic and apoptosis-related proteins. These results have important implications for understanding the mechanisms by which MDR1 gene therapy can protect normal tissues from radiation- or chemotherapy-induced damage during tumor treatment. (c) 2006 by Radiation Research Society
    Type of Publication: Journal article published
    PubMed ID: 16953664
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