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  • 1
    Keywords: CELLS ; EXPRESSION ; CELL ; COMBINATION ; Germany ; human ; MODEL ; MODELS ; THERAPY ; SYSTEM ; GENE ; PROTEIN ; PROTEINS ; EFFICIENCY ; gene therapy ; TRANSDUCTION ; GENE-TRANSFER ; INFECTION ; fibroblasts ; GLYCOPROTEIN ; PARTICLES ; virus ; VECTOR ; leukemia ; EFFICIENT ; HEMATOPOIETIC-CELLS ; GENE-THERAPY ; HEMATOPOIETIC STEM-CELLS ; GUIDELINES ; LEADS ; TRANSFECTION ; LENTIVIRAL VECTOR ; LEVEL ; methods ; PROTEIN-A ; ENVELOPE ; HUMAN-CELLS ; TROPISM ; pseudotyping ; lentiviral vectors
    Abstract: Objective. Lentiviral vectors are increasingly used for preclinical models of gene therapy and other forms of experimental transgenesis. Due to the broad tropism and the ability for concentration by ultracentrifugation, most lentiviral vector preparations are produced using the vesicular stomatitis virus glycoprotein (VSV-g) protein as envelope. Recently, Hanawa and colleagues have demonstrated that the ecotropic envelope protein of murine leukemia viruses allows efficient pseudotyping of HIV-1-derived vector particles. However, this method has found little acceptance, despite potential advantages. Materials and Methods. We produced lentiviral vectors pseudotyped with murine ecotropic envelope using a four-plasmid transient transfection system and evaluated their performance in murine fibroblasts and hematopoietic cells. Results. Titers of lentiviral "ecotropic" supernatants were only slightly lower than those produced with VSV-g, could be concentrated by overnight centrifugation (13,000g), and efficiently transduced murine fibroblasts and hematopoietic cells but not human cells. Our Institutional Biosafety Committee agreed on the production and use of replication-defective lentiviral vectors pseudotyped with murine ecotropic envelope under biosafety level 1 (BL1) conditions with additional BL2 practices. We also obtained useful guidelines for the work with human infectious lentiviral vectors. Conclusions. For the researcher, "ecotropic" lentiviral vectors significantly improve the convenience of daily work, compared to the conditions required for lentiviral pseudotypes that are capable of infecting human cells. High efficiency and superior biosafety in combination with convenient handling will certainly boost the potential applicability of this important vector system. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc
    Type of Publication: Journal article published
    PubMed ID: 16647564
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  • 2
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    Abstract: Hematopoietic stem cells (HSCs) sustain lifelong blood cell regeneration by balancing their ability for self-renewal with their ability to differentiate into all blood cell types. To prevent organ exhaustion and malignant transformation, long-lived HSCs, in particular, must be protected from exogenous and endogenous stress, which cause severe DNA damage. When DNA is damaged, distinct DNA repair mechanisms and cell fate controls occur in adult HSCs compared with committed cells. Growth arrest and DNA damage-inducible 45 alpha (GADD45A) is known to coordinate a variety of cellular stress responses, indicating the molecule is an important stress mediator. So far, the function of GADD45A in hematopoietic stem and progenitor cells is controversial and appears highly dependent on the cell type and stress stimulus. Recent studies have analyzed its role in cell fate decision control of prospectively isolated HSCs and have revealed unexpected functions of GADD45A, as discussed here. The upregulation of GADD45A by DNA damage-causing conditions results in enhanced HSC differentiation, probably to efficiently eliminate aberrant HSCs from the system. These findings, in concert with a few studies on other stem cell systems, have led us to propose DNA damage-induced differentiation as a novel DNA damage response mechanism in stem cells that circumvents the fatal consequences of cumulative DNA damage in the stem cell compartment.
    Type of Publication: Journal article published
    PubMed ID: 27262218
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  • 5
    Keywords: RECEPTOR ; IN-VITRO ; PROTEIN ; IDENTIFICATION ; LYSOPHOSPHATIDIC ACID ; INTERNAL TANDEM DUPLICATION ; MOTILITY ; KINASE DOMAIN MUTATIONS ; PROBE LEVEL DATA ; LYSOPHOSPHOLIPASE-D ACTIVITY
    Abstract: Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D by converting lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which signals via G-protein-coupled receptors and has important functions in cell migration and proliferation. This study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant downregulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression, and induced chemotaxis in all tested human leukemic cell lines and human CD34(+) progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80%, demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.
    Type of Publication: Journal article published
    PubMed ID: 23377000
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  • 6
    Abstract: The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has recently been "repurposed" to efficiently mutate mammalian genomes in a sequence-specific manner. The application of this genome-editing technology to hematology has afforded new approaches for functional genomics and even the prospect of "correcting" dysfunctional HSCs in the treatment of serious genetic hematological diseases. In this Perspective, we provide an overview of three recent CRISPR/Cas9 methods in hematology: gene disruption, gene targeting, and saturating mutagenesis. We also summarize the technical considerations and advice provided during the May 2017 International Society of Experimental Hematology New Investigator Committee webinar on the same topic.
    Type of Publication: Journal article published
    PubMed ID: 28757433
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  • 7
    Abstract: Integration-deficient lentiviruses (IdLVs) deliver genes effectively to tissues but are lost rapidly from dividing cells. This property can be harnessed to express transgenes transiently to manipulate cell biology. Here, we demonstrate the utility of short-term gene expression to improve functional potency of hematopoietic stem and progenitor cells (HSPCs) during transplantation by delivering HOXB4 and Angptl3 using IdLVs to enhance the engraftment of HSPCs. Constitutive overexpression of either of these genes is likely to be undesirable, but the transient nature of IdLVs reduces this risk and those associated with unsolicited gene expression in daughter cells. Transient expression led to increased multilineage hematopoietic engraftment in in vivo competitive repopulation assays without the side effects reported in constitutive overexpression models. Adult stem cell fate has not been programmed previously using IdLVs, but we demonstrate that these transient gene expression tools can produce clinically relevant alterations or be applied to investigate basic biology.
    Type of Publication: Journal article published
    PubMed ID: 28911908
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  • 8
    Keywords: THERAPY ; ACTIVATION ; REPLICATION ; insertional mutagenesis ; HUMAN-IMMUNODEFICIENCY-VIRUS ; INTEGRATION ; REPOPULATING CELLS ; MARKING ; SCID-X1 ; SITE ANALYSIS
    Abstract: Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat-driven gamma-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus-long terminal repeat-driven gamma-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of linear amplification-mediated PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for linear amplification-mediated PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with gamma-retroviral long terminal repeat vectors.
    Type of Publication: Journal article published
    PubMed ID: 22989760
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  • 9
    Keywords: PEPTIDE ; CELLS ; tumor ; BLOOD ; CELL ; Germany ; human ; PHASE-I ; NEW-YORK ; GENE ; EFFICIENCY ; LINES ; TIME ; TRANSDUCTION ; gene transfer ; GENE-TRANSFER ; primary ; MR ; CELL-LINES ; SUSCEPTIBILITY ; TARGET ; virus ; MUTANT ; VECTORS ; DIFFERENCE ; VECTOR ; CELL-LINE ; leukemia ; EFFICIENT ; STEM-CELLS ; PROGENITOR CELLS ; specificity ; HEMATOPOIETIC PROGENITOR CELLS ; PERIPHERAL-BLOOD ; RECOMBINANT ADENOASSOCIATED VIRUS ; VIRAL VECTORS ; adeno-associated virus ; AAV ; TUMOR-CELL-LINES ; TRANSDUCTION EFFICIENCY ; cord blood ; RECOMBINANT ; RE ; INCREASE ; LENTIVIRAL VECTOR ; stem cells ; methods ; progenitor ; MUTANTS ; USA ; MOBILIZED PERIPHERAL-BLOOD ; STEM ; VIRUS VECTORS ; viral ; MEDICINE ; transfer efficiency ; serotype ; 2-MEDIATED TRANSDUCTION ; BETA-GLOBIN GENE ; MILD LUNG-DISEASE ; THALASSEMIC MICE
    Abstract: Objective. Currently standard recombinant adeno-associated virus serotype 2(rAAV2)-based vectors lack the efficiency for gene transfer into primary human CD34(+) peripheral blood progenitor cells (PBPC). Materials and Methods. An advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34(+) PBPC cells. Results. On a panel of leukemia cell lines (CML/AML), significantly higher gene transfer efficiency of the rAAV capsid mutants (up to 100% gene transfer) was observed compared to standard rAAV2 vectors. A higher transduction efficiency in the imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S and a pronounced difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S were particularly striking. On solid tumor cell lines, on the other hand, rAAV2 was more efficient than the capsid mutants, suggesting an increased specificity of our capsid mutants for hematopoietic progenitor cells. On primary human CD34(+) PBPC significantly higher (up to eightfold; 16% green fluorescent protein-positive) gene transfer could be obtained with the newly generated vectors compared to standard rAAV2 vectors. Conclusion. These novel vectors may enable efficient gene transfer using rAAV-based vectors into primary human blood progenitor cells for a future clinical application. (c) 2008 ISEH Society for Hematology and Stem Cells. Published by Elsevier Inc
    Type of Publication: Journal article published
    PubMed ID: 18495326
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  • 10
    Keywords: EXPRESSION ; IN-VITRO ; SURVIVAL ; IN-VIVO ; GENE-EXPRESSION ; BONE-MARROW ; CD34(+) CELLS ; HEMATOPOIETIC PROGENITOR CELLS ; SCID MICE ; ACUTE MYELOID-LEUKEMIA ; CANCER STEM-CELLS ; ALDEHYDE DEHYDROGENASE-ACTIVITY ; COMBINED IMMUNODEFICIENT MICE ; CORD-BLOOD
    Abstract: Objective. Leukemia-initiating cells can retrospectively be defined by tumorigenicity in immunodeficient mice and be characterized by surface markers. The latter still being discussed for acute myeloid leukemia (AML), nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were used to evaluate long-time reconstitution and expansion of AML subpopulations. Materials and Methods. Bone marrow cells from patients with AML were separated according to CD34 expression, aldehyde dehydrogenase (ALDH) activity, and divisional kinetics in comparison to cord blood - derived CD34(+) hematopoietic stem cells, evaluating survival and expansion in NOD/SCID mice. The AML long-term surviving capacity of subpopulations recovered from NOD/SCID mice was confirmed by ex vivo survival. Results. AML mononuclear cells were detected in bone marrow and spleen of NOD/SCID mice 12 weeks after transplantation. The majority of recovered cells were CD34(+) and significantly more CD34(+) cells were recovered after application of ALDH(bright) (high ALDH activity), CD34(+), or slowly dividing (PKHbright) than after ALDH(dim), CD34(-), or fast dividing (PKHdim) cell application. CD123(+), CD63(+), and CD44v7(+) cells were also more abundant after the transfer of ALDH(bright) or CD34(+) AML mononuclear cells. In the spleen, large AML cell clusters were only recovered after ALDH(bright), CD34(+), or PKHbright cell transfer. Importantly, in secondary long-term in vitro cultures, quite exclusively CD34(+) AML mononuclear cells survived and expanded. Conclusions. Separation of ALDH(bright), CD34(+), or PKHbright cells enriches for AML long-term surviving capacity, which reside in the CD34(+) subpopulation, as rather exclusively CD34(+) cells survived and expanded in vivo and ex vivo. Long-term survival capacity may be supported by CD44v7 expression.
    Type of Publication: Journal article published
    PubMed ID: 21087653
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