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  • 11
    Abstract: BACKGROUND AND AIMS: Because of their pluripotency, human CD34(+) peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC. METHODS: A panel of pseudotyped AAV vectors (designated AAV2/x, containing the vector genome of serotype 2 and capsid of serotype x, AAV2/1-AAV2/6) was screened on primary human granulocyte-colony-stimulating factor (G-CSF)-mobilized CD34(+) PBPC to determine their gene transfer efficacy. Additionally, double-stranded self-complementary AAV (dsAAV) were used to determine possible second-strand synthesis limitations. RESULTS: AAV2/6 vectors proved to be the most efficient [12.8% (1.8-25.4%) transgene-expressing PBPC after a single transduction], being significantly more efficient (all P〈0.005) than the other vectors [AAV2/2, 2.0% (0.2-7.3%); AAV2/1, 1.3% (0.1-2.9%); others, 〈; 1% transgene-expressing PBPC]. In addition, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using pseudotyped dsAAV vectors: for dsAAV2/2 [9.3% (8.3-20.3%); P〈0.001] and dsAAV2/6 [37.7% (23.6-61.0%); P〈0.001) significantly more PBPC expressed the transgene compared with their single-stranded counterparts; for dsAAV2/1, no significant increase could be observed. CONCLUSIONS: We have shown that clinically relevant transduction efficiency levels using AAV-based vectors in human CD34(+) PBPC are feasible, thereby offering an efficient alternative vector system for gene transfer into this important target cell population.
    Type of Publication: Journal article published
    PubMed ID: 19929455
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  • 12
    Keywords: IN-VITRO ; GENE-EXPRESSION ; DIFFERENTIATION ; bone marrow ; ADHESION ; STEM-CELLS ; cord blood ; BONE-MARROW-CELLS ; adipose tissue ; EX-VIVO EXPANSION ; mesenchymal stromal cells ; OSTEOBLASTIC DIFFERENTIATION ; OSTEOGENIC DIFFERENTIATION ; impedance monitoring ; MATRIX-MEDIATED RETENTION
    Abstract: Background aims. For their wide mesodermal differentiation potential, mesenchymal stromal/stem cells (MSC) are attractive candidates for tissue engineering. However, standardized quality control assays monitoring differentiation that are non-invasive and continuous over time are lacking. Methods. We employed a non-invasive assay, using two different systems, to discriminate osteogenic and adipogenic differentiation of MSC by monitoring impedance. Fibroblasts and keratinocytes served as nonspecific controls. Impedance profiles were recorded comparing MSC from bone marrow and adipose tissue, either non-induced or induced for osteogenesis or adipogenesis, for 5-14 days, and correlated with differentiation markers assessed by reverse transcription-quantitative polymerase chain reaction and Western blot. Additionally, differentiation modulating effects of extracellular matrix components were analyzed. Results. Adhesion and growth-related impedance profiles of non-induced MSC roughly resembled those of fibroblasts, whereas keratinocytes differed significantly. Distinct from that, osteogenic induction of MSC revealed initially rapid and continuously rising impedance, corresponding to mineralized calcium matrix formation. Conversely, adipogenic induction caused shallower initial slopes and eventually declining profiles, corresponding to more compact, adipocyte-like cells with numerous lipid vacuoles. Pre-coating with either collagen type I or IV apparently favored osteogenesis and fibronectin adipogenesis. Impedance recordings correlated well with the extent of differentiation evaluated by histochemical staining and protein and gene expression. Conclusions. Overall, our data demonstrate that impedance profiling offers a basis for standardized real-time, non-invasive high-throughput screening of MSC properties. It enables further testing of the influence of diffusible factors or extracellular matrix composites on MSC differentiation or maintenance of stemness, thus substantiating therapeutic application
    Type of Publication: Journal article published
    PubMed ID: 21619493
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