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Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood

100%

Ołdak T., Kruszewski M., Machaj E. K., Gajkowska A., Pojda Z.

2002

vol. 49

issue 3

625-632

Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34+ hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34+ hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34+ cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 μF, 550 V/cm, and 10 μg of DNA per 500 μl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34+ hematopoietic cells derived from human umbilical cord blood.

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy

73%

Markowicz S., Niedzielska J., Kruszewski M., Ołdak T., Gajkowska A., Machaj E. K., Skurzak H., Pojda Z.

2006

vol. 53

issue 1

203-212

Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR+ cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34+ cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor α (TNF-α). Transfection of CD34+ cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.

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2 Acta Biochimica Polonica

1 2006

1 2002

2 Gajkowska A.

2 Kruszewski M.

2 Machaj E. K.

2 Ołdak T.

2 Pojda Z.

1 Markowicz S.

1 Niedzielska J.

1 Skurzak H.

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Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy