title: 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.
abstract: 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 alpha (TNF-alpha). 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.
authors.affiliation: The Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warszawa, Poland. sergiusz@coi.waw.pl
authors.forename: Sergiusz
authors.initials: S
authors.lastname: Markowicz
authors.forename: Joanna
authors.initials: J
authors.lastname: Niedzielska
authors.forename: Marcin
authors.initials: M
authors.lastname: Kruszewski
authors.forename: Tomasz
authors.initials: T
authors.lastname: Ołdak
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Gajkowska
authors.forename: Eugeniusz K
authors.initials: EK
authors.lastname: Machaj
authors.forename: Henryk
authors.initials: H
authors.lastname: Skurzak
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Pojda
mesh_terms: D018952:Antigens, CD34 / Q000096:biosynthesis; D016895:Culture Media, Serum-Free / Q000378:metabolism; D003713:Dendritic Cells / Q000166:cytology* / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism; D009369:Neoplasms / Q000628:therapy*; D019089:Stem Cell Factor / Q000378:metabolism; D014162:Transfection; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology*
chemical_list: D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D019089:Stem Cell Factor; D014409:Tumor Necrosis Factor-alpha; C084881:flt3 ligand protein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor