Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood.
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 microF, 550 V/cm, and 10 microg of DNA per 500 microl. 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.
Ołdak
Tomasz
T
Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warszawa, Poland. tomaszo@coi.waw.pl
Kruszewski
Marcin
M
Machaj
Eugeniusz Krzysztof
EK
Gajkowska
Agnieszka
A
Pojda
Zygmunt
Z
D018952:Antigens, CD34 / Q000097:blood*; D002470:Cell Survival; D018274:Electroporation / Q000379:methods*; D005312:Fetal Blood / Q000166:cytology / Q000502:physiology*; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D014162:Transfection / Q000379:methods*
D018952:Antigens, CD34; D008164:Luminescent Proteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D049452:Green Fluorescent Proteins; D013926:Thrombopoietin