High-efficiency electrotransfection of human primary hematopoietic stem cells.
A major obstacle to gene transfer into hematopoietic stem cells, a key step for many gene therapy and tissue replacement applications, is its low efficiency. High cell mortality is responsible for the low efficiency of electrotransfection when this technique is applied to certain 'refractory' cell types such as hematopoietic stem cells. Using human primary CD-34+ cells from peripheral blood as a model, we found that transfection-induced apoptosis and, to a lesser extent, postpulse colloidal-osmotic swelling are two main factors for the poor transfection of these cells. By applying caspase inhibitors (B-D-Fluomethyl Ketone and Z-VAD-FMK) to reduce apoptosis, and by using the postpulse pelleting method to suppress colloidal-osmotic swelling, we achieved a transfection efficiency of ~20%, regardless of the presence of cytokines in the suspension medium. This effort brings the ex vivo electrotransfection efficiency within the reach of therapeutic applications.
Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
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D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D018952:Antigens, CD34 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D061945:Caspase Inhibitors; D002470:Cell Survival; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018274:Electroporation / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009996:Osmotic Fragility; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019089:Stem Cell Factor / Q000494:pharmacology; D014162:Transfection / Q000379:methods*
D000590:Amino Acid Chloromethyl Ketones; D018952:Antigens, CD34; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D019089:Stem Cell Factor; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D049452:Green Fluorescent Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor