Gene transfer in purified human hematopoietic peripheral-blood stem cells by means of electroporation without prestimulation.
Gene transfer in hematopoietic stem cells (HSCs) is an important tool, exploring regulation of the hematopoietic system and understanding the development and expansion of malignant cell clones. It is also a mandatory step for gene therapy of hematopoietic disorders. Although retroviral transduction of HSCs is effective, prestimulation of cells is generally required, also inducing differentiation of HSCs. Furthermore, the risk of viral recombination and insertional mutagenesis cannot be ruled out. Potential advantages of nonviral transfection are biosafety and easy management. However, experience in nonviral methods for transfecting peripheral-blood stem cells (PBSCs) is limited. To avoid differentiation, we evaluated the efficiency of gene transfer by means of electroporation without cytokine prestimulation. Compared with prestimulated (stem-cell factor, granulocyte-colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin-3, interleukin-6, erythropoietin, and monoclonal antibody to transforming growth factor-beta), transfection of thawed nonstimulated PBSCs was equally efficient, with a median transfection rate of 3.7%, transfection efficiency of 0.8%, and survival of 19.5% (n = 5). With freshly isolated HSCs, the rate of transfected cells could be increased to a median of 27.0% (range 8.3%-31.0%), transfection efficiency of 6.9% (range 4.5%-12.6%), and survival of 43% (range 22%-64%) (n = 5). However, the percentage of transfected cells declined with time; almost no cells were detectable by day 11. One cause for the lack of long-term expression of the heterologous gene in this system was induction of apoptosis in transgenic PBSCs, shown by up-regulation of CD95 (FAS antigen).
Weissinger
Florian
F
Medizinische Poliklinik der Julius-Maximilians-Universität, Würzburg, Germany. f.weissinger@medizin.uni-wuerzburg.de
Reimer
Peter
P
Waessa
Tanja
T
Buchhofer
Stefan
S
Schertlin
Tobias
T
Kunzmann
Volker
V
Wilhelm
Martin
M
D036541:ADP-ribosyl Cyclase / Q000032:analysis; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D017209:Apoptosis; D001781:Blood Component Removal; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002469:Cell Separation; D002470:Cell Survival; D016207:Cytokines / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics; D019014:fas Receptor / Q000235:genetics
D015703:Antigens, CD; D018952:Antigens, CD34; D016207:Cytokines; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D019014:fas Receptor; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1