High efficiency electroporation of human umbilical cord blood CD34+ hematopoietic precursor cells.
Human umbilical cord blood provides an alternative source of hematopoietic cells for purposes of transplantation or ex vivo genetic modification. The objective of this study was to evaluate electroporation as a means to introduce foreign genes into human cord blood CD34+ cells and evaluate gene expression in CD34+/CD38(dim) and committed myeloid progenitors (CD33+, CD11b+). CD34+ cells were cultured in X-VIVO 10 supplemented with thrombopoietin, stem cell factor, and Flt-3 ligand. Electroporation efficiency and cell viability measured by flow cytometry using enhanced green fluorescent protein (EGFP) as a reporter indicated 31% +/- 2% EGFP+ /CD34+ efficiency and 77% +/- 3% viability as determined 48 hours post-electroporation. The addition of allogeneic cord blood plasma increased the efficiency to 44% +/- 5% with no effect on viability. Of the total CD34+ cells 48 hours post-electroporation, 20% were CD38(dim)/EGFP+. CD34+ cells exposed to interleukin-3, GM-CSF and G-CSF for an additional 11 days differentiated into CD33+ and CD11b+ cells, and 9% +/- 3% and 8% +/- 7% were expressing the reporter gene, respectively. We show that electroporation can be used to introduce foreign genes into early hematopoietic stem cells (CD34+/CD38(dim)), and that the introduced gene is functionally expressed following expansion into committed myeloid progenitors (CD33+, CD11b+) in response to corresponding cytokines. Further investigation is needed to determine the transgene expression in functional terminal cells derived from the genetically modified CD34+ cells, such as T cells and dendritic cells.
Wu
M H
MH
Section of Hematology-Oncology, Department of Medicine and Cancer Research Center, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA.
Smith
S L
SL
Dolan
M E
ME
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000276:immunology*; D000943:Antigens, Differentiation / Q000276:immunology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D018274:Electroporation / Q000379:methods*; D005312:Fetal Blood / Q000166:cytology*; D015870:Gene Expression; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase / Q000276:immunology; D010949:Plasma / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D007377:Interleukin-3; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D049452:Green Fluorescent Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1