title: High efficiency electroporation of human umbilical cord blood CD34+ hematopoietic precursor cells.
abstract: 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.
authors.affiliation: Section of Hematology-Oncology, Department of Medicine and Cancer Research Center, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA.
authors.forename: M H
authors.initials: MH
authors.lastname: Wu
authors.forename: S L
authors.initials: SL
authors.lastname: Smith
authors.forename: M E
authors.initials: ME
authors.lastname: Dolan
mesh_terms: 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
chemical_list: 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