Efficient expression of foreign genes in human CD34(+) hematopoietic precursor cells using electroporation.
Introduction of foreign genes into human CD34(+) hematopoietic precursor cells offers a means to correct inborn errors or to protect human stem cells from chemotherapeutic damage. Electroporation is a non-chemical, nonviral, highly reproducible means to introduce foreign genes into mammalian cells that has been used primarily for rapidly dividing cells. CD34(+) cells isolated from mobilized peripheral blood of patients were cultured for 48 h in serum-free culture medium supplemented with Flt-3 ligand, stem cell factor and thrombopoietin. Cell cycle analysis showed an increase in % S-phase from 2% on day 0 to 28% on day 2 without significant loss of mean fluorescence intensity (MFI). Optimal electroporation conditions for CD34(+) cells were 550 V/cm, 38 ms, 30 microg DNA/500 microl at cell densities between 0.2 x 10(6) and 10 x 10(6) cells/ml resulting in transient EGFP gene expression in 21% (+/- 1%) of CD34(+) precursor cells, as determined by flow cytometry 48 h after electroporation. The more primitive cells were also found to be EGFP(+) as determined by subset analysis using Thy1, CD38, AC133 and c-kit conjugated monoclonal antibodies. Methylcellulose assays on electroporated CD34(+) cells yielded 20% (+/- 7%) EGFP(+) colonies (CFU-GM, BFU-E and CFU-mix) and 22% (+/- 5%) EGFP(+) long-term colony-initiating cells (LTC-IC). The reporter gene was found to be integrated into the LTC-IC genomic DNA as determined by inverse PCR and DNA sequencing. These results suggest that electroporation has the potential to effectively and stably deliver exogenous genes into human hematopoietic precursor cells.
Wu
M H
MH
Section of Hematology-Oncology and Cancer Research Center, Department of Medicine, University of Chicago, 5841 S Maryland Avenue, Box MC2115, Chicago, IL 60637, USA.
Liebowitz
D N
DN
Smith
S L
SL
Williams
S F
SF
Dolan
M E
ME
D018952:Antigens, CD34 / Q000032:analysis*; D018929:Cell Culture Techniques; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015894:Genome, Human; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology*; D006801:Humans; D014162:Transfection
D018952:Antigens, CD34