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 μg DNA/500 μl at cell densities between 0.2 × 10⁶ and 10 × 10⁶ 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.