Efficient expression of foreign genes in human CD34(+) hematopoietic precursor cells using electroporation

Introduction of foreign genes into human CD34(+) hematopoietic precursor ce lls 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 t hat has been used primarily for rapidly dividing cells. CD34(+) cells isola ted from mobilized peripheral blood of patients were cultured for 48 h in s erum-free culture medium supplemented with Flt-3 ligand, stem cell factor a nd 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 mug DNA/500 mul at cell densities between 0.2 x 10(6) a nd 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 af ter electroporation. The more primitive cells were also found to be EGFP(+) as determined by subset analysis using Thy1, CD38, AC133 and c-kit conjuga ted monoclonal antibodies. Methylcellulose assays on electroporated CD34(+) cells yielded 20% (+/- 7%) EGFP(+) colonies (CFU-GM, BFU-E and CFU-mix) an d 22% (+/- 5%) EGFP(+) long-term colony-initiating cells (LTC-IC). The repo rter gene was found to be integrated into the LTC-IC genomic DNA as determi ned by inverse PCR and DNA sequencing. These results suggest that electropo ration has the potential to effectively and stably deliver exogenous genes into human hematopoietic precursor cells.