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

Gene Therapy volume 8, pages 384–390 (2001)Cite this article

Abstract

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 determind 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.

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Acknowledgements

This work was supported by United States Public Health Service grants CA81485, CA71627 (MED), and a breast cancer planning grant awarded to The University of Chicago Cancer Center CA66132.

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  1. DN Liebowitz

    Present address: Immunex Corporation, 51 University Street, Seattle, 98101-2936, WA, USA

Authors and Affiliations

  1. Department of Medicine, Section of Hematology-Oncology and Cancer Research Center, University of Chicago, 5841 S Maryland Avenue, Box MC2115, Chicago, 60637, IL, USA

    MH Wu, DN Liebowitz, SL Smith, SF Williams & ME Dolan

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Wu, M., Liebowitz, D., Smith, S. et al. Efficient expression of foreign genes in human CD34+ hematopoietic precursor cells using electroporation. Gene Ther 8, 384–390 (2001). https://doi.org/10.1038/sj.gt.3301393

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