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Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation

Bone Marrow Transplantation volume 27, pages 1201–1209 (2001)Cite this article

Abstract

The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (⩾90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 ± 0.9%, 1.9 ± 0.3% and 1.0 ± 0.6% of the selected CD34+ cells were in G0–G1, S-phase, or G2–M, respectively. After 2–4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 ± 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased the percentage of CD34+ cells in S-phase to 36.3 ± 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38−, HLA-DR−, and c-kitlow, but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1–2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30–40 positive colonies (16 ± 7%) per 1 × 105 electroporated CD34+ cells. Bone Marrow Transplantation (2001) 27, 1201–1209.

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Acknowledgements

This work was supported by United States Public Health Service grants CA57725 (MED) and CA71627 (MED), and the breast cancer planning grant awarded to University of Chicago Cancer Center CA661324 (MED). We would like to thank Susan Barker and Kahadijah James from the Apheresis Facility and Kristi Hollingsworth and Janine Kobar from the Stem Cell Cryolab at the University of Chicago for providing the apheresis blood products used in these studies.

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Author notes

  1. SL Smith & DN Liebowitz

    Present address: Clinical Services-Stem Cell Department, Institute for Transfusion Medicine, 1205 N Milwaukee Ave, Glenview, IL 60025, USA

  2. SF Williams

    Present address: Hematology Oncology Associates of Illinois, 676 N St Clair, Suite 2140, Chicago, IL 60611, USA

  3. MH Wu and SL Smith: MHW and SLS contributed equally to this work

Authors and Affiliations

  1. Department of Medicine, Section of Hematology-Oncology and Cancer Research Center, University of Chicago, Chicago, IL, USA

    MH Wu, SL Smith, AM Lin, SF Williams & ME Dolan

  2. Howard Hughes Medical Institute and Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA

    GH Danet

  3. Immunex Corp., 51 University St, Seattle, WA 98101, USA

    DN Liebowitz

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Wu, M., Smith, S., Danet, G. et al. Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation. Bone Marrow Transplant 27, 1201–1209 (2001). https://doi.org/10.1038/sj.bmt.1703054

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