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

M. H. Wu, S. L. Smith, G. H. Danet, A. M. Lin, S. F. Williams, D. N. Liebowitz, M. E. Dolan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1201-1209
Number of pages9
JournalBone Marrow Transplantation
Volume27
Issue number11
DOIs
StatePublished - 2001
Externally publishedYes

Keywords

  • CD34 cells
  • Cell cycle
  • EGFP
  • Flt-3L
  • SCF
  • TPO

ASJC Scopus subject areas

  • Hematology
  • Transplantation

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