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

The ability to culture CD34(+) stem cells, while maintaining their pluripot ency, is essential for manipulations such as gene transfection for therapeu tic trials. Human peripheral blood (PB) CD34(+) cells (greater than or equa l to 90% purity) were cultured for up to 4 days in serum-free culture mediu m supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 lig and (Flt-3L), with or without PIXY321 (IL-3 /GM-CSF fusion protein) and hum an serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide, P rior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of th e selected CD34(+) cells were in GO-GI, S-phase, or G2-M, respectively. Aft er 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the pe rcent 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-formi ng units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6, T here is an increase from day 0 to day 4 in the percentages of CD34(-) with CD38(-), HLA-DR-, and c-kit(low), but not Thy-1(+) cells. Electroporation w ith 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 efficien t transfection, Flow cytometry analysis demonstrated that 22% of the viable cells are CD34(+)/EGFP(+) 48 h post electroporation, The introduced report er gene appears to be stable as determined by EGFP(-)/LTC-IC (long-term col ony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34(+) cells.