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

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

“…[39][40][41] Nonviral gene transfer is also considered to be less efficient in nondividing cells 42 and prestimulation of CD34 + cells for 48 h has been found to enhance transfection with a conventional plasmid by electroporation. 31 Thus, the apparent lack of requirement of prestimulation for efficient transfection with pEPI-eGFP provides this vector with a strong advantage, since it offers the possibility to transfect freshly isolated CD34 + cells without the need for prolonged ex vivo culture. In addition to that, we observed no survival deficit and no impairment of clonogenic capacity of transfected hematopoietic progenitors, which suggests that no toxic effect is exerted on cell viability by either the electroporation procedure or the plasmid DNA itself (data not shown).…”

“…Primary human fibroblast-like cells, pEPI-eGFP is detected in semisolid colonies derived from transfected CD34 + cells Next, we transfected CD34 + -enriched cells isolated from umbilical cord blood using electroporation. [28][29][30][31][32] Transfection efficiency was between 10 and 30% (mean: 18.478%, n ¼ 5), as estimated by flow cytometric evaluation of the percentage of eGFP-expressing CD34 + cells. Cell survival was in all cases above 60% (mean: 66.876%, n ¼ 5).…”

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

“…6 Immediately after electroporation, the CD34 + cells were resuspended in cell culture medium containing TPO/SCF/Flt-3L with or without IL-3, PIXY321, CM, and serum in 24-well plates at 1 × 10 6 cells/well. The cells were incubated at 37°C and 5% CO 2 for an additional 48 h prior to transfection efficiency and viability analyses by flow cytometry.…”

“…Previous studies have demonstrated that electroporation can be a reasonable means of hematopoietic cell gene transfer, and the genes delivered are integrated into the host chromosome. [6][7][8][9][10] Although cell synchronization and electroporation studies have led to controversial conclusions on the requirement for S-phase, [11][12][13] it appears that efficient electroporation of hematopoietic cells requires prestimulation, which results in an apparent increase in the S-phase sub-population. 6,14,15 Ideal culture conditions would promote a high efficiency of gene transfer to ensure not only short-term, but also long-term engraftment and subsequent bone marrow repopulation with each hematopoietic lineage carrying the transgene.…”

“…[6][7][8][9][10] Although cell synchronization and electroporation studies have led to controversial conclusions on the requirement for S-phase, [11][12][13] it appears that efficient electroporation of hematopoietic cells requires prestimulation, which results in an apparent increase in the S-phase sub-population. 6,14,15 Ideal culture conditions would promote a high efficiency of gene transfer to ensure not only short-term, but also long-term engraftment and subsequent bone marrow repopulation with each hematopoietic lineage carrying the transgene. In vitro manipulation of human stem cells for gene transfer by electroporation involves three primary goals: (1) optimization of short-term culture conditions that will increase the percentage of cells in S-phase and enhance successful incorporation of exogenous gene delivered; (2) maintenance of the pluripotent stem cells for full bone marrow engraftment and long-term reconstitution after transplant; and (3) optimization of the electroporation conditions for efficient transfection.…”