High efficiency electroporation of human umbilical cord blood CD34(+) hematopoietic precursor cells

Human umbilical cord blood provides an alternative source of hematopoietic cells for purposes of transplantation or ex vivo genetic modification. The objective of this study was to evaluate electroporation as a means to intro duce foreign genes into human cord blood CD34(+) cells and evaluate gene ex pression in CD34(+/)CD38(dim) and committed myeloid progenitors (CD33(+), C D11b(+)). CD34(+) cells were cultured in X-VIVO 10 supplemented with thromb opoietin, stem cell factor, and Flt-3 ligand. Electroporation efficiency an d cell viability measured by flow cytometry using enhanced green fluorescen t protein (EGFP) as a reporter indicated 31% +/- 2% EGFP(+)/CD34(+) efficie ncy and 77% +/- 3% viability as determined 48 hours post-electroporation. T he addition of allogeneic cord blood plasma increased the efficiency to 44% +/- 5% with no effect on viability. Of the total CD34+ cells 48 hours post -electroporation, 20% were CD38(dim)/EGFP(+). CD34(+) cells exposed to inte rleukin-3, GM-CSF and G-CSF for an additional 11 days differentiated into C D33(+) and CD11b(+) cells, and 9% +/- 3% and 8% +/- 7% were expressing the reporter gene, respectively. We show that electroporation can be used to in troduce foreign genes into early hematopoietic stem cells (CD34(+)/CD38(dim )), and that the introduced gene is functionally expressed following expans ion into committed myeloid progenitors (CD33(+), CD11b(+)) in response to c orresponding cytokines. Further investigation is needed to determine the tr ansgene expression in functional terminal cells derived from the geneticall y modified CD34(+) cells, such as T cells and dendritic cells.