EVALUATION OF A CYTOKINE COMBINATION INCLUDING THROMBOPOIETIN FOR IMPROVED TRANSDUCTION OF A RETROVIRAL GENE INTO G-CSF-MOBILIZED CD34(-CELLS() HUMAN BLOOD)

We examined cell culture conditions with various combinations of cytok ines including thrombopoietin (TPO) to obtain the most efficient trans duction of recombinant retrovirus vectors into G-CSF-mobilized blood C D34(+) cells which were obtained from children and purified with an Is olex 50 system (Baxter; Deerfield, IL), Three different 4-day culture conditions for the stimulation of CD34(+) cells were compared in terms of a cell-cycle analysis by fluorometry and gene transduction efficie ncy as determined by resistance to G418 and Neo(R) polymerase chain re action (PCR) for individual colony-forming unit-granulocyte/macrophage (CFU-GM) grown in a methylcellulose culture system, The cytokines tes ted were: A) interleukin (IL)-6 + stem cell factor (SCF); B) IL-3 + IL -6 + SCF, and C) IL-3 + IL-6 + SCF + TPO, Without a cell culture, the percentage of CD34(+) cells in the cell cycle (the percentage of cells in phases S and G(2)/M) was 4.6%, After a four-day culture (n = 5), t his value increased with the addition of IL-3 (22%) or IL-3 + TPO (27% ,p < 0.05) as compared to that with the baseline cocktail of IL-6 + SC F (15%), The cell number uniformly increased approximately 10-fold in each culture condition, The average efficiency of gene transfer into i ncubated CD34(+) cells with the corresponding combinations of cytokine s was, respectively, 57%, 47%, and 30% for G418-screened CFU-GM and 72 %, 68%, and 51% for polymerase chain reaction-positive CFU-GM, A stati stically significant difference (p < 0.01) was found for G418/CFU-GM w ith IL-3 + IL-6 + SCF (57%) versus IL-3 + IL-6 + SCF + TPO (30%), Henc e, it is likely that the increased cell proliferation produced by the addition of TPO was not necessarily translated into an increased rate of retroviral-mediated gene transduction, possibly because TPO prefere ntially induced the differentiation of stem cells into mature progenit ors in these culture systems.