Evaluation of Cytokine Combination to Improve the Transduction of Retroviral-mediated NeoR Gene into CD34+ Cells Purified from Umbilical Cord Blood / 대한소아혈액종양학회지

ABSTRACT

PURPOSE: Retrovirs-mediated transduction of target genes into bone marrow progenitor cells or peripheral lymphocytes has been less optimal due to low efficiency and minimal expression on long-term analysis. This study aims to establish an efficient 4-day culture condition for the increased transduction efficacy into CD34+ cells selected on umbilical cord blood by comparing combination of various cytokines. METHODS: CD34+ cells from umbilical cord blood selected by Isolex-50R were incubated with supernatant containing XM5/PA317 vector for 96 hours. Cytokine combinations were used including IL-6+SCF, IL-6+IL-3+SCF, and IL-6+IL-3+SCF+TPO. Methylcellulose colony assay was done after culture. The data were expressed as mean+/-SD with 3 experiments. The efficiency of gene transfer was assessed by the ability of transduced CFU-GM to grow in the presence of G418 and PCR analysis of individual CFU-GM. RESULTS: The mean recovery rate of CD34+ cells after purification was 22%, and the purity of the final CD34+-enriched fraction was 82+/-13% (mean+/-SD). After a 4-day culture, the cell number increased 5~10 fold in each culture condition. The transduction efficiency evaluated by both G418-screened CFU-GM and PCR-positive CFU-GM with the above cytokine combinations was 46% and 64%, 41% and 57%, and 28% and 45%. However, there were no significant differences of colony counts between the cytokine combinations. CONCLUSION: We were unable to establish the best recipe of cytokine combination as the number of experiments was small and we tried only a fixed concentration of cytokines. For the future, the study of developing a novel vector, a better condition of transduction, and better combination of cytokines is warranted to attain the goal of highly effective, long-lasting method of gene transfer.