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

Kook, Hoon; Yoon, Won Sang; Nah, Jung A; Park, Anna; Hwang, Tai Ju

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.