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KMID : 0387820020090020220
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Clinical Pediatric Hematology-Oncology
2002 Volume.9 No. 2 p.220 ~ p.225
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Evaluation of Cytokine Combination to Improve the Transduction of Retroviral-mediated NeoR Gene into CD34 Cells Purified from Umbilical Cord Blood
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Kook Hoon
Yoon Won-Sang
Na Jung-A
Park An-Na
Hwang Tai-Ju
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Abstract
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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.
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KEYWORD
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Gene transfer, CD34+ cells, Umbilical cord blood, Cytokine
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