Evaluation of a Cytokine Combination Including Thrombopoietin for Improved Transduction of a Retroviral Gene into G‐CSF‐Mobilized CD34 + Human Blood Cells

Abstract

We examined cell culture conditions with various combinations of cytokines including thrombopoietin (TPO) to obtain the most efficient transduction of recombinant retrovirus vectors into G‐CSF‐mobilized blood CD34⁺ cells which were obtained from children and purified with an Isolex 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 efficiency as determined by resistance to G418 and Neo^R polymerase chain reaction (PCR) for individual colony‐forming unit‐granulocyte/macrophage (CFU‐GM) grown in a methylcellulose culture system. The cytokines tested 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 G₂/M) was 4.6%. After a four‐day culture (n = 5), this 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 + SCF (15%). The cell number uniformly increased approximately 10‐fold in each culture condition. The average efficiency of gene transfer into incubated CD34⁺ cells with the corresponding combinations of cytokines was, respectively, 57%, 47%, and 30% for G418‐screened CFU‐GM and 72%, 68%, and 51% for polymerase chain reaction‐positive CFU‐GM. A statistically significant difference (p < 0.01) was found for G418/CFU‐GM with IL‐3 + IL‐6 + SCF (57%) versus IL‐3 + IL‐6 + SCF + TPO (30%). Hence, 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 preferentially induced the differentiation of stem cells into mature progenitors in these culture systems.