BACKGROUND: Hematopoietic stem cells(HSC) remain one of the most promising target cells for gene therapeutic approaches, but is currently limited by a number of issues, including the low gene transfer efficiency.
METHODS: We evaluated the effect of
recombinant fibronectin fragment (retronectin) and cytokines during retroviral transduction of CD34(+) cells and analyzed the characteristics of transduced cells. To rapidly characterize and isolate transduced cells, we used a retroviral vector coding
for the truncated form of the low-affinity none growth factor receptor (deltaLNGFR).
RESULTS:The total number of CD34(+) cells(1.5~6.3-fold) and the amount of cycling (S+G(2)/M) cell fractions(2~6-fold) were increased after cytokine prestimulation,
especially using thrombopoietin (TPO)-based cytokines. The immunophenotype of CD34(+) cells showed no significant differences according to the cytokines. However, CD34(+)AC133(+) cells were more effectively maintained in the presence of TPO. The
transduction efficiency of CD34(+) cells was significantly increased in the presence of retronectin (6.7+/-2.3% vs 23.1+/-4.4%). Immunomagnetic selection of the transduced cells allowed us to enrich these effectively(6.7+/-2.3% --> 86.3+/-6.5%).
Compared with control, deltaLNGFR transduction did not influence on the immunophenotype and cloning efficiency of CD34(+) cells. Among the deltaLNGFR(+)-derived colonies, 85.0% were shown to contain an integrated deltaLNGFR gene.
CONCLUSION: These data
show that retronectin and TPO-based cytokines can be used to facilitate retroviral transduction of CD34(+) cells. Also, deltaLNGFR transduced cells could be rapidly characterized by FACS analysis and effectively enriched by immunomagnetic selection
method. Further improvement of transduction conditions for stimulation of HSC proliferation without differentiation and development of new vectors that obviate the requirement for cell division are likely to enhance transduction of primitive HSC.

Keywords: deltaLNGFR retroviral vector, CD34(+) cells, Transduction efficiency, Recombinant fibronectin fragment (retronectin), Cytokines