The TRIP vector allows efficient transduction of slow-dividing multipotent CD34⁺ CB cells.

(A) EGFP expression was studied as a function of cell divisions. The results of one representative experiment (Table 1, experiment 1) out of 2 experiments are illustrated here. CD34⁺PKH26^bright cells were sorted on day 0 and transduced during 24 hours with 500 ng P24/mL TRIP viral stock. On day 1 the PKH26 fluorescence profile of the transduced CD34⁺cells (indicated by a solid line) was compared to that of the nondivided colcemid-treated CD34⁺ cells (indicated by a broken line), demonstrating that at the end of the vector exposure, more than 95% of the cells had not undergone division. A histogram profile of the PHK26 fluorescence in the cells exposed to the vector after an additional 48 hours of culture in lymphomyeloid conditions is shown (day 3). At this time point, cells have undergone between 0 and 5 divisions (gates 0 to 5). The dot-plot on the right shows EGFP expression in each of the division peaks indicated in the histogram. (B) Flow cytometry analysis of the progeny of a single TRIP-transduced CD34⁺ cell, sorted using the gate corresponding to the slow-dividing population. Briefly, slow-dividing CD34⁺cells were sorted (the sorting gate corresponds to gates 0 and 1 in the histogram in panel A) and cultured at one cell per well in culture conditions allowing the differentiation of both lymphoid (B and NK) and myeloid cells. Three weeks later, cells from clones containing at least 500 cells were labeled with mAbs directed against CD19 (B cells), CD56 (NK cells), CD15 (granulocytes), CD14 (myelomonocytic cells), and CD1a (dendritic cells). Each clone was analyzed individually, and the forward scatter and side scatter criteria (FSC/SSC) profiles were carefully monitored. Expressions of markers in cells present in the specific FSC/SSC morphological gates (gates 1 to 4) are shown.