Multilineage in vitro differentiation of transduced CB CD34⁺ cells. (A) In vitro differentiation of CB CD34⁺ cells transduced with TPO-, SCF-, or TPO/SCF-displaying HIV-derived vectors for 72 hours at an MOI of 20. Control incubations with VSV-G–displaying HIV vectors in the absence (–) or presence of recombinant cytokines (rTPO = 10 ng/mL; rSCF = 50 ng/mL; Flk-3 = 100 ng/mL) for 72 hours were performed. The obtained cell lineages are the results of a 2-week in vitro lymphoid culture in Iscove medium on MS5 cells. In this culture, the total number of transduced B cells (CD19⁺ cells) and residual progenitors (CD34⁺ cells) for 3 independent experiments are shown. The absolute number of transduced differentiated cells is calculated, for example, for B cells as percent CD19 in total population × percent transduction × expansion of the cells after culture. The number of transduced granulo-monocytes in a lymphoid/myeloid culture on MS5 in the presence of FCS, IL-2, IL-15, and SCF is shown for 2 independent experiments. (B) In vitro differentiation of CB CD34⁺ cells transduced for 72 hours with TPO-displaying LVs or VSV-G–displaying vectors in the presence of rTPO (10 ng/mL). Differentiation was performed in 2 steps: (1) lymphoid/myeloid culture in presence of FCS, SCF, IL-15, and IL-2 on MS5 cells for 15 days, followed by (2) culture in the presence of Epo and IL-3 for 5 days. After step 1 of culture, differentiation into natural killer cells (CD56⁺) and B cells (CD19⁺) is shown. After step 2 of culture, maturation into erythrocytes (GPA⁺) and residual progenitors (CD34⁺) are indicated. For each cell lineage the GFP⁺ cells are indicated in the upper right quadrant (data presented are representative of 3 different experiments).