LTC-ICs indicate long-term culture-initiating cells; CB, cord blood; EGFP, enhanced green fluorescent protein.

In each experiment, 60 slow-dividing cells were sorted and cultured at one cell per well. Cultures were terminated after 5 weeks, and the contents of each well were individually plated in clonogenic assays. Out of 60 input cells, 9 cells (15%) in experiment 1 and 16 cells (27%) in experiment 2 were retrospectively identified as LTC-ICs.

One hundred twenty slow-dividing cells were sorted and cultured at one cell per well. In experiments 1 and 2, 61 and 67 clones, respectively, contained enough cells (more than 500 cells) after 2 to 4 weeks to allow phenotyping by flow cytometry. In experiments 1 and 2, 29 of 61 clones and 37 of 67 clones, respectively, contained only cells from one lineage defining monopotent progenitors (CD19⁺ B, CD56⁺ NK, or CD14⁺ CD15⁺ myeloid cells). Similarly, 14 of 61 input cells in experiment 1 and 14 of 67 input cells in experiment 2 were bipotent progenitors (B+NK, B+M, and NK+M), and 18 (29.5%) of 61 input cells and 16 (24%) of 67 input cells, respectively, were tripotent cells (B+NK+M). These proportions are similar to those we have previously observed with unmanipulated CD34⁺ CB cells.19 

Transduction efficiency was evaluated as the percentage of slow-dividing CD34⁺ cells expressing EGFP 48 hours after exposure to TRIP vector particles (Figure 4).