Relative amplification of total stem cells and human cell engraftment into NOD/SCID mice. (A) After 1 month of cell culture, CD34⁺ cells transduced with SALL4A or SALL4B had 1780- and 1463-fold increases, respectively, relative to control cells. Values are means + SD. (B) Furthermore, SALL4-transduced cells showed 9.32-fold increases for SALL4A and 8.88-fold increases for SALL4B versus controls for the total number of LTC-ICs after 1 month. Values are means + SD. (C) Overall, SALL4A-transduced cells had a total fold CD34⁺/CD38⁻ stem cell expansion 16 776 over control, whereas SALL4B-transduced cells showed 13 320-fold increases. Values are means + SD. (D) Representative flow cytometry analysis 4 weeks after injection for CD45⁺ human leukocytes from peripheral blood of NOD/SCID recipients transplanted with SALL4A- or SALL4B-transduced HSCs. (E) Representative flow cytometry profile 4 weeks after injection of a mouse exhibiting multilineage repopulation of human cells by engrafted cells. Although the negative control animal showed no engraftment of human cells, the experimental animal showed both CD15⁺ myeloid and CD19⁺ lymphoid human cell engraftment. (F) Flow analysis of secondary and tertiary bone marrow transplant NOD/SCID mice. The animals were positive for CD45⁺ cells in both secondary (2.74%) and tertiary (3.29%) transplants (left panel). The threshold bars for positive and negative populations were set according to independent staining controls. When the CD45⁺ population in the tertiary transplant was analyzed further for specific lineages, CD33 myeloid and CD19/CD3 lymphoid cells were positively measured (right panel). (G) Amount of human engraftment in the peripheral blood of NOD/SCID mice transplanted with 20 000 (SALL4A, ■; SALL4B, ▴, or GFP, ♦) or 40 000 (SALL4A, □; SALL4B, ▵; or GFP, ◊) initial human CD34⁺ cells. (H) Limiting dilution analysis of CD34⁺ bone marrow cells injected into NOD/SCID mice (n = 72) after lentiviral transfection with SALL4A (□), SALL4B (▴), or GFP (○).