Engraftment and in vivo reconstitution of NOD-SCID animals by CD34⁺CD38⁻ CB cells is inhibited by enforced GATA-2 expression in a level-dependent manner. (A) GFP expression level after 3 days in vitro culture, showing GFP intensity and transduction efficiency of transplanted GATA-2–transduced populations. Staining for human CD45 and GFP signal are shown for 1 representative animal from each group at 4 and 8 weeks (same animal sampled at 4 weeks is shown at 8 weeks). All animals are represented in Figure S1C. (B) High GATA-2–mediated expansion defect in NOD-SCID animals. Mean proportions of engrafted GFP⁺ cells falling in the upper gate shown in panel A, with high expression of GFP, are plotted for all engrafted animals at 8 weeks (n = 4 for each transgene). Error bars indicate SEM. (C) Expression level-dependent inhibition of hematopoiesis in NOD-SCID mice is not due to alterations in homing. GFP expression level after 3 days in vitro culture and in vivo 4 weeks after intraosseous injection. Engrafted cells expressing high levels of GATA-2 are not observed at the same frequency as in the initial transplant material. Cells were injected directly into the bone marrow cavity after only 5 hours of exposure to lentivirus. Staining for human CD45 and GFP signal are shown. (D) Proportions of engrafted human GFP⁺ cells falling in the upper GFP gate shown in panel C are plotted for the populations given as a transplant (dark gray) and from all engrafted animals at 4 weeks after intraosseous injection (black; n = 2 for vector-transduced cells; n = 1 for GATA-2). (E) Similar to panel C, cells were injected directly into the bone marrow cavity after 5 hours of transduction, and engraftment was assessed 12 weeks later. (F) Similar to panel D, proportions of engrafted human GFP⁺ cells falling in the upper GFP gate shown in panel E are plotted for the transplant (dark gray) and from all engrafted animals at 12 weeks after intraosseous injection; n = 2 for both groups. (G) Relative lymphoid and myeloid reconstitution is affected by enforced GATA-2 expression. For each animal, the proportion of human CD45⁺GFP⁻ and human CD45⁺ GFP⁺ cells falling in CD19⁺ lymphoid and CD33⁺ myeloid gates are plotted; n = 4 animals in each group. Vector-transduced and GFP⁻ cells show no significant difference in lineage distribution, whereas GATA-2–transduced GFP⁺ cells read out predominantly in the myeloid lineage. (H) Ki-67 expression of engrafted GFP-positive cells sorted from the bone marrow of NOD-SCID mice. Many more vector-transduced cells express the proliferation marker Ki-67 than GATA-2–transduced cells. Plots are shown from 1 of 2 representative experiments, where 1 of 2 vector-engrafted animals is compared with a single GATA-2–engrafted animal. (I) Plots showing proportions of quiescent cells lacking expression of Ki-67 from all animals in the experiment represented in panel H.