Constitutive Id2 expression induces neutrophil differentiation. CD34⁺ cells were transduced with eGFP or Id2 and cultured in the presence of G-CSF for 17 days. (A) The percentages of eGFP-positive cells were determined by FACS analysis after 7, 10, 14, and 17 days of differentiation. Results are expressed as an average of 3 independent experiments plus or minus SEM. The difference between the percentage of eGFP-positive cells after 17 and 7 days of differentiation is depicted in the right panel. (B) Proliferation was determined by counting the eGFP-positive cells. Results are expressed as fold increase in cell numbers compared with day 7 as an average of 3 independent experiments plus or minus SEM. (C) Proliferation was determined by performing ³H-thymidine incorporation assays. Data were depicted as a ratio between cells transduced with empty vector alone and cells transduced with Id1. Data are expressed as an average of 3 independent experiments plus or minus SEM. (D) The percentage of apoptotic, transduced cells was determined with annexin V–PE by FACS analysis after 7, 10, 14, and 17 days of differentiation. Data are expressed as an average of 3 independent experiments plus or minus SEM. (E) CD34⁺ cells were transduced with eGFP or Id2. After 14 and 17 days of neutrophil differentiation, transduced cells were separated from the nontransduced cells, and cytospins were made. Cytospins were stained with May-Grünwald-Giemsa solution. (F) Neutrophil differentiation was expressed as a percentage of maturing neutrophils within the neutrophil lineage. Data are expressed as an average of 5 independent experiments plus or minus SEM. (G) Neutrophil differentiation was also analyzed by staining the granule protein lactoferrin after 17 days of culture. Data were depicted as the ratio of the levels (arithmetric mean) of lactoferrin between cells transduced with empty vector alone or cells transduced with Id2. Data are expressed as an average of 3 independent experiments plus or minus SEM. (H) CD34⁺ cells, cultured in the presence of the cytokines SCF, FLT-3 ligand, and TPO, were transduced with empty vector alone or with Id2. Afer 3 days of culture, cells were injected into β2-microglobulin^(–/–) NOD/SCID mice. Six weeks after injection, mice were killed, eGFP-positive human myeloid cells were sorted, and cytospins were made. Cytospins were stained with May-Grünwald-Giemsa solution.