Conditional deletion of Hif-2α specifically within the hematopoietic system has no major impact on steady-state HSC maintenance and posttransplantation self-renewal. (A) Total numbers (per 2 femurs and 2 tibias) of BM B-lymphoid (CD19⁺B220⁺), myeloid (CD11b⁺Gr1⁺), and erythroid (Ter119⁺) cells and thymocytes from Hif-2α^fl/fl;Vav-iCre mice (n = 4) and control mice (Vav-iCre–negative; n = 6). Data are mean ± standard error of the mean (SEM). Analyses shown in panels A-F were performed on sex-matched 9- to 12-week-old mice. (B) Colony-forming cell (CFC) assay using M3434 media (StemCell Technologies) was performed on total BM cells from Hif-2α^fl/fl;Vav-iCre mice and control mice. Colony-forming units (CFUs) of granulocytes, erythroid cells, macrophages, and megakaryocytes (Mix); granulocytic-macrophagic CFU (GM); megakaryocytic CFU (Mk); erythroid burst-forming units (E); granulocytic CFU (G); and macrophagic CFU (M) colonies were counted and scored 10 days after initial plating (mean ± SEM; n = 6 mice per group). This CFC assay was repeated in an additional 2 independent experiments. (C-D) Total numbers of LK and LSK cells in the BM of Hif-2α^fl/fl;Vav-iCre mice (n = 4) and control mice (n = 5). Data are mean ± SEM. (E) Total numbers of HSCs and primitive progenitor cell populations from Hif-2α^fl/fl;Vav-iCre mice (n = 4) and control mice (n = 5). Data are mean ± SEM. (F) Representative dot plots indicating frequencies of HSCs (LSKCD48⁻CD150⁺ cells) and primitive progenitors (LSKCD48⁺CD150⁺ and LSKCD48⁺CD150⁻ cells) from Hif-2α^fl/fl;Vav-iCre mice (n = 4) and control mice (n = 5). Values are means ± SEM. (G) Experimental design of serial transplantation experiments. Lethally irradiated B6.SJL primary recipient mice were transplanted with 100 CD45.2⁺ HSCs from Hif-2α^fl/fl;Vav-iCre mice or control mice together with 200 000 cells from WT CD45.1⁺ unfractionated BM. LSK cells from primary recipients were then sorted 16 weeks after transplantation and transplanted into lethally irradiated secondary recipient mice. (H) Peripheral blood (PB) chimerism (percentage of CD45.2⁺ cells in PB) in primary recipients of control and Hif-2α–deficient HSCs at indicated time points after transplantation. Data are mean ± SEM (n = 5 per group). The data are representative of 3 independent transplantation experiments. (I) Frequencies of CD45.2⁺ cells in BM mononuclear cells (BM) and LK, LSK, and HSC compartments of the primary recipient mice at 16 weeks after transplantation. Data are mean ± SEM (n = 5 per group). The data are representative of 3 independent transplantation experiments. (J) FACS plots showing the percentage of CD45.2⁺ cells in LSKCD48⁻CD150⁺ HSCs, LSKCD48⁺CD150⁺ and LSKCD48⁺CD150⁻ cell fractions of the primary recipient mice. Data are mean ± SEM (n = 5 per group). The data are representative of 3 independent transplantation experiments. (K) Representative gel showing polymerase chain reaction (PCR) amplification of genomic DNA from CD45.2⁺ fraction of the BM from primary recipient mice transplanted with Hif-2α^fl/fl;Vav-iCre cells and control cells (as shown in panels G-J). Samples were taken 16 weeks after transplantation before the secondary transplantation. (L) Secondary transplantation experiment. LSK cells from primary recipients of Hif-2α–deficient and control cells were sorted 16 weeks after transplantation and transplanted (2000 cells/mouse) into lethally irradiated secondary recipient mice (together with 200 000 WT support BM cells). Chimerism was determined in PB 16 weeks after transplantation. Error bars indicate SEM (n = 6 per group). The data are representative of 2 independent transplantation experiments. (M) HSC numbers in Hif-2α^fl/fl;Vav-iCre mice and control mice after injection with 5-FU. Ten-week-old Hif-2α^fl/fl;Vav-iCre mice and control mice received a single intraperitoneal dose of 5-FU (150 mg/kg). LSKCD48⁻CD150⁺ HSC numbers were determined at indicated time points. Error bars indicate SEM (n = 3-5 mice per group per time point). Δ, excised allele; DN, CD4⁻CD8⁻ double-negative cells; DP, CD4⁺CD8⁺ double-positive cells; fl, undeleted conditional allele; WT, wild-type allele.