Nrf2^−/− mice have abnormalities in BM and HSPC function. (A) Nrf2 and target gene expression is highest in HSPCs. Expression of the Nrf2 target genes glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and heme oxygenase 1 (HO-1) were quantified in pooled BM from 5 Nrf2^+/+ mice. Cells from the KSL, myeloid (c-Kit⁺Sca1⁻Lin⁻), and lymphoid (c-Kit^dimSca1⁺Lin⁻) progenitor compartments were isolated by FACS and quantitative RT-PCR performed in triplicate. Gene expression was normalized to β-actin and quantified relative to KSL cells. (B) Whole BM from Nrf2^−/− mice has decreased stem cell activity. Whole BM cells (666 000) from Nrf2^+/+ and Nrf2^−/− were mixed with 333 000 CD45.1 competitor normal BM cells (2:1 ratio) and injected into lethality irradiated CD45.1 hosts. Peripheral blood was analyzed for donor chimerism at the indicated time points. Data are the combined averages from 6 total mice per genotype from 2 separate experiments. *P < .05. (C) Phenotypic HSPCs from Nrf2^−/− show defective transplantation. Five hundred KSL cells from Nrf2^+/+ and Nrf2^−/− mice were mixed with 250 000 CD45.1 competitor normal BM cells and injected into lethally irradiated CD45.1 hosts. Peripheral blood was analyzed for donor chimerism at the indicated time points. Data represent the average peripheral blood engraftment from 10 total recipient mice per genotype in 2 separate experiments.*P < .05. (D) BM cellularity in Nrf2^+/+ and Nrf2^−/− mice. Average number of cells per tibia. n = 6 mice each group. *P = .019. (E) Progenitor and stem cell numbers are increased in Nrf2^−/− mice. Relative frequency of KSL, LT-HSC (CD34⁻ c-Kit⁺Sca1⁺Lin⁻), and ST-HSC (CD34⁺ c-Kit⁺Sca1⁺Lin⁻) cells. (F) HSPCs in Nrf2^−/− mice are more proliferative as shown by BrdU uptake in vivo. Mice were injected with 100 mg/kg of BrdU to mark actively proliferating cells, and BM was harvested 14 hours later and assessed for BrdU labeling by flow cytometry. Data represent the average number of BrdU-positive KSL cells for each genotype. P = .06. (G) Greater rates of spontaneous apoptosis in Nrf2^−/− HSPCs. Freshly isolated BM cells were analyzed by flow cytometry for annexin V positivity to identify apoptotic cells. Data are average values of annexin V⁺ cells as a percentage of the KSL compartment. *P < .01. (H) Myeloid progenitor frequency. Nrf2^−/− BM cells were examined by flow cytometry. Increased common myeloid progenitors (FcRγ^lowCD34⁺c-Kit⁺Sca1⁻Lin⁻) and decreased granulocyte-monocyte progenitors (FcRγ^highCD34⁺c-Kit⁺Sca1⁻Lin⁻) were observed, with a trend (P < .1) toward statistical significance. (I) Early myeloid engraftment is impaired in mice transplanted with Nrf2^−/− BM. Percentage of CD45.2⁺ cells in peripheral blood expressing myeloid lineage markers Gr1⁺/Mac1⁺ from mice transplanted with Nrf2^+/+ and Nrf2^−/− BM as in Figure 1C. *P = .007. (J) In vitro myeloid colony formation is impaired in Nrf2^−/− mice. One hundred KSL cells were plated in methylcellulose medium supplemented with cytokines, and the colonies were scored at 14 days. *P < .05