All experiments using the mice were performed in accordance with our institutional guidelines for the use of laboratory animals and approved by the review board for animal experiments of Chiba University (approval ID: 21–150).
Generation of Mice
To generate tissue-specific Bmi1
-transgenic mice, we used the plasmid R26StopFL
, a modified version of pROSA26-1 with a loxP
-stop cassette, an frt
cassette, and a bovine polyadenylation sequence 
. We cloned a cDNA encoding a flag-tagged Bmi1
upstream of the IRES
). R1 ES cells were transfected, cultured, and selected as previously described 
. For conditional expression of Bmi1
, the RosaStopFLBmi1
mice were crossed with Tie2-Cre
mice. C57BL/6 (CD45.2) mice were purchased from Japan SLC (Shizuoka, Japan). C57BL/6 mice congenic for the Ly5 locus (CD45.1) were purchased from Sankyo-Lab Service (Tsukuba, Japan). Mice were bred and maintained in the Animal Research Facility of the Graduate School of Medicine, Chiba University in accordance with institutional guidelines. This study was approved by the institutional review committees of Chiba University (approval numbers 21–65 and 21–150).
Flow Cytometric Analysis and Cell Sorting
Mouse CD34–LSK HSCs were purified from BM of 8–12-week-old mice. Mononuclear cells were isolated on Ficoll-Paque PLUS (GE Healthcare). Cells were stained with an antibody cocktail consisting of biotinylated anti-Gr-1, Mac-1, interleukin (IL)-7Rα, B220, CD4, CD8α, and Ter119 monoclonal antibodies. The monoclonal antibodies were purchased from eBioScience or BioLegend. Lineage-positive cells were depleted with goat anti-rat IgG microbeads (Miltenyi Biotec) through an LS column (Miltenyi Biotec). Cells were further stained with Alexa Fluor® 647 or eFluor® 660-conjugated anti-CD34, phycoerythrin (PE)-conjugated anti-Sca-1, and phycoerythrin/Cy7 (PE/Cy7)-conjugated anti-c-Kit antibodies. Biotinylated antibodies were detected with allophycocyanin/Cy7 (APC/Cy7)-conjugated streptavidin. Dead cells were eliminated by staining with Propidium iodide (1 µg/ml, Sigma). Analysis and sorting were performed on a FACS Aria II (BD Bioscience).
Cell Cycle Analysis
Fresh BM cells (1×107, CD45.2) were transplanted into 8-week-old CD45.1 mice irradiated at a dose of 9.5 Gy without competitor cells. Four months later, BM mononuclear cells were isolated on Ficoll-Paque PLUS. Cells were stained with an antibody cocktail consisting of biotinylated anti-Gr-1, Mac-1, IL-7Rα, B220, CD4, CD8α, Ter119, and CD45.1 monoclonal antibodies. Cells were further stained with Alexa Fluor® 700-conjugated anti-CD34, pacific blue-conjugated anti-Sca-1, and APC-conjugated anti-c-Kit antibodies. Biotinylated antibodies were detected with APC/Cy7-conjugated streptavidin. Analysis was performed on a FACS Aria II. To analyze the cell-cycle status, cells were incubated with 1 µg/ml Pyronin Y (Sigma) at 37°C for 45 min with protection from light. Bulk sorted CD34-LSK cells were incubated in SF-O3 supplemented with 50 µM?2-β-mercaptoethanol, 0.2% BSA, 1% GPS, 50 ng/ml SCF, 50 ng/ml TPO for 10 days at 37°C in a 5% CO2 atmosphere. At day 10 of culture, the cell cycle profiles of culture cells were analyzed using an APC BrdU Flow Kit (BD Pharmingen). The cells were incubated with 10 µM BrdU at 37°C for 30 min and then stained with an antibody cocktail consisting of biotinylated anti-Gr-1, Mac-1, IL-7Rα, B220, CD4, CD8α, and Ter119 monoclonal antibodies. Cells were further stained with PE-conjugated anti-Sca-1, and PE/Cy7-conjugated anti-c-Kit antibodies. Biotinylated antibodies were detected with APC/Cy7-conjugated streptavidin. Analysis was performed on a FACS Canto II (BD Bioscience).
Colony assays were performed in methylcellulose-containing Iscove’s modified Dulbecco’s medium (Methocult M3234; Stemcell Technologies) supplemented with 20 ng/ml mouse SCF, 20 ng/ml mouse IL-3, 20 ng/ml human TPO, and 3 U/ml human EPO (Peprotech), and incubated at 37°C in a 5% CO2 atmosphere. The number of HPP- and LPP-colony-forming cells (CFCs), which generate a colony with a diameter ≥1 mm and <1 mm, respectively, were evaluated by counting colonies at day 10–14 of culture. Colonies were individually collected, cytospun onto glass slides, and subjected to Hemacolor (MERCK) staining for morphological examination. To evaluate the proliferative and differentiation capacity of Tie2-Cre;R26StopFLBmi1 HSCs in vitro, single CD34-LSK HSCs were clonally sorted into 96-microtiter plates containing 100 µl SF-O3 (Sanko Junyaku) supplemented with 50 µM 2-β-mercaptoethanol, 10% FBS, 1% L-glutamine, penicillin, streptomycin solution (GPS; Sigma), 10 ng/ml mouse SCF, 10 ng/ml human TPO, 10 ng/ml mouse IL-3, and 3 unit/ml human EPO (PeproTech). At day 14 of culture, the colonies were counted and individually collected for morphological examination. To evaluate the tolerance of test cells against oxidative stress, CD34-LSK cells were cultured in the presence of DL-Buthionin-(S,R)-sulfoximine (BSO, Sigma) or N-Acetyl-L-cysteine (NAC, Sigma) for the indicated time periods, then subjected to colony assays or flow cytometric analyses.
Serial Transplantation and CRU Assays
Fresh BM cells (5×105, CD45.2) or 10-day cultured CD34-LSK cells (CD45.2) corresponding to 20 initial CD34-LSK cells were transplanted into 8-week-old recipient mice (CD45.1) irradiated at a dose of 9.5 Gy together with 5×105 and 2×105 BM competitor cells from 8-week-old CD45.1 mice, respectively. For serial transplantation, BM cells were collected from all recipient mice at 12–20 weeks after transplantation and pooled together. Then, 5×106 BM cells were transplanted into 8-week-old B6-CD45.1 mice irradiated at a dose of 9.5 Gy without competitor cells. Third and fourth transplantation were similarly performed using 5×106 pooled BM cells. Peripheral blood (PB) cells of the recipient mice were analyzed with a mixture of antibodies that included PE/Cy7-conjugated anti-CD45.1, pacific blue-conjugated anti-CD45.2, PE-conjugated anti-Mac-1 and anti-Gr-1, APC-conjugated anti-B220, and APC/Cy7-conjugated anti-CD4 and anti-CD8α antibodies. Cells were analyzed on a FACS Canto II. Percent donor chimerism was calculated as (% donor cells) ×100/(% donor cells + % recipient cells). To obtain the competitive repopulating units (CRUs), CRU assays were performed with a limiting number of test cells and the data were analyzed using L-Calc software (StemCell Technologies). Peripheral blood cell counts were made using an automated cell counter, Celltec α (Nihon Kohden).
Bulk sorted CD34-LSK cells were incubated in SF-O3 supplemented with 50 µM?2-β-mercaptoethanol, 0.2% BSA, 1% GPS, 50 ng/ml SCF, 50 ng/ml TPO for 10 days at 37°C in a 5% CO2 atmosphere. At day 10 of culture, the cultured cells were incubated with APC-conjugated anti-Annexin V (BD Pharmingen) and propidium iodide at room temperature for 15 min with protection from light. Analysis was performed on FACS Canto II.
Immunostaining of γH2A.X
Cells were incubated in a culture medium drop on slide glasses pre-treated with poly-L-lysine (Sigma) for 2 hours. After fixation with 2% paraformaldehyde and blocking in 4% sheep serum for 30 min at room temperature, cells were incubated with purified anti-phospho-Histone H2A.X (Ser139) antibody (Cell Signaling Technology) for 12 hours at 4°C. The cells were then washed and incubated with Alexa Flour 555-conjugated anti-rabbit IgG goat polyclonal antibody (Invitrogen) for 60 min at room temperature. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were taken with a Keyence BZ-9000 fluorescence microscope.
Total RNA was isolated using TRIZOL LS solution or TRIZOL solution (Invitrogen) and reverse transcribed by the ThermoScript RT-PCR system (Invitrogen) with an oligo-dT primer. Real-time quantitative polymerase chain reaction (PCR) was performed with an ABI prism 7300 Thermal Cycler (Applied Biosystems) using FastStart Universal Probe Master (Roche). The combination of primer sequences and probe numbers are as follows: for p16Ink4a, probe #91, 5′-AATCTCCGCGAGGAAAGC-3′, and 5′-GTCTGTCTGCAGCGGACTC-3′; for p19Arf, probe #106, 5′-GGGTTTTCTTGGTGAAGTTCG-3′, 5′- TTGCCCATCATCATCACCT-3′, and for Bmi1, probe #95, 5′-AAACCAGACCACTCCTGAACA-3′ and 5′-TCTTCTTCTCTTCATCTCATTTTTGA-3′.
Total cell lysate was resolved by SDS-PAGE and transferred to a PVDF membrane. The blots were probed with a mouse anti-Bmi1 (clone 8A9, kindly provided by Dr. N. Nozaki, MAB Institute, Co. Ltd., Japan), and a horseradish peroxidase-conjugated secondary antibody. The protein bands were detected with an enhanced chemiluminescence reagent (SuperSignal, Pierce Biotechnology).
Detection of ROS
Cells were stained with an antibody cocktail consisting of biotinylated anti-Gr-1, Mac-1, IL-7Rα, B220, CD4, CD8α, and Ter119 monoclonal antibodies. Cells were further stained with PE-conjugated anti-Sca-1, and PE/Cy7-conjugated anti-c-Kit antibodies. Biotinylated antibodies were detected with APC/Cy7-conjugated streptavidin. After staining with antibodies, cells were incubated with CellROX™ Deep Red Reagent (5 µM, Invitrogen) at 37°C for 30 min with protection from light. Dead cells were eliminated by staining with propidium iodide (1 µg/ml, Sigma). Analysis was performed on a FACS Aria II.