Flow-cytometry and isolation of HSCs
Bone marrow cells were either flushed from the long bones (tibias and femurs) or isolated by crushing the long bones (tibias and femurs), pelvic bones, and vertebrae with mortar and pestle in Hank’s buffered salt solution (HBSS) without calcium and magnesium, supplemented with 2% heat-inactivated bovine serum (GIBCO, Grand Island, NY). Cells were triturated and filtered through nylon screen (45 μm, Sefar America, Kansas City, MO) or a 40μm cell strainer (Fisher Scientific, Pittsburg, PA) to obtain a single-cell suspension. For isolation of CD150+CD48−CD41−lineage−Sca-1+c-kit+ HSCs, bone marrow cells were incubated with PE-conjugated anti-CD150 (TC15-12F12.2; BioLegend), FITC-conjugated anti-CD48 (HM48-1; BioLegend), FITC-conjugated anti-CD41 (MWReg30; BD Biosciences), biotin- or APC- or PerCP-Cy5.5- conjugated anti-Sca-1 (Ly6A/E; E13-6.7), and biotin- or APC-conjugated anti-c-kit (2B8) antibody, in addition to antibodies against the following FITC-conjugated lineage markers: Ter119, B220 (6B2), Gr-1 (8C5), and CD2 (RM2-5), CD3 (KT31.1) and CD8 (53-6.7). Unless otherwise mentioned, antibodies were obtained from BioLegend, BD Biosciences, or eBioscience (San Diego, CA). Biotin-conjugated antibodies were visualized using streptavidin-conjugated APC-Cy7. HSCs were sometimes pre-enriched by selecting Sca-1+ or c-kit+ cells using paramagnetic microbeads and autoMACS (Miltenyi Biotec, Auburn, CA). Nonviable cells were excluded from sorts and analyses using the viability dye 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/ml). Apoptotic cells were identified using APC Annexin V (BD biosciences). Flow cytometry was performed with FACSAria II or FACSCanto II flow-cytometers (BD Biosciences).
For isolation of lineage−Sca-1+c-kit+ cells (LSKs) and lineage−Sca-1−c-kit+CD34+CD16/CD32+ GMPs, whole bone marrow cells were incubated with FITC conjugated anti-CD34 (RAM34) for 90 minutes on ice followed by addition of PE-conjugated monoclonal antibodies to lineage markers including B220 (6B2), CD3 (KT31.1), CD4 (GK1.5), CD8 (53-6.7), Gr-1 (8C5), Mac-1 (M1/70), and Ter119 in addition to APC-conjugated anti-Sca-1 (Ly6A/E; E13-6.7), biotin-conjugated anti-c-kit (2B8) and Alexa Fluor 700 conjugated anti-CD16/32 (93) antibodies. Biotin-conjugated c-kit staining was visualized using streptavidin APC-Cy7.
B-cells were analyzed using FITC-conjugated anti-B220, PE-conjugated anti-CD43 (S7) and APC-conjugated anti-sIgM. T-cells were analyzed using FITC-conjugated anti-CD4, PE-conjugated anti-CD8 and APC-conjugated anti-CD3. Myeloid cells were analyzed using FITC-conjugated anti-Ter119, PE-conjugated anti-Gr-1 and APC-conjugated anti-Mac-1.
To measure mitochondrial mass the HSC stain was modified to make the APC channel available for Mitotracker Deep Red staining (Molecular Probes, Eugene, OR). After antibody staining cells were incubated with 1 nM Mitotracker Deep Red and 50 μM verapamil (Sigma, St. Louis, MO) for 15 min at 37 °C followed by flow-cytometry.
To measure ROS levels the HSC stain was modified to make the FITC channel available for DCFDA staining (2′-7′-dichlorofluorescein diacetate, Molecular Probes, Eugene, OR). To do this, antibodies for HSC isolation were PE/Cy5-conjugated anti-CD150 (TC15-12F12.2; BioLegend), PE-conjugated anti-CD48 (HM48-1; BioLegend), PE-conjugated anti-CD41 (MWReg30; BD PharMingen), APC-conjugated anti-Sca-1 (Ly6A/E; E13-6.7), biotin-conjugated anti-c-kit (2B8) antibody, and PE-conjugated antibodies against lineage markers. Biotin-conjugated c-kit staining was visualized using streptavidin APC-Cy7. After antibody staining cells were incubated with 5μM DCFDA for 15 min at 37 °C followed by flow-cytometry.
To measure mitochondrial membrane potential, the HSC stain was modified to make the PE channel available for tetramethyl rhodamine methyl ester (TMRM; Molecular Probes, Eugene, OR)39
. Antibodies for HSC isolation were PE/Cy5-conjugated anti-CD150 (TC15-12F12.2; BioLegend), FITC-conjugated anti-CD48 (HM48-1; BioLegend), FITC-conjugated anti-CD41 (MWReg30; BD BioSciences), APC-conjugated anti-Sca-1 (Ly6A/E; E13-6.7), and biotin-conjugated anti-c-kit (2B8) antibody and FITC-conjugated antibodies against lineage markers. Biotin-conjugated c-kit staining was visualized using streptavidin APC-Cy7. After antibody staining cells were incubated with 25 nM TMRM for 15 min at 37 °C followed by flow-cytometry.
Cell cycle analysis
BrdU incorporation in vivo was measured by flow-cytometry using the APC BrdU Flow Kit (BD Biosciences, San Jose, CA). Mice were given an intraperitoneal injection of 1 mg of BrdU (Sigma, St. Louis, MO, St. Louis, MO) per 6 g of body mass in PBS and maintained on 1 mg/ml of BrdU in the drinking water for 24 hours. Cell cycle analysis in vitro was performed as follows. 500 HSCs were sorted into SF-O3 medium containing SCF and TPO (see below) and cultured for 3 days. BrdU (10 μM final concentration) was added for an hour before cells were cytospun to a slide. Slides were fixed with cold methanol for 5 minutes at −20 °C, then washed with PBS containing 0.01 % NP-40 and treated with 2N HCl for 20 minutes. Slides were blocked in PBS containing 4 % goat serum, 4 mg/ml BSA and 0.1% NP-40 followed by staining overnight at 4 °C with antibodies against BrdU (BU1/75, 1:100, Abcam, Cambridge, MA) and phospho-Histone H3 Serine10 (3H10, 1:2500, Millipore, Temecula, CA) diluted in blocking buffer. Primary antibody staining was developed with secondary antibodies conjugated to Alexa fluor 488 or 594 (Invitrogen, Eugene, OR) together with DAPI (2 μg/ml). Slides were analyzed on an Olympus microscope equipped with 40× objective lens.
For Ki-67/propidium iodide staining, HSCs were sorted into 70% ethanol and kept at - 20°C for at least 24 hours. Ki-67 staining was performed using a FITC Ki-67 kit (BD Biosciences), followed by staining with 50μg/ml propidium iodide (Molecular Probes, Eugene, OR) and analyzed by flow-cytometry.
Long-Term Competitive Repopulation Assay
Adult recipient mice (CD45.1) were irradiated with an Orthovoltage X-ray source delivering approximately 300 rad/min in two equal doses of 540 rad, delivered at least 2 hr apart. Cells were injected into the retro-orbital venous sinus of anesthetized recipients. Beginning 4 weeks after transplantation and continuing for at least 16 weeks, blood was obtained from the tail veins of recipient mice, subjected to ammonium-chloride potassium red cell lysis, and stained with directly conjugated antibodies to CD45.2 (104), CD45.1 (A20), B220 (6B2), Mac-1 (M1/70), CD3 (KT31.1), and Gr-1 (8C5) to monitor engraftment.
The same number of cells (25,000-35,000) from each population to be analyzed were sorted into Trichloroacetic acid (TCA) and adjusted to a final concentration of 10% TCA. Extracts were incubated on ice for 15 minutes and spun down for 10 minutes at 16.1 rcf at 4°C. The supernatant was removed and the pellets were washed with acetone twice then dried. The protein pellets were solubilized with Solubilization buffer (9 M Urea, 2% Triton X-100, 1% DTT) before adding LDS loading buffer (Invitrogen, Carlsbad, CA). Proteins were separated on a Bis-Tris polyacrylamide gel (Invitrogen) and transferred to a PVDF membrane (Millipore, Billerica, MA). Antibodies were anti-Lkb1 (#3047), anti-phospho-AMPKα (Thr172) (#2535), anti-AMPKα (#2532), anti-phospho-Acetyl-CoA Carboxylase (Ser79) (#3661), anti-phospho-S6 (#2215), anti-phospho-4EBP1 (#2855), anti-phospho-eIF4G (#2441) (all from Cell Signaling Technology, Danvers, MA) and anti-ß-actin (A1978, Sigma).
Caspase activity and ATP measurement
The same numbers of cells (approximately 5000 cells, depending on the experiment) were sorted into microcentrifuge tubes containing HBSS with 2% calf serum and pelleted. Cell pellets were lysed using Caspase-Glo2 reagent (Promega, Madison, WI) and luminescence was measured using a luminometer. Background luminescence from HBSS plus 2% calf serum was measured and the value was subtracted from sample values, then the values were divided by the cell number used to calculate the caspase activity/cell. Cellular ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche Applied Science, Indianapolis, IN). Cells were sorted in PBS and boiled in the presence of 100 mM Tris, 4 mM EDTA then luciferase reagents were added. Background was measured using buffer without cells and subtracted from the values of each cell sample. ATP level/cell was calculated by dividing the measured value with the cell number used in the assay.
Methylcellulose culture of bone marrow cells, HSCs and GMPs were performed as described36
. Primary colonies were resuspended and replated in secondary cultures and counted 14 days later.
Cell culture for analysis of mitotic spindles and chromosome numbers
HSCs were sorted into SF-O3 media (Sankyo Junyaku, Japan) supplemented with 1% Heat-inactivated fetal bovine serum, 1% Penicillin-Streptomycin-Glutamine (GIBCO, Grand Island, NY), 50 μM 2-Mercaptoethanol, 50 ng/ml SCF and 50 ng/ml TPO (both from Peprotech, Rocky Hill, NJ). Similar result were obtained by culturing in STIF medium consisting of StemSpan serum-free medium (StemCell Technologies) supplemented with 10 μg/mL heparin (Sigma, St. Louis, MO, St Louis, MO), 10 ng/mL mouse SCF, 20 ng/mL mouse TPO, 20 ng/mL mouse IGF-2 (R&D Systems, Minneapolis, MN), and 10 ng/mL human FGF-1 (Peprotech). LSKs and GMPs were cultured in STIF supplemented with 10 ng/ml IL-3 and IL-6 (both from Peprotech). Single HSCs were sorted into each well and their cell numbers were monitored as indicated. To prepare cytospins for immunostaining, 500 to 1000 cells were sorted into each well and cultured for 3 days. Annexin-V staining was performed after 3 days of culture.
For chromosome counts, LSK or GMP cells were cultured in STIF medium supplemented with 10 ng/ml IL-3 and IL-6 for 2 days then arrested in metaphase by a 2 h incubation with 100 ng/ml colcemid (KaryoMAX solution, GIBCO). Cells were treated with hypotonic solution (0.56 % KCl) for 15 minutes at 37 °C, then fixed with 3:1 methanol:glacial acetic acid and spread on a slide to prepare metaphase spreads.
Cytospin slides prepared as above without acid treatment were stained overnight at 4°C with antibodies against α-tubulin (clone YL1/2, 1:100), γ-tubulin (clone C-11, 1:100, both from Santa Cruz Biotechnology, Santa Cruz, CA) and phospho-Histone H3 Serine10 (3H10) diluted in blocking buffer. Primary antibody staining was developed with secondary antibodies conjugated to Alexa fluor 488, 555 and 647 together with DAPI (2 μg/ml). Slides were analyzed on a Leica confocal microscope.
Quantitative real-time (reverse transcription) PCR
HSCs and WBM cells were sorted into Trizol (Invitrogen) and RNA was isolated according to manufacturer’s instructions. cDNA was made with random primers and SuperScript III reverse transcriptase (Invitrogen). Quantitative PCR was performed using a SYBR Green Kit and a LightCycler 480 (Roche Applied Science). Each sample was normalized to β-actin. Primers to quantify Lkb1 cDNA levels were Lkb1 F, 5′-CACACTTTACAACATCACCA-3′, Lkb1 R, 5′-CTCATACTCCAACATCCCTC-3′, Prkaa1 F, 5′- CACCCTCACATCATCAAACTG-3′, Prkaa1 R, 5′- CTCCTCCAGAGACATATTCCA-3′, Prkaa2 F, 5′- CTTAAACTCTTTCGTCATCCTC-3′, Prkaa2 R, 5′- AACAATTCACCTCCAGACAC-3′, β-actin F, 5′-CGTCGACAACGGCTCCGGCATG-3′ and β-actin R, 5′- GGGCCTCGTCACCCACATAGGAG-3′. To quantify mitochondrial DNA copy number, cells were sorted into Trizol and DNA was isolated according to manufacturer’s instructions. Quantitative PCR was performed with primers for mtND4 (mtND4 F, 5′-ggaaccaaactgaacgccta-3′ and mtND4 R, 5′- atgagggcaattagcagtgg-3′) and β2 microglobulin intron (B2m F, 5′-tcattagggaggagccaatg-3′ and B2m R, 5′- atcccctttcgtttttgctt-3′).
PCR of genomic DNA for genotyping
To assess the degree of Lkb1 excision in genomic DNA from donor cells after transplantation, approximately 1000 donor Gr-1+ cells were sorted into alkaline lysis buffer (25 mM NaOH, 0.2 mM EDTA) and boiled, then neutralized by addition of an equal volume of neutralizing buffer (40 mM Tris-HCl). The neutralized extract was used for PCR with the following primers; R1 5′-CTGTGCTGCCTAATCTGTCG-3′, F2 5′-TTCACCATCCCTTGTGACTG-3′ and F4 5′-ATCGGAATGTGATCCAGCTT-3′. To genotype tail DNA from mice for the presence of the Lkb1fl allele primers R1 and F2 were used.