Bone marrow cells were plated in methylcellulose medium (MethoCult3434; Stem Cell Technologies) at a density of 20,000 cells/ml media according to the manufacturer’s instructions. Differential colony counts were scored 7–10 days after plating. For the serial replating assay, SmoNull or SmoWT bone marrow was retrovirally transduced with MLL-AF9 MSCV-GFP as previously described. Whole bone marrow cells were then plated at a concentration of 20,000 cells/mL. Colonies were counted after 7 days, removed from the plate by dilution with RPMI containing 10% FBS and 1% penicillin/streptomycin, washed in PBS with 2% FBS, and replated onto a new plate for up to five rounds.
For the pharmacological experiments, hematopoietic colony assays were carried out in complete methylcellulose medium (MethoCult GF, Stem Cell Technologies) according to the instructions of the manufacturer in the presence of vehicle, rSHH (final concentration 100 ng/ml), and/or the Hedgehog pathway antagonist HhAntag (final concentration 300 nM). Colonies were scored on day 8 after plating, and the experiment was carried out in triplicate.
Cell Staining and Flow Cytometry Analysis
The contribution of definitive hematopoietic cells of bone marrow, spleen, and peripheral blood was verified by staining with monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE), or peridinin-chlorophyll-protein complex (perCP), all from BD Biosciences. The following antibodies were used: CD45, CD3, B220, CD4, CD8, Gr-1, Mac-1, c-kit, CD34, CD41, Ter119, CD45.1, and CD45.2. Surface marker expression was visualized with a FACSCalibur (Becton Dickinson) and was analyzed with FlowJo software. Stem and progenitor cell population analysis was done by lineage depletion of whole bone marrow cells using unconjugated murine lineage antibodies CD3, CD4, CD8, Gr-1, B220/CD45R, CD19, IL-7R, or Ter119, which were labeled using goat anti-rat PECy5. The following antibodies were used for the LSK compartment (containing long-term and short-term HSCs) and progenitor staining: CD34, FcRgII, c-kit, and Sca-1. LSK cells were defined by Sca-1+ and c-kit+ expression. Progenitor cells were defined by CD34+, FcRgII dim (CMP), CD34 dim, FcRgII dim (MEP), and CD34+, FcRgII+(GMP). Flow analysis for LSK or progenitor cells populations was done by using FACSAria (Becton Dickinson) and analyzed as above. For cell-cycle analysis, Pyronin Y and Hoescht staining were used as described elsewhere (Cheng et al., 2000
Smoothened conditional knockout mice (Smotm2Amc
), noted as SmoC/C
(Long et al., 2001
), were obtained from Jackson Laboratories. Mice were housed in a sterile barrier facility approved by the IUCAC at Children’s Hospital Boston. SmoC/C
mice were backcrossed to C57/Bl6 for eight generations and then crossed with Mx1-Cre, also in a C57/Bl6 background (Kuhn et al., 1995
Routine PCR genotyping of the Smo alleles was performed on tail DNA by using a Promega PCR Master Mix kit. The total PCR volume was 13 μl, including 1 μlof 1:10 diluted tail DNA. The following primers were used to amplify the Smo floxed target gene: Smo-1 forward, 5′-GCAAGCTCGTGCTCTGGTC-3′; and Smo-2 reverse, 5′-CCGGTGGATGTGGAATGTG-3′, which generated a 250 bp band. For the Mx1-Cre genotyping, QIAGEN PCR Core kit was used. The primers used were Cre reverse, 5′-ACGACCGGCAAACGGACAGAAGCA-3′; and Mx1 forward, 5′-CCCAACCTCAGTACCAAGCCAAG-3′. The total PCR volume was 25 μl, including 1 μl of undiluted tail DNA.
Induction of Mx1-Cre Expression with pIpC
Animals were treated with polyinosine-polycytidylic acid (pIpC, Sigma) by intraperitoneal (i.p.) injection at ~10 weeks of age. Doses up to 25 μg/g mouse weight (or maximum dose 600 μg, whichever was less) were given every other day for a total of three doses. Mice were analyzed between 1 and 18 months after the first injection.
Detection of SmoC Excision
DNA was isolated from whole peripheral blood or from frozen single-cell suspensions of spleen, thymus, and bone marrow using the QIAamp DNA blood mini kit or tissue kit (QIAGEN). DNA was quantified and diluted to a standard concentration of 25 ng for PCR.
Semiquantitative Three-Primer PCR for Excision Analysis
A three-primer PCR was performed with the following primers: common reverse primer (CCATCACGTCGAACTCCTGGC) at a 1× concentration in the PCR reaction; forward primer for amplification of nonexcised (“floxed”) SmoC allele (CCGATTCGCAGCGCAT) at a 0.5× concentration; and forward primer for amplification of excised SmoC allele (GGCCTGCGCTGCTCAACA TGG) at a 0.5× concentration. Annealing temperature was 60°C.
Mouse Analysis and Organ Collection
Peripheral blood was collected from the retroorbital cavity using a heparinized glass capillary tube. Complete peripheral blood count analysis including a differential blood count was obtained by using Hemavet (Drew Scientific). For histological analysis, peripheral blood smears were stained with a standard Wright-Giemsa stain. Mice were euthanized, and all relevant organs were collected, fixed in 10% formalin, and subsequently paraffin embedded. Histological sections (4 μm) were stained by hematoxylin and eosin (H&E) in a histopathology core facility (Brigham and Women’s Hospital).
Bone Marrow Transplant Experiments
For the competitive bone marrow transplant experiment, SmoWT/CD45.2 or SmoNull/CD45.2 donor bone marrow was transplanted in various ratios together with a competitor WT bone marrow from B6.SJL F1CD45.1/CD45.2 mice into B6.SJLCD45.1 recipients. A total of 2 × 106 cells were transplanted with donor to competitor ratios of 1:0, 3:1, 1:1, 1:3, and 0:1, respectively. This approach enabled differentiation between donor, competitor, and recipient bone marrow populations using flow cytometry analysis with fluorochrome-labeled antibodies for CD45.1 or CD45.2. Helper bone marrow cells were generated by crossing C57/Bl6 and SJL mice and using offspring from the first generation that were CD45.1/CD45.2 double positive.
For retroviral bone marrow transplant experiments, SmoNull
B6 F8 mice were injected i.p. with pIpC 25 μg/g every other day for three doses. Three weeks after the first pIpC injection, peripheral blood was collected. DNA was extracted to confirm Smo
excision by using the three-primer PCR technique previously described. Mice were then treated with one dose of intraperitoneal 5-FU 150 mg/kg on day −8. Bone marrow cells were harvested on day −2 and “spin infected” twice with a MSCV retrovirus expressing MLL-AF9/GFP (kindly provided by Dr. S. Armstrong) (Krivtsov et al., 2006
; Scholl et al., 2007
). Retrovirally transduced SmoNull
bone marrow was then transplanted into lethally irradiated B6.SJL WT recipient mice. A subset of bone marrow cells was used for serial replating on methylcellulose as described above. Peripheral blood was collected 4 weeks after transplantation to evaluate peripheral blood counts. Mice were euthanized at the clinical onset of leukemia determined by signs of distress, such as decreased activity and hepatosplenomegaly. Peripheral blood and all relevant mouse organs were collected as described above and used for comprehensive analysis, including CBC with differential, organ weight, histopathology, and flow cytometric analysis.
Serial 5-FU Experiment
5-FU was administered weekly at a dose of 150 mg/kg i.p. to SmoNull or SmoWT mice. Sequential blood counts prior to 5-FU therapy (day 1) and on days 5, 7, 11, and 14 were obtained, and survival was monitored.
Pharmacological Experiments with HhAntag
Female 6- to 8-week-old C57/BL6 mice were obtained from Charles River Laboratories (Wilmington, MA). All mice were housed and maintained according to the animal use guidelines of Genentech, Inc., conforming to State of California legal and ethical practices. Mice were treated with either control (MCT; 0.5% methyl cellulose, 0.2% tween-80 at 10 ml/kg) or 100 mg/kg HhAntag (10 mg/ml solution in MCT) twice daily for 21 days by oral gavage and blood collected 6 hr following the final dose by cardiac puncture for analysis. Analysis was performed on a Cell Dyn 3700 System from Abbott Laboratories, Inc. (Abbott Park, IL).
Hh pathway genes were quantitatively assessed in frozen tissues by TaqMan using standard techniques. Transcript levels were normalized to the ribosomal protein L19 (RPL19) and results expressed as normalized expression values (= 2 – ΔCt).
Determination of HhAntag Concentration in Plasma
HhAntag was detected in plasma following oral administration 6 hr following the last daily dose. Whole blood was collected via cardiac puncture, placed in heparinized Eppendorf tubes on ice, and centrifuged at 10,000 × g for 4 min. The plasma supernatant was added to internal standard solution, mixed by vortexing, and centrifuged. Sample extract was assayed for HhAntag by LC/MS/MS analysis. Plasma levels were calculated using an authentic standard curve.