G csf

CD34⁺ HPCs were infected at an MOI of 2 for 48 h. Following infection, pure populations of viable, infected (GFP-positive) CD34⁺ HPCs were isolated by Wolf Cell Sorter (Nanocellect Biomedical, Inc.) using 7AAD Viability Staining Solution (Invitrogen eBioscience) and PE-conjugated antibody specific to human CD34 (clone 581, Biolegend). Long-term cultures were maintained in Myelocult supplemented with hydrocortisone in transwells above an irradiated M2-10B4 and S1/S1 stromal cell monolayer for 12 days (53 (link)). After 12 days in long-term bone marrow culture, infected hematopoietic cell populations were serially diluted twofold in reactivation medium: RPMI supplemented with 20% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomomycin, 15 ng/mL G-CSF, and 15 ng/mL GM-CSF (all cytokines purchased from R&D Systems, Minneapolis, MN, USA). Fibroblasts were examined microscopically for GFP expression up to 3 weeks. To differentiate between virus made as a result of reactivation and pre-existing virus in the cell cultures, an equal number of cells was serially diluted and plated on fibroblasts after being mechanically disrupted. The frequency of infectious center production during the culture period was measured using an extreme limiting dilution assay as previously described (53 (link)).

Frozen bone marrow (BM) blasts from newly diagnosed AML patients were obtained at the National University Hospital in Singapore with informed consent and approved by Institutional Review Board of National University Hospital. Written consents were obtained from the participants. Immediately after recovery, primary AML cells were cultured in IMDM supplemented with 10% FBS (Invitrogen), FLT3 ligand (20 ng/ml), SCF (20 ng/ml), IL-3 (20 ng/ml), G-CSF (50 ng/ml) and thrombopoietin (TPO; 50 ng/ml) (R&D Systems).

All murine studies were performed with approval of the Animal Care and Use Committees of Northwestern University and Jesse Brown VA Medical Center. Mice with disruption of the IRF8 gene (Icsbp^−/− mice) were obtained from Dr. Keiko Ozato (NIH, Bethesda, MD) [27 (link)].
Mononuclear cells were isolated from femurs of wild type or Icsbp^−/− C57/BL6 mice and cultured in DME supplemented with 10% FCS, 1% pen-strep, 10 ng/ml GM-CSF, 10 ng/ml IL-3, 100 ng/ml Scf (R&D Systems Inc., Minneapolis MN). Lin^-ckit⁺CD34⁺ cells were isolated from these cultures by magnetic bead affinity technique (Miltenyi Biotechnology, Auburn, CA) (referred to as “myeloid progenitor conditions”). Lin^-ckit⁺CD34⁺ cells (with increased βcatenin expression) represent the LSC population in murine CP-CML [27 (link)]. Some cells were differentiated with 10 ng/ml G-CSF for 24 hrs (R&D Systems Inc., Minneapolis MN) (> 70% of cells are Gr1⁺ under these conditions; not shown).
Bcr-abl retrovirus and shRNA expressing lentiviruses were prepared by transfecting 293T cells with the relevant plasmid and the Ecopack plasmid [14 (link)]. Viral supernatants collected 48 hours post-transfection were titered in NIH3T3 cells. Murine bone marrow cells were transduced by incubation with viral supernatant (~10⁷ pfu/ml) supplemented with polybrene (6 μg/ml) [14 (link)]. Transgene expression was confirmed by PCR and/or %GFP⁺ cells.

Neurons were plated at a density of 5×10⁴ cells per well in 24-well multidishes, and stimulated with 1–100 ng/ml G-CSF (R&D Systems) 3 h before oAβ_1–42 stimulation. Cells were treated with 0.3–30 µM BIX02189 as an ERK5/MEK5 inhibitor (Selleck, Houston, TX, USA) or 0.1–10 µM DL-thiorphan as a neprilysin inhibitor (Enzo Life Sciences, Farmingdale, NY, USA) 1 h before G-CSF stimulation. After 24-h stimulation of oAβ_1–42, neurons were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After blocking with 5% goat serum for 1 h at room temperature, cells were stained with rabbit polyclonal anti-microtubule–associated protein (MAP)-2 antibody (1∶1000, Millipore, Billerica, MA, USA), and Aβ was stained with mouse monoclonal anti-Aβ antibody (clone 4G8, 1∶1000, Millipore). Images were analyzed with a deconvolution fluorescent microscope system (BZ-8000; Keyence, Osaka, Japan).