Gm csf

Monocyte isolation and DC differentiation were performed as described previously.⁴ (link) Briefly, monocytes were obtained using the adherence method, where PBMCs were plated in adherent plastic plates (Sarstedt, Nümbrecht, Germany) in medium (X-Vivo 15 medium, Lonza, Basel, Switzerland) supplemented with 5% human serum, 2 mM L-glutamine and 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), 1,000 U/mL (100 ng/mL) IL-4, and 800 U/mL (50 ng/mL) Granulocyte-macrophage colony-stimulating factor (GM-CSF) (both from STEMCELL Technologies) and incubated in a CO₂, 37°C incubator for 7 days. On day 4, the medium was replaced with fresh medium supplemented with IL-4 and GM-CSF. On day 7, DCs were matured with maturation medium containing 1,000 U/mL (100 ng/mL) IL-4, 800 U/mL (50 ng/mL) GM-CSF, 10 ng/mL tumor necrosis factor alpha (TNF-α) (STEMCELL Technologies), 1 μg/mL PGE2 (Sigma), 10 ng/mL IL-1β (Feldan, Quebec City, QC, Canada), and 100 ng/mL IL-6 (Miltenyi Biotec, Bergisch Gladbach, Germany) and loaded with the desired peptide (1 μg/mL LMP2_426–434, [CLGGLLTMV] or 1 μg/mL WT1_37–45 [VLDFAPPGA], both from JPT Peptides (Berlin, Germany). Last, DC medium was supplemented with interferon γ (IFN-γ; 1,000U/mL, Feldan) for the last 24 h of maturation.

Hematopoietic colony formation was assessed by seeding 20,000 unfractionated mouse femur bone marrow cells into MethoCult M3334 or MethoCult M3234 supplemented with 10 ng/ml GM-CSF (STEMCELL Technologies). The cultures were incubated at 37°C for 10 days and then colonies were counted under the microscope.

All cell cultures were performed in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO₂. For liquid and methylcellulose cultures, PU.1 eYFP low and high SLAM cells were purified from mice that were treated with either PBS or 0.5 µg IL-1β (Peprotech, Rocky Hill, NJ, USA) 24 h prior to harvest. PU.1 low and high were selected based on median fluorescent intensity (MFI), where approximately the lowest 25% were designated PU.1 low and the highest 25% designated as PU.1 high. Pools of 400 cells per well were sorted for liquid culture into Stempro 34 medium (Gibco, Waltham, MA, USA, 10639011) and supplemented with antibiotic–antimycotic (Gibco, 15240062), 2 mM l-glutamine (Gibco, 25030024), 25 ng/mL IL-11 (Peprotech), 25 ng/mL SCF (Peprotech), 25 ng/mL TPO (Peprotech), 25 ng/mL Flt3L (Peprotech), 10 ng/mL IL-3 (Peprotech), 10 ng/mL GM-CSF (Peprotech), and 4 U/mL EPO (Peprotech). Half of the medium was replenished every two days, and cells were harvested for flow cytometry analysis and cell counts on days 4 and 8. Methylcellulose cultures were executed using Methocult M3231 Base Media (Stemcell Technologies, Cambridge, MA, USA 03231) containing antibiotic–antimycotic, IMDM, 25 ng/mL IL-11, 25 ng/mL SCF, 25 ng/mL TPO, 25 ng/mL Flt3L, 10 ng/mL IL-3, 10 ng/mL GM-CSF, and 4 U/mL EPO. Colonies were counted and phenotyped at day 8.

Human tissue was obtained with the required ethical approval from the NHS National Research Ethics Committee and informed consent from patients. Patient bone marrow biopsies were obtained, and the AML cells purified using lymphoprep followed by CD34 MACS bead enrichment. Patient mutation details are in Table 1. Primary cells and PDX cells (patient 5 only) were cultured on human mesenchymal stem cells, in SFEMII (StemCell Technologies) supplemented with 1% Pencillin/Streptomycin, 1 µM UM729 (Stemcell Technologies), 750 nM SR1 (Stemcell Technologies), 150 ng/ml SCF, 100 ng/ml TPO, 10 ng/ml FLT3, 10 ng/ml IL3, 10 ng/ml GM-CSF (all cytokines from Peprotech). Where primary cells were frozen prior to use, they were allowed to recover for a week before performing phenotypic assays but sorted directly from defrost for gene expression analysis. Healthy CD34+ cells (Amsbio) were cultured in SFEMII with StemSpan CD34 Expansion Supplement (Stem Cell Technologies) and 500 nM UM729 for 1 week, then moved into the t(8;21) media for 24 h prior to setting up assays.

JAK1^GOF and XY control iPSC cells were split using ReLeSR (STEMCELL Technologies) onto ultra-low adhesion plates. EBs were grown in culture with bFGF-free stem cell medium containing 300 ng/mL FLT3 (PeproTech), 300 ng/mL SCF (PeproTech), 50 ng/mL BMP-4 (R&D Systems), 50 ng/mL G-CSF (R&D Systems), 10 ng/mL IL-3 (PeproTech), and 10 ng/mL IL-6 (R&D Systems) as described previously (50 (link), 51 (link)). RNA was extracted from collected EBs on day 7 using QIAGEN RNeasy Plus Mini Kit. After 16 days, EBs were dissociated into single cells using 1 mg/mL Collagenase B, nonenzymatic cell dissociation buffer (Thermo Fisher Scientific) and mechanical sheering. Cells were then plated onto methylcellulose (MethoCult H4434 containing SCF, EPO, IL-3, and GM-CSF; STEMCELL Technologies) and cultured for 14 days (50 (link)). Throughout the experimental period, samples were cultured in 21% O₂, 5% CO₂ at 37°C.