Fms like tyrosine kinase 3 ligand

Peripheral blood mononuclear cells (PBMCs) were collected at Ramathibodi Hospital (October 2023) and used to assess drug toxicity. Briefly, 10 mL of whole blood was mixed with anticoagulant citrate dextrose solution and then diluted in a 1 : 1 ratio with PBS containing 2% FBS. This mixture was subsequently layered over Ficoll Paque Plus (GE Healthcare, Chicago, IL, USA) and centrifuged at 400 g for 30 min. Subsequently, PBMCs were collected, washed with PBS, cultured in StemSpan™ SFEM (STEMCELL Technologies, Vancouver, Canada), and supplemented with 50 μg·mL⁻¹ of stem cell factor, FMS‐like tyrosine kinase 3 ligand, interleukin‐3, and interleukin‐6 (all from PeproTech, Cranbury, NJ, USA). For cell viability and cell counting assays, PBMCs were seeded at densities of 2 × 10⁴ or 1 × 10⁵ cells and treated with specified ceftriaxone concentrations for 48 h. Cell viability was determined using the CellTiter‐Glo assay, and these data were subsequently used to construct dose–response curves. Viable cells, identified by their negative trypan blue staining, were counted using a hemocytometer.

Human CD34⁺ BM cells were liquid cultured for nine days in StemSpan SFEM medium II (STEMCELL Technologies) containing human stem cell factor (100 ng/ml), FMS-like tyrosine kinase 3 ligand (100 ng/ml), and thrombopoietin (50 ng/ml) (all from Peprotech) with or without LJP-1586. After nine days, the cells were stained with anti-CD38 and anti-CD34 antibodies, washed using DMEM, centrifuged and resuspended in 100 µl DMEM. ROS were detected by DHR-123 reagent (Molecular Probes). For this, DHR-123 was diluted in DMSO and kept as a 5 mM stock solution at  – 20 °C for single use. The aliquots were thawed, diluted 160 times (30 µM) just before adding 12.5 µl to the HSC suspended in 100 µl DMEM to a final concentration of 3 µM. The cells were then incubated for 10 min at 37 °C and followed by activation with Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich. The stock solution of PMA was frozen at 1 mg/ml in DMSO, freshly thawed and diluted 500 times in order to add 12.5 µl to a final concentration of 200 ng/ml. After 20 min at 37 °C, the cells were washed with PBS, resuspended and analyzed by flow cytometry. The red DHR123 turns to green after oxidation. CD38⁻ and CD34⁺ positive cells were gated and fluorescence intensity of oxidized DHR-123 was measured from the filter channel 530 nm/30 nm using LSR Fortessa instrument (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Human CD34⁺ cells were cultured in HSC medium, consisting of StemSpan ACF (StemCell Technologies) supplemented with 100 ng ml⁻¹ human stem cell factor (PeproTech), 100 ng ml⁻¹ FMS-like tyrosine kinase 3 ligand (PeproTech), 50 ng ml⁻¹ thrombopoietin (PeproTech), 10 μg ml⁻¹ human low-density lipoprotein (StemCell Technologies) and 1× Glutamax (Thermo Fisher Scientific) with or without UM171 at 35 nM.
Primary murine haematopoietic cells were cultured in StemPro-34 SFM (Life Technologies) with supplement and 50 ng ml⁻¹ SCF, 25 ng ml⁻¹ TPO, 30 ng ml⁻¹ Flt3-Ligand (all PeproTech), 100 U ml⁻¹ penicillin–streptomycin and 2 mM l-glutamine (Thermo Fisher).

SCD patient CD34⁺ HSPCs were isolated from unmobilized PB following receiving Boston Children’s Hospital institutional review board approval and informed patient consent. The SCD CD34⁺ HSPCs were enriched using the Miltenyi CD34 Microbead kit (Miltenyi Biotec, Auburn, CA, USA). CD34⁺ cells were prestimulated for 36–40 h at 1 × 10⁶ cells/mL in Stem Cell Growth Medium (CellGenix, Portsmouth, NH, USA) supplemented with SCF, FMS-like tyrosinekinase 3 ligand, and TPO, all from Peprotech. Cells were then enumerated and transduced with the vector at an MOI as indicated in presence of LentiBOOST enhancer (SIRION Biotech, Gräfelfing, Germany) for 24 h before downstream processing.

LKS⁺ cells purified from the BM of BALB/c mice were cultured in serum-free S-Clone SF-03 medium (Sanko Junyaku) supplemented with 1% BSA, 100 ng/mL stem cell factor (PeproTech), 100 ng/mL thrombopoietin, 25 ng/mL fms-like tyrosine kinase-3 ligand (PeproTech), 10 ng/mL IL6 (PeproTech), and 10 ng/mL IL3 (PeproTech) for 24 hours. Cultured LKS⁺ cells were infected with the retrovirus carrying MSCV-MLL-AF9-ires-GFP using a ViroMag R/L kit (OZ Bioscience) to obtain leukemia-initiating cells. The resultant cells (50–100 GFP⁺ cells included in 30,000 KLS⁺ cells) were intravenously transplanted into 5 Gy X-irradiated recipient BALB/c mice along with 3 × 10⁵ normal BM cells in a 200 µL volume. Total BM cells were harvested from primary AML mice and stored in liquid nitrogen. A frozen stock of primary AML cells was cultured in semisolid methylcellulose-based medium (Methocult GF M3534, STEMCELL technologies). To prepare the mouse AML model, 2 × 10⁵ colony-forming cells were intravenously transplanted into sublethally irradiated secondary recipient mice along with 1 × 10⁶ normal BM cells.