Stem cell factor (scf)

Bulk blast crisis CML cells recovered from the spleen of terminally ill recipient mice that were initially transplanted with BCR-ABL⁺/NUP98-HOXA9⁺ KLS cells isolated from REM2 mice were either irradiated (0, 5, or 10 Gy) in PBS with glucose and cultured in X-Vivo15 media (Lonza) supplemented with 50 μM 2-mercaptoethanol, 10% (vol/vol) fetal bovine serum, SCF (100 ng/ml, R&D Systems) and TPO (20 ng/ml, R&D Systems) for 7 h or treated with imatinib (0.5 or 5 μM) or control DMSO for 7 h in X-Vivo15 media (Lonza) supplemented with 50 μM 2-mercaptoethanol, 10% (vol/vol) fetal bovine serum, SCF (100 ng/ml, R&D Systems) and TPO (20 ng/ml, R&D Systems). Cells were then washed and stained with antibodies against lineage markers. Apoptosis assays were performed by staining cells with Annexin-V (BD Pharmingen).

Hematopoietic progenitor cells (HPCs) were generated as previously described [23 (link)]. Briefly, iPSCs were dissociated into single cells and quickly re-aggregated in Essential 8 Medium (Thermo Fisher Scientific) containing 80 ng/ml BMP4 (R&D SYSTEMS), 80 ng/ml VEGF (R&D SYSTEMS), 2 μM CHIR99021 and 30 μM Y-27632) (9000 cells/well) using a Nunclon Sphera Microplates 96U-Well Plate. Embryoid bodies were cultured at 37 °C, 5% CO₂ and 5% O₂ during differentiation. On day 2, the medium was removed, and Essential 6 Medium (Thermo Fisher Scientific) containing 80 ng/ml VEGF, 25 ng/ml bFGF (R&D SYSTEMS), 50 ng/ml SCF (R&D SYSTEMS) and 2 μM SB431542 (Tocris) was added. On day 4, the medium was replaced with Stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich) containing 50 ng/ml SCF (R&D SYSTEMS), 20 ng/ml TPO (R&D SYSTEMS), 40 ng/ml VEGF and 50 ng/ml IL-3 (R&D SYSTEMS). HPCs were harvested by sorting with PE Mouse Anti-Human CD34 (BECKMAN COULTER, Brea, CA, USA #A07776) and FITC Mouse Anti-Human CD43 (Thermo Fisher Scientific #11-0439-42) at day 12 using BD FACSAria II (BD Biosciences).

Embryoid bodies (EBs) were generated from the three hiPSC lines via forced aggregation as previously described [16 (link)]. The hiPSC lines were enzymatically treated and plated in 96-well plates at a density of 3,000–5,000 cells per well in serum free medium (SFM) supplemented with 10 ng/mL FGF-basic, 10 ng/mL BMP-4, 10 ng/mL VEGF, and 50 ng/mL SCF (R&D System). The cells were aggregated by centrifugation at 3,000rpm for 5 minutes and placed in a humidified incubator at 37°C with 5% CO₂. The medium was replaced with fresh SFM containing cytokines every 3 days. Ten days after mesoderm induction, blood-like cells (BLCs) surrounding the EBs were observed. Fourteen days after mesoderm induction, single cells from the three hiPSC lines were collected by filtration through a 70 μm filter. Cell phenotype was analyzed by flow cytometry using CD34 and CD45 as markers of hematopoietic stem cells and progenitor cells. Hematopoietic cells derived from the hiPSC lines were further cultured in suspension in SFM supplemented with 10 ng/mL Flt3, 10 ng/mL IL-3, 10 ng/mL TPO, 50 mg/mL, GM-CSF, and 50 ng/mL SCF plus 3 units/mL EPO (R & D System). After 5 days of hematopoietic differentiation, expression of CD34 (555824, BD Biosciences), CD45 (555483, BD Biosciences), CD15 (11-0159-42, eBioscience), CD33 (555450, BD Biosciences), and CD235a (559943, BD Biosciences) was analyzed by flow cytometry.

HSCs were cultured under ‘expansion' conditions in U-bottom 96-well plates for 5 days. Cultures were maintained in Stemline II (Sigma) supplemented with 10 μg ml⁻¹ Heparin (Sigma), 100 ng ml⁻¹ SCF (R&D Systems), 2 ng ml⁻¹ Flt3 ligand (R&D), 20 ng ml⁻¹ TPO (R&D Systems), 10 ng ml⁻¹ FGF-1 (Invitrogen), 500 ng ml⁻¹ IGFBP2 (R&D Systems) and 100 ng ml⁻¹ AngL-3 (R&D Systems)31 (link). At the end of the culture period, cells were stained with TMRM and analysed or sorted by flow cytometry. To induce differentiation, HSCs were cultured in a basal medium (Stemline II containing 100 ng ml⁻¹ SCF and 2 ng ml⁻¹ Flt3 ligand) supplemented with 20 ng ml⁻¹ IL-3 (R&D Systems) and 100 ng ml⁻¹ IL-6 (R&D Systems). For some experiments, 5 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma) was added at the medium. FCCP stock solution was prepared by dissolving the powder in ethanol at 10 mM concentration.