Murine scf

Bone marrow eosinophil cultures were prepared as described previously⁴¹ with slight modification. Bone marrow cells were harvested from femurs and tibias of WT and Ccl6^-/- mice. After removing RBCs, c-Kit⁺ hematopoietic progenitor cells were enriched by positive selection with APC-conjugated c-Kit antibody and anti-APC MicroBeads (Miltenyi Biotec). Cells were resuspended to a density of 1 × 10⁶/mL in Iscove’s Modified Dulbecco’s Medium (Gibco) supplemented with 10% FBS (Invitrogen), 2 mM L-glutamine (GlutaMAX™, Gibco), 5 × 10⁻⁵ M β-ME (Sigma-Aldrich), MEM Non-Essential Amino Acids (11140050, Gibco), 1 mM sodium pyruvate (11360070, Gibco), and 100 U/mL penicillin/streptomycin. The cytokines murine SCF (100 ng/mL, PeproTech) and murine Flt3-Ligand (100 ng/mL, PeproTech) were supplemented to the culture for 4 days. On day 4 and day 8, cells were washed and treated in the presence of IL-5 (10 ng/mL, R&D systems) for the duration of the culture. For BX471 treatment, 1 μM BX471 was added on days 0, 4, and 8 (blue arrow in Fig. 6a), or days 4 and 8 (red arrow in Fig. 6a). Flow cytometry analyses of eosinophils were performed on days 4 and 8–10. The number of total cells was counted with a cell counter.

The primary lineage-negative (Lin-) murine HSPCs were obtained through isolating bone marrow cells from femur and tibia of wildtype BALB/C mice, followed by depletion of mature hematopoietic cells using a lineage cell depletion kit (Miltenyi Biotec, 130-090-858) as described before^{3 (link),4 (link)}. Lin- HSPCs were then subject to cytokine stimulation for ex vivo expansion in the Opti-MEM medium supplemented with 15% of FBS (Invitrogen, 16000-044), 1% of antibiotics, 50 μM of β-mercaptoethanol, 25 ng/mL of murine SCF (PeproTech, 250-03) and 10 ng/ml of murine Flt3 ligand (Sigma, SRP3198). After 3–4 days of stimulation, spinoculation was performed for retroviral transduction. Briefly, murine HSPCs were mixed with concentrated retrovirus (Retro-X™ Concentrator; Takara, 631456) in the presence of 8 ug/ml polybrene in a fibronectin-coated 6-well plate, followed by centrifugation at 1,500 g for 1 h. Drug selection with 2 μg/mL puromycin or 500 μg/mL G418 started 2 days post-infection. For routine liquid culture of transduced HSPCs, we used a home-made medium recipe, which uses the culture supernatants of an mSCF-producer cell line (mSCF-CHO cells, gift of MP Kamps, UCSD) and an mFlt3l-producer cell line (SP2.0-mFlt3L cells, gift of R. Rottapel, University of Toronto) as source of murine SCF and Flt3L, respectively^{5 (link),61 (link),62 (link)}.

Briefly, mouse BM cells were flushed from the femur and tibia and were then sorted for Lin^-c-Kit⁺ cells by flow cytometry after staining with anti-lineage marker cocktail and anti-c-Kit antibodies. Lin^-c-Kit⁺ cells were grown in 10% FBS-supplemented RPMI-1640 medium with 2 mM L-glutamine, penicillin/streptomycin, and 20 ng/mL murine stem cell factor (SCF, Peprotech; Rocky Hill, NJ, USA) at 37°C under 5% CO₂ for 2 days. The cells were then cultured in the presence of 20 ng/mL murine SCF and 200 ng/mL murine TPO (mTPO, Peprotech) for the indicated time points to obtain murine megakaryocytes as described previously 19 (link), 20 (link).

Bone marrow aspirates from three AML mice were combined, isolated, and cultured in RPMI containing 20% fetal bovine serum (FBS), streptomycin/penicillin, 50 µM 2-mercaptoethanol (RPMI-20), and supplemented with murine SCF (10 ng/mL) and IL-3 (10 ng/mL) (Peprotech or BioLegend). The cells were serially passaged for ~1 month. Inhibitor library screening to evaluate drug sensitivity was performed as previously described [5 (link)]. Briefly, cultured AML mouse-derived cells were counted and seeded into four 384-well plates at a concentration of 2000 cells/well. The cells were then subjected to titrations of 188 unique small-molecule inhibitors (SMIs) in culture for 72 h. MTS reagent (CellTiter96 AQueous One; Promega) was added and the optical density was read at 490 nm to assess viability.

BM lineage-negative (Lin^-) cells were isolated from 8-week-old 182WT and 182KO mice (Stemcell Technologies) five days after 5-fluorouracil treatment and cultured in StemSpan SFEM (Stemcell Technologies) supplemented with murine SCF (50 ng/mL, PeproTech), TPO (50 ng/mL, PeproTech), and FLT3 ligand (50 ng/mL, PeproTech) overnight. Furthermore, Lin^- cells were retrovirally transduced with MSCV-green fluorescent protein (GFP)-internal ribosome entry site (IRES)-MLL-AF9 4 (link) or MSCV-GFP-IRES-AML1-ETO(9a) 34 (link) by two rounds of spinoculation at 2000×rpm for 2 h 35 (link). GFP⁺ cells were sorted by fluorescence-activated cell sorting and were intravenously injected into lethally irradiated C57BL/6J mice (Beijing Vital River Laboratory, Beijing, China) plus radioprotective BM cells (4×10⁵ for every mouse). The sample size in vivo study was estimated according to prior experience in our laboratory. All animal procedures and care are performed according to national and international policies and institutional guidelines of the ethics committee of the First Affiliated Hospital of Wenzhou Medical University.

For establishment of a murine B-ALL model by overexpressing the N-Myc oncogene in hematopoietic stem progenitor cells, an MSCV-N-Myc-IRES-GFP plasmid together with the pCL-ECO packaging plasmid were transfected into HEK293T cells, followed by collection of the supernatant containing retroviruses 48–72 h after transfection. Lin⁻ fetal liver cells were purified and spin infected with N-Myc retroviruses. The infected cells were then incubated in Stemspan medium (StemCell) in the presence of 10 ng/mL murine SCF (Peprotech) and 10 ng/mL murine IL-7 (Peprotech) for 2 days. A total of 1–3 × 10⁵ infected Lin- BM cells were transplanted into lethally irradiated (10 Gy) recipient mice by retro-orbital injection. GFP⁺ BM B-ALL cells collected from primary recipient mice were sorted and transplanted into lethally irradiated 6- to 8-week-old recipients. The frequency of GFP⁺ cells in the peripheral blood of recipient mice was analyzed by flow cytometric analysis at the indicated time points post-transplantation to evaluate B-ALL development. In another set of experiments, 5 × 10⁵ B-ALL cells were cultured in 12-well plates with 500 μL of StemSpan serum-free medium (STEMCELL Technologies) containing 10 ng/mL mouse SCF (Peprotech), 10 ng/mL murine IL-7 (Peprotech), and 30 μg of ANGPTL2-containing SEVs, ANGPTL2-mut#2-containing SEVs or control SEVs for 6 h before transplantation.