Mouse scf

The fetal liver and AGM were dissected microscopically from embryos at E11.5 using forceps and then dissociated into cells by applying friction between the frosted ends of slide glasses. The suspension in HemEx Type-9A (Cell Science & Technology Institute Inc. Sendai, Miyagi, Japan) was seeded at 10⁴ cells/well in 48 well plates coated with human fibronectin (40 μg/mL, Corning Inc, Corning, NY, USA) and cultured in serum-free conditions in the presence of mouse TPO (100 ng/mL; PeproTech) and mouse SCF (10 ng/mL; PeproTech) for 20 days, during which half of the medium was changed every 3 days after 7 days of culture. Proliferating hematopoietic progenitors were harvested using a pipette on day 20 and suspended in α-minimum essential medium. Suspensions seeded at a concentration of 7.4 × 10⁴ cells/well in 48 well plates were cultured with murine SCF (10 ng/mL), murine M-CSF (20 ng/mL), and murine RANKL (100 ng/mL) for osteoclast differentiation.

Growth time was 7 days. The medium was complete DMEM/F-12, 1% penicillin-streptomycin-glutamine (Gibco), PVA 87% hydrolyzed (P8136, 363081 or 363146), 1× insulin-transferrin-selenium-ethanolamine (Gibco), 1× HEPES (Gibco), 100 ng ml⁻¹ mouse TPO (PeproTech) and 10 ng ml⁻¹ mouse SCF (PeproTech)^{59 (link)}.

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.

Lin⁻ mouse BM cells were isolated by flushing femurs, tibias, and iliac crests of 6- to 8-week-old CD45.2 C57BL/6 or CD45.2 Berkeley SCD mice (BERK-SCD, JAX stock #003342) followed by lineage depletion using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin− cells were pre-stimulated at 1 × 10⁶ cells/mL in Stem Cell Growth Medium (CellGenix) supplemented with mouse SCF (100 ng/mL), hTPO (100 ng/mL), mouse IL-3 (mIL-3) (20 ng/mL), and hFlt3-L (100 ng/mL), all from Peprotech. Following a 36- 40-h pre-stimulation, cells were transduced at a density of 1 × 10⁶ cells/mL in the presence of LentiBOOST enhancer, and transduced cells (without sorting) were transplanted by retro-orbital injection into lethally irradiated (7 + 4 Gy, split dose) CD45.1 recipients (B6.SJL-Ptprca Pepcb/BoyJ, Jax Strain #002014) 24 h after transduction. PB samples were collected at weeks 4, 8, 12, and 16 to measure engraftment by flow cytometry (CD45.2/CD45.1), determine RBC indices, and quantitate sickled cells. At week 16, mice were euthanized, and BM cells were used to measure engraftment by flow cytometry (CD45.2/CD45.1), VCN, and mRNA expression, spleens were collected to weigh. All animal experiments were approved by the Boston Children's Hospital's Institutional Animal Care and Use Committee.

NK cell differentiation from HPCs was performed as previously described. In brief, to isolate HPCs, Lin⁻ (B cell (B220), T/NK cells (CD2), granulocytes (Gr-1), monocytes (CD11b), NK/NKT cells (NK1.1), and erythrocytes (TER-119)) cells were purified using the MACS Cell Seperation kit (Miltenyi Biotec). c-Kit⁺ cells from the Lin⁻ cells were positively selected using the CD117 (c-Kit) microbeads (Miltenyi Biotec). The purified HPCs were plated into a 24-well plate (BD) at 1 × 10⁶ cells/well and cultured for 7 d in complete RPMI1640 medium supplemented with a mixture of mouse Flt3L (50 ng/ml, Peprotech), mouse SCF (30 ng/ml, Peprotech), mouse IL-7 (0.5 ng/ml, Peprotech), Indometacin (2 ug/ml, Sigma), and gentamycin (20 ug/ml, Sigma). The cell were refreshed with the same media on day 3. To generate the mNK cells, 7 d-HPCs were maintained with IL-15 (30 ng/ml, Peprotech) for 6~7 days.