Recombinant mouse scf

B-cell lineage colony-forming units (CFU-proB) of efficiency WT, Vav3^−/−and Rac2^−/− p190-BCR-ABL⁺ B-cell progenitor transduced with empty or Bmi1 or Phlpp2 lentiviral vector were quantified after 9 days culture of leukemic BM cells or sorted p190-BCR-ABL expressing B-cell progenitors in M3134 methylcellulose (Stem Cell Technologies, Vancouver, BC) containing 20 ng/mL of recombinant mouse IL-7 and 100 ng/mL of recombinant mouse SCF (PeproTech, Rocky Hill, NJ) and supplemented with 30% FBS (Hyclone GE Healthcare, Marlborough, MA), 2 mM l-glutamine (Invitrogen, Waltham, MA), 1% antibiotics (penicillin-streptomycin; Invitrogen), 100 μM β-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA), and 1% BSA (Sigma-Aldrich, St. Louis, MO). CFU-proB containing clusters of more than 40 cells were counted on day 9 of culture.

A total of 2 × 10⁶ DM murine leukemic cells were stained for CD11b, Gr-1, CD55, CD41 and CD34 (Supplementary Table 9). Granulocyte-like (CD11b^highGr-1⁺), erythroid/basophil-like (CD55^highCD41⁻) and CD34⁺ fractions were subsequently FACS-sorted (BD Influx; BD Biosciences). One thousand cells of each sorted population were seeded in 1 ml methylcellulose medium (M3434, STEMCELL Technologies) supplemented with recombinant mouse SCF (PeproTech) and mouse IL-3 (PeproTech) with recombinant IL-6 and Epo (R&D Systems) in duplicate. Methylcellulose cultures were maintained at 37 °C and 5% CO₂; total colony forming units (CFUs) were enumerated 7 days later. Photographs of colonies were obtained with the STEMvision instrument and software (STEMCELL Technologies). Data are presented as a fold change of average CFUs per 1,000 cells seeded, relative to the CD34⁺ fraction. Ten thousand granulocyte-like, erythroid/basophil-like or CD34⁺ DM cells were also maintained in standard culture conditions for 21 days, and the number of cells was counted every 7 days. The total cell number is presented as a fold to the corresponding CD34⁺ counterpart for each time point. P values were calculated using a two-way ANOVA (Prism v.9.1, GraphPad Software).

RCM (R cell, mutant Kit) and R cells were established from splenocytes of DO11.10 mice by repeated stimulation with ovalbumin peptides in vitro. The cell lines exhibited mast cell-like surface phenotype, c-Kit⁺ FcεRI⁺, and mast cell-like expression profiles of proteases. Moreover, RCM and R cells can secrete biologically active product on stimulation in vitro and in vivo. They do not express SCF. We were unable to find Kit(wt) by cDNA sequencing in RCM cells. For culture of R cells, we used culture supernatants from T-cell lines stimulated with an anti-T cell receptor antibody as a cytokine cocktail. R cells were cultured in 0.25% cytokine cocktail. RCM cells proliferated without the cocktail and developed tumours in vivo. HMC-1.2 (referred to henceforth as HMC-1), RBL-2H3 and pt18 cells were fro m Dr Hirohisa Saito and Dr Kenji Matsumoto (National Center for Child Health and Development), Dr Ko Okumura (Juntendo University) and Dr Ryo Goitsuka (Tokyo University of Science), respectively. These cells and RCM cells were cultured at 37 °C in RPMI1640 medium supplemented with 10% fetal calf serum (FCS), penicillin, streptomycin and 50 μM 2-mercaptoethanol. HMC-1 cells were grown in suspension at 37 °C in α-MEM containing 10% FCS, penicillin and streptomycin. For stimulation, cells were starved for at least 3 h and then treated with 50 ng ml⁻¹ recombinant mouse SCF (PeproTech).

The co-culture system was developed by modifying the HSC expansion system [24] (link). WT-PαS cells were freshly sorted by FACS and plated at 2000/well in a 96-well plate (Corning Inc.). After PαS cells were cultured in maintenance medium until sub-confluent growth, the medium was changed to osteogenic induction medium in the presence or absence of OSM. At day 7 of osteogenic induction, the medium was removed, rinsed, and replaced with 200 µl of HSC culture medium containing 5,000 LSK cells. The HSC culture medium consists of StemSpan SFEM medium (Stemcell Technologies, Inc., Vancouver, British Columbia, Canada) supplemented with 50× penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA), 10 ng/mL recombinant mouse SCF (Pepro Tech, Inc., Rocky Hill, NJ, USA). After co-culture, cells were inoculated at 37°C under 4% O₂ and 5% CO₂ conditions for 7 days. One hundred microliters of medium were replaced with fresh HSC culture medium every 2–3 days. At day 7 of co-culture, after treatment with 0.25% trypsin-EDTA, nucleated cells were enumerated using a hemocytometer after staining with Turk's solution (Wako). Re-analyses of the LSK fraction in expanded cells were performed using the BD FACS Canto II system (BD Biosciences). Data were analyzed using the FlowJo software.

Retroviral and lentiviral transduction have been described previously [73 (link)]. In brief, mouse low-density (LD) bone marrow (BM) (LDBM) cells or Lineage-/c-kit+/Sca1+ (LSK) cells were transduced with bicistronic retroviral vectors (MIEG3) encoding the BCR-ABL1 isoform p190 in the presence of 20 ng/mL of recombinant mouse IL-7 (PeproTech, Rocky Hill, NJ), 10 ng/mL of recombinant mouse SCF (PeproTech), and the recombinant fragment of fibronectin, CH296 (RetroNectin, Takara Bio Inc.) for 16 hours at 37° C. For the Klf5 or Gstm1 expression, lentiviral particles encoding either full-length mKlf5 cloned in a pCDH1-MCS1-EF1-copGFP vector (System Biosciences, Palo Alto, CA), mGstm1 (GeneCopoeia, Catalog #: EX-Mm02884-Lv166) or related empty vectors, were used for stable expression in p190-BCR-ABL+ B-cell lymphoid precursors. B-ALL cell lines were transduced with lentiviral particles expressing either full-length hKLF5 cDNA (GeneCopoeia, Catalog #: EX-T1795-Lv166) or the control bicistronic mCherry-expressing empty vector driven by the promoter EF1 at a multiplicity of infection of 10 for 6 hours at 37° C.