Myelocult

Cell lines were maintained in complete media as indicated in Supplemental Methods. YTS, BLCL, K562, 721.221, Raji, P815, and YAC-1 cell lines were cultured in RPMI-1640 (Thermo Fisher Scientific), HEK293T cells in DMEM (Thermo Fisher Scientific), and NK92 cell lines in Myelocult (Stemcell Technologies) (52 (link)). BLCL were generated from patient PBMCs by adding EBV supernatant. Cell lines were confirmed mycoplasma negative before their use in experiments.

Visually confirmed single cells were added onto irradiated UG26 feeder cells in flat-bottomed wells containing 200 μl of MyeloCult (STEMCELL Technologies) supplemented with 10⁻⁶ M hydrocortisone (Sigma-Aldrich) in 96-well plates and cultures maintained with weekly half-medium changes (Woehrer et al., 2013 (link)). After 6 weeks, fresh SFM containing FBS plus SF plus IL-3 plus IL-6 plus Epo was added to the wells, and those containing clusters of >25 nonadherent cells 12 days later were counted as positive.

2000 FACS-sorted cells from stem and progenitor subpopulations were plated in duplicate in Methocult optimum (H4034, Stem Cell Technologies). Following incubation at 37° for 10–12 days, colonies were counted. For replating, 50 individual primary colonies per sample were picked and re-suspended in 200 μl Methocult in 96 well plates, incubated at 37° for 10–12 days before positive wells counted. Primary CP-CML samples were sorted for Lin⁻CD34⁺CD93^−/+ cells and cultured overnight. Cells were washed and inoculated into pre-prepared long-term cultures comprising a stromal feeder layer (1:1 mix of irradiated (80 Gy) SL/SL and M210B4 murine fibroblasts) in long-term myeloid culture medium (MyeloCult supplemented with hydrocortisone; Stem Cell Technologies) [21 (link)]. Cultures were maintained for 5 weeks with 50% media changes performed weekly. Each well was harvested, counted, and seeded into Methocult to perform CFC assays.

For LTC-IC assay, 1 × 10⁴ cells/mL UCB CD34⁺ were plated into six-well plates containing AFT024-hKirre cells or AFT024 control cells treated in LTC medium (Myelo-Cult, StemCell Inc., Vancouver, BC, Canada) along with 10 M hydrocortisone sodium hemisuccinate (Sigma Chemical Co., St. Louis, Missouri, USA). Half of medium was replaced weekly with fresh medium as described previously [17 (link)]. Adherent and nonadherent cells were harvested weekly during 5–8 weeks of coculture and transferred to 1.8% methylcellulose medium (it was dissolved in distilled water at the concentration of 1.8%, autoclaved the solution, and then put it at 4°C for gradual and complete salvation) containing 2.8% BSA, 30% FBS (Hyclone), 50 ng/mL SCF, 20 ng/mL IL-3, 20 ng/mL GM-CSF, 20 ng/mL IL-6, and 3 U/mL EPO at 37°C with 5% CO₂ in humidified incubator. After 14–16 d of cultures, colonies with greater than 50 cells were counted to assess LTC-IC activities. Colony-forming units (CFU-Cs) and lineage-specific colonies including colony-forming unit-erythrocyte (BFU-Es), colony-forming unit granulocyte-macrophages (CFU-GMs), and colony-forming unit mix (CFU-Mix's) were counted using an inverted microscope (Nikon; Sesto Fiorentino, Italy) and calculated the frequency of LTC-IC according to the manufacturer's instructions (StemCell Inc.).