Bit 9500 serum substitute

Kasumi-1 cells used in this study were maintained at 37°C with 5% CO₂. They were cultured in RPMI-1640 (Cellgro) supplemented with 10% heat-inactivated (HI) FetaPlex serum (GEMINI). SKNO-1 cells were cultured in RPMI-1640 supplemented with 10% Stasis FBS (GEMINI) and 10 ng/ml GM-CSF (R&D Systems). RPMI media was supplemented with 2 mM l-glutamine, 100 I.U/ml penicillin, and 100 μg/ml streptomycin. Primary human t(8;21) AML patient cells were cultured with IMDM supplemented with 20% BIT 9500 Serum Substitute (StemCell Technologies), 50 ng/ml SCF (PeproTech), 10 ng/ml IL-6 (PeproTech) and 10 ng/ml IL-3 (PeproTech).

CD34-enriched cord blood cells and 8227 cells were transduced with pLBC2-BS (from J.E.D.’s laboratory) vectors containing NLS1–HK2 driven by the SFFV promoter and BFP driven by a chimaeric EF1α/SV40 promoter. Transduction of 8227 cells and CD34-enriched cord blood cells was performed as described in a recent publication^{53 (link)}. Twenty-four-well plates were coated with retronectin for 2 h at room temperature. The plates were then blocked with 2% BSA (wt/vol) for 30 min at room temperature. After the removal of BSA, 0.5 ml of concentrated virus particles in HBBS along with 25 mM HEPES was added to each well and the plates were centrifuged at 1,600g for 5 h at room temperature to aid in the attachment of viral particles. After centrifugation, the viral particle solution was removed and 5 × 10⁵ cells were added to each well in 1 ml X-VIVO 10 medium supplemented with 20% BIT 9500 serum substitute (Stem Cell Technologies) and growth factor cocktail. The plates were centrifuged again at 600g for 10 min and transferred to a 37 °C incubator for 24 h. The cells were then resuspended in fresh medium at a concentration of 1 × 10⁶ cells ml⁻¹ and seeded in 24-well plates (1 ml per well). After an additional 3 d, the transduction efficiency (percentage of BFP⁺ cells) was determined by flow cytometry and expression was confirmed by confocal microscopy.

CD9⁺Lin⁻CD34⁺CD45RA⁻ and CD9⁻Lin⁻CD34⁺CD45RA⁻ HSPCs were cultured in 96-well plates with StemSpan SFEM II medium (09655, STEMCELL Technologies, Vancouver, BC, Canada) supplemented with recombinant human stem-cell factor (rhSCF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-3 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-6 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-9 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh–G colony-stimulating factor (G-CSF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh–GM colony-stimulating factor (GM-CSF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh-thrombopoietin (TPO; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), and 20% BIT 9500 Serum Substitute (09500, STEMCELL Technologies, Vancouver, BC, Canada). Besides, 24-well Transwells (3470, Corning Incorporated, Corning, NY, USA) were used for the indirect co-culture of immune cell progenitors and CD9⁺Lin⁻CD34⁺CD45RA⁻ HSPCs, and additional rhIL-7 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA) was added in the transwell co-culture system. Lin⁻CD34⁺CD45RA⁺CD38⁺CD9⁺CD10⁺ and Lin⁻CD34⁺CD45RA⁺CD38⁺CD161⁺ were used for pre-B cells and NK/Tp enrichment, respectively (Supplemental Fig. 6d).^{68 (link)} All cultures were incubated at 37 °C in a humidified chamber under 5% carbon dioxide.