Rhil 9

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.

CD34 + HSPCs were differentiated to the megakaryocyte lineage by culturing for 11 days in IMDM based media containing 30 ng/mL rhTPO (PeproTech), 1 ng/mL rhSCF (PeproTech), 7.5 ng/mL rhIL-6 (PeproTech), 13.5 ng/mL rhIL-9 (PeproTech), 20% BIT supplement (StemCell Technologies), 40 µg/mL LDL (Millipore-Sigma), 0.05 mM beta mercaptoethanol (Millipore-Sigma), and 1x Penicillin/Streptomycin (ThermoFisher Scientific).