Human thrombopoietin tpo

HSCs were collected according to the manufacturer's protocol for the magnetic bead‐based EasySep Mouse SCA1 Positive Selection Kit (StemCell Technologies, Vancouver, British Columbia, Canada). Briefly, bone cells were collected from the femur and tibia of C57BL/6 mice and made into a suspension of 1×10⁶ cells mL⁻¹ in StemSpan Serum‐Free Expansion Medium (SFEM) (StemCell Technologies) containing human IL‐6 (100 ng mL⁻¹, StemCell Technologies, Canada), human fms‐like tyrosine kinase‐3 (Flt3) ligand (100 ng mL⁻¹, StemCell Technologies), murine stem‐cell factor (SCF) (50 ng mL⁻¹, StemCell Technologies), and human thrombopoietin (TPO) (20 ng mL⁻¹, PeproTech, Cranbury, New Jersey, USA). Mouse SCA1 PE Labeling Reagent (50 µL mL⁻¹, StemCell Technologies) was first added to the cell suspension, and the mixture was kept in the dark at room temperature for 15 min. Then, a PE Selection Cocktail (70 µL mL⁻¹, StemCell Technologies) was added to the mixture and kept in the dark at room temperature for 15 min. After vortexing, dextran RapidSphere (50 µL mL⁻¹, StemCell Technologies) was added to the mixture and stored at room temperature in the dark for 10 min. After the mixture was incubated in the magnet for 5 min, the supernatant was discarded and SFEM was added to resuspend the cells. This step was repeated three times to obtain HSCs, and the cells were used in passages 3–5.

Human iPS cells were dissociated into single cells with Accutase (Merck-Millipore, Burlington, MI). The cells were resuspended in differentiation medium I (mTeSR1 (STEMCELL Technologies, Vancouver, Canada), 50 μg/ml ascorbic acid, 0.45 mM MTG, 2 mM l-glutamine, and antibiotics) supplemented with 10 ng/ml human BMP4, 1 ng/ml Activin A, and 10 μM Y-27632 and were then plated on low-attachment dishes (day 0). On day 2, cell aggregates were cultured in differentiation medium II (Iscove’s modified Dulbecco’s medium (IMDM), 50 μg/ml ascorbic acid, 0.45 mM MTG, 2 mM L-glutamine, and antibiotics) supplemented with 10 ng/ml human BMP4 and 5 ng/ml human VEGF. On day 4, half of the medium was changed to differentiation medium II supplemented with 10 ng/mL human BMP4, 5 ng/mL human VEGF, and 10 μM SB431542. On day 6, cell aggregates were cultured in differentiation medium II supplemented with 2 ng/ml human BMP4, 5 ng/ml human VEGF, 10 ng/ml human stem cell factor (SCF) (Peprotech, Rocky Hill, NJ), and 10 ng/ml human thrombopoietin (TPO) (Peprotech). On day 10, differentiated cells were analyzed by flow cytometry with an LSR Fortessa flow cytometer.