Recombinant human vascular endothelial growth factor

Patient-specific iPSCs were differentiated into hematopoietic stem cells (HSCs) under defined, serum-free and feeder-free conditions as previously reported.^{20 (link)} Briefly, TrypLE (Invitrogen)-dissociated iPSCs were seeded onto 6-well plates that were precoated with 3 mg cm⁻² human plasma fibronectin (Invitrogen) in mTeSR1 supplemented with an inhibitor of Rho-associated kinase (H1152, Sigma). After 1 day, mTeSR1 was replaced with a hematopoietic commitment medium containing Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 1 × HIT (Stem Cell Technologies), 450 μM monothioglycerol (Sigma), 50 ng ml⁻¹ recombinant human BMP4 (R&D Systems), 50 ng ml⁻¹ recombinant human vascular endothelial growth factor (Invitrogen), 0.1 mM NEAA (Invitrogen) and 2 mM L-glutamine (Invitrogen). After 6 days, the medium was changed to a hematopoietic maturation medium consisting of Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 5 U ml⁻¹ heparin (Sigma), 25 ng ml⁻¹ thrombopoietin (Invitrogen), 25 ng ml⁻¹ human recombinant stem cell factor (Invitrogen), 25 ng ml⁻¹ human recombinant FLT3L (Peprotech, Rocky Hill, NJ, USA), 10 ng ml⁻¹ interleukin-3 (Invitrogen) and 10 ng ml⁻¹ interleukin-6 (Invitrogen). The cells were incubated under hypoxic conditions (5% O₂ balanced with nitrogen).