Differentiation of hiPSCs to Platelets

According to a protocol previously established by Takayama et al. [29 (link)], hiPSC colonies were removed from MEF feeders using a dissociation buffer (0.25% trypsin, 1 mg/ml collagenase IV, 20% KSR, 1 mmol/l CaCl₂ in PBS), transferred onto irradiated C3H10T1/2 cells and differentiated with IMDM medium containing 10 mg/l insulin, 5.5 mg/l transferrin, 6.7 mg/ml selenium, 2 mmol/l L-glutamine, 15% fetal bovine serum (all Gibco), 0.45 μmol/l α-monothioglycerol (Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma-Aldrich) and 20 ng/ml recombinant human vascular endothelial growth factor (Invitrogen). On day 15, hiPSC-Sacs were disrupted with a cell scraper, crushed with a pipette and passed through a 40 μm cell strainer (BD Falcon). The yielded cells were transferred onto irradiated C3H10T1/2 cells and cultured in the same medium without vascular endothelial growth factor containing 100 ng/ml human TPO (R&D), 50 ng/ml human SCF (R&D), and 25 U/ml heparin (Sigma-Aldrich). Medium was changed every 3 days. According to Takayama et al. [29 (link)], floating cells from days 24 to 30 were collected for platelet and MK analysis.