organism: Homo sapiens
description: iPSCs were differentiated in 24 days into SMCs using a small molecule-based monolayer differentiation protocol1. In brief, iPSC monolayers of 85% cell confluence were dissociated into single cells by Accutase (Thermo Fisher Scientific), seeded on Matrigel-coated dish at 105 cells/cm2 and cultured for 36 hours in E8BAC medium (E5 medium2 plus 5 ng/ml BMP4, 25 ng/ml Activin A, 19.4 mg/l insulin, 10 mM Y27632 and 1 mM CHIR99021). Cells were dissociated and seeded on a new dish coating Matrigel at 1.6 x 104 cells/cm2 in E6T medium (E5 medium supplemented with 19.4 mg/l insulin, 1.7 ng/ml TGF-b1 and 10 mM Y27632) for 18 hours and then cultured with E5F medium (E5 medium supplemented with 19.4 mg/l insulin and 100 ng/ml FGF2) (day 3-7). From day 8 to day 11, cells were treated with FVR medium (E5 medium supplemented with 19.4 mg/l insulin, 50 ng/ml VEGF and 5 mM RESV) and changed every other day. From day 12, cells were cultured in E6-TP medium (E5 medium supplemented with 19.4 mg/l insulin, 5 mM RESV, 10 ng/ml PDGFbb and 1.7 ng/ml TGF-b1). On day 16, cells were cryopreserved when necessary or split in a new Matrigel-coated dish at 1x105 cells/cm2 cell density. After day 24, iPSC-SMCs were maintained in DMEM supplemented with 5 mg/ml insulin (Thermo Fisher Scientific, Waltham, MA, USA9), 0.5 ng/ml EGF (Thermo Fisher Scientific, Waltham, MA, USA), 2 ng/ml FGF (Thermo Fisher Scientific, Waltham, MA, USA) and 5% FBS. 5% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). The cells were passaged with 0.25% Trypsin/EDTA solution (Thermo Fisher Scientific, Waltham, MA, USA).