Fibroblast growth factor 2 fgf 2

The undifferentiated hiPSCs (BJ1) were maintained in mTeSR^TM 1 (STEMCELL Technologies, Vancouver, BC, Canada) on 5% matrigel at 37 °C, 5% CO₂. Differentiation of hiPSCs into ECs was performed following a protocol published by Lee et al.^{64 (link)} Briefly, for mesodermal differentiation, enzymatically dissociated clumps of hiPSCs (lower than passage 60) were cultured on 0.01% collagen-coated plates in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Gibco BRL, Gaithersburg, MD, USA) including 20% Serum Replacement (SR, Gibco BRL) with CHIR99021 (3 μM) (STEMCELL Technologies) and fibroblast growth factor 2 (FGF2, 5 ng ml⁻¹) (R&D systems, Minneapolis, MN, USA) for 3 days. The mesodermally differentiated cells were cultured in DMEM/F12 medium including 20% SR with VEGF-A (10 ng ml⁻¹) (R&D systems), delta-like ligand 4 (DLL4, 25 ng ml⁻¹) (R&D systems), epidermal growth factor (EGF, 5 ng ml⁻¹) (R&D systems), and FGF2 (5 ng ml⁻¹) for another 11 days, and hiPSC-derived CDH5 + cells were purified by magnetic-activated cell sorting (MACS, Miltenyi Biotec, Auburn, CA, USA)^{64 (link)}.

iPSCs were maintained in feeder-free conditions in mTeSR1 medium (STEMCELL Technologies, 05850) on hESC-Qualified Matrix (Corning, 8774552), using Gentle Cell reagent for passaging (STEMCELL Technologies, 07174). For targeting, iPSCs were replated on mitotic inactivated mouse embryonic fibroblast (MEF) feeder cells in human iPS medium [20% knockout serum (Life Technologies), GlutaMAX (Life Technologies), nonessential amino acids (Life Technologies), fibroblast growth factor 2 (FGF2) (10 ng/ml; R&D Systems), 100 μM β-mercaptoethanol (Life Technologies), and Primocin (InvivoGen) in Dulbecco’s modified Eagle’s medium/D12 (Life Technologies)]. iPSCs on feeders were passaged with collagenase type 4 (1 mg/ml; Gibco). All iPSCs displayed a normal karyotype when analyzed by G-banding (Cell Line Genetics). Additional details pertaining to iPSC derivation, characterization, and culture are available for free download at https://crem.bu.edu/cores-protocols/. iPSCs used here are available upon request from the CReM iPSC repository at https://stemcellbank.bu.edu.

Human recombinant Transforming Growth Factor-beta 1 (TGF-β1) and Platelet-Derived Growth Factor-BB (PDGF-BB) were obtained from Biochrom GmbH (Berlin, Germany). Fibroblast Growth Factor 2 (FGF2) and Insulin-like Growth Factor-I (IGF-I) were purchased from R&D Systems Europe (Abingdon, UK), while Epidermal Growth Factor (EGF) from Sigma (St. Louis, MO, USA). The TGF-β type I receptor (TGFβR1) kinase inhibitor SB431542 was from Sigma, while the PDGF receptor (PDGFR) kinase inhibitor STI571 from Novartis AG (Basel, Switzerland). The FGF receptor 1 (FGFR1) kinase inhibitor SU5402, the IGF-I receptor (IGFIR) kinase inhibitor tyrphostin I-OMe-AG538, and the EGF receptor (EGFR) kinase inhibitor tyrphostin AG1478 were obtained from Calbiochem-Merck KGaA (Darmstadt, Germany). The p38 MAPK inhibitor SB203580, and the NF-κB inhibitor BAY117082 were from Sigma, while the Sp1 inhibitor mithramycin from Cayman Chemical (Ann Arbor, MI, USA).