Materials and Methods Mouse Embryonic Stem Cell Culture, Establishment and Treatment of EBs Murine C57BL/6x129SvEv-derived else (passage 3�C12) embryonic stem cells (kindly provided by N. Gale, Regeneron Pharmaceuticals, Tarrytown, N.Y., USA) were cultured on mitotically inactivated primary mouse embryonic fibroblasts (passage 2�C5; Institute of Laboratory Animal Science, University of Zurich, Switzerland) in Dulbecco’s modified Eagle medium (Gibco, Eggenstein, Germany), supplemented with 18% fetal bovine serum (Gibco), 100 nM sodium pyruvate (Sigma, Buchs, Switzerland), MEM vitamins, 2 mM L-glutamine, streptomycin and penicillin (all from Gibco), 10 mM 2-mercaptoethanol and 2,000 U/ml recombinant leukemia inhibitory factor (LIF; Chemicon International, Temecula, Calif., USA).
Primary mouse embryonic fibroblasts and LIF were removed and murine embryonic stem cells were transferred to suspension culture for EB formation as described [29]. After 3 or 4 days, EBs of the same size (approximately 500 ��m in diameter) were transferred into 12-well dishes (1 EB per well; BD Bioscience, San Diego, Calif., USA) and cultured for 14 days without LIF. Then, EBs were incubated with or without the following factors for 2, 4, 6, 8, 10, 12 or 14 days: 20 ng/ml recombinant human VEGF-A (VEGF-A 165; kindly provided by the National Cancer Institute, Bethesda, Md., USA); 200 ng/ml recombinant human VEGF-C (R&D Systems, Minneapolis, Minn.
, USA); 20 ng/ml human fibroblast growth factor-2 (kindly provided by the National Cancer Institute); 1 mg/ml hyaluronic acid sodium salt from human umbilical cord (Fluka, Buchs, Switzerland); 100 ng/ml recombinant human IGF-1 (R&D Systems); 25 ng/ml recombinant human IL-3 (Chemicon International); 30 ng/ml human hepatocyte growth factor (R&D Systems); 20 ng/ml human platelet growth factor (R&D Systems); 50 ng/ml human growth hormone (R&D Systems); 20 ng/ml recombinant human IL-7 (Chemicon International); 100 ��M S-nitroso-N-acetylpenicillamine (Sigma); 10 mM human endothelin-3 (R&D Systems); 1, 2.5, 5, 10 or 100 ��M all-trans-RA (Sigma); 10 ��M 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (Sigma); 1 or 10 ��M 13-cis-RA (Sigma); 0.5 mM N-6,2��-O-dibutyryl-adenosine 3��,5��-cyclic monophosphate (cAMP; Fluka); 10 ��M Ro 41-5253 (BioMol International, Plymouth Meeting, Pa.
, USA); 10 ��M N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide, Di-HCl salt (H89; Calbiochem, San Diego, Calif., USA). EBs were fixed in ?20��C cold 100% methanol or in 4% paraformaldehyde at 4��C for 10 min. Endothelial Cell Culture, Real-Time RT-PCR, FACS and Immunostains Human umbilical vein endothelial cells (HUVECs), obtained from ScienceCell Research Labs (San Diego, Calif., USA) were seeded into fibronectin-coated culture dishes (10 ��g/ml; BD Biosciences, Bedford, Mass., USA) and were cultured in endothelial cell basal medium Carfilzomib (Cambrex Bio Science, Walkersville, Md.