Human recombinant vegf a165

To obtain endothelial differentiation of mouse ES cells, cells were mildly trypsinized and suspended in Iscove's modified Dulbecco medium with 15% FBS, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 450 μM monothioglycerol, 10 μg ml⁻¹ insulin, 50 ng ml⁻¹ human recombinant VEGF-A165 (Peprotech Inc.), 2 U ml⁻¹ human recombinant erythropoietin (Cilag AG) and 100 ng ml⁻¹ human basic fibroblast growth factor (bFGF) (Genzyme). Cells were seeded in Petri dishes and cultured for 4 or 7 days at 37 °C with 5% CO₂ and 95% relative humidity.

MSCs were isolated, characterized, and differentiated from Yucatan microswine femurs as reported previously by our group [22 (link)]. All animal procedures were in compliance with applicable federal, state, and local laws and regulations, and institutional policies. The Institutional Animal Care and Use Committee of Creighton University approved the animal research protocol. Cells used for experiments in this study were between passages 3 and 5. The isolated MSCs were CD14^–CD45^–CD44⁺CD90⁺CD105⁺. The growth media used to harvest and culture MSCs was Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. The differentiation media (DM) used for differentiation was endothelial growth media 2 (EGM-2) containing 2 ng/ml, 25 ng/ml, or 50 ng/ml recombinant human VEGF-A₁₆₅ (Peprotech, Rocky Hill, NJ, USA), and/or 2 ng/ml, 25 ng/ml, or 50 ng/ml recombinant human Ang II (Sigma; St. Louis, MO, USA). Basic EGM-2 was used as negative control DM. Stimulation began when MSCs were at 50% confluency and continued for 10 days. The cell cultures were maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide. Media containing varying concentrations of VEGF-A, Ang II, and ATR inhibitor were changed every 48 hours.

All antibodies used in this study, their origin, and working concentrations are listed in Table 1. Recombinant human VEGFA₁₆₅ (PeproTech) and human VEGFC (Sigma-Aldrich) were used for in vivo intradermal injections and in vitro experiments.

A total of 3 × 10⁴/well GFP-MCs were seeded in a 24-well plate in full-growth MC medium and grown for 3 days. MC medium was refreshed every other day. On day 3, cells were either given fresh MC medium or cultured in basal MC medium (1% FBS) or in basal MC medium supplemented with 50 ng/mL recombinant human VEGF-A₁₆₅ (100-20; Peprotech, London, UK) and culture for another 3 days.