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In vitro monolayer endothelial differentiation of murine iPSCs

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  1. Protocol


  1. Resource Description
    To induce endothelial differentiation, approximately 1x105 undifferentiated iPSCs were seeded in each well of Matrigel-coated 6-well plates and cultured in differentiation medium containing RPMI and B-27 supplement minus insulin (Life Technologies) with 5 μM CHIR-99021 (a glycogen synthase kinase [GSK]-3 inhibitor; Selleck Chemicals, Houston, TX, USA) for 2 days, followed by RPMI and B-27 supplement minus insulin with 2 μM CHIR-99021 for 2 additional days. The medium was then changed to RPMI and B-27 supplement minus insulin with 50 ng/mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 10 ng/mL fibroblast growth factor basic (FGFb; R&D Systems), and 10 μM Y-27632 (a rho-associated protein kinase [ROCK] inhibitor; Sigma-Aldrich, Saint Louis, MO, USA) for 3 days. For the subsequent 7 days, the medium was changed to RPMI and B-27 supplement (with insulin) with 50 ng/mL VEGF, 10 ng/mL FGFb, 10 μM Y-27632, and 1 μM SB 431542 (a transforming growth factor [TGF]-β inhibitor; Sigma-Aldrich). At day 14, iPSC-ECs were sorted for CD31+/CD144+ markers using FACS and expanded on 0.2% gelatin coated plates. After sorting, iPSC-ECs were cultured in EBM-2 Basal Medium supplemented with the EGM-2 BulletKit (Lonza, Basel, Switzerland) at 37°C, 20% O2, and 5% CO2 in a humidified incubator with medium changes every 48 hours, and cells were passaged once they reached 80-90% confluence. Murine iPSCECs used for in vitro and in vivo characterizations were between passages 3 and 5.
  2. Used by
    Rabinovitch Lab
  3. Related Publication or Documentation
    Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism
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