Cd201 pe

VECs were differentiated as previously described (Pan et al., 2016 (link); Wu et al., 2018 (link)). In brief, human ESC clones were seeded in matrigel coated 6-well plates in mTeSR medium at day 0. ESCs were washed with IMDM medium (Gibco) and cultured in EGM-2 medium (Lonza) supplemented with 25 ng/mL BMP4 (R&D), 3 μmol/L CHIR99021 (Selleck), 3 μmol/L IWP2 (Selleck) and 4 ng/mL FGF2 (JPC) for 3 days. At day 4, Cells were rinsed with IMDM medium and cultured in EGM-2 medium supplemented with 50 ng/mL VEGF (HumanZyme), 10 ng/mL IL6 (Peprotech) and 20 ng/mL FGF2 (JPC) for 3 days. Endothelial cells were sorted with CD201-PE (Biolegend, 351904, 1:300), CD34-FITC (BD biosciences, 555821, 1:100). IgG-FITC (BD biosciences, 555748) and IgG-PE (BD biosciences, 555749) were used as isotype controls.

Unmanipulated UCB, BM or mobilized PB CD34⁺ cells or cells from cultures treated with cytokines alone or cultures treated with VPA were stained with anti-human CD34-APC (BD Pharmingen), CD90-FITC (Thermo Fisher Scientific), CD201-PE (Bio-Legend), CD45RAef-506 (Thermo Fisher Scientific), CD38 SB-702 (Thermo Fisher Scientific) for 30 min at 4°C, washed and analyzed using an Attune flow cytometer (Thermo Fisher Scientific). Analyses were performed with Attune software (Thermo Fisher Scientific) and FlowJo Software (BD Biosciences). Compensation parameters are assessed by using AbC^TM Total Antibody Compensation Bead Kit (Thermo Fisher Scientific) according to the manufacturer instructions. Doublet exclusion was performed by FSC-H/FSC-A selection followed by SSC-H/SSC-A selection. Gating of positive/negative population was performed by using fluorescence minus one control (FMO) strategy (Papa et al., 2020b (link)). Purification of the various CD34⁺ cell subpopulations is performed in media supplemented with cytokines or cytokines and VPA using a BD FACSAria (BD Bioscience).

Both RELA^+/+ and RELA^–/– human embryonic stem cells (hESCs) were differentiated into hVECs.^{31 (link),91 (link)–94} In brief, hESCs were picked into 6-well plate coated with Matrigel and cultured with mTeSR on day 0. hESCs were washed with IMDM (Gibco) and cultured with EGM-2 medium (Lonza) supplemented with 25 ng/mL BMP4 (R&D), 3 μM CHIR99021 (Selleck), 3 μM IWP2 (Selleck) and 4 ng/mL FGF2 (JPC) on day 1 through day 3. The medium was then replaced by EGM-2 medium supplemented with 50 ng/mL VEGF (HumanZyme), 10 ng/mL IL6, and 20 ng/mL FGF2 (JPC) for another 3 days. CD201-PE (Biolegend, 351904, 1:300) and CD34-FITC (BD biosciences, 555821, 1:100) were used to sort and purify hVECs.