4.1. iPSC Culture
We used iPSCs derived from
Normal Human Dermal Fibroblasts Neonatal (NHDF-Neo, Lonza, Milan, Italy) using the Sendai virus technology (
https://hpscreg.eu—IRFMNi001-B, released on 17 September 2020) and its subclone knockout for CIITA that we had recently generated using the CRISPR/Cas9 system (
https://hpscreg.eu—IRFMNi001-B-1, released on 25 June 2021). The cells were routinely cultured on Matrigel-coated dishes (Matrigel hESC-Qualified Matrix, Corning, Sial S.r.l., Rome, Italy) in mTeSR Plus medium (Stemcell Technologies, Cologne, Germany), detached using Accutase (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) twice a week, and plated at a density of 20,000–60,000 cells/cm
2.
4.2. Human iPSC CRISPR/Cas9-Mediated Genome Editing
For B2M knockout, 106 CIITA knockout iPSCs were nucleofected with 10 µg (52 pmol)-Cas9-GFP-protein (Sigma-Aldrich, St. Louis, MO, USA) precomplexed with 390 pmol sgRNA B2M custom (Sigma-Aldrich, St. Louis, MO, USA) (RNP:sgRNA = 1:7.5) according to the manufacturer’s instructions. Nucleofection was conducted using the A023 program with the Human Stem Cell Nucleofector Kit 2 and Nucleofector2b Device (Lonza, Milan, Italy). Transfected cells were plated on Matrigel-coated 6-well plates in mTeSR Plus with 10 µM Y-27632. After 24 h, GFP+ single cells were isolated through cell sorting (FACSAria IIu; Becton Dickinson Italia S.p.A., Milan, Italy), plated on MEF feeder-coated plates with a density of 500 cells/cm2 and then the single-cell-derived colonies were expanded for another 8 days. Thirty emergent clones were picked manually and plated on Matrigel-coated wells in mTeSR Plus medium.
4.8. Differentiation of iPSCs into Endothelial-like Cells
For expansion, CD144+ cells were grown in EGM-2 medium (Endothelial Cell Growth Medium-2, Lonza, Milan, Italy) without hydrocortisone, supplemented with 20% defined fetal bovine serum (FBS HyClone, Euroclone, Milan, Italy) and 10 µM SB431542 (Sigma–Aldrich St. Louis, MO, USA) (EC expansion medium). All experiments described in the manuscript were performed using iPSC-derived ECs between passages three and five.