Macs sorter

iPSC-ECs from healthy control (control 1) and HLHS patient (HLHS 3) were dissociated using Accutase (Gibco) and resuspend in EGM-2 medium to maintain cell viability >90%. Human fetal heart was micro-dissected into left and right ventricular free wall, minced, and digested using Liberase (Sigma). Following digestion for 10 min, EGM-2 medium was added to stop the reaction and cells were transferred through a 100 μm cell strainer and further purified to CD144⁺ and CD144⁻ populations using the MACS sorter (Miltenyi Biotec). Cells with viability>90% were sent to Stanford Functional Genomics Facility (SFGF) for 10X chromium single-cell RNA-seq paired end library preparation using V2 version (10X Genomics). All samples were uniquely indexed, mixed, and evenly distributed into the Illumina HiSeq 4000 for sequencing.

We differentiated ECs from iPSCs based on a previous published protocol reported by our group (9 (link)). Briefly, iPSCs (over passage 15) were cultured to 70% confluence and placed in differentiation medium (RPMI and B-27 supplement minus insulin, Life Technologies) with 6 μM CHIR-99021 (Selleck Chemicals) for two days, followed by 3 μM CHIR-99021 for another two days. The medium was then changed and 50 ng/mL vascular endothelial growth factor (VEGF; Gemini), and 25 ng/mL basic fibroblast growth factor (FGFb; Gemini) were added for four additional days. On the eighth day, iPSC-ECs were sorted for CD144⁺ using antibody-coated beads and a magnetic-activated cell sorter (MACS) sorter (Miltenyi), and expanded on 0.2% gelatin coated plates. After sorting, iPSC-ECs were cultured in Endothelial Cell Basal Medium-2 (EBM-2) supplemented with the EGM-2 BulletKit (Lonza) at 37°C, 21% O₂, and 5% CO₂ in a humidified incubator with medium changes every 48 hours. Cells were passaged once they reached 80%–90% confluence. iPSC-ECs used for experiments were between passages 2 and 5.