Y 27632

NCRM5 and NCRM5-AAVS1-CAG-EGFP (NCRM5 iPSCs with CAG-EGFP integrated at Chr.19 AAVS1 safe harbor locus) were established by the iPSC core facility of the National Heart, Lung, and Blood Institute in NIH. Mart1-iPSC were kindly provided by Riken BBC, in Japan. SCD-iPSC line was established from bone marrow stromal cells isolated from a sickle cell disease patient.³⁴ (link) All hiPSCs were cultured on Matrigel (Corning) or iMatrix-511 (Nippi) coated dishes in xeno-free hiPSC medium Essential 8 (Invitrogen) or StemFit Basic02 (Ajinomoto Co., Inc). They were routinely passaged as small clumps/single cells using 0.5 mM EDTA in phosphate buffered saline (PBS) with the split ratio of 1:6 to 1:10 every 3 to 4 days after reaching 65%–80% confluence. After EDTA treatment, hiPSCs were transferred to new Matrigel or iMatrix-511 coated dishes in hiPSC medium supplemented with ROCK inhibitor Y-27632 (10 μM, R&D Systems Inc). Next day, the medium was changed to hiPSC medium without ROCK inhibitor.

E7VI medium: DF3S base media (Gibco #ME110262L1), SB341542 (5 µM), VEGFA165 (50 ng/mL), FGF2 (100 ng/mL), Transferrin (10.7 µg/mL), Insulin (20 µg/mL).
E8BAC medium: E8 base media (Gibco #A1517001), BMP4 (5 ng/mL), Activin A (25 ng/mL), CHIR 99021 (1 µM).
Differentiation of endothelial cells (ECs) was carried out following our previously published protocol.^{73 (link)}
Human induced pluripotent stem cells (iPSCs) were grown in E8 media on Matrigel coated plate. Next, cells were dissociated using Versene (Gibco #15040066) and incubate for 3–5 min at 37°C until colonies detach. Cells were cultured in Matrigel coated wells of 6 well plates in E8BAC media for 2 days (to 100% confluence) with the addition of 10 µM Y27632 (R&D Systems #1254/10). Cells were then grown in E7VI medium for an additional 3 days. Cells were then harvested with Accutase (Gibco #A1110501) to produce a single cell suspension and centrifuged at 300×g for 5 min and resuspend 1 × 10⁸ cells in 300 µL of cold MACS buffer consisting of 1× PBS, 0.5% BSA (Fisher #BP1600), and 2 mM EDTA (Invitrogen #15575020). A 100 µL of FcR blocking reagent and 100 µL of CD34 microbeads (Miltenyi Biotec #130-046-702) were added and the mixture was incubated at 4°C for 30 min. Endothelial cells (ECs) were then isolated with CD34 microbeads by auto MACS (Miltenyi) to yield purified populations of CD34⁺CD31⁺ cells, which were cryopreserved.