Isolation of cord blood and in vitro cell culture

Normal human CB was provided by CORD:USE, a cord blood banking company in Orlando, FL, and Soonchunhyang University Bucheon/Cheonan Hospital in South Korea. This study was approved by local Institutional Review Boards (IRBs; 2016-04-008-004 and 2018-05-037-002). Fully informed consent was obtained from all patients prior to donation. Mononuclear cells were isolated by density gradient centrifugation over Ficoll-Plaque Plus (GE Healthcare, Marlborough, MA, USA) according to the manufacturer’s protocol. CD34⁺ cells were obtained through immunomagnetic selection (Miltenyi Biotec, Auburn, CA, USA) over two sequential columns. This yielded CD34⁺ cells with 90–98% purity. CD34⁺ cells were then cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 ng/mL stem cell factor (SCF), thrombopoietin (TPO), and FMS-LIKE TYROSINE KINASE 3 LIGAND (FLT3L) to expand cell numbers (28).