Isolation and Expansion of Umbilical Cord Blood-Derived Stromal Cells

CB was collected from the umbilical cord vein with informed consent of the mother (21 (link)). USSCs were generated from 94 (40.3%) of 233 CB. The mononuclear cell fraction was obtained by Ficoll (Biochrom) gradient separation followed by ammonium chloride lysis of RBCs. Cells were plated out at 5–7 × 10⁶ cells/ml in T25 culture flasks (Costar). Two different media were used to initiate growth of the adherent USSC colonies: myelocult medium (StemCell Technologies) and low glucose DMEM (Cambrex) with 30% FCS, dexamethasone (10⁻⁷ M; Sigma-Aldrich), penicillin (100 U/ml; Grünenthal), streptomycin (0.1 mg/ml; Hefa-pharma), and ultraglutamine (2 mM; Cambrex). Expansion of the cells was performed in the same media but with a lower concentration or in the absence of dexamethasone. Cells were incubated at 37°C in 5% CO₂ in a humidified atmosphere. When cells reached 80% confluency, they were detached with 0.25% trypsin (Cambrex) and replated 1:3 as outlined in Table S1 available at http://www.jem.org/cgi/content/full/jem.20040440/DC1. USSC karyotyping was performed by cytogenetic standard protocols.