Umbilical cord blood-derived hematopoietic stem cells that are expanded ex-vivo on a feeder layer of mesenchymal precursor cells.
The cord-blood unit with the lowest dose of total nucleated cells is thawed, and equal fractions are placed into 10 flasks containing mesenchymal stromal cells with 50 ml of Good Manufacturing Practices (GMP)–grade serum-free medium containing stem-cell factor, Flt3 ligand, thrombopoietin and granulocyte colony-stimulating factor (100 ng/ml).
After 7 days of coculture, the nonadherent cells are transferred to 10 1-liter culture bags containing 450 ml coculture medium with cytokines and cultured for an additional 7 days. On day 7 of culture, the flasks are replenished with 50 ml coculture medium containing the cytokines, and cultured for another 7 days. On day 14 of culture, all the nonadherent cells in the flasks and bags are pooled, washed, and infused.