Ex vivo expansion and gene therapy using cord blood CD34+ cells

CD34+ cells can be efficiently isolated from cord blood by sequential Percoll, Ficoll-Hypaque, and immunomagnetic bead separation. This population, containing up to 25% progenitor cells and 0.2-1% stem cells, defined by a long-term culture-initiating cell (LTC-initiating cell) assay, can be expanded in vitro in the presence of a specific combination of cytokines resulting in a > 100,000-fold increase in cells, a > 2,000-fold increase in progenitors, and an 18- to 20-fold increase in LTC-initiating cells. These latter populations can be efficiently transfected with a retroviral vector expressing a mutated dihydrofolate-reductase gene that confers methotrexate resistance.