DENSITY ENRICHMENT AND CHARACTERIZATION OF HEMATOPOIETIC PROGENITORS AND STEM-CELLS FROM UMBILICAL-CORD BLOOD

Umbilical cord blood (UCB) is being used for hematopoietic rescue afte r myeloablative therapy in a rapidly growing number of patients, Recen t developments of cord blood banking, ex vivo progenitor expansion and gene therapy techniques have raised the issue of efficient progenitor and stem cell enrichment procedures using UCB, We have used discontin uous density gradient techniques to analyze progenitor distribution in the mononuclear cell fraction of cord blood, This resulted in establi shment of a highly reproducible, rapid, cost-effective single-step den sity separation method that generates a light density fraction, which when compared to conventional mononuclear cells has a high number of c lonogenic progenitors, can be extensively expanded in vitro for up to 21 days and has the ability to sustain long-term hematopoiesis when in oculated on a preformed stromal layer, It can also serve as an efficie nt target for retrovirally mediated gene transfer, utilizing a vector expressing a mutated dihydrofolate reductase gene that confers methotr exate resistance.