BACKGROUND: During the last few years there has been increasing interest, from both biologic and clinical points of view, in the ex vivo expansion of umbilical cord blood (UCB)-derived hematopoietic cells. This has brought about the need to characterize different cell populations present in UCB, and to explore different ex vivo approaches for the culture, expansion and biologic manipulation of these cells. METHODS: By using a negative-selection method, two UCB cell populations were obtained that were enriched for primitive lineage-negative (Lin-) cells, including those expressing the CD34 Ag (35-93% of the total cells in each fraction). Population I was enriched for CD34+ Lin- cells, whereas population II was enriched for CD34+ CD38- Lin- cells. Both populations were cultured in serum-free liquid cultures supplemented with different combinations of early and late-acting recombinant cytokines (all of them added at 10 ng/mL). Every 5-7 days proliferation, expansion and differentiation capacities of each population were determined, for a total period of 25-42 days. RESULTS: Both cell populations showed extensive proliferation and expansion capacities; however, population II [2300- and 232-fold increase in nucleated and colony-forming cell (CFC) numbers, respectively] was clearly superior in both parameters compared with population I (1120- and 20-fold increase in nucleated and CFC numbers, respectively). Depending on the cytokine combination used, granulocytes, macrophages and erythroblasts were preferentially produced. We also observed that both populations were highly sensitive to the inhibitory effects of tumor necrosis factor-alpha, even in the presence of stimulatory cytokines. DISCUSSION: This study demonstrates that the two progenitor cell-enriched populations obtained by negative selection possess extensive proliferation and expansion potentials in vitro, generating significant numbers of both primitive and mature cells. These cells may be a good alternative to purified CD34+ cells, obtained by positive selection, for pre-clinical and clinical protocols aimed at the ex vivo expansion of UCB cells.
BACKGROUND: During the last few years there has been increasing interest, from both biologic and clinical points of view, in the ex vivo expansion of umbilical cord blood (UCB)-derived hematopoietic cells. This has brought about the need to characterize different cell populations present in UCB, and to explore different ex vivo approaches for the culture, expansion and biologic manipulation of these cells. METHODS: By using a negative-selection method, two UCB cell populations were obtained that were enriched for primitive lineage-negative (Lin-) cells, including those expressing the CD34 Ag (35-93% of the total cells in each fraction). Population I was enriched for CD34+ Lin- cells, whereas population II was enriched for CD34+ CD38- Lin- cells. Both populations were cultured in serum-free liquid cultures supplemented with different combinations of early and late-acting recombinant cytokines (all of them added at 10 ng/mL). Every 5-7 days proliferation, expansion and differentiation capacities of each population were determined, for a total period of 25-42 days. RESULTS: Both cell populations showed extensive proliferation and expansion capacities; however, population II [2300- and 232-fold increase in nucleated and colony-forming cell (CFC) numbers, respectively] was clearly superior in both parameters compared with population I (1120- and 20-fold increase in nucleated and CFC numbers, respectively). Depending on the cytokine combination used, granulocytes, macrophages and erythroblasts were preferentially produced. We also observed that both populations were highly sensitive to the inhibitory effects of tumor necrosis factor-alpha, even in the presence of stimulatory cytokines. DISCUSSION: This study demonstrates that the two progenitor cell-enriched populations obtained by negative selection possess extensive proliferation and expansion potentials in vitro, generating significant numbers of both primitive and mature cells. These cells may be a good alternative to purified CD34+ cells, obtained by positive selection, for pre-clinical and clinical protocols aimed at the ex vivo expansion of UCB cells.