Abstract
Background During long-term culture of primitive hematopoietic cells large numbers of mature cells are generated that, on the one hand, consume nutrients and cytokines present in the medium and, on the other hand, may produce or elicit the production of soluble factors that limit the growth of primitive cells. Thus it is possible that under standard culture conditions hematopoietic stem and progenitor cells are unable to display their true proliferation and expansion potentials.
Methods Hematopoietic cell populations, enriched for CD34+ cells, were obtained from both umbilical cord blood (UCB) and mobilized peripheral blood (MPB), and cultured in cytokine-supplemented liquid culture, under continuous removal of mature cells by means of weekly reselection of primitive, lineage-negative (Lin−) cells. Proliferation and expansion capacities of such cells were determined weekly for a 42-day culture period.
Results As expected, based on our previous studies in standard liquid cultures, throughout the culture period there was a continuous decrease in the proportion of progenitor cells; however, after every reselection on days 7, 14 and 21, there was a significant enrichment for both CD34+ cells and colony-forming cells (CFC). As a result of such an enrichment, the cumulative increase in the numbers of total cells and CFC in cultures with two, three or four selections was significantly higher than the increments observed in standard cultures, in which only a single selection was performed on day 0. Cultures of UCB cells showed consistently higher levels of both total cells and CFC than cultures of MPB cells.
Discussion Taken together, these results indicate that continuous removal of mature cells from liquid cultures of primitive progenitors results in higher increments in the levels of both total cells and CFC.