Growth kinetics of progenitor cell-enriched hematopoietic cell populations in long-term liquid cultures under continuous removal of mature cells

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.

Introduction

Understanding the biology of hematopoietic stem and progenitor cells has become a major goal for scientists working in the hematology and stem cell arenas [1., 2., 3.]. Such an interest has derived not only because these cells represent excellent in vitro and in vivo models for the study of cellular processes, such as proliferation, differentiation and plasticity, but also because of their relevance in understanding the origin of some hematologic disorders (such as leukemias, myelodysplasia and aplastic anemia) and because of their current and future clinical applications [4, 5].

Several in vitro systems have been developed for the study of hematopoietic cells. They include semi-solid cultures, for the identification and enumeration of progenitors capable of forming hematopoietic colonies (colony-forming cells; CFC [6]), and liquid cultures, established in the presence or absence of stromal cells in which proliferation and expansion of primitive hematopoietic cells can be sustained for several weeks [7, 8]. In this regard, stroma-free liquid cultures supplemented with combinations of early- and late-acting stimulatory cytokines have proven particularly important, because they allow and promote the expansion of progenitor cells [9, 10]. Interestingly, this procedure has already been used in clinical settings with promising results; indeed, different groups have demonstrated that in vitro-expanded hematopoietic cells can be safely introduced into patients for the treatment of hematologic and non-hematologic disorders [11., 12., 13., 14.].

The adequate input cell population for the in vitro culture of hematopoietic cells depends on the particular objectives of each study; hence both mononuclear (MNC) and CD34⁺ cells are widely used. It is noteworthy that in the vast majority of studies aimed at the expansion of primitive progenitors, input cells consist of pure populations of CD34⁺ cells (>95% of total cells in the selected fraction) obtained by positive selection via cell sorting or immunomagnetic columns [15]. Such an approach has resulted in significant expansion of progenitor cells, although there is controversy as to whether this in vitro system is effective in expanding more primitive (stem) cells [16, 17]. More recently, and in trying to explore alternative approaches for the culture and expansion of hematopoietic cells, we have reported on the characterization of hematopoietic cell populations obtained from BM, umbilical cord blood (UCB) and mobilized peripheral blood (MBP) by means of negative selection [18, 19]. Such populations were enriched for CD34⁺ cells (40–90% of total cells in the selected fraction) and their proliferation and expansion potentials were assessed in serum- and stroma-free liquid cultures supplemented with combinations of stimulatory cytokines. In those studies, we demonstrated that, in spite of not being a pure population of CD34⁺ cells, the selected cells showed extensive proliferation and expansion capacities. Considering that the actual cell numbers recovered from the negative selection procedure may be even higher than those recovered after cell sorting, such populations may represent a good alternative for pre-clinical and clinical expansion protocols.

It is noteworthy, however, that in this in vitro system selection of primitive cells is performed only once, at culture onset, and further (secondary, tertiary, etc.) cultures are established with a small fraction of the total (unselected) cells generated every 5 or 7 days. As the culture period proceeds, there is generation of large numbers of mature, lineage-positive cells, mostly myeloid, which, 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 [20., 21., 22., 23., 24., 25.]. Thus it is possible that, under such culture conditions, hematopoietic stem and progenitor cells are unable to display their true proliferation and expansion potentials. With this in mind, the major goal of the present study was to assess the proliferation and expansion capacities of hematopoietic cell populations, enriched for CD34⁺ cells, in cytokine-supplemented liquid cultures under continuous removal of mature cells by means of reselection of primitive, lineage-negative (Lin⁻) cells.