Comparative in vitro analysis of different hematopoietic cell populations from human cord blood: in search of the best option for clinically oriented ex vivo cell expansion.

BACKGROUND: Ex vivo expansion of hematopoietic stem and progenitor cells has become a priority in the experimental hematology arena. In this study we have obtained different hematopoietic cell populations from umbilical cord blood and simultaneously assessed their proliferation and expansion kinetics. Our main goal was to determine which one of these cell populations would be more suitable for clinical-grade ex vivo expansion. STUDY DESIGN AND METHODS: By using immunomagnetic-negative selection and cell sorting, five cell populations were obtained: unseparated mononuclear cells (MNCs; I); two lineage-negative cell populations, one enriched for CD34+ CD38+ cells (II) and the other enriched for CD34+ CD38- cells (III); and two CD34+ cell fractions purified by fluorescence-activated cell sorting, one containing CD34+ CD38+ cells (IV) and the other containing CD34+ CD38- cells (V). The kinetics of such populations were analyzed in both relative and absolute terms. RESULTS: No expansion was observed in Population I; in contrast, significant increments in the numbers of both progenitor and stem cells were observed in cultures of Populations II to V. Population V (reaching 12,800-fold increase in total cells; 1280-fold increase in CD34+ cells; 490-fold increase in colony-forming cells; and 12-fold increase in long-term culture-initiating cells) showed the highest proliferation and expansion potentials. CONCLUSION: Our study suggests that the cell fraction containing greater than 98% CD34+ CD38- cells would be the ideal one for large-scale ex vivo expansion; however, based on our data, it seems that, except for MNCs, all other cell populations could also be used as input cell fractions.

Individual and combined effects of mesenchymal stromal cells and recombinant stimulatory cytokines on the in vitro growth of primitive hematopoietic cells from human umbilical cord blood.

BACKGROUND AIMS: We have previously characterized the in vitro growth of two cord blood-derived hematopoietic cell populations in liquid cultures supplemented with recombinant cytokines. In the present study, we assessed the effects of bone marrow-derived mesenchymal stromal cells (MSC) on the growth of such cells. METHODS: CD34(+) CD38(+) Lin(-) and CD34(+) CD38(-) Lin(-) cells were obtained by negative selection, and cultured in the presence of marrow-derived MSC and/or early- and late-acting cytokines. Hematopoietic cell growth was assessed throughout a 30-day culture period. RESULTS: In the presence of MSC alone, both populations showed significant proliferation. Direct contact between MSC and CD34(+) cells was fundamental for optimal growth, especially for CD34(+) CD38(-) Lin(-) cells. In the presence of early-acting cytokines alone, cell growth was significantly higher than in cultures established with MSC but no cytokines. In cultures containing both MSC and early-acting cytokines, a further stimulation was observed only for CD34(+) CD38(-) Lin(-) cells. The cytokine cocktail containing both early- and late-acting cytokines was significantly more potent at inducing hematopoietic cell growth than the early-acting cytokine cocktail. When cultures were supplemented with early- and late-acting cytokines, MSC had no further effect on the growth of hematopoietic cells. CONCLUSIONS: MSC seem to play a key role, particularly on more primitive (CD34(+) CD38(-) Lin(-)) cells, only in the absence of cytokines or the presence of early-acting cytokines. When both early- and late-acting cytokines are present in culture, MSC seem to be unnecessary for optimal development of CFC and CD34(+) cells.