Ex Vivo Expansion of Hematopoietic Progenitors from CD34⁺-Selected Cells on Mouse Embryonic Fibroblast Feeders in a Non-Contact Co-Culture Using Matrigel or Fibronectin-Coated Inserts.

Adult pluripotent stem cells can be characterized as cells with both the capacity to self-renew and the ability to differentiate into distinct hematopoietic cell lineages and also into other tissue types. Because of the important course to stem cell research and clinical application the development of ex vivo culture systems is crucial for an effective expansion of these hematopoetic progenitor cells. After positive selection, CD34⁺-cells from peripheral G-CSF stimulated blood of healthy donors were cultured for 2 weeks in a serum-free medium with 2% of cord blood serum and the addition of early-acting cytokines (SCF, IL-3, Flt3-ligand, LIF, and TPO). The cells were grown in 6-well inserts on a feeder layer of mouse embryonic fibroblastic cells (MEF). Both cell types were separated from each other by a microporous membrane allowing only diffusion of soluble factors like cytokines but not cell migration. Additionally, the membrane was coated with Matrigel, a basement membrane matrix equivalent (MG), or with fibronectin (FN). Cultures were sampled at days 4, 7, 10 and 14 for cell count, colony-forming units-assay and immunophenotyping by flow cytometry. After two weeks of culture, the highest proliferation of stem cells was found in the control (medium without MEF, Matrigel and FN; 364-fold) followed by MEF- and FN-cultures (341- and 306-fold, respectively). The lowest clonal production was estimated in the cultures with MG (MG alone: 124,4-fold and MEF+MG: 48,2-fold, respectively). Examining the cells for colony-forming units the highest number of colony-forming unit-granulocyte-erythrocyte-macrophage (CFU-GEM), burst-forming unit-erythrocyte (BFU-E), colony-forming unit-granulocyte-macrophage (CFU-GM) and colony-forming unit-erythrocyte (CFU-E) were found in cultures with MEF and MEF+FN. We found multipotential colonies (CFU-GEM and BFU-E) at the highest expansion rate with FN (13,3-fold) and MEF (8,2-fold) at the fourth day of culture and with both additives in a MEF+FN-culture at the seventh day (17,2-fold). In addition to these results, there were sporadic undifferentiated progenitor cell colonies in MEF+MG-culture up to the day 14, despite an initially lower growth rate than that on MEF alone. The percentages of CD34⁺-cells estimated by flow cytometry were downregulated continiously during the culture period. At day 7, the highest number of CD34⁺-cells was found in the cultures with MG (46,0%) and MEF+MG (42,0%). The maximum number of CD34⁺-cells expressing no CD38 but CD61 was found at day 4 in FN culture (20,4%) and MG culture (15,9 %), respectively. In summary, we recognized in cultures with MG as a factor similar to the natural microenvironment a correlation between the diminished proliferation rate of stem cells in favor of an increased generation of multipotential colonies combined with their delayed differentiation into granulocyte-macrophage and erythrocyte colonies. Appropiate feeder cells as well as matrix proteins like Matrigel or fibronectin may helpful in improving the ex vivo expansion of hematopoietic progenitor cells.