In vitro expansion and lineage commitment of the human umbilical cord blood myeloid progenitors

Abstract

In vitro proliferation and myeloid differentiation of humanumbilical cord blood (UCB) CD34+ cells with/without bonemarrow multipotential mesenchymal stromal cells (BMMMSCs)feeding layer in serum-free medium supplementedwith IL-3, IL-6, SCF and Flt3/l were investigated.Mononuclear cells (MCs) were isolated from the healthydonor UCB specimens (n = 4), CD34-enriched MCs wereobtained by positive immunomagnetic selection (CD34+-rich).Both cell fractions (MCs and CD34+-rich) were expanded fortwo weeks in vitro. Cells were seeded to the new vesselswhen necessary. The percentage of CD34/CD45-positive cellswas accounted by flow cytometry, subpopulations of myeloidprogenitors were assessed in colony-forming tests.Upon two weeks of culture the number of CD34+ cells inMC fraction augmented by 70±29 and 980±414-fold withand without BM-MMSC feeding layer respectively and wasnot significantly differed from that in CD34+-rich fraction.Progenitor cell subpopulations were not altered during thefirst week of culture but granulocytopoiesis was superiorover erythropoiesis during the second week. CD34 andCD45 expression patterns revealed two hematopoietic cellpopulations, one of them differentiating on the first week ofexpansion, and the other - on the second one. BM-MMSCfeeder promoted significantly higher (p < 0,05) expansionrates of CD34+ cells and myeloid progenitors in comparisonwith non-feeding culture. BM-MMSC feeder also causedprogenitor cells redistribution between suspension andadhesive fractions by predominantly binding erythroid andmultipotential progenitors. Optimum expansion protocol formultipotential progenitors was culturing CD34+-rich cellpopulation with BM-MMSC feeding layer for a week whichresulted in the increase the amount of the nucleated cells,CD34+ cells, multi-potential progenitors, and colony-formingunits by 56, 36, 45, and 60-fold respectively.

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