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The Impact of Progenitor Enrichment, Serum, and Cytokines on the Ex Vivo Expansion of Mobilized Peripheral Blood Stem Cells: A Controlled Trial

The aim of this study was to verify, and possibly improve, culture conditions to expand human mobilized peripheral blood stem cells (PBSCs). We investigated the role of three parameters: A) the culture medium (serum‐free versus serum‐dependent); B) the initial cell population (Ficoll‐separated mononucleated cells versus CD34+‐selected cells), and C) the low concentration of recombinant cytokines, flt3 ligand, and thrombopoietin in association with a basic cocktail of stem cell factor, interleukin (IL)‐6, IL‐3, GM‐CSF, and erythropoietin. Eighteen leukapheresis samples were monitored in static culture for 15 days. The expansion potential was assessed at day 10 and 15 by total nuclear cells, colony‐forming‐units (CFUs) (burst‐forming units‐erythroid [BFU‐E], colony‐forming units‐granulocyte‐macrophage [CFU‐GM], and colony‐forming units‐granulocyte‐erythroid‐macrophage‐megakaryocyte [CFU‐GEMM]), and flow cytometry immunophenotyping (CD34+/CD38−, CD38+, CD33+, CD41+, GlyA+ progenitor cells). The results, evaluated by multivariate analysis of variance, emphasize that some variables affected the outcome of stem and progenitor cell expansion. CD34+ enrichment increased expansion of total nuclear cells, number of CD38+ and CD33+ late precursors, and number of the CFU‐GM compartment. Interestingly, however, quantitative expansion of GlyA+ and the early progenitor cells (CD34+/CD38−, CFU‐GEMM, BFU‐E) are favored by the use of unselected mononucleated cells. Regarding the role of serum, no significant difference was observed except for expansion of total nuclear cells, CFU‐GM, and BFU‐E. Cytokine combinations, in particular the use of flt3 ligand, stimulated expansion of almost all the cellular subsets, reaching a statistical significance for total nuclear cells and CFU‐GM. Our study indicates that progenitor and late precursor multilineage cell compartments of mobilized PBSCs may be significantly expanded in short‐term cultures by well‐defined experimental conditions. Furthermore, these data might be useful when evaluating ex vivo expansion of hematopoietic cells for clinical purposes.