Production of functional myeloid cells from CD34-selected hematopoietic progenitor cells using a clinically relevant ex vivo expansion system.

There is increasing clinical interest focused on ex vivo manipulation and expansion of hematopoietic cells. In this study, we demonstrate that a simple combination of growth factors can expand progenitors to yield functional myeloid cells. Furthermore, this system can produce mature, functionally competent cells in the absence of fetal bovine serum (FBS), which will enhance the clinical utility of this approach. Hematopoietic progenitor cells obtained from normal bone marrow and from leukapheresis products were studied. The mononuclear fraction was enriched for CD34 cells using the Ceprate CD34 biotin kit (CellPro #LC34-1 or LC34-2). The selected cells were expanded for two weeks in Iscove's medium supplemented with 20% FBS and various combinations of interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF) and interleukin -6 (IL-6) added either simultaneously or sequentially. The optimal combination of these factors identified for myeloid expansion was simultaneous addition of IL-3, SCF and G-CSF (at 50 ng/ml each), resulting in an average 773 +/- 133-fold expansion of nucleated cells (n = 5). When corrected for the purity of CD34 cells in the starting population, the mean fold expansion with IL-3, SCF and G-CSF was 2,265 +/- 729. A mean of 74.7 +/- 10.5% (n = 3) of the expanded cells was positive for CD11b; 86-91% (n = 2) of the cells were promyelocytes or more mature granulocytes. Functional assays demonstrated normal phagocytosis and intracellular killing of Staphylococcus aureus (S. aureus) by the expanded cell population. Studies performed using cells expanded in defined serum-free media demonstrated that fold expansion was decreased and that the cells produced were less mature and functionally less competent than cells expanded with FBS. The decreased expansion could be partially reversed, and the functionality almost completely restored by the addition of autologous plasma.

Identification of conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit critical for function. Evidence for formation of a heterodimeric receptor complex prior to ligand binding.

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues, a tryptophan-serine motif (WSXWS) in the extracellular domain, and a proline-rich cytoplasmic domain. The high affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (hGM-CSFR) consists of two subunits, alpha (hGM-CSFR alpha) and beta (hGM-CSFR beta), both of which are members of the receptor superfamily. In this study, we prepared mutations in conserved amino acids of the receptor subunit necessary for GM-CSF binding (hGM-CSFR alpha) and analyzed mutant receptors for low affinity binding, internalization, and high affinity binding when complexed with the beta subunit. Mutations in the cytoplasmic domain did not affect GM-CSF binding or receptor internalization. Mutation of a single conserved serine residue within the WSXWS motif diminishes cell surface receptor expression but not ligand binding. Mutation of either the second or third conserved cysteine residue of hGM-CSFR alpha resulted in complete loss of low affinity binding; however, co-expression of the cysteine 2 mutant with hGM-CSFR beta yielded a high affinity receptor complex. Since neither the cysteine 2 mutant nor the beta subunit can bind ligand alone, this result suggests that hGM-CSFR alpha and hGM-CSFR beta exist in a preformed heterodimeric protein complex on the plasma membrane.

Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts.

Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients. After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived. The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF. In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d. In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts. Culture with fibroblasts alone did not support eosinophil survival. The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts. Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition. In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S. mansoni larvae than the freshly isolated, normodense cells from the same donor. Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes. In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.