Jensen, Bettina M6; Frandsen, Pernille7; Raaby, Ellen Margrethe Nedergaard4; Oluf Schiøtz, Peter5; Skov, Per S5; Poulsen, Lars K.6
1 Department of Immunology and Microbiology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Københavns Universitet2 Department of Clinical Medicine, Department of Clinical Medicine, Faculty of Health and Medical Sciences, Københavns Universitet3 Aalborg University4 Institut for Klinisk Medicin - Børneafdeling A, SKS5 unknown6 Department of Clinical Medicine, Department of Clinical Medicine, Faculty of Health and Medical Sciences, Københavns Universitet7 Aalborg University
Different protocols exist for in vitro development of HuMCs from hematopoietic stem cells, which results in distinct mast cells regarding molecular markers and activation patterns. Here, we introduce a SR profile using immunological, neurogenic, and pharmacological stimuli to characterize cellular functionality. Mast cells were obtained from three culture protocols using two types of PBdMCs (CD34(+) PBdMC or CD133(+) PBdMC) and one type of CBdMC (CD133(+) CBdMC). We analyzed resting cells for specific mast cell markers at protein and mRNA levels, thereby creating a molecular profile. To characterize the SR profile, we stimulated cells with anti-IgE, C3a, C5a, Substance P, or Compound 48/80 and measured the release of histamine and cytokines (IL-10, IL-13, GM-CSF, TNF-α). Molecular profiling revealed that CD133(+) CBdMC expressed less chymase, FcεRIα, and CD203c but more CD117 compared with CD34(+) and CD133(+) PBdMC. The SR profile for histamine release illustrated a functional heterogeneity between PBdMC and CBdMC. PBdMC released >10% histamine upon stimulation with anti-IgE, C3a, Substance P, and Compound 48/80, whereas CBdMC only reacted to C3a. Cytokine secretion was only detected after anti-IgE stimulation. Here, the SR profile identified the CD133(+) PBdMC as the most active cells regarding secretion of IL-10, IL-13, GM-CSF, and TNF-α. Cells from all three culture protocols, however, produced IL-10 spontaneously at comparable levels. We recommend validating mast cell cultures by means of molecular and SR profiles to characterize the mast cells and enhance consensus among studies.
Journal of Leukocyte Biology, 2014, Vol 95, Issue 6, p. 893-901