Abstract
Eosinophil differentiation is a complex series of events regulated by cytokines at multiple levels, including proliferation, survival, and maturation. The development of an ex vivo eosinophil differentiation model, using the current knowledge on factors involved in this process, has facilitated efforts to understand the molecular mechanisms underlying human eosinophil development. Differentiation of human hematopoietic progenitor cells, isolated by density centrifugation and immunomagnetic cell separation, towards mature eosinophils, involves a 17-day culture period in the presence of a mixture of cytokines. At early stages of differentiation, these cells can be retrovirally transduced resulting in modulation of the expression of genes of interest to examine their role in eosinophil development. Eosinophil maturation can be analyzed by combining three different methods: histochemical analysis, flow cytometric analysis, and Luxol Fast Blue staining. In addition to this ex vivo differentiation model, human hematopoietic progenitors can be transplanted into immune-deficient mice resulting in the development of all human hematopoietic lineages in the mouse bone marrow, including eosinophils. Although the ex vivo differentiation model can be used separately, combining it with the transplantation model will give insight into not only regulation of human eosinophil development but also hematopoiesis in general.
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References
Majeti R, Park CY, Weissman IL (2007) Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell 1(6):635–645, Pubmed: 18371405
Hoebeke I, De Smedt M, Stolz F, Pike-Overzet K, Staal FJ, Plum J, Leclercq G (2007) T-, B- and NK-lymphoid, but not myeloid cells arise from human CD34(+)CD38(-)CD7(+) common lymphoid progenitors expressing lymphoid-specific genes. Leukemia 21(2):311–319, Pubmed: 17170726
Manz MG, Miyamoto T, Akashi K, Weissman IL (2002) Prospective isolation of human clonogenic common myeloid progenitors. Proc Natl Acad Sci U S A 99:11872–11877, Pubmed: 12193648
Mori Y, Iwasaki H, Kohno K, Yoshimoto G, Kikushige Y, Okeda A, Uike N, Niiro H, Takenaka K, Nagafuji K, Miyamoto T, Harada M, Takatsu K, Akashi K (2009) Identification of the human eosinophil lineage-committed progenitor: revision of phenotypic definition of the human common myeloid progenitor. J Exp Med 206(1):183–193, Pubmed: 19114669
Sitnicka E, Buza-Vidas N, Larsson S et al (2003) Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells. Blood 102:881–886, Pubmed: 12676789
Yamaguchi Y, Suda T, Suda J et al (1988) Purified interleukin 5 supports the terminal differentiation and proliferation of murine eosinophilic precursors. J Exp Med 167:43–56, Pubmed: 3257253
Sanderson CJ, Warren DJ, Strath M (1985) Identification of a lymphokine that stimulates eosinophil differentiation in vitro. Its relationship to interleukin 3, and functional properties of eosinophils produced in cultures. J Exp Med 162:60–74
Buitenhuis M, Baltus B, Lammers JW, Coffer PJ, Koenderman L (2003) Signal transducer and activator of transcription 5a (STAT5a) is required for eosinophil differentiation of human cord blood-derived CD34+ cells. Blood 101(1):134–142, Pubmed: 12393707
Buitenhuis M, Verhagen LP, Cools J, Coffer PJ (2007) Molecular mechanisms underlying FIP1L1-PDGFRA-mediated myeloproliferation. Cancer Res 67(8):3759–3766, Pubmed: 17440089
Buitenhuis M, van Deutekom HW, Verhagen LP, Castor A, Jacobsen SE, Lammers JW, Koenderman L, Coffer PJ (2005) Differential regulation of granulopoiesis by the basic helix-loop-helix transcriptional inhibitors Id1 and Id2. Blood 105(11):4272–4281, Pubmed: 15701714
Ulfman LH, Kamp VM, van Aalst CW, Verhagen LP, Sanders ME, Reedquist KA, Buitenhuis M, Koenderman L (2008) Homeostatic intracellular-free Ca2+ is permissive for Rap1-mediated constitutive activation of alpha4 integrins on eosinophils. J Immunol 180(8):5512–5519, Pubmed: 18390735
Buitenhuis M, Verhagen LP, van Deutekom HW, Castor A, Verploegen S, Koenderman L, Jacobsen SE, Coffer PJ (2008) Protein kinase B (c-akt) regulates hematopoietic lineage choice decisions during myelopoiesis. Blood 111(1):112–121, Pubmed: 17890457
Beekman JM, Verhagen LP, Geijsen N, Coffer PJ (2009) Regulation of myelopoiesis through syntenin-mediated modulation of IL-5 receptor output. Blood 114(18):3917–3927, Pubmed: 19654410
Geest CR, Zwartkruis FJ, Vellenga E, Coffer PJ, Buitenhuis M (2009) Mammalian target of rapamycin activity is required for expansion of CD34+ hematopoietic progenitor cells. Haematologica 94(7):901–910, Pubmed: 19535348
Geest CR, Buitenhuis M, Laarhoven AG, Bierings MB, Bruin MC, Vellenga E, Coffer PJ (2009) p38 MAP kinase inhibits neutrophil development through phosphorylation of C/EBPalpha on serine 21. Stem Cells 27(9):2271–2282, Pubmed: 19544470
de Bruin AM, Buitenhuis M, van der Sluijs KF, van Gisbergen KP, Boon L, Nolte MA (2010) Eosinophil differentiation in the bone marrow is inhibited by T cell-derived IFN-gamma. Blood 116(14):2559–2569, Pubmed: 20587787
Kollet O, Peled A, Byk T, Ben-Hur H, Greiner D, Shultz L, Lapidot T (2000) beta2 microglobulin-deficient (B2m(null)) NOD/SCID mice are excellent recipients for studying human stem cell function. Blood 95(10):3102–3105, Pubmed: 10807775
Glimm H, Eisterer W, Lee K, Cashman J, Holyoake TL, Nicolini F, Shultz LD, von Kalle C, Eaves CJ (2001) Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice. J Clin Invest 107(2):199–206, Pubmed: 11160136
Kikly KK, Bochner BS, Freeman SD, Tan KB, Gallagher KT, D’alessio KJ, Holmes SD, Abrahamson JA, Erickson-Miller CL, Murdock PR, Tachimoto H, Schleimer RP, White JR (2000) Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells and basophils. J Allergy Clin Immunol 105:1093–1100, Pubmed: 10856141
Floyd H, Ni J, Cornish AL, Zeng Z, Liu D, Carter KC, Steel J, Crocker PR (2000) Siglec-8: a novel eosinophil-specific member of the immunoglobulin superfamily. J Biol Chem 275:861–866, Pubmed: 10625619
Acknowledgements
The author thanks the current and past members of the departments of Respiratory Medicine and Immunology at the University Medical Center Utrecht (the Netherlands), the Department of Hematology at the Erasmus MC (the Netherlands), and the Hematopoietic Stem Cell Laboratory at the Lund Strategic Research Center for Stem Cell Biology and Cell Therapy in Sweden for helping develop these protocols.
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Buitenhuis, M. (2014). Cell Signalling During Human Eosinophil Differentiation. In: Walsh, G. (eds) Eosinophils. Methods in Molecular Biology, vol 1178. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1016-8_6
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DOI: https://doi.org/10.1007/978-1-4939-1016-8_6
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