Cell Signalling During Human Eosinophil Differentiation
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.
Key wordsHuman eosinophil differentiation Hematopoietic progenitors CD34 Retroviral transduction Flow cytometric analysis β2-Microglobulin (−/−) NOD/SCID mice Histochemical analysis
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.
- 2.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: 17170726PubMedCrossRefGoogle Scholar
- 4.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: 19114669PubMedCentralPubMedCrossRefGoogle Scholar
- 5.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: 12676789PubMedCrossRefGoogle Scholar
- 11.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: 18390735PubMedCrossRefGoogle Scholar
- 18.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: 11160136PubMedCentralPubMedCrossRefGoogle Scholar
- 19.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: 10856141PubMedCrossRefGoogle Scholar