Gene Expression Accompanied by Differentiation of Cord Blood-Derived CD34+ Cells to Eosinophils

Abstract

To clarify the relation between the expression of genes such as eosinophil-specific granular proteins and cytokine receptors and the pathogenesis of allergic disease, cord blood-derived CD34+ cells were cultured and differentiated into eosinophils. Gene expression in the cells during the differentiation was determined by real-time reverse transcription PCR (ABI PRISM 7700). CD34+ mononuclear cells cultured with stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5 in Iscove’s MEM, and proliferated until the 2nd week, when the cell number reached a plateau. Under these conditions, more than 90% of the cells differentiate into mature eosinophils in 3 weeks. The expression of major basic protein and eosinophil-derived neurotoxin in the treated cells increased until week 2 and decreased between week 2 and 3. However, the expression of membrane receptor genes, such as IL-5 receptor (α chain), IL-3 receptor (α chain), GM-CSF-α receptor, GM-CSF-β receptor, CC chemokine receptor 3, interferon-γ receptor, platelet-activating factor receptor and leukotriene D₄ receptor, increased until the 3rd week of eosinophil maturation. Our study suggests that the in vitro eosinophil differentiation and maturation model is useful for clarifying the relation between eosinophil-specific gene expression during allergic diseases and the progression of the disease.

Gene expression screening of human mast cells and eosinophils using high-density oligonucleotide probe arrays: abundant expression of major basic protein in mast cells

“…Indeed, it has been reported by others 34 and by us 28 that the level of MBP transcripts in CB-derived developing eosinophils decreases after complete maturation. We also used Genechip to measure the transcripts of major granule proteins in eosinophils derived from 6 patients with atopic dermatitis.…”

“…Because immature CB-derived eosinophils express MBP mRNA but lose it after maturation, 28,34 PB eosinophils seem to be mature enough to produce more MBP. A similar dissociation between protein and mRNA after maturation is also found for histidine decarboxylase 38 and chymase 26 in human MCs.…”

“…The immature eosinophils were obtained by culturing CB CD34 ϩ cells in the presence of IL-5 plus IL-3, as described previously. 27,28 The RNA was DNase-treated, converted to cDNA by reverse transcription (RT) using a kit from Invitrogen (San Diego, CA), and subjected to polymerase chain reaction (PCR) amplification. The primer sequences for MBP were as follows: 5Ј-GTG CTA AGA CGC TGC CTG AG-3Ј for the 5Ј primer and 5Ј-TTT CAG TGG GTT GAC GGC-3Ј for the 3Ј primer, spanning a fragment of 440 bp.…”

Signal transducer and activator of transcription 5a (STAT5a) is required for eosinophil differentiation of human cord blood–derived CD34+ cells

“…Several studies have used similar ex vivo differentiation protocols for eosinophil differentiation from CD34 ϩ progenitor cells derived from cord blood or peripheral blood. [46][47][48] Recently, Hashida et al 49 demonstrated that 80% of the cord blood-derived CD34 ϩ cells in their study developed into mature eosinophils after 17 days of differentiation. However, they only distinguished between blast cells and mature eosinophils without mentioning the other stages of eosinophil differentiation.…”

Leukotriene B4 pathway regulates the fate of the hematopoietic stem cells

“…Crooks et al, 1998). Recently, it has been reported that cysteinyl leukotriene receptor is involved in differentiation of HSCs into eosinophils (Hashida et al, 2001). In addition, several eicosanoids have been reported to be involved in stem cell differentiation or proliferation (Desplat et al, 2000;Braccioni et al, 2002).…”