Pediatr Allergy Respir Dis.  1999 Dec;9(4):412-420.

Characterization of IL-3, IL-5, GM-CSF-induced Eosinophils from Human CD34+ Cord Blood Cells

• Lee KS
• Ahn KM
• Nam SY
• Son DY
• Ham SC
• Han MY
• Lee SI

Abstract

PURPOSE
Eosinophils are cells of the granulocyte lineage that participate in host defence against parasitic disease and mediate allergic inflammation. In this study by using the combination of cytokines IL-3, IL-5 and GM-CSF, we explore the characterization of cultured eosinophils from CD34+ CBCs.
METHODS
Mononuclear cells were isolated heparinized umbilical cord blood by Ficoll-paque (1.077 g/ml) density gradient centrifugation method. The CD34-bearing hematopoietic progenitor cells were collected by elution after their adhesion to a magnetic cell sepatation (MACS) column. The CD34+ cells were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and were cultured in the presence of IL-3, IL-5, and GM-CSF in a 6-well plate bottomed tissue culture plate at 37 degrees C for 7-28 days in humidified 5% CO2 and 95% air atmosphere. For the identification of cultured eosinophils Wright's and Giemsa staining, RT-PCR, Southern blotting and FACS analysis are used.
RESULTS
We analyzed the cultured eosinophils Wright's and Giemsa staining, the total cell number of cells increased 50-fold by days 28 of culture. Also, using the sensitive RT-PCR technique, we monitered the appearance of mRNA transcrips of EPO. Identity of each RT-PCR product was confirms by southern blotting with independent gene-specific oligonucleotide probes and we found increasing of hybridization signals for EPO at 7th culture days. In addtion, we identified eosinophils in cultured CD34+ CBCs by flow cytometry. As s results, we succeeded in developing of pure eosinophils efficiently from CD34+ of CBCs in the presence of IL-3, IL-5 and GM-CSF.
CONCLUSION
The in vitro growth of CD34+ CBCs may provide a useful system to study growth factor and stage-dependent adhesion molecule expression, as well as function on developing eosinophils.

Keyword

Eosinophil; CD34+ cord blood cells; Cytokines

MeSH Terms

Atmosphere
Azure Stains
Blotting, Southern
Cell Count
Centrifugation, Density Gradient
Cytokines
Eosinophils*
Fetal Blood*
Flow Cytometry
Granulocyte-Macrophage Colony-Stimulating Factor
Granulocytes
Hematopoietic Stem Cells
Heparin
Humans*
Inflammation
Interleukin-3*
Interleukin-5*
Oligonucleotide Probes
Parasitic Diseases
RNA, Messenger
Azure Stains
Cytokines
Granulocyte-Macrophage Colony-Stimulating Factor
Heparin
Interleukin-3
Interleukin-5
Oligonucleotide Probes
RNA, Messenger