Clin Pediatr Hematol Oncol.  2006 Oct;13(2):208-217.

Generation of Functional Dendritic Cells from Human Umbilical Cord Blood CD34+ Cells

Affiliations
  • 1Department of Microbiology, Gachon Medical School, Incheon, Korea.
  • 2Department of Pediatrics, Ewha Womans University College of Medicine, Ewha Medical Research Center, Korea.
  • 3Department of Microbiology, Ewha Womans University College of Medicine, Ewha Medical Research Center, Korea.
  • 4Department of Pediatrics, Seoul National University College of Medicine, Cancer Reseach Institute, Seoul, Korea. hsahn@snu.ac.kr

Abstract

PURPOSE: The aim of this study was to generate dendritic cells (DCs) from CD34+ cells of human umbilical cord blood (UCB) and to characterize the requirements for maximizing antigen uptake and the stimulatory function of DCs for effective antigen presentation.
METHODS
The CD34+ cells from UCB were cultured at a density of 1.0 x 10(5) cells/mL in IMDM supplemented with 10% FBS. Cultures were expanded for 7 days (1st-step culture) with stem cell factor (SCF), flt3-ligand (FL) and thrombopoietin (TPO) and were differentiated into DCs for 5 days (2nd-step culture) with interleukin (IL)-4 and granulocyte macrophage-colony stimulating factor (GM-CSF) with or without tumor necrosis factor (TNF)-alpha. Immunophenotypical characterization of the cultured cells were analysed with various MoAb on a FACSCalibur flow cytometer. These cells were exposed to stimulation with Lipopolysaccharide (LPS), TNF-alpha, N-formyl-L-methionyl-L-leucyl-L-phenylalanin (FMLP) and LPS+TNF-alpha+FMLP for the DCs maturation. These cells were also incubated with fluorescent beads and FITC-conjugated C. albicans for the evaluation of phagocytic activity of generated DCs.
RESULTS
During the 2nd-step culture, the increase of total cell numbers was 2 times higher in the presence of TNF-alpha than without it. Exposure to stimulants resulted in an up-regulation of costimulatory proteins (CD80, CD86), HLA-DR, CD40 and adhesion molecules (CD11c, CD11b) and the stimulants combination of LPS+TNF+FMLP was superior to LPS, TNF-alpha, or FMLP on their own. Expansion of CD34+ cells with SCF, FL, TPO, IL-4 and GM-CSF/or TNF-alpha yielded morphologically and phenotypically mature DCs with phagocytic activity against the beads and C. albicans.
CONCLUSION
CD34+ cells derived from UCB differentiated into morphologically, phenotypically and functionally mature DCs.

Keyword

Umbilical cord blood; CD34+ cells; Dendritic cells

MeSH Terms

Antigen Presentation
Cell Count
Cells, Cultured
Dendritic Cells*
Fetal Blood*
Granulocytes
HLA-DR Antigens
Humans*
Interleukin-4
Interleukins
Stem Cell Factor
Thrombopoietin
Tumor Necrosis Factor-alpha
Umbilical Cord*
Up-Regulation
HLA-DR Antigens
Interleukin-4
Interleukins
Stem Cell Factor
Thrombopoietin
Tumor Necrosis Factor-alpha
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