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IMPROVED METHOD FOR THE GENERATION OF LONG LIVED AND FUNCTIONALLY ACTIVE DENDRITIC/LANGERHANS CELLS FROM CD34+ PROGENITOR CELLS.
Seyoum Ayehunie, Sarah Lamore, Kristen Bellevance, John Sheasgreen, and Mitchell Klausner.
MatTek Corporation, Ashland, MA.
Presented at LC Symposium-Tokyo (2003).
Keywords: Allogenic T cell proliferation, Autologous T cell proliferation, Birbeck granules, CD11c-, CD123+, CD1a, CD34+ progenitor cells, DC, Dendritic cells, Gene transfection, HIV-1, HLA-DR, IL-12, IL-6, INF-gamma, Immunological experiments, Keyhole limpet hemocyanin (KLH), LPS, Langerhans cells, MIP-1a, MIP-3a, PDC, PMA, Plasmacytoid, Plasmacytoid dendritic cells, T-cell, TNF-a, TNF-alpha, Tetanus toxoid, Umbilical cord blood, Viral infection studies, pDC
Endpoints: IL-12, IL-6, MIP-1a, MIP-3a
Materials Tested: IFN-gamma, KLH, LPS, PMA, Tetanus toxoid
Summary:
The difficulty in harvesting large number of cells, short survival time, and rapid phenotypic changes in culture have prevented the widespread use of human dendritic cells (DC)/Langerhans cells (LC) for fundamental studies involving these key immunological cells.
Here MatTek scientists report on an improved method of generating DC from CD34+ progenitor cells derived from human umbilical cord blood (HUCB).
The method resulted in an average of 205 ± 86.02 fold increase (n = 15) in DC number.
These DC express CD1a, HLA-DR, and costimulatory molecules, and can be cultured for over 36 days with no significant change in cell number or phenotype.
Transmission electron microscopy showed the presence of Birbeck granules, a key ultrastructural marker of DC, over the duration of the culture period.
These cells contain plasmacytoid and myeloid DC populations and can be frozen and recovered with a viability of 80-90%.
Upon pulsing with external stimuli such as LPS, PMA, and IFNγ, the DC showed a reproducible (n = 4), high level of gene and protein responsiveness in terms of IL-12, MIP-1α, MIP-3α, IL-6, and TNF-α expression.
Functionally, the DC can:
1) be induced to express foreign genes following transfection,
2) initiate allogeneic T cell proliferation,
3) induce autologous T-cell proliferation in response to the neo-antigen, keyhole-limpet hemocyanin (KLH) or the recall-antigen, tetanus toxoid,
4) induce primary and secondary T-cell responses to a strong allergen (FITC) but not for an irritant (SDS), and
5) be infected with HIV-1.
In conclusion, MatTek scientists have developed a method to harvest and culture functional DC that have longer life span in culture. These cells are likely to be useful in a broad variety of immunological experiments, study of pathogenic microbes, and gene transfection experiments.
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