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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 Bellavance, John Sheasgreen, and Mitchell Klausner. MatTek Corporation, Ashland, MA.
Abstract

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.

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, Tetanus toxoid, Umbilical cord blood, Viral infection studies, pDC

Materials Tested

IFN-gamma, KLH, LPS, PMA, Tetanus toxoid

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