IMPROVED METHOD FOR THE GENERATION OF LONG LIVED AND FUNCTIONALLY ACTIVE DENDRITIC/LANGERHANS CELLS FROM CD34+ PROGENITOR CELLS.

• TR Number: 305
• Authors: Seyoum Ayehunie, Sarah Lamore, Kristen Bellavance, John Sheasgreen, and Mitchell Klausner. Mattek Corporation, Ashland, MA.
• Materials Tested: IFN-gamma, KLH, LPS, PMA, Tetanus toxoid

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.