IN-VITRO EXPANSION AND CHARACTERIZATION OF DENDRITIC CELLS DERIVED FROM HUMAN BONE-MARROW CD34(+) CELLS

Dendritic cells (DC), as professional antigen-presenting cells, play a major role in stimulating naive T cell responses iv vivo and in vitro , and may exacerbate or modulate T lymphocyte-mediated reactions, such as interactions between a hematopoietic graft and the recipient, eg G VHD and graft-versus-leukemia. Here, we describe a two-stage cell cult ure system for expansion of functionally active human DC from CD34(+) marrow precursors, Optimal outgrowth was achieved by initially culturi ng CD34(+) cells for 5 days in medium containing GM-CSF, MGF and TNF-a lpha. Substitution of CD40L and IL-4 for TNF-alpha during a subsequent 5-day subculture increased DC content, such that by 10 days the cultu res contained approximately 40% DC as determined by immunophenotype an d morphology. An increase in DC purity to 84% at 10 days was achieved by immunomagnetic separation for CD1a(+) cells from 5-day cultures and subculturing these cells in medium with IL-4 and CD40L, Reversing the sequence of growth factors during culture and subculture decreased th e yield and purity of DC, Expression of CD80 and CD86 was enhanced by adding CD40L and IL-4, and the DC showed stimulatory activity in MLC. In conclusion, we have described a simple two-stage culture system to generate functional DC from CD34(+) marrow precursors.