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Published online before print January 17, 2007

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(Journal of Leukocyte Biology. 2007;81:957-967.)
© 2007 by Society for Leukocyte Biology

Efficient generation of CD34+ progenitor-derived dendritic cells from G-CSF-mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment

Sophie Paczesny^*,,1,2, Yin-Ping Li^,,1, Na Li, Véronique Latger-Cannard^||, Luc Marchal^¶, Jing-Ping Ou-Yang, Pierre Bordigoni, Jean-François Stoltz and Assia Eljaafari^,#

^* Hematology Department, Children’s Hospital,
Laboratory of Mechanobiology and Engineering of Cells and Tissues, CNRS UMR 7563, and Unit of Cellular and Tissue Therapy,
^¶ Department of Electron Microscopy, Faculty of Medicine, and
^|| Laboratory of Hematology, CHU Nancy, France;
Department of Pediatrics, University of Michigan Cancer Center, Ann Arbor, Michigan, USA;
Department of Pathology and Pathophysiology, Wuhan University, Wuhan, China; and
^# Immunogenomics Mixed Unit, HCL-BioMerieux, Lyon, France

² Correspondence: Department of Pediatrics, University of Michigan Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0942, USA. E-mail: sophiep{at}umich.edu

As a result of their potent antigen-presentation function, dendritic cells (DC) are important tools for cell therapy programs. In vitro-generated DC from enriched CD34+ hematopoietic stem cells (HSC; enriched CD34 DC) have already proven their efficiency in Phase I/II clinical trials. Here, we investigated whether enrichment of CD34+ HSC before the onset of culture was absolutely required for their differentiation into DC. With this aim, we developed a new two-step culture method. PBMC harvested from G-CSF-mobilized, healthy patients were expanded for 7 days during the first step, with early acting cytokines, such as stem cell factor, fetal liver tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin. During the second step, expanded cells were then induced to differentiate into mature DC in the presence of GM-CSF, Flt-3L, and TNF- for 8 days, followed by LPS exposure for 2 additional days. Our results showed that the rate of CD34+/CD38+/lineage^neg cells increased 19.5 ± 10-fold (mean±SD) during the first step, and the expression of CD14, CD1a, CD86, CD80, and CD83 molecules was up-regulated markedly following the second step. When compared with DC generated from enriched CD34+ cells, which were expanded for 7 days before differentiation, DC derived from nonenriched peripheral blood stem cells showed a similar phenotye but higher yields of production. Accordingly, the allogeneic stimulatory capacity of the two-step-cultured DC was as at least as efficient as that of enriched CD34 DC. In conclusion, we report herein a new two-step culture method that leads to high yields of mature DC without any need of CD34+ HSC enrichment.

Key Words: ex vivo expansion • immunotherapy • cell vaccine • lipopolysaccharide