RRC ID 54576
Author Sumimoto H, Tsuji T, Miyoshi H, Hagihara M, Takada-Yamazaki R, Okamoto S, Ikeda Y, Takahashi T, Kawakami Y.
Title Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells.
Journal J. Immunol. Methods
Abstract Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-alpha), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC.
Volume 271(1-2)
Pages 153-65
Published 2002-12-20
PII S0022175902003423
PMID 12445738
MeSH Antigens, CD34 / immunology* Antigens, Neoplasm Bone Marrow Cells / cytology Bone Marrow Cells / immunology* Cell Differentiation / immunology Coculture Techniques Dendritic Cells / cytology Dendritic Cells / immunology* Endocytosis / immunology Fetal Blood Genetic Therapy HIV / genetics HIV / immunology Humans Interferon-gamma / immunology Interleukin-4 / immunology Lentivirus / genetics* Lipopolysaccharides / immunology MART-1 Antigen Membrane Proteins / immunology Neoplasm Proteins / genetics Neoplasm Proteins / immunology Stem Cell Factor / immunology Stromal Cells / cytology Stromal Cells / immunology Thrombopoietin / immunology Transduction, Genetic / methods* Tumor Necrosis Factor-alpha / immunology
Resource
DNA material CS-CDF-CG-PRE (RDB04379)