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
DOI 10.1016/s0022-1759(02)00342-3
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
IF 1.913
Times Cited 14
DNA material CS-CDF-CG-PRE (RDB04379)