Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells.
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.
Sumimoto
Hidetoshi
H
Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan.
Tsuji
Takashi
T
Miyoshi
Hiroyuki
H
Hagihara
Masao
M
Takada-Yamazaki
Rie
R
Okamoto
Shin-ichiro
S
Ikeda
Yasuo
Y
Takahashi
Tsuneo
T
Kawakami
Yutaka
Y
D018952:Antigens, CD34 / Q000276:immunology*; D000951:Antigens, Neoplasm; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology*; D002454:Cell Differentiation / Q000276:immunology; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D004705:Endocytosis / Q000276:immunology; D005312:Fetal Blood; D015316:Genetic Therapy; D006678:HIV / Q000235:genetics / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D015847:Interleukin-4 / Q000276:immunology; D016086:Lentivirus / Q000235:genetics*; D008070:Lipopolysaccharides / Q000276:immunology; D058965:MART-1 Antigen; D008565:Membrane Proteins / Q000276:immunology; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology; D019089:Stem Cell Factor / Q000276:immunology; D017154:Stromal Cells / Q000166:cytology / Q000276:immunology; D013926:Thrombopoietin / Q000276:immunology; D014161:Transduction, Genetic / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D018952:Antigens, CD34; D000951:Antigens, Neoplasm; D008070:Lipopolysaccharides; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D019089:Stem Cell Factor; D014409:Tumor Necrosis Factor-alpha; C084881:flt3 ligand protein; D015847:Interleukin-4; D007371:Interferon-gamma; D013926:Thrombopoietin