Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Hidetoshi Sumimoto, Takashi Tsuji, Hiroyuki Miyoshi, Masao Hagihara, Rie Yamazaki, Shinichiro Okamoto, Yasuo Ikeda, Tsuneo Takahashi, Yutaka Kawakami

Research output: Contribution to journalArticle

12 Citations (Scopus)

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-α (TNF-α), 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.

Original languageEnglish
Pages (from-to)153-165
Number of pages13
JournalJournal of Immunological Methods
Volume271
Issue number1-2
DOIs
Publication statusPublished - 2002 Dec 20

Fingerprint

Mesenchymal Stromal Cells
Dendritic Cells
Genes
Fetal Blood
Stem Cell Factor
Bone Marrow
Blood Cells
HIV
Melanoma-Specific Antigens
Thrombopoietin
Adaptive Immunity
Granulocyte-Macrophage Colony-Stimulating Factor
Transgenes
S Phase
Innate Immunity
Interleukin-4
Genetic Therapy
Lipopolysaccharides
Tumor Necrosis Factor-alpha
Fluorescence

Keywords

  • Bone marrow stromal cell
  • DC
  • Ex vivo expansion
  • Gene therapy
  • Hematopoietic stem cell
  • HIV lentivirus vector

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

Cite this

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells. / Sumimoto, Hidetoshi; Tsuji, Takashi; Miyoshi, Hiroyuki; Hagihara, Masao; Yamazaki, Rie; Okamoto, Shinichiro; Ikeda, Yasuo; Takahashi, Tsuneo; Kawakami, Yutaka.

In: Journal of Immunological Methods, Vol. 271, No. 1-2, 20.12.2002, p. 153-165.

Research output: Contribution to journalArticle

Sumimoto, Hidetoshi ; Tsuji, Takashi ; Miyoshi, Hiroyuki ; Hagihara, Masao ; Yamazaki, Rie ; Okamoto, Shinichiro ; Ikeda, Yasuo ; Takahashi, Tsuneo ; Kawakami, Yutaka. / Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells. In: Journal of Immunological Methods. 2002 ; Vol. 271, No. 1-2. pp. 153-165.
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