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 language | English |
|---|---|
| Pages (from-to) | 153-165 |
| Number of pages | 13 |
| Journal | Journal of Immunological Methods |
| Volume | 271 |
| Issue number | 1-2 |
| DOIs | |
| Publication status | Published - 2002 Dec 20 |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells
AU - Sumimoto, Hidetoshi
AU - Tsuji, Takashi
AU - Miyoshi, Hiroyuki
AU - Hagihara, Masao
AU - Yamazaki, Rie
AU - Okamoto, Shinichiro
AU - Ikeda, Yasuo
AU - Takahashi, Tsuneo
AU - Kawakami, Yutaka
PY - 2002/12/20
Y1 - 2002/12/20
N2 - 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.
AB - 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.
KW - Bone marrow stromal cell
KW - DC
KW - Ex vivo expansion
KW - Gene therapy
KW - Hematopoietic stem cell
KW - HIV lentivirus vector
UR - http://www.scopus.com/inward/record.url?scp=0037146782&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037146782&partnerID=8YFLogxK
U2 - 10.1016/S0022-1759(02)00342-3
DO - 10.1016/S0022-1759(02)00342-3
M3 - Article
C2 - 12445738
AN - SCOPUS:0037146782
VL - 271
SP - 153
EP - 165
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -