title: Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment.
abstract: The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.
authors.affiliation: First Department of Internal Medicine, Faculty of Medicine, Niigata University, Niigata, Japan.
authors.forename: Aichun
authors.initials: A
authors.lastname: Liu
authors.forename: Masuhiro
authors.initials: M
authors.lastname: Takahashi
authors.forename: Miwako
authors.initials: M
authors.lastname: Narita
authors.forename: Zhiyin
authors.initials: Z
authors.lastname: Zheng
authors.forename: Naoko
authors.initials: N
authors.lastname: Kanazawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Abe
authors.forename: Kohji
authors.initials: K
authors.lastname: Nikkuni
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Furukawa
authors.forename: Ken
authors.initials: K
authors.lastname: Toba
authors.forename: Ichiro
authors.initials: I
authors.lastname: Fuse
authors.forename: Yoshifusa
authors.initials: Y
authors.lastname: Aizawa
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D000001:Calcimycin / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D005312:Fetal Blood / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; D007476:Ionophores; D000001:Calcimycin