Cytokine-based high log-scale expansion of functional human dendritic cells from cord-blood CD34-positive cells

Abstract

Dendritic cells (DCs) play a crucial role in maintaining the immune system. Though DC-based cancer immunotherapy has been suggested as a potential treatment for various kinds of malignancies, its clinical efficacies are still insufficient in many human trials. Issues that limit the clinical efficacy of DC-based immunotherapy, as well as the difficulty of the industrial production of DCs, are largely due to the limited number of autologous DCs available from each patient. We here established a possible breakthrough, a simple cytokine-based culture method to expand the log-scale order of functional human DCs. Floating cultivation of cord-blood CD34(+) cells under an optimized cytokine cocktail led these progenitor cells to stable log-scale proliferation and to DC differentiation. The expanded DCs had typical features of conventional myeloid DCs in vitro. Therefore, the concept of DC expansion should contribute significantly to the progress of DC immunotherapy.

Figure 1. Cytokine-based expansion of CB CD34⁺ cell-derived CD11c⁺ cells.

(a) The regimen of cell expansion from CB CD34⁺ cells. (b) Growth curve of total cells from CB CD34⁺ cells. After 4 weeks of cultivation under GM/SCF (black line), culture medium was subsequently replaced with GM/IL-4 (red line) for 1 week. GM/IL-4 could not expand the cells. (c) CD11c-positive ratios of expanded cells under GM-CSF and SCF. The ratio of proliferating cells during cultivation under GM/SCF gradually increased, exceeding 80% after 5 weeks' cultivation. The cells cultured under GM/IL-4 for a week after 4 weeks' cultivation under GM/SCF also showed a high CD11c-positive ratio. (d) Growth curve of CD11c⁺ cells from CB CD34⁺ cells. These experiments were repeated seven times in 5 individuals (GM/SCF) and three times in 3 individuals (GM/IL-4). (e) The numbers of CD11c⁺/CD14⁺ and CD11c⁺/CD14^- DC generated from each culture. (f) FACS profiles of the DC products. These experiments were repeated at least three times in 3 individuals.

Figure 2. In vitro characterization of expanded human DCs.

(a) Morphology of rSeV-activated DCs. Wright-Giemsa–stained cytocentrifuge preparations are shown. Bars in panels = 30 um. (b) FACS analyses assessing the expression of typical surface markers. DCs with or without further stimulus were subjected to FACS analyses. A bar graph indicating the corresponding mean fluorescent intensity (MFI) containing data from four independent experiments is shown.

Figure 3. Assessment of functions that are typically seen in DCs.

(a) Expression of typical human inflammatory cytokines/chemokines of monocyte-derived DCs and expanded DCs in response to various stimuli. These six panels were assessed by the Cytometric Bead Array (CBA) system and contain data from three independent experiments. (b) FITC-dextran uptake assay assessing endo-/phagocytotic activity, a typical feature of antigen-presenting cells like DCs. DCs were stimulated by each stimulus and then exposed to 1 mg/ml of FITC–dextran for 30 min at 4 or 37 degrees Celsius. The uptake was expressed MFI between cell samples incubated at 37 and 4 degrees Celsius. This experiment was performed three times. (c) A graph showing MLR activity for allo-antigen by each immature DC or activated DC by SeV/dF, LPS, or OK-432. This experiment was performed three times.