Abstract
OBJECTIVE This research was to induce dendritic cells (DCs) from mice embryonic stem cells and bone marrow mononuclear cells in vitro, and then compare the biologic characteristics of them.
METHODS Embryonic stem cells (ESCs) suspending cultured in petri dishes were induced to generate embryonic bodies (EBs). Fourteen-day well-developed EBs were transferred to histological culture with the same medium and supplemented 25 ng/ml GM-CSF and 25 ng/ml IL-3. In the next 2 weeks, there were numerous immature DCs outgrown. Meantime, mononuclear cells isolated from mice bone marrow were induced to derive dendritic cells by supplementing 25 ng/ml GM-CSF and 25 ng/ml IL-4, and then the morphology, phenotype and function of both dendritic cells from different origins were examined.
RESULTS Growing mature through exposure to lipopolysaccharide (LPS), both ESC-DCs and BM-DCs exhibited dramatic veils of cytoplasm and extensive dendrites on their surfaces, highly expressed CD11c, MHC-II and CD86 with strong capacity to stimulate primary T cell responses in mixed leukocyte reaction (MLR).
CONCLUSION ESC-DC has the same biologic characteristics as BM-DC, and it provides a new, reliable source for the functional research of DC and next produce corresponding anti-tumor vaccine.
keywords
Introduction
Dendritic cells (DCs) are the most potent antigen presenting cells which are responsible for priming native T cells in the immune response. Immunotherapy of DC/tumor vaccine has become an important therapeutic strategy against tumors. However, the basic researches and clinical applications of DCs have been limited by the low yield and purity of DCs generated by traditional bone marrow derivation. Embryonic stem cells (ESCs) have the ability of self-renewal, the potency of poly-differentiation to kinds of tissue cells and immunity tolerance, so it is likely to be used in different individual. In this study, we induced mouse bone marrow mononuclear cells and embryonic stem cells to generate dendritic cells in vitro, and then compared the biologic characteristics of them.
Materials and Methods
Materials
Main reagents
Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF), Recombinant mouse interleukin-3 (IL-3), Recombinant mouse interleukin-4 (IL-4), Recombinant mouse leukemia inhibitory factor (LIF) (PeproTech), Lipopolysaccharide (LPS) (Sigma), Mitomycin C (MMC), and Methabenzthiazuron (MTT) (Duchfo).
Solutions
ESC culture medium: high glucose DMEM plus 15% fetal calf serum, and rmLIF 10 μg/L while no feeder line. Embryoid bodies culture medium: same as ESC culture medium but without rmLIF. ESC derived dendritic cells (ESC-DC) culture medium: high glucose DMEM plus 15% fetal calf serum plus 25 mg/L rmGM-CSF and rmIL-3. Mice bone marrow derived dendritic cells (BM-DC) culture medium: high glucose DMEM plus 15% fetal calf serum plus 25 mg/L rmGM-CSF and rmIL-4.
Cell line and mice
The E14.1 mice ESC line was contributed by Stem Cell Research Center of Sun Yat-Sen University. Six to 8 week old female BALB/c and 129 mice were purchased from Animal Center of Sun Yat-Sen University (Guangzhou, China).
Methods
Induction of dendritic cell from ESC
El4.1 cells with the concentration of l × 105/ml were planted into feeder layer cells dealing with Mitomycin C. ESCs demanded complete medium with 5% CO2 at 37°C and were passaged every 2~3 days at the ratio of l:3 or 1:6 in order to encourage the formation of EBs from the ESC line. We firstly removed most of the embryonic fibroblasts. It was best achieved by passaging the ESC twice in a source of exogenous LIF to circumvent the need for feeder cells. The amount of the ESC may be achieved after substantially reducing the proportion of fibroblasts to a very low density in petri dishes, and maintained in suspension culture with EB culture medium. In order to encourage the differentiation of DC in further step, l2~14 day EBs should be harvested by transferring to 6-well plate with 4~5 EBs per well and DC culture medium. LPS was added at the 15th day and the suspended cells were harvested 24 h later.
Induction of dendritic cell from mouse bone marrow
Bone marrow was flushed out of femurs and tibias of 129 mice. Mononuclear cells at the interface were collected by centrifugation over a lympholyte gradient. After being washed, they were cultured in 6-well plate for 2 h with the density of 5 × 104/ml. Then suspended cells were removed and the adhered cells were cultured with BM-DC culture medium. LPS was added at the 7th day and the suspended cells were harvested 24 h later.
Observation of morphology
Cells were observed under microscope with the cover on the well plates every day. Suspended cells were identified by electro-microscopy.
Cell surface marker analysis
Cells were collected and placed into centrifuge tube, mixed with FITC-CD11c, MHC II-PE, FITC-CD86 and MHC II-PE, respectively, and then were kept in centrifuge tube at 4°C for 30 min. After being washed twice by PBS, cells were assayed by flow cytometry.
Mixed lymphocyte reactions (MLR)
Two source of derived dendritic cells were collected respectively, 25 μg/ml MMC, as a stimulator being added for 45 min and washed thrice, as stimulator. Nylon wool purified T-cells as responders were added allocated in 96-well round bottomed plates at various ratios for 4 days. At the same time, control group was set, which didn’t include stimulators. Four hours before harvesting, cells were pulsed with 20 μl MTT (5 mg/ml), plus 100 μl DMSO, and then the absorbency at a wavelength of 490 nm measured by using an ELISA plate reader. The stimulating index was calculated as: (absorbency of study group subtracted absorbency of control)/absorbency of control group.
Statistical analysis
The data was calculated using mean ± SD and checked by t test. P < 0.05 was considered significant.
Results
Morphologic characteristics of two source of derived DCs
The cells belonging to the DC lineage first appeared as early as day 5 after placing EBs in an appropriate cytokine milieu. The DC may be easily identified by the feature of their distinctive location around the perimeter of most colonies (Fig. 1A), forming a “halo” that was highly refringent under darkfield illumination. The population expanded rapidly over time, the cells had irregular shape but with no characteristic veils of cytoplasm (Fig. 1B). ESC-DCs grew mature after overnight incubation by adding 1 μg/ml of LPS and histological culture. A proportion of them were released from the tissue culture plate forming a population of free-floating cells with dramatic veils of cytoplasm and extensive dendrites, and initially formed clusters (Fig. 1C). Compared with ESC-DCs, immature BM-DCs have already had irregular dendrites after being cultured for 5~7 days (Fig. 1D). After 1 μg/ml of LPS was added for 24 h, the dramatic veils of cytoplasm and extensive dendrites were more distinct (Fig. 1E), just as the mature ESC-DCs. Besides, 10~20 cell conglomerate could be seen (Fig. 1F). By electro-microscopy the dramatic veils of cytoplasm and extensive dendrites of both ESC-DCs and BM-DCs were clearly observed.
The margin of a colony of differentiated cell types derived from a single EB showing the early appearance of immature ESC-DCs as a “halo” of phase bright cells (A, 30×). The immature ESC-DCs had no characteristic veils of cytoplasm (B, 100×). When it was induced mature by adding LPS, the dendrites of ESC-DCs became apparent, and formed clusters (C, 200×). Immature BM-DCs had a few irregular dendrites (D, 200×), and were more distinct when becoming mature (E, 200×). 10~20 cell conglomerate could be seen (F, 200×). By electro-microscopy, the dramatic veils of cytoplasm and extensive dendrites of both ESC-DCs (G, 4000×) and BM-DCs (H, 4000×) could be seen clearly.
Analysis of surface phenotype
DCs derived from both ESC and BM were induced and became mature through exposure to LPS and highly expressed surface molecules MHC-II and CD86 which associated with antigen presenting. The expression of peculiar surface molecule CD11c in BM-DCs was more evident than that of ESC-DCs. In the stage of immature, all surface molecules in both ESC-DCs and BM-DCs were at low level, especially in ESC-DCs (Table 1).
Phenotype of two sources of derived DC.
Mixed lymphocyte reactions (MLR)
Following exposure to LPS, two sources derived DCs were induced and became mature demonstrating their capacity that they could stimulate potent primary T cell responding in mixed leukocyte cultures (Table 2).
The stimulating index for primary T cell of two source of derived DC.
Discussion
DCs are unique among populations of antigen presenting cells by the virtue of their capacity to direct the outcome of antigen recognition by naive T cells[1]. DCs in the periphery capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses[2]. Numerous studies have shown that DCs play crucial roles in controlling tumor growth, graft rejection and autoimmunity. At present, we have already induced DCs from bone marrow et al.[3-5], and will produce anti-tumor vaccine by gene transfection, tumor-associated antigen impaction and cell fusion[6-8]. The effects of research in experimental animal were excited. Furthermore, DC immunotherapy has been introduced in the clinic. It has been proven to be feasible, non-toxic and effective in some cancer patients, and particularly it is true when the DCs are completely mature and activated[9]. The results of the first clinical trial have been published, and unequivocally demonstrated that only mature DCs are capable of inducing potent anti-KLH-specific T-cell and B-cell response[10].
The life cycle of DC includes several stages characterized by distinct functions and mechanisms of regulation[11]. The DCs derived from different sources or in different life stages may have different biologic characteristics. ESCs are derived from the inner cell mass of the trophoblast, and have the ability to differentiate into all the tissues of the fetus[12]. Prior studies have shown that ESCs not only mice derived but human derived could be induced into DCs in vitro[13,14].
In the current study, we induced DCs differentiated from BM mononuclear cell and ESC line E14.1 respectively. The results have showed that they could all be induced into DCs with normal surface phenotype in vitro by additional appropriate signals in the form of growth factors and cytokines. The dramatic veils of cytoplasm and extensive dendrites were seen on their surface when they were induced and became mature by adding LPS and expressed the myeloid peculiar surface molecule CD11c, MHC-II and CD86 which associated with antigen presenting. Compared with ESC-DCs, the surface morphologic characteristics of BM-DCs were more evident. They had already a few irregular dendrites before becoming mature by adding LPS, and expressed lower level CD11c, MHC-II, and CD86. In induced mature BM-DCs by LPS, the characteristics were more evident. However, ESC-DCs have no analogic characteristics before induced and being mature by adding LPS. They had no dendrites on surface, and the expressions of CD11c, MHC-II, and CD86 were at very low level. Even though the characteristics were in evidence after being mature, the expression of CD11c which represents the specific surface molecule of DC was lower than that of BM-DCs.
We conclude that DCs spontaneously become mature when induced from BM mononuclear cells, and highly express CD11c, MHC-II and CD86 gradually. Compared with BM-DCs, ESC-DCs keep immature without stimulation of extra antigen, so that the capacity for expansion of ESC-DCs greatly surpass that of their bone marrow-derived counterparts[15]. Two sources of derived mature DCs had demonstrated their capacity that they could stimulate potent primary T cell responses in mixed leukocyte reactions and there was no statistical difference between them. By this research, we can conclude that ESC-DC has analogical biologic characteristics compared with those in BM-DC which has been applied in basic and clinical research. The source of ESC-DC is abundant. And the ESC-DC derived process can be manipulated easily, so it could provide a reliable source for the research of anti-tumor vaccine produced by DC in next step.
Footnotes
This work was supported by a grant from Program for New Century Excellent Talents in University, China (No. NECT-05-0820).
- Revision received August 13, 2008.
- Accepted December 30, 2008.
- Copyright © 2009 by Tianjin Medical University Cancer Institute & Hospital and Springer
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