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Stem Cells, Vol. 18, No. 1, 53-60, January 2000
© 2000 AlphaMed Press

Development of Mouse Dendritic Cells from Lineage-Negative c-kit<low Pluripotent Hemopoietic Stem Cells In Vitro

Biao Feng, Muneo Inaba, Zhexiong Lian, Yunze Cui, Junko Toki, Tomoki Ito, Tienan Jin, Tianxue Fan, Guoxiang Yang, Chengze Yu, Taketoshi Kushida, Susumu Ikehara

First Department of Pathology, Kansai Medical University, Moriguchi City, Osaka, Japan

Key Words. c-kit<low • Dendritic cells • Hemopoietic stem cells

Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara{at}takii.kmu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) are essential for the presentation of antigens in the primary immune response. To examine the generation of DCs from hemopoietic stem cells in the bone marrow (BM), lineage-negative (Lin–)/CD71– bone marrow cells (BMCs) from C57BL/6 mice were separated into major histocompatibility complex (MHC) class Ihigh/ c-kitlow and MHC class Ihigh/c-kit<low (phenotypically c-kit-negative, but c-kit message only detected by reverse transcriptase-polymerase chain reaction) populations. A large number of cells with the morphological, phenotypical, and functional characteristics of DCs was generated from both c-kitlow and c-kit<low populations when cultured with a combination of cytokines (GM-CSF, tumor necrosis factor-a [TNF-a], interleukin 7 [IL-7], IL-3, stem cell factor [SCF], and flt3 ligand); the cytokine combination studies revealed that SCF and IL-3 in addition to GM-CSF and TNF-a are essential for DCs to be generated from these primitive populations. To our surprise most (>80%) generated cells expressed high levels of DC surface markers such as DEC205 and MHC class II, and they were potent stimulators in the primary allogeneic T cell activation. The development of DCs from c-kit<low cells was slower than that from c-kitlow cells. These results indicate that c-kit<low cells are more primitive than c-kitlow cells, although both c-kitlow cells and c-kit<low cells can differentiate into DCs. It should be noted that the combination of these cytokines selectively induces DCs from both c-kit<low and c-kitlow cells in vitro, suggesting that the ex vivo expansion of DCs using these primitive cells would be applicable to immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs) form a heterogeneous population, these cells being widely distributed throughout both lymphoid and nonlymphoid tissues. DCs are specialized to present antigens in a major histocompatibility complex (MHC)-restricted manner and to initiate the primary immune response more efficiently than other types of antigen-presenting cells such as B cells and macrophages [1-5]. DCs are classified on the basis of their tissue distribution: thymic DCs in the thymus, interdigitating DCs in the lymphoid organs, veiled cells in the afferent lymphatics, interstitial DCs in the nonlymphoid organs, Langerhans' cells in the epidermis, and blood DCs in the peripheral blood [1-11].

Substantial numbers of functional DCs have been generated from human peripheral blood mononuclear cells or human bone marrow (BM) precursors using various cytokines including GM-CSF, interleukin 4 (IL-4), tumor necrosis factor-{alpha} (TNF-{alpha}) and stem cell factor (SCF) [6, 12-16]. DCs can be generated from the common precursors shared by granulocyte- and macrophage- lineage cells and also generated in vitro from murine MHC class II-negative bone marrow cells (BMC) in a suspension culture in response to GM-CSF [17, 18]. However, a relatively high cell concentration has been required for the induction of DCs in the system, suggesting that one or more additional factors may be necessary for the generation of DCs. The differentiation and maturation of DCs from human CD34+ hemopoietic progenitors have essentially required the addition of human TNF-{alpha} [19-25]. Moreover, TNF-{alpha} can also induce the terminal differentiation of blood monocyte-derived DCs that are generated by stimulation with GM-CSF and IL-4 [15, 16]. Thus, TNF-{alpha} is necessary for the development of DCs. Although both GM-CSF and TNF-{alpha} are known to stimulate the development of mature DCs from progenitors, SCF and flt3 ligand (FL) also have the ability to stimulate the proliferation of DCs from progenitors in vitro and in vivo without affecting DC morphology and functions [26, 27]. These cytokines therefore seem to play a critical role in the DC development from progenitors.

The differentiation pathway of DCs from hemopoietic stem cells (HSCs) has not been fully clarified. It is therefore intriguing to know whether highly purified murine early hematopoietic progenitor cells (HPCs) can generate DCs in vitro in response to cytokines. Recent reports have shown that DCs can be generated from murine Lin–/c-kit+ HPCs and single adult human CD34+/Lin–/CD38– progenitors [26, 28]. We have recently shown that pluripotent HSCs (P-HSCs) are c-kit<low (phenotypically c-kit–, but the c-kit message is only detectable by reverse transcriptase - polymerase chain reaction; [RT-PCR]) by assessing their long-term repopulating activity after serial BM transplantation (BMT) and colony-forming units-spleen (CFU-S) formation [29, 30]. The purified P-HSCs (Lin–/CD71–/MHC class Ihigh/c-kit<low cells) show the capacity not only to differentiate into multilineage cells but also to self-renew for more than 1.5 years [29]. However, the generation of DCs from P-HSCs has not been determined yet. In the present study, we compare the generation of DCs derived from c-kit<low P-HSCs with that from c-kitlow HSCs by culturing them with various cytokines and analyzing their morphological, phenotypical and functional characteristics.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female C57BL/6 and BALB/c mice (8-10 wks old) were purchased from CLEA Japan (Osaka, Japan), and kept under specific pathogen-free conditions in our animal facility until use.

Antibodies
Purified rat monoclonal antibodies (mAbs) against CD4 (GK1.5), CD8 (53-6.72), CD45R (B220, RA3-6B2), granulocytes (Gr-1, RB6), macrophages (CD11b: Mac-1: M1/70), erythroid lineage cells (TER119) and transferrin receptor (CD71) were purchased from PharMingen (San Diego, CA). These mAbs were used to deplete myeloid/lymphoid/erythroid lineage cells and CD71+ cells in combination with magnetic beads conjugated with sheep anti-rat IgG Ab (Dynabeads® M-450, Dynal A.S.; Oslo, Norway). Fluorescein isothiocyanate (FITC)-coupled anti-H-2Kb mAb and phycoerythrin (PE)-coupled anti-c-kit mAb (ACK4) from PharMingen were used to further purify the HSCs. FITC-coupled mAbs against CD4, CD8, CD11b, CD80, CD86, CD54, CD40, Gr-1, MHC class II (I-Ab) and Thy 1.2, and biotin-coupled NLDC145 (mAb against DEC 205), which were also from PharMingen, were used to analyze surface phenotypes.

Purification of HSCs
BMCs were collected from the tibias and femurs of C57BL/6 mice that had been treated with 5-fluorouracil (150 mg/kg) three days before sacrifice. They were rinsed and suspended in phosphate-buffered saline (PBS) containing 2% heat-inactivated fetal calf serum (FCS) (PBS-FCS). BMCs were applied to Percoll (Pharmacia Fine Chemicals; Uppsala, Sweden) discontinuous density gradient centrifugation. After centrifugation, cells with low density of 1.066 < p < 1.077 were collected, as reported previously [30, 31]. The low-density cells were then incubated with a mixture of mAbs against CD4, CD8, CD11b, Gr-1, B220, TER119, and CD71 for 30 min on ice, and then washed twice with PBS-FCS, followed by sheep anti-rat IgG-conjugated magnetic-beads (Dynabeads®) to deplete the cells bearing myeloid/ lymphoid/erythroid lineage markers and CD71 molecules. The cells thus prepared (Lin–/CD71– cells) were stained with PE-anti-c-kit mAb and FITC-anti-H-2Kb mAb, and cells with MHC class Ihigh/c-kitlow and MHC class Ihigh/c-kit- (phenotypically c-kit–, but slightly detected by RT-PCR, termed as c-kit<low) were sorted using a FACStar® (Becton Dickinson & Co; San Jose, CA). These two populations were cultured with a mixture of cytokines.

Culture of HSCs
After the sorting, the cells (5 x 103) were resuspended in RPMI 1640 medium with 10% fetal bovine serum and cultured in 24-well-culture dishes (Flow Laboratories, Inc.; McLean, VA) supplemented with 30 ng/ml of recombinant human (rHu) FL (Genzyme; Cambridge, MA), 30 ng/ml of recombinant murine (rMu) SCF (PharMingen), 10 ng/ml of rMu IL-3 (PharMingen), 100 ng/ml of rMu GM-CSF, 25 ng/ml of rMu TNF-{alpha} (PharMingen) and 10 ng/mL of rMu IL-7. Half of the medium was replaced by fresh medium containing the cytokines on day 3. From day 7, the medium was changed into RPMI 1640 with 10% FCS supplemented with 100 ng/ml of rMu GM-CSF, and 25 ng/ml of rMu TNF-{alpha} until the end of the experiments. Every three days, half of the medium was aspirated and replaced with fresh medium containing the cytokines for maintaining the concentration of the cytokines.

To detect the effect of different cytokines on DC generation from HSCs, five different combinations of cytokines were used as follows. Group 1: rMu GM-CSF (100 ng/ml) , rMu TNF-{alpha} (25 ng/ml ), rMu IL-7 (10 ng/mL), rMu IL-3 (10 ng/ml), rMu SCF (30 ng/ml ), and rHu FL (30 ng/ml ); Group 2 (-FL): GM-CSF, TNF-{alpha}, IL-7, IL-3, and SCF; Group 3 (-FL/-SCF): GM-CSF, TNF-{alpha}, IL-7, and IL-3; Group 4 (-FL/-SCF/-IL-3): GM-CSF, TNF-{alpha}, and IL-7; Group 5 (-FL/-SCF/-IL-3/-IL-7): GM-CSF and TNF-{alpha}.

Cell Morphology
Nonadherent cells were collected seven days after the culture and low-density cells were isolated using lymphocyte-isolating solution. These cells were washed twice in PBS-FCS solution, and CD11c and MHC class II-positive cells were sorted by a FACStar®. The sorted cells were mounted onto slides with a CYTOSPIN 3 (Shandon; Runcorn, UK), and the cells on the glass were then stained by Giemsa's solution. The morphology of these cells was observed under the optical microscope.

Electron Microscope (EM) Studies
For electron transmission microscopy, cells were fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) and postfixed with 1% OsO4. After dehydration with graded ethanol, they were embedded in Epon. Ultrathin sections were stained with lead citrate and uranyl acetate, and studied using a Hitachi H-700-{alpha} electron microscope (Hitachi; Ibaragi, Japan).

Flow Cytometric Analyses
The BM HSC-derived DCs were assayed on days 7 and 14. The cultured cells were applied on an iso-osmotic Nycodenz medium and the low-density cells were collected after the centrifugation. They were resuspended in PBS-FCS and stained for flow cytometry using the following mAbs: biotin-coupled mAb against MHC class II, biotin-coupled NLDC145 (mAb against DEC205), PE-conjugated mAb against CD11c, FITC-conjugated mAbs to CD4, CD8a, CD86,CD80, CD54, CD40, Gr-1, CD11b, B220, and Thy1.2. Streptavidin-RED670 was used as the second-stage reagent for all biotin-conjugated mAbs. The appropriate isotype-matched control mAbs (PharMingen) were used in all experiments to determine the levels of background staining. The stained cells were analyzed by a FACScan® (Becton Dickinson & Co.).

Mixed Leukocyte Reaction (MLR)
The primary MLR was conducted as previously described [32] with some modification. Briefly, stimulator cells (CD11c and MHC class II-positive cells) in the low-density fraction of cultured cells were isolated by a FACStar®. After irradiation (10 Gy), these cells were incubated in graded doses of 2 x 105 allogeneic CD4 T cells in 96-well U-bottom tissue culture plates (0.2 ml of medium per well). Cell proliferation was measured by adding the 0.5 µCi of [methyl 3H] thymidine to the cultures during the last 12 h of three-day cultures. The cells were harvested onto filters, and radioactivity was measured in a scintillation counter. Results are presented as the mean cpm of triplicate cultures.

Each experiment was carried out three or more times and reproducible results were obtained. Representative data are shown in the Figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differentiation of DCs from HSCs after Culture with Cytokines
To determine whether HSCs can differentiate into DCs in vitro, Lin–/MHC class Ihigh/c-kitlow cells and Lin–/MHC class Ihigh/c-kit<low cells were sorted, and 5,000 cells of each were cocultured with a mixture of cytokines (GM-CSF, TNF-{alpha}, IL-7, IL-3, SCF and FL), as described in Materials and Methods. Each sorted population showed >98% purity by reanalysis using a FACScan. After coculture with the combination of cytokines for seven days, the cell number in cultures of c-kitlow cells had increased to 800-fold, while that of c-kit<low cells had increased to 80-fold (Fig. 1Go). On day 14, the cell number in cultures of c-kitlow cells had decreased to 300-fold, while that of c-kit<low cells had increased to 360-fold, the level being comparable with that of c-kitlow cells. Under phase-contrast microscope observation, on day 3, no sheet-like cells were found, but many round cells appeared in the cultures of the c-kitlow cells. In contrast, only a few round cells were found in the c-kit<low cells. On day 5, colonies began to form in both groups. More cells were generated from the c-kitlow cells than from the c-kit<low cells (Figs. 2A and 2BGoGo). Sheet-like cells appeared in the cultures of both c-kit<low and c-kitlow cells. Thereafter, the number of round cells increased in the cultures, and some aggregate cells were observed (Figs. 2C and 2DGoGo). On day 7, cells generated from the c-kitlow and c-kit<low cells showed the obvious generation of a sheet-like membrane, suggesting the typical DC morphology (Figs. 2E and 2FGoGo). EM studies revealed that the cells were indeed DCs showing the long cell process and prominent tubular-vesicular system with dense bodies (Fig. 2GGo).



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  		Figure 1. Time course of cell growth after culturing c-kitlow and c-kit<low cells. c-kitlow and c-kit<low cells were cultured in 24-well plates with the mixture of cytokines: rMu GM-CSF (100 ng/ml), rMu TNF-{alpha} (25 ng/ml), rMu IL-7 (10 ng/ml), rMu IL-3 (10 ng/ml), rMu SCF (30 ng/ml), and rHu FL (30 ng/ml). The cytokines were added at the start of the cultures, and half the medium was replaced with medium containing the fresh cytokine mixture every three days. From the seventh day, half the medium was replaced every three days with the medium containing fresh GM-CSF and TNF-{alpha}. The nonadherent cells were counted.

 


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  		Figure 2. Morphology of cells derived from c-kitlow and c-kit<low cells by culturing with a combination of cytokines (GM-CSF, TNF-{alpha}, IL-7, IL-3, SCF and FL). Phase-contrast microscopical findings of cells generated from c-kitlow and c-kit<low cells (A to D) or CD11c+/MHC class II+ cells stained by Giemsa (E and F). c-kitlow and c-kit<low cells were stimulated by the cytokine cocktail for five or seven days. (A) x 100; (B) x 200; (C) and (D) x 100; (E) and (F) x 400; (G) x 7,000.

 
Surface Markers of DCs
Mature DC-like cells were developed from c-kitlow and c-kit<low cells by culturing with the mixture of cytokines (Fig. 2Go). We next examined the surface phenotypes of these cells. Most (>80%) of the cells generated from the c-kitlow and c-kit<low cells possessed DEC 205 (expressed only on nonlymphoid DCs) and MHC class II molecules on days 7 and 14 of the culture, as shown in Figure 3Go. Extensive analyses of cell surface markers were carried out on these cells, and two populations were separated out, based on the expression of CD11c: CD11c– and CD11c+ (Fig. 4Go). Most of the generated cells were moderately positive for CD80, CD86, and CD40, and highly positive for CD54, CD11b and Thy1.2, but negative for CD8 and CD4low (Fig. 4Go). These cells expressed neither B220 nor Gr-1 (data not shown). Based on these findings, it can be concluded that most of the generated cells were DCs.



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  		Figure 3. Fluorescence-activated cell sorter (FACS) analysis of nonadherent cells generated from c-kitlow and c-kit<low cells. Nonadherent cells generated from c-kitlow and c-kit<low cells after culturing with a mixture of cytokines (GM-CSF, TNF-{alpha}, IL-7, IL-3, SCF, and FL) on days 7 and 14 were stained with biotinylated NLDC145 (DEC205) and FITC-coupled MHC class II mAb.

 


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  		Figure 4. Further FACS analysis of cells generated from c-kitlow and c-kit<low cells. c-kitlow and c-kit<low cells were cultured with a mixture of the cytokines (GM-CSF, TNF-{alpha}, IL-7, IL-3, SCF, and FL). The cells recovered from the cultures (on days 7 and 14) were stained with the mAbs indicated in the figure. The closed profile indicates the cells stained with isotype-matched control Abs. Each histogram shows the representative result of five different cell preparations.

 
Stimulatory Activity of Generated DCs
The stimulatory activity of DCs was next examined in the primary MLR. DCs generated from c-kitlow and c-kit<low cells (harvested on days 7 and 14) were irradiated and added in graded doses to a fixed number of CD4+ T cells (2 x 105 cells) from BALB/c mice. The DCs generated from both c-kitlow and c-kit<low cells after culture for 14 days showed potent stimulatory activity to allogeneic CD4+ T cells in MLR. The DCs generated after culture for seven days, however, did not show significant stimulator activity, which was similar to the response observed in the syngeneic responder/stimulator combination (Fig. 5Go). This seems to be due to the change in the degree of adhesion/costimulatory molecule expression; the expression of CD54, CD86, and CD40 gradually increased during the culture periods (particularly from 7 to 14 days), though the expression of MHC class II was unchanged.



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  		Figure 5. Stimulatory activity of DCs in the primary MLR. Allogenic MLR was performed using purified CD4+ T cells (2 x 105 cells) as responder cells. c-kitlow and c-kit<low cells were cultured with a mixture of the cytokines for 7 and 14 days, and, CD11c+/MHC class II+ cells were sorted by a FACS. They were irradiated and used as stimulators in the indicated cell numbers. The data represent the mean cpm ± SD of triplicated cultures.

 
Effects of Cytokines on Generation of DCs from HSCs
The role of cytokines in the generation of DCs was next assessed by sequential deletion of cytokines from the cytokine mixture (GM-CSF, TNF-{alpha}, IL-7, IL-3, SCF, and FL). The DCs generated from the c-kitlow and the c-kit<low cells were counted during different periods of culture. The number of cells generated from the c-kitlow cells with the cytokine mixture reached a maximum on day 7 in Group 1. However, the cells generated from the c-kit<low cells with the cytokine mixture reached a maximum on day 14 in Group 2 (Figs. 6A and 6BGoGo). The deletion of FL from the cytokine mixture did not affect the generation of DCs in the cultures of both c-kitlow and c-kit<low cells when examined on days 7 and 14 (Group 1 versus Group 2 in Figs. 6A and 6BGoGo). The deletion of SCF from the cytokine mixture significantly decreased the generation of DCs in either c-kitlow or c-kit<low cells on days 7 and 14 (Group 3), and the further deletion of IL-3 completely abolished the DC development (Group 4). Only a few cells were grown on days 7 and 14 from the c-kitlow and c-kit<low cells cultured with GM-CSF and TNF-{alpha} (Group 5) which are essential to the DC development from DC precursors [26]. The deletion of GM-CSF and TNF-{alpha} from the mixture of all cytokines did not generate DCs from the c-kitlow or c-kit<low cells (data not shown). These results indicate that SCF and IL-3 are necessary for the proliferation and early differentiation of c-kitlow and c-kit<low cells. There was no significant difference in the DC morphology and phenotypes generated between c-kit<low and c-kitlow cells (data not shown).




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  		Figure 6. Effects of different cytokine combinations on generation of DCs from c-kitlow and c-kit<low cells. c-kitlow (closed bar) and c-kit<low (striped bar) cells (4 x 103 cells/well) were cultured in 24-well plates with different cytokine combinations as follows; Group 1: rMu GM-CSF (100 ng/ml) , rMu TNF-{alpha}(25 ng/ml), rMu IL-7 (10 ng/mL), rMu IL-3 (10 ng/ml), rMu SCF (30 ng/ml ), and rHu FL (30 ng/ml); Group 2 (-FL): GM-CSF, TNF-{alpha}, IL-7, IL-3, and SCF; Group 3 (-FL/-SCF): GM-CSF, TNF-{alpha}, IL-7, and IL-3; Group 4 (-FL/-SCF/-IL-3): GM-CSF, TNF-{alpha}, and IL-7; Group 5 (-FL/-SCF/-IL-3/-IL-7): GM-CSF and TNF-{alpha}. Cytokines were added at the start of the cultures, and half the medium was supplemented with the fresh cytokine cocktail every three days. From seven days, half the medium was replaced with a fresh medium containing only fresh GM-CSF and TNF-{alpha}every three days. The number of DCs (class II+/DEC-205+) was counted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DCs, a rare population of hemopoietic origin, reside in both lymphoid and nonlymphoid tissues. Recently, several reports have shown that large numbers of DCs can be generated from murine and human progenitors with cytokines [12, 14, 15, 18-21, 33]. In these reports GM-CSF is essential for DCs to generate from mouse BM progenitors (proliferation and formation of aggregates) [18, 33], while both GM-CSF and TNF-{alpha} are necessary for human DCs to generate from CD34+ progenitors [20, 21]. In the present study, SCF was found to be important for the generation of DCs from primitive c-kit<low and c-kitlow cells (Fig. 6Go). It has been reported that SCF promotes the survival of very early multipotent progenitors and has a synergistic effect with late-acting growth factors and cytokines on the promotion of proliferation and terminal maturation of hemopoietic cells, though SCF alone has little colony-stimulating activity on the BMC [34]. This was confirmed in the present study (Figs. 6A and BGoGo); SCF seems to act as a survival-promoting factor on c-kitlow and c-kit<low cells. SCF is thus important in the early phase of the DC development. Furthermore, IL-3 was found to be also important to maintain early development of DCs (Fig. 6Go). It has been shown that FL stimulates the proliferation of purified c-kit+ mouse BMC, and that it shows a synergistic action with IL-3 [35]. In our experiment FL was not critical for the generation of DCs when SCF and IL-3 were present. Furthermore, IL-7, which has been reported to play a role in the development of thymic DCs [36], was also not critical for the generation of DCs. Furthermore, we have recently shown that the transition from human c-kit<low cells to c-kitlow cells occurs within a few days of the culture in the presence of hemopoietic stimuli including FL and IL-7 [37]. From these findings, it can be speculated that, under the influence of the hemopoietic cytokines used in this experiment, the c-kit molecules are acquired on the primitive c-kit<low cells at the start of the culture period, as the first maturational step of DC development (transition from c-kit<low cells to c-kitlow cells), after which the resultant c-kitlow cells can differentiate into DC progenitors under the influence of SCF or IL-3, and that these progenitors can then develop into mature DCs under the influence of late-acting (DC-inducible) cytokines (GM-CSF and TNF-{alpha}).

It has been shown that DCs can be generated from mouse Lin–/c-kit+ cells and also single adult human CD34+/Lin–/CD38– progenitors [26, 28]. We have recently shown that mouse P-HSCs are c-kit<low by a long-term repopulating assay after serial BMT [29] and CFU-S formation assay [30]. The c-kit<low P-HSCs show the capacity not only to differentiate into multilineage cells but also to self-renew for more than 3.5 years (M. Inaba et al., manuscript in preparation). However, the development of DCs from these c-kit<low cells has not been clarified. Our present results indicate that cells with irregular shape, sheet-like projections and veil processes (Fig. 2Go) are generated from c-kit<low cells, and that they have the features of DCs, expressing high levels of DEC205, MHC class II and costimulating molecules. Moreover, we have found that they show the strong stimulatory activity in the primary MLR, suggesting that these cells also function as antigen-presenting cells. The DCs generated from our culture did not express CD8, a marker of lymphoid DC, but express Thy 1.2 and strongly express Mac-1 (CD11b). It has been shown that GM-CSF alone induces the differentiation from mouse BMC into DCs which express a high level of MHC class II, but not Thy 1 [18]. However, Zhang et al. have recently reported that DCs generated from c-kit+ cells express a high level of Thy 1 [26], which is compatible with our present results.

In conclusion, mature DCs can be selectively generated from both c-kitlow and c-kit<low cells in the mouse BMC by culturing with a mixture of cytokines (GM-CSF, TNF-{alpha}, IL-7, IL-3, SCF and FL) for 14 days. This method for the expansion of DCs would be useful for analyzing the role of DCs in tumor immunity.


   Acknowledgments
 
We thank Mr. F. Ishida (Research Center of Kansai Medical University) for flow cytometry studies, and Ms. K. Ando for preparing the manuscript.

This work was supported by grants from the Ministry of Health and Welfare of Japan, the Ministry of Education, Science and Culture, and the Japan Private School Promotion Foundation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol 1997;9:10-16.[CrossRef][Medline]

  2. Steinman RM, Pack M, Inaba K. Dendritic cells in the T-cell areas of lymphoid organs. Immunol Rev 1997;156:25-37.[CrossRef][Medline]

  3. Ardavin C. Thymic dendritic cells. Immunol Today 1997;18:350-361.[CrossRef][Medline]

  4. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991;9:271-296.[CrossRef][Medline]

  5. Steinman RM. Dendritic cells and immune-based therapies. Exp Hematol 1996;24:859-862.[Medline]

  6. Peters JH, Gieseler R, Thiele B et al. Dendritic cells: from ontogeneic orphans to myelomonocytic descendants. Immunol Today 1996;17:273-278.[CrossRef][Medline]

  7. Steinman RM, Kaplan G, Witmer MD et al. Identification of a novel cell type in peripheral lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro. J Exp Med 1979;149:1-16.[Abstract/Free Full Text]

  8. Kraal G, Breel M, Janse M et al. Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody. J Exp Med 1986;163:981-997.[Abstract/Free Full Text]

  9. Vremec D, Zorbas M, Scollay R et al. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med 1992;176:47-58.[Abstract/Free Full Text]

  10. Ardavin C, Wu L, Li CL et al. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 1993;362:761-763.[CrossRef][Medline]

  11. Inaba K, Witmer-Pack M, Inaba M et al. The tissue distribution of the B7-2 costimulator in mice: abundant expression on dendritic cells in situ and during maturation in vitro. J Exp Med 1994;180:1849-1860.[Abstract/Free Full Text]

  12. Inaba K, Steinman RM, Witmer-Pack M et al. Identification of proliferating dendritic cell precursors in mouse blood. J Exp Med 1992;175:1157-1167.[Abstract/Free Full Text]

  13. Santiago-Schwarz F, Divaris N, Kay C et al. Mechanisms of tumor necrosis factor-granulocyte-macrophage colony-stimulating factor-induced dendritic cell development. Blood 1993;82:3019-3028.[Abstract/Free Full Text]

  14. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor-{alpha}. J Exp Med 1994;179:1109-1118.[Abstract/Free Full Text]

  15. Romani N, Gruner S, Brang D et al. Proliferating dendritic cell progenitors in human blood. J Exp Med 1994;180:8393.

  16. Akagawa KS, Takasuka N, Nozaki Y et al. Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes. Blood 1996;88:4029-4039.[Abstract/Free Full Text]

  17. Inaba K, Inaba M, Deguchi M et al. Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II -negative progenitor in mouse bone marrow. Proc Natl Acad Sci USA 1993;90:3038-3042.[Abstract/Free Full Text]

  18. Inaba K, Inaba M, Romani N et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 1992;176:1693-1702.[Abstract/Free Full Text]

  19. Reid CDL, Stackpoole A, Meager A et al. Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow. J Immunol 1992;149:2681-2688.[Abstract]

  20. Caux C, Dezutter-Dambuyant C, Schmitt D et al. GM-CSF and TNF-{alpha} cooperate in the generation of dendritic Langerhans cells. Nature 1992;360:258-261.[CrossRef][Medline]

  21. Szabolcs P, Moore MAS, Young JW. Expansion of immunostimulatory dendritic cells among the myeloid progeny of human CD34+ bone marrow precursors cultured with c-kit ligand, granulocyte-macrophage colony-stimulating factor, and TNF-{alpha}. J Immunol 1995;154:5851-5861.[Abstract]

  22. Young JW, Szabolcs P, Moore MAS. Identification of dendritic cell colony-forming units among normal human CD34+ bone marrow progenitors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-{alpha}. J Exp Med 1995;182:1111-1119.[Abstract/Free Full Text]

  23. Rosenzwajg M, Canque B, Gluckman JC. Human dendritic cell differentiation pathway from CD34+ hematopoietic precursor cells. Blood 1996;87:535-544.[Abstract/Free Full Text]

  24. Strunk D, Rappersberger K, Egger C et al. Generation of human dendritic cells/Langerhans cells from circulating CD34+ hematopoietic progenitor cells. Blood 1996;87:1292-1302.[Abstract/Free Full Text]

  25. Caux C, Vanbervliet B, Massacrier C et al. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF-{alpha}. J Exp Med 1996;184:695-706.[Abstract/Free Full Text]

  26. Zhang Y, Mukaida N, Wang J et al. Induction of dendritic cell differentiation by granulocyte-macrophage colony-stimulating factor, stem cell factor, and tumor necrosis factor-{alpha} in vitro from lineage phenotypes-negative c-kit+ murine hematopoietic progenitor cells. Blood 1997;90:4842-4853.[Abstract/Free Full Text]

  27. Jacobsen SEW, Okkenhaug C, Myklebust J et al. The flt3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors. J Exp Med 1995;181:1357-1363.[Abstract/Free Full Text]

  28. Miller JS, McCullar V, Punzel M et al. Single adult human CD34+/Lin–/CD38– progenitors give rise to natural killer cells, B-lineage cells, dendritic cells and myeloid cells. Blood 1999;93:96-106.[Abstract/Free Full Text]

  29. Doi H, Inaba M, Yamamoto Y et al. Pluripotent hemopoietic stem cells are c-kit<low. Proc Natl Acad Sci USA 1997;94:2513-2517.[Abstract/Free Full Text]

  30. Lian Z, Feng B, Sugiura K et al. c-kit<low pluripotent hemopoietic stem cells form CFU-S on day 16. STEM CELLS 1999;17:39-44.[Abstract/Free Full Text]

  31. Ogata H, Bradley WG, Inaba M et al. Long-term repopulation of hematolymphoid cells with only a few hematopoietic stem cells in mice. Proc Natl Acad Sci USA 1995;92:5945-5949.[Abstract/Free Full Text]

  32. Freudenthal PS, Steinman RM. The distinct surface of human blood dendritic cells, as observed after an improved isolation method. Proc Natl Acad Sci USA 1990;87:7698-7702[Abstract/Free Full Text]

  33. Scheicher C, Mehlig M, Zecher R et al. Dendritic cells from mouse bone marrow: in vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor. J Immunol Meth 1992;154:253-264.[CrossRef][Medline]

  34. Zsebo KM, Wypych J, McNiece IK et al. Identification, purification and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium. Cell 1990;63:195-201.[CrossRef][Medline]

  35. Lyman SD, James L, VandenBos T et al. Molecular cloning of a ligand for the flt3/flk2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 1993;75:1157-1167.[CrossRef][Medline]

  36. Varas A, Vicente A, Sacedon R et al. Interleukin-7 influences the development of thymic dendritic cells. Blood 1998;92:93-100.[Abstract/Free Full Text]

  37. Sogo S, Inaba M, Ogata H et al. Induction of c-kit molecules on human CD34+/c-kit<low cells: evidence for CD34+/c-kit<low cells as primitive hemopoietic stem cells. STEM CELLS 1997;15:420-429.[Abstract/Free Full Text]

accepted for publication December 21, 1999.




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