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a Department of Cell Biology, Beijing Institute of Basic Medical Sciences, Beijing, China;
b Department of Urology, PLA General Hospital, Beijing, China.
Key Words. Mesenchymal progenitor cells • Mixed leukocyte reaction • Immune regulation
Ning Mao, M.D., Department of Cell Biology, Beijing Institute of Basic Medical Sciences, 27 Tai-ping Road, Beijing 100850, People’s Republic of China. Telephone: 86-10-66931320; Fax: 86-10-68213039; e-mail: maoning{at}nic.bmi.ac.cn or maoning2002{at}hotmail.com
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ABSTRACT |
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INTRODUCTION |
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In recent studies, MPCs from humans and baboons have been shown to exhibit low immunogenicity and demonstrate significant suppressive activity in cell cultures containing alloreactive T cells [6, 7]. Immunologically, MPCs express surface markers including vascular cell adhesion molecule-1, intercellular adhesion molecule-1, lymphocyte function-associated antigen-3, CD44, and CD117 (c-kit) that have been proven to be involved in T-cell interactions [4, 8, 9]. The in vitro phenomenon that immature thymocytes selectively adhere to bone marrow stromal cells further implies that MPCs have an important role in lymphopoiesis [10]. From these accumulated data, it has been proposed that MPCs may play a critical role in modulating T-cell development and proliferation. However, the relationship between MPCs and the immune response remains unclear.
MPCs have been successfully isolated from human [4], feline [11], canine [12], rabbit [13], rat [14], chicken [15], sheep, goat, and pig [16] bone marrow by selecting the clonally growing adherent cells in plastic culture. The purity of MPCs in marrow adherent cells differs among species. Human and canine marrow adherent cells are relatively homogeneous and contain a high percentage of MPCs [5, 17]. In many respects, the mouse is an ideal model to study the cell biology and biochemical characteristics of MPCs. However, the standard method of plastic adherence has failed to yield relatively pure MPC populations from normal murine bone marrow because of several reasons: A) Marrow harvests contain low frequencies of MPCs (2-5/106 bone marrow nucleated cells [18]); B) mature hematopoietic cells exist in the center of the bone marrow, whereas stromal cells are present near the surface of the bone [19]. Recently, this has been reconfirmed by studies in humans and mice, which showed that MPCs are located in the endosteum or compact bone [20, 21]. Because of this deep location, it is difficult to attain enough MPCs even after strong flushing during marrow cell preparation; C) Murine bone marrow adherent cells contain a high frequency of non-MPCs and hematopoietic cells; and D) Most of the adherent stromal cells from murine bone marrow manifest differentiated phenotypes, even in the absence of differentiation-inducing culture conditions [22]. Due to the lack of appropriate methods to purify murine MPCs, the cellular characteristics and conditions required for growth and differentiation of these cells have not been clearly defined.
The ideal method to culture murine MPCs undoubtedly will vary depending on the goal of the study. From immortalized transgenic mouse models, or using retroviral selection of cycling adherent bone marrow cells, it is possible to isolate and culture-expand gene-transformed MPCs from murine bone marrow [22, 23]. In the present study, we wanted to evaluate the immunomodulatory effect of MPCs on lymphocytic proliferation. Since a major concern with gene-transformed cells is the introduction of a foreign gene that might render the cells abnormal, we enriched a relatively pure population of fresh MPCs by adding bone fragments and bone-marrow-conditioned medium to the culture. Additionally, the immunomodulatory effects of MPCs on T lymphocyte proliferation in vitro were studied.
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MATERIALS AND METHODS |
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Preparation of Bone-Marrow-Conditioned Medium
Femur and tibia bone fragments flushed off marrow were cultured in alpha-modified minimal essential medium (
-MEM) supplemented with 20% selected fetal bovine serum (FBS) (HyClone Lab, Inc.; Logan, UT; http://www.hyclone.com). Three days later, the medium was collected by centrifugation and used as bone-marrow-conditioned medium for MPC culture.
Culture and Expansion of MPCs
Six-week-old C57BL/6 mice were sacrificed by cervical dislocation. Their femurs and tibiae were carefully cleaned of adherent soft tissue, the epiphyses were removed with a rongeur, and the marrow was harvested by inserting a syringe needle (20-gauge) into one end of the bone and flushing with -MEM. After being drawn and expelled from the same syringe three times to disperse the marrow, cells were inoculated into 25-cm2 plastic flasks at a concentration of 106 nucleated cells per cm2. The four residual bone fragments from bone marrow preparation were also placed into the tissue culture flasks. The cells and bone fragments were cultured in complete medium (-MEM containing 10% selected FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin) at 37°C in a humidified atmosphere containing 95% air and 5% CO2 and allowed to attach for 4 days, at which point, nonadherent cells were removed by changing the culture medium.
Subsequent medium changes were performed every 4 days. When primary cultures became nearly confluent, the cells were detached with 0.025% trypsin containing 0.02% EDTA for 1 minute at room temperature. The action of trypsin was stopped by adding one-half the volume of FBS. After being washed twice with phosphate-buffered saline (PBS), the cells were suspended in complete medium supplemented with 10% bone-marrow-conditioned medium and then subcultured into culture flasks. Cells resulting from this replating were designated first-passage cells. At each passage, cells were typically diluted 1:2. After the fourth passage, cells were cultured in -MEM complete medium supplemented with 10 ng/ml basic fibroblast growth factor (bFGF) (PeproTech; Rocky Hill, NJ; http://www.peprotech.com). At about the eighth passage, cells were harvested for the experiments described below.
Flow Cytometry Analysis
Trypsinized cells (2 x 105) were washed with fluorescence-activated cell sorting (FACS) buffer (2% FBS, 0.1% NaN3 in PBS), incubated on ice for 30 minutes, and stained with fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse stem cell antigen (Sca)-1 (eBioscience; San Diego, CA; http://www.ebioscience.com), CD45, CD34 (Becton Dickinson; San Jose, CA; http://www.bd.com), phycoerythrin (PE)-conjugated rat anti-mouse H-2Kb, c-kit (CD117), CD44 (Becton Dickinson), major histocompatibility complex (MHC) class II I-A (b), CD80, and CD86 (eBioscience). Cells were also stained with purified rat anti-mouse CD29, CD105, and CD31 (Becton Dickinson) and then counterstained with FITC-conjugated goat anti-rat IgG. The cells stained with FITC- or PE-labeled goat anti-rat IgG were used as negative controls. After washing twice with FACS buffer, cells were fixed with 1% paraformaldehyde in PBS. At least 10,000 events were collected and further analyzed with a FACSCalibur cytometer and CellQuest software (Becton Dickinson).
Differentiation Assays of MPCs
Osteogenic differentiation was induced by culturing cells with osteoinductive medium (-MEM supplemented with 10% FBS, 10 mM ß-glycerol phosphate, 50 µM ascorbate-2-phosphate, 10-7 M dexamethasone, and 100 ng/ml recombinant human bone morphogenic protein [rhBMP]-2 [R&D Systems; Minneapolis, MN; http://www.rndsystems.com] [24]) and examining for alkaline phosphatase (ALP) activity and extracellular matrix (ECM) calcification.
To induce adipocytic differentiation, cells were cultured as monolayers, allowed to become near confluent, and then maintained in adipogenic induction medium (-MEM supplemented with 10% FBS, 10-7 M dexamethasone, and 10 µg/ml insulin) for 2 weeks with a medium change every other day [25]. Then, cells were assessed using Oil Red-O staining as an indicator of intracellular lipid accumulation.
Chondrogenic differentiation was performed using the micromass culture technique, as previously described [25, 26]. Briefly, 10 µl of a concentrated cell suspension (8 x 106 cells/ml) were plated into the center of each well of a six-well culture plate and allowed to attach at 37°C for 2 hours. Chondrogenic medium (-MEM supplemented with 1% FBS, 6.25 µg/ml insulin, 50 nM ascorbate-2-phosphate, 10 ng/ml transforming growth factor [TGF]-ß1 [PeproTech], and 1% antibiotic) was gently overlaid so as not to detach the cell nodules, and cultures were maintained in chondrogenic medium for 2 weeks prior to analysis.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from MPCs cultured in control or osteoinductive medium for 9 days using Trizol (GIBCO/BRL; Grand Island, NY; http://www.invitrogen.com) according to the manufacturer’s instructions and then treated with DNase (Promega; Madison, WI; http://www.promega.com). RT-PCR was performed by using the mRNA selective PCR kit (Takara Shuzo; Kyoto, Japan; http://www.takara.co.jp). A tenfold dilution of each sample was PCR amplified to achieve signals within the linear amplification range. The primers used were as follows: hypoxanthine phosphorybosyl-transferase, 5'-GCTGGTGAAAAGGACCTCT-3' and 5'-CACAGGACTA GAACACCTGC-3' [27]; osteocalcin (OCN), 5'-TCTGACA AAGCCTTCATGTCC-3' and 5'-AAATAGTGATACCGTA GATGCG-3'; and osteopontin (OPN), 5'-ACACTTTCACTC CAATCGTCC-3' and 5'-TGC CCTTTCCGTTGTTGTCC-3' [28]. Cycles for each primer pair were empirically determined so as to yield a product within the early exponential phase of synthesis to assure comparative analyses. Amplified DNA fragments were separated on a 2% agarose gel containing 0.1 µg/µl ethidium bromide and visualized under UV light.
Histochemical and Cytological Staining
Alkaline Phosphatase Assay The BCIP/NBT kit (Zhongshan Company; Beijing, China; http://www.zsbio.com) was used to assess ALP activity according to the manufacturer’s instructions.
von Kossa Staining Mineralization of ECM was visualized by von Kossa staining. Briefly, cells incubated in osteogenic medium for 4 weeks were fixed with 4% paraformaldehyde for 60 minutes at room temperature. After being rinsed with distilled water, cells were overlaid with 1% weight by volume (w/v) silver nitrate solution in the absence of light for 30 minutes. Then, cells were washed several times with distilled water and developed under UV light for 60 minutes. Excess silver staining was removed by washing several times with 5% sodium hyposulfite solution.
Oil Red-O Staining Cells were fixed for 60 minutes at room temperature in 4% formaldehyde/1% calcium, washed with 70% ethanol, and then stained for 10-15 minutes in a fresh working solution consisting of three parts Oil Red-O stock solution (0.5% Oil Red-O in 99% isopropyl alcohol) and two parts distilled water, which was mixed for at least 5 minutes and filtered before use [29]. Excess stain was removed by washing with 70% ethanol followed by several changes of distilled water prior to observation.
Alcian Blue Staining Cell nodules were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed with several changes of PBS. Studies have shown specific staining of sulfated proteoglycans, present in cartilaginous matrices, at pH levels of 1 and below [30]. In light of this, the fixed cells were incubated for 30 minutes with 1% (w/v) Alcian blue (Sigma A-3157; Sigma; St. Louis, MO; http://www.sigmaaldrich.com) in 0.1 M hydrochloric acid (HCl) (pH 1.0) and washed with 0.1 M HCl for 5 minutes to remove excess stain.
Allogeneic Mixed Lymphocyte Reaction (MLR) and Mitogen Proliferative Assays
Woolen purified BALB/c spleen T lymphocytes were incubated in triplicate in 96-well round-bottom plates (2 x 105/well) with a similar number of irradiated (20 Gy) spleen cells from the C57BL/6 strain or third-party (Kunming) mice in RPMI 1640 medium supplemented with 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10-5 M 2-mercaptoethanol. The total volume was 200 µl/well. Proliferation was assessed after 5 days by pulsing the cells with 1 µCi/well [3H]-labeled thymidine for the last 16 hours. Cells were harvested over fiberglass filters, and the ß irradiation was measured. Results are expressed as mean counts per minute (cpm) ± standard deviation (SD). Twenty Gray irradiated C57BL/6-derived MPCs were administered at different doses in the MLR on day 0. In mitogen proliferation assays, 2 x 105 responding spleen T cells from C57BL/6 mice were cocultured with 20 µg/ml concanavalin-A (ConA), with or without autologous MPCs present in the culture.
Statistical Analysis
Differences in the proliferation responses of responder cells were analyzed using the two-tailed Student’s t-test. p values less than 0.05 were considered statistically significant.
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RESULTS |
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), yet they replicated rapidly and could be expanded mitotically. Between day 4 and day 8, the number of MPC colonies increased, and a few large colonies could be observed. Some adherent round cells, which seemed to be macrophages, were also present in the primary culture, but they firmly adhered to the plate and were not as easily trypsinized. By the end of the fourth passage, normal cultures had very low frequencies of round cells (1%-2% of total cells) and consisted of greater than 95% bipolar fibroblastic cells (Fig. 1B).
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. Results show that more than 90% of the cells were strongly stained by monoclonal antibodies directed against Sca-1, which is a hallmark of hematopoietic and mesenchymal stem/progenitor cells, CD44, which mediates cell attachment to hyaluronan and osteopontin, and CD29, which represents the receptor for ECM. Additionally, cells were stained by the monoclonal antibodies to CD105 and c-kit. MPCs were also positive for H-2Kb, but were negative for MHC class II I-A (b). They did not express the costimulatory molecules CD80 and CD86 and they were also negative for CD45 (pan-hematopoietic marker), CD34, and CD31.
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Figures 1C and 1D
illustrate the ALP activity on day 8 of subcultured MPCs grown in the presence or absence of osteoinductive medium. A significantly greater ALP activity was observed after 8 days of osteoinductive treatment. These cultures were also studied for their ability to elaborate mineralized ECM. As shown in Figures 1E and 1F, MPCs grown in osteoinductive medium showed a substantial calcium deposition on day 28, while no calcium deposition was detected by von Kossa staining in control MPC cultures. Expressions of the osteoblast marker genes OCN and OPN during osteoblast differentiation were also investigated. OCN expression was positive in the osteoinductive cultures, while remaining negative in the control. Although a low-level expression of OPN could be detected in cultures grown in control medium, MPCs cultured in osteoinductive medium displayed a much higher level of OPN expression (Fig. 3).
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Chondrogenic differentiation can be induced in vitro using a micromass culture technique in which cellular condensation (a critical first event of chondrogenesis) is duplicated. Enhanced differentiation can be obtained by treating cells with dexamethasone and TGF-ß [32]. When cultured with these agents under micromass conditions, MPCs formed cell nodules associated with a well-organized ECM rich in sulfated proteoglycans [4, 33]. These sulfated proteoglycans can be specifically detected using Alcian blue staining under acidic conditions [30]. As shown in Figures 1I and 1J, murine MPC nodules cultured in chondrogenic-inductive medium were associated with an Alcian-blue-positive ECM, indicative of the presence of sulfated proteoglycans within the matrix, whereas no obvious staining was observed in undifferentiated MPCs. This suggests that MPCs possess the capacity to differentiate toward the chondrogenic lineage.
Alloresponse of MPCs
To test whether MPCs could elicit a proliferative response of allogeneic lymphocytes, T cells were cultured with allogeneic MPCs at various doses (Fig. 4). Although responding BALB/c T cells were able to proliferate in the presence of irradiated spleen cells of the MPC donor, C57BL/6, proliferation levels in the presence of purified MPCs from the same source were comparable with the autologous control, indicating that sorted T cells did not proliferate in response to allogeneic MPCs.
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). This could be suppressed in a dose-dependent way by the addition of increasing numbers of MPCs to the cultures, resulting in a >90% lower response at an MPC:T cell ratio of 1:2 (mean incorporation of 2,107.333 ± 430.2291 cpm, p = 0.000497) and a >50% lower response (mean incorporation of 5,637.429 ± 2,908.68 cpm) at a 1:20 ratio. The suppression of proliferation did not appear to be dependent on the source of MPCs. As shown in Figure 5, when splenocytes from third-party (Kunming) mice were used as stimulators, similar results were found. This shows that MPCs can inhibit proliferation independent of whether they are from the same source as the stimulator or a third party. Similarly, T-cell proliferation elicited by means of nonspecific polyclonal activators, such as ConA, could be suppressed when autologous MPCs were present (a >50% lower response at an MPC:T cell ratio of 1:2, Fig. 6). These results suggest that MPCs can suppress T-lymphocyte proliferation induced by cellular and nonspecific mitogenic stimuli.
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DISCUSSION |
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Culture Expansion of MPCs
Murine bone marrow stroma contains mesenchymal progenitors that may serve as precursors for bone, cartilage, fat, and muscle. However, it is more difficult to isolate and culture-expand fresh MPCs from murine bone marrow using classical plastic adherent methods than from humans and other animals. Also, the optimal culture conditions for murine MPCs remain undetermined. During the pathophysiology of bone fracture, MPCs present in bone marrow expand rapidly so as to differentiate into osteoblasts. MPCs also contribute to the marrow microenvironment with inductive/regulatory signals for the development of hematopoietic cells as well as for stromal cells, including the MPCs themselves, by constitutively secreting interleukin (IL)-6, IL-7, IL-8, IL-11, IL-12, IL-14, and IL-15, macrophage-CSF, leukemia inhibitory factor (LIF), Flt-3 ligand, and stem cell factor [9, 34], and are inducible with IL-1 to produce IL-1, LIF, G-CSF, and GM-CSF [35]. The conditioned medium recovered from cultured regeneration marrow contains these growth factors as well as fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor-1 [36], which have recently been shown to be vital to culture-expand pluripotent mesenchymal stem cells from murine bone marrow [37]. We found that using bone fragments and conditioned medium, especially in the early phase of culture, enabled the cells to grow under better conditions, and long-term cell expansion could be attained.
On day 3, MPC colonies appeared as fibroblast-like cells, bipolar in morphology, contrasting with contaminating monocyte colonies, which appeared as large, round cells with irregular boundaries. The number of contaminating macrophages decreased with cell passaging, and by the end of the fourth passage, most of the cells were bipolar fibroblast-like in morphology. The phenotype analysis showed that they were strongly stained with Sca-1 while negative for CD45, implying that these cells were mesenchymal progenitors without contamination of hematopoietic cells.
However, the most prominent characteristic of MPCs is their multiple differentiation capacity when exposed to the appropriate induction media. Murine MPCs exhibited multilineage potential in vitro, differentiating toward adipogenic, osteogenic, and chondrogenic lineages when cultured in the presence of lineage-specific differentiation factors. These results were consistent with those observed in a variety of species, including rats [38], dogs [12], cats [11], and humans [4].
The data presented here demonstrate that the murine bone-marrow-derived adherent cells cultured in our system gave rise to a morphologically homogeneous population of cells that exhibited phenotypic and functional features of mesenchymal progenitors. The method used in our study proved to be a novel and reliable protocol to prepare a relatively homogeneous population of murine MPCs.
Immunomodulatory Capacity of MPCs
In the current study, we investigated the immunogenicity of MPCs. When allogeneic MPCs were added to the T-cell culture, the proliferation results were similar to those of the syngeneic control, consistent with the immunophenotype that MPCs displayed MHC I but were negative for both MHC II and the T-cell costimulatory molecules CD80 and CD86. This demonstrates that murine MPCs were not substantially immunogenic. Furthermore, a dramatically lower T-cell proliferation was obtained when mixed cultures of T lymphocytes stimulated by irradiated allogeneic splenocytes were performed in the presence of irradiated allogeneic or third-party MPCs. These immunosuppressive characteristics are consistent with their counterparts in baboons and humans [6, 7]. Human MPCs also do not express MHC II antigen or the T-cell costimulatory molecule B7 [39], and can exert a tolerizing effect on umbilical cord blood graft lymphocytes during in vitro expansion [40]. Ongoing MLR can be suppressed by the addition of responder, stimulator, or, third-party MPCs, and human MPCs may actually inhibit both primary and secondary MLRs [6]. MPCs also exert an in vivo immunoregulatory effect, contributing to the prolonged survival of allogeneic skin grafts in baboons and the decreased incidence of graft-versus-host disease after bone marrow transplantation in humans [7, 41]. These accumulated data provide evidence of the potential immunomodulatory properties of these cells.
Murine MPCs expressed molecules involved in T-cell interactions, such as CD44 and CD117. The expressions of these surface molecules, together with previous findings that MPCs share surface markers with thymic epithelium [8, 9], suggest that MPCs may set up an extrathymic site for T-cell lymphopoiesis within the bone marrow microenvironment. The close relationship between T cells and MPCs has been well described. Early-phase thymocytes preferentially adhere to bone marrow stromal cells in vitro [10]. In addition, after bone marrow transplantation plus bone grafts, donor stromal cells can migrate from bone marrow to thymus, where they participate in positive selection of thymocytes [42]. As murine marrow MPCs can be induced to differentiate into bone marrow stromal elements, such as osteoblasts, chondrocytes, and adipocytes, it is possible that these cells may also play an important role in both hematopoietic engraftment and the induction of immune tolerance. All these developments illuminate a wide prospect for MPCs as a useful tool in allograft transplantation.
At present, the mechanisms of why MPCs can suppress lymphocytic proliferation are not well defined. Soluble factors such as TGF-ß1 and hepatic growth factor may play a role in this regulatory effect [6]. However, studies carried out in murine models are required to further investigate this area.
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ACKNOWLEDGMENT |
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FOOTNOTES |
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