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Departments of
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Medicine-Hematology/Oncology,
Pediatrics, and
Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey; and
University of Medicine and Dentistry of New Jersey-Graduate School of Biomedical Sciences, Newark, NJ 07103
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LHSC can trans-differentiate into nonhemopoietic cells (12, 13). MSC similarly possess trans-differentiation potential and may prove to be clinically useful (3, 7). MSC might be the progeny of a more primitive mesenchymal stem cell, described as multiple adult progenitor cell (3, 14). Regardless, MSC and/or their precursor(s) might withstand the test of science in the long term as the best candidate stem cells for application in repair medicine (3, 15). MSC can differentiate into different cell lineages, including BM stroma (5). The ability of MSC to form stroma gives these cells the potential to be adjuvant cells in BM transplantation to improve engraftment of the LHSC (2, 16).
MSC show plasticity, based on their ability to trans-differentiate into cells of other germ layers (17). MSC have been reported to form neurons (18, 19, 20, 21), hepatocytes (14), muscle cells (22), and endothelia (23, 24). The lineage commitment and trans-differentiation properties of MSC make these stem cells candidates for use in repair/molecular medicine and tissue engineering. MSC have the potential to repair bone and could conceivably be used to replace osteoblasts in patients with osteogenesis imperfecta (2).
Morphologically, MSC could be confused with BM fibroblasts/stroma or at times even neurons. However, MSC are symmetrical, while stromal cells are asymmetrical. LHSC, and their progenies can be distinguished from MSC by expression of nonoverlapping markers. MSC are negative for CD34, CD45, and CD14 (16) and express endoglin (SH2 and SH3), CD29, CD44, CD71, CD90, CD106, and CD120a (7). Both endothelial cells and MSC express endoglin (25); however, despite the close anatomical association of MSC with the vasculature, MSC are negative for the endothelial cell marker, CD31 (7).
At this time it is uncertain how the potential expression of MHC class II might affect implantation or transplantation of MSC. Most of the cell types derived from trans-differentiated MSC will not express MHC class II, unless there is an inflammatory response. For example, neural cells can express MHC class II under certain pathophysiological conditions (26). Current studies do not provide definitive answers regarding the differentiation stage at which MSC will be transplanted into patients. The question that arises is whether MSC should be implanted in an injured tissue as partly or fully trans-differentiated cells or if chemotactic mechanisms should be applied to the site of injury to attract autologous MSC.
Low expression of MHC class II on MSC gives these cells the potential to elicit weak allogeneic responses. Thus, it is possible that MSC could mediate alteration of the immune system during the interval between transplantation and trans-differentiation. Reports have shown that MSC are low allogeneic stimulators and might even suppress an ongoing immune response (27). Therefore, the design of clinical strategies to use MSC in BM transplantation and repair medicine cannot ignore their allogeneic properties, which could be solved if MSC show properties of veto functions.
Veto cells suppress CTL precursor function against Ags present on the veto cell surface, but not against those on third-party allogeneic cells (28, 29). Veto cells have been shown to prevent the rejection of allogeneic BM grafts in an Ag-specific manner (30). Veto cells are ubiquitously distributed, including in the BM (28, 29, 30, 31). In the present study we showed immune-suppressive effects of MSC during allogeneic responses and also demonstrated veto-like properties of MSC. This report explores questions that provide further insight into the biology of MSC, and the results have important implications about their clinical utility.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BM aspirates and peripheral blood (10–20 ml) were taken from healthy subjects ranging in age from 20 to 30 years. BM aspirates were taken from the posterior iliac crest and immediately placed in medium containing preservative-free heparin. The protocol for human subjects adhered to guidelines outlined by the institutional review board of the University of Medicine and Dentistry of New Jersey (Newark, NJ). Allogeneic differences of peripheral blood and BM donors were determined in one-way MLR, described below.
Reagents
The following were purchased from Sigma-Aldrich (St. Louis, MO): sodium heparin, dexamethasone, 
-glycerophosphate, silver nitrate, Ficoll-Hypaque, paraformaldehyde (PFA), DMEM with high glucose, RPMI 1640, BSA, 
-MEM, DMEM, Con A, LPS, hydrocortisone, and glutamine. FCS and horse serum (HyClone, Logan, UT) were heat-inactivated for 45 min at 56°C. PBS, PHA, and colcemid were purchased from Life Technologies (Grand Island, NY). L-ascorbic acid-2-phosphate was purchased from Wako Chemicals (Osaka, Japan).
Antibodies
The following Abs were purchased from BD PharMingen (San Jose, CA): PE-conjugated HLA-DR mAb, PE-conjugated CD14 mAb, PE-rat anti-mouse 
, polyclonal FITC-conjugated anti-mouse IgG, isotype control IgG, and FITC-conjugated CD45 mAb. CD44 mAb was purchased from T Cell Diagnostics (Cambridge, MA). CD31 and prolyl-4-hydroxylase mAbs were purchased from DAKO (Carpinteria, CA). FITC-conjugated nonimmune rabbit IgG was purchased from Sigma-Aldrich.
The SH2 hybridoma (HB-10743) was purchased from American Type Culture Collection (Manassas, VA) and grown according to their instructions. SH2 cells were injected i.p. in BALB/c mice that were >6 mo old. Mice were housed in an American Association for Accreditation of Laboratory Animal Care-accredited animal facility, and the use of mice for propagation of ascites was approved by the University of Medicine and Dentistry of New Jersey institutional animal care and use committee. SH2 Abs were collected as ascites from mice and then purified by affinity on a Sepharose G column. Purified IgG was tested against MSC in dose-binding immunofluorescence assays. Nonspecific binding was determined with BM fibroblasts. For immunofluorescence assays, SH2 mAb was used at 50 ng/ml of purified IgG.
Culture of MSC
Immediately after taking BM aspirates from a donor, 2 ml was added to 15 ml of DMEM with 10% FCS in Falcon 3003 petri dishes (Fisher Scientific, Springfield, NJ). Plates were incubated at 37°C for 3 days, after which RBC and granulocytes were removed by Ficoll-Hypaque density-gradient centrifugation. Cultures were reincubated until confluence, which occurred at 
2–3 wk after removal of RBC. MSC are sensitive to the toxic effects of Ficoll-Hypaque and were quickly collected and replaced in the cell culture flask following density-gradient centrifugation. FCS is crucial for optimal propagation of MSC and required proper quality control screening before its use to culture MSC. The FCS used in our studies ensured stable phenotype expression by cultured MSC for more than seven passages. Cells were passaged at least three times before being used in the assays (described below). Cells used in all assays were negative for prolyl-4-hydroxylase, CD14, CD31, and CD45 and positive for SH2 and CD44 (32) (Table I).
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Stromal cultures were established with unfractionated BM aspirates as previously described (33). Briefly, BM aspirates were placed in -MEM containing 12.5% FCS, 12.5% horse serum, 10-7 M hydrocortisone, 10-4 M 2-ME, and 1.6 mM glutamine. After 3 days at 33°C, RBC and granulocytes were removed by Ficoll-Hypaque density gradient centrifugation, and cultures were reincubated with weekly replacement of 50% medium until cells reached confluence. BM fibroblasts were prepared by passaging trypsinized BM stroma at least five times in -MEM with 20% FCS. Fibroblast cultures were examined for purity by immunofluorescence and cytochemical staining as previously described (33).
One-way MLR
PBMC were isolated by Ficoll-Hypaque density gradient centrifugation of peripheral blood. PBMC were washed and then resuspended at 2 x 106/ml in RPMI 1640 with 10% FCS and 5 x 10-7 M 2-ME (R10 medium). MSC were deadhered by trypsinization or gentle scraping with a disposable cell scraper. PBMC and MSC were resuspended in R10 medium at 2 x 106 and 105/ml, respectively. Stimulators (PBMC and MSC) were gamma-irradiated (200 cGy) with a cesium source. After irradiation cells were washed once, recounted, and then resuspended at the same concentrations, as stated above. Dose-response assays determined 105 MSC/ml to be the optimal concentration for one-way MLR with MSC stimulators.
MLR were performed in 96-well, flat-bottom plates (Corning, Corning, NY). Equal volumes (0.1 ml) of stimulators (gamma-irradiated PBMC or MSC) and responders (PBMC) were added to each well, and cultures were performed in triplicate. The cultures were pulsed with 1 µCi/well of [methyl-3H]TdR (70–90 Ci/mmol; NEN, Boston, MA) during the last 16 h of a 4-day culture. Cells were harvested with a PhD cell harvester (Cambridge Technologies, Cambridge, MA) onto glass-fiber filters (Cambridge Technologies). [3H]TdR incorporation was quantified in a scintillation counter (Beckman, Fullerton, CA). The results are expressed as the stimulation index (S.I.), which is the mean dpm of experimental cultures (responders + gamma-irradiated stimulators)/dpm of responder cells with only medium.
Modified MLR
Two modifications of MLR were performed. 1) MSC were fixed with 0.4% PFA, and 104 cells were used as stimulators with PBMC responders. PFA fixation was performed by resuspending 104 MSC/ml in 0.4% PFA (solution prepared in PBS, pH 7.4) for 5 min. After this, cells were washed twice in PBS and then resuspended in R10 medium. 2) One-way MLR was performed with nonirradiated PBMC as responders and gamma-irradiated allogeneic PBMC as stimulators. Parallel cultures contained third-party allogeneic (nonirradiated or gamma-irradiated) MSC or BM fibroblasts. MSC and BM fibroblasts were cultured from BM aspirates of the same donor. Allogeneic differences among BM donors as well as donors of responder and stimulator PBMC were pretested in the one-way MLR (described above).
Effects of MSC on cytotoxic responses
51Cr release assay. For use as target cells, PBMC from healthy donors were resuspended at 107 cells/ml in R10 medium, and 1 ml was added to 4 ml of R10 medium in 25-cm2 tissue culture flasks (Costar; Corning). Cells were stimulated with PHA (final dilution, 1/1000), and flasks were incubated in an upright position. On day 5 cells were washed three times, and 107 cells in 1 ml of R10 medium were labeled with 200 µCi of 51Cr (401 mCi/mg; NEN) for 2 h. Cells were washed five times and then resuspended in R10 medium at 105 cells/ml for use in the cytotoxicity assay.
Cytotoxic effector cells were prepared to ascertain that the cells were primed against the alloantigens of the donors’ target cells (described above). Therefore, effector PBMC showed allogeneic differences from target PBMC. Stimulators (106/ml) were irradiated with 200 cGy and cocultured with an equal number of nonirradiated responder PBMC in upright 25-cm2 tissue culture flasks (5 x 106 cells total/flask) at 37°C for 5 days.
Cytotoxic assay. Effector cells were harvested and then washed three times. Viable cells were resuspended at 107 cells/ml in R10 medium. Assays were performed in 96-well, round-bottom, tissue culture plates (Costar) at E:T cell ratios of 100:1, 50:1, 25:1, and 12.5:1 with or without MSC (103–105 cells total). The following controls were performed in sextuplet: target cells incubated in medium alone to determine spontaneous release of 51Cr from targets, and target cells incubated in medium containing 1% Triton X-100 to determine maximal isotope release from targets. Typically, spontaneous release was <10%, and maximum release was >90% of the absolute counts per minute of target cells. After 4 h cell-free supernatants were collected, and the amounts of 51Cr released through cell lysis were determined in a gamma counter. The percent cell lysis was calculated according to the following equation: (experimental point (dpm) - spontaneous release (dpm))/(total release (dpm) - spontaneous release (dpm)) x 100.
Cytokine array
The human cytokine protein array II was purchased from Ray Biotech (Norcross, GA) and used according to the manufacturer’s instructions. Briefly, membranes were incubated for 30 min in 5% BSA in 0.01 M Tris buffer with 0.15 M NaCl, pH 7.6. After this, membranes were subjected to three 5-min washes with 1x TBS/0.1% Tween, followed by two 5-min washes with TBS. Fresh culture medium or cell-free supernatants from confluent MSC were added to the membrane, which were incubated for 1 h with biotin-conjugated cytokines (provided with the kit). After incubation, membranes were washed twice with TBS and then incubated for 30 min with HRP-conjugated streptavidin. Unbound reagents were washed, and the membranes were developed with the ECL system (NEN).
Immunofluorescence
Immunofluorescence studies were performed microscopically and by FACScan. For microscopy, MSC or fibroblasts were cultured on coverslips that were placed in a 35-mm petri dish. At 80% confluence, nonadherent cells were aspirated, and the adherent cells washed with PBS, pH 7.4. Primary Abs were diluted in PBS with Tween and then added to coverslips. Cells were incubated with Abs for 30 min at room temperature, washed three times with PBS/Tween, and secondary Abs were added for 30 min. Control slides were incubated with secondary Ab alone, fluorescein-conjugated primary Ab alone, or a fluorescein-conjugated isotype control Ab. Cells were examined for fluorescence intensity using Probis microscope (Olympus, New Hyde Park, NY). For FACScan, cells were trypsinized and similarly labeled while in suspension.
Karyotyping
MSC were seeded into Falcon 3003 petri dishes. After 12 h, cells were 40–50% confluent. At this time, 50 µl of colcemid (10 µg/ml) was added to each dish to yield a final concentration of 0.1 µg/ml, and cultures were reincubated overnight at 37°C. Cells were collected by trypsinization, washed, lysed with 0.75 M hypotonic KCl, and fixed with acid/alcohol (3/1). Chromosomes in metaphase were analyzed following Wright’s staining.
Telomerase activity
Telomerase activities from MSC and BM fibroblasts were determined with the Telo TAGGG telomerase PCR ELISA Plus kit (Roche, Indianapolis, IN). The technique followed the manufacturer’s instructions. Briefly, cells at 50–75% confluence were trypsinized and washed with PBS, pH 7.4, and 2 x 105 cells were lysed with the lysis reagent provided in kit. Cell-free supernatants were transferred to another tube and then kept on ice until 2 vol of lysate (3 and 10 µl) were analyzed in PCR for telomerase activity, which is proportional to the density of telomeric repeats.
Adipogenic differentiation
Adipogenic differentiation was performed with the Adipogenic hMSC Differentiation Bullet Kit (Cambrex BioScience, Walkersville, MD). The method followed the manufacturer’s recommendations. Briefly, MSC at 80% confluence were trypsinized and then replated at 5 x 105 cells/well in six-well tissue culture plates. At cell confluence, adipogenic differentiation was performed with three cycles of induction/maintenance medium. Each cycle consisted of 3-day culture in adipogenic induction medium, followed by 1–3 days with adipogenic maintenance medium. Control wells were cultured, and medium was replaced on the same schedule using adipogenic maintenance medium. After the three-cycle schedule, cells were grown in adipogenic maintenance medium for 7 days, with change of medium every 3 days. After this, cells were washed with PBS, fixed with 10% formalin, stained with Oil Red O, and counterstained with hematoxylin.
Osteogenic differentiation
MSC were trypsinized, replated at 2 x 104 cells/well of a six-well plate, and allowed to adhere for 24 h. Osteogeneic differentiation was induced in DMEM containing 10% FCS, 100 nM dexamethasone, 10 mM -glycerophosphate, and 50 µM L-ascorbic acid-2-phosphate. Differentiation medium was replaced every 3–4 days for 3 wk. Controls wells were grown and replaced with MSC medium on the same schedule. Osteogenic differentiation was assessed by mineral deposits by von Kossa stain. Cells were fixed with 10% formalin, stained for 10 min in the dark with 2% silver nitrate solution, washed, and exposed for 15 min to light.
Statistical analysis
Data were analyzed using ANOVA and Tukey-Kramer multiple comparisons test. A value of p <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1A shows photomicrographs of MSC cultures taken at different growth confluencies. Morphologically, MSC are spindle-shaped and symmetrical with respect to the nuclei. The morphology of MSC was the same regardless of the degree of confluence or the number of cell passages (sparse growth, Fig. 1A, left; confluent culture, Fig. 1A, right). In our hands we could not alter the formulation of the culture medium used to grow MSC (described in Materials and Methods). A change in the culture medium resulted in cells that were morphologically different from those shown in Fig. 1A. The symmetrical, linear morphology of MSC (Fig. 1A) could be distinguished from the asymmetry and polarization in multiple directions exhibited by BM fibroblasts (Fig. 1B). To differentiate BM fibroblasts (BM stroma) from MSC is relevant because BM fibroblasts are differentiated progenies of MSC (34).
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We previously established cultures of BM fibroblast with mononuclear cells from BM aspirates and -MEM containing 20% FCS (33). With recent phenotypic definition of MSC (7), we re-evaluated our technique for culturing fibroblasts (7) and found <5% contamination of cells that are positive for SH2 (not shown). We now show that purified BM fibroblasts (no detection of SH2+ cells; Table I) can be obtained by subculture with five passages of confluent BM stromal cells (35). Immunofluorescence staining of these fibroblasts at passage 5 showed that in addition to a distinct morphology (Fig. 1B), fibroblasts could be distinguished from MSC by immunofluorescence analyses (Table I).
Multiple passages of MSC and in vitro manipulation of BM cells could cause changes in chromosomes, such as whole or partial and/or chromosomal fusion. Therefore, we performed cytogenetic analyses on representative MSC cultures. For each experiment the karyotype was normal (representative karyotype shown in Fig. 1C). High telomerase activity is characteristic of stem cells (36). We therefore studied telomerase activities in MSC and in differentiated cells of mesenchymal origin, i.e., fibroblasts. In seven analyses, using a different donor, the results show 4-fold increases in telomerase activity in MSC compared with passaged BM fibroblasts. The high telomerase activity demonstrated in MSC is consistent with their stem cell property (36).
Osteogenic and adipogenic differentiation were performed as further verification that the cells shown in Fig. 1A have stem cell properties (7). In three differentiation studies (each with a different donor), the cultures stained positive for Oil Red O (Fig. 1D) and von Kossa stain (Fig. 1E), indicating that the cells have adipogenic and osteogenic potential.
MHC class II expression on MSC
The level of MHC class II expression on MSC is undefined (2, 7). Since MSC are proposed as a potential source for tissue regeneration (3, 24), clarification of MHC class II expression is important. Therefore, we studied the expression of MHC class II by FACS analysis with purified MSC labeled with PE-conjugated HLA-DR mAb. The results showed a shift in >70% MSC to the right after adjustment with the isotype control (overlapping region of Fig. 2A). Based on the broad peak shown in the fluorescence labeling (Fig. 2A), we next studied the expression of HLA-DR at a single-cell level by immunofluorescence microscopy. The results (Fig. 2B) showed bright fluorescence at the cell membrane. The fluorescence was observed in a punctated distribution pattern in areas close to the nuclei, but dim to undetectable on the projections that were distant from the nuclei. Isotype control staining did not show any fluorescence (not shown). The data show that MSC express HLA-DR, and that the expression is concentrated in areas of the cell membrane close to the nuclei.
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MSC have been reported to exhibit a capacity to stimulate only meager responses in allogeneic PBMC (27). However, since our results showed dense expression of MHC class II expression on MSC (Fig. 2), our next sets of studies were designed to determine whether MSC could elicit allogeneic responses. This question was addressed in one-way MLR with gamma-irradiated MSC as stimulators obtained from five different BM donors. Stimulator MSC from each donor was cultured with responder PBMC from seven different donors. One-way MLR verified that stimulators and responders were allogeneic with each other (not shown). Each bar in Fig. 3 represents the mean S.I. ± SD of seven responders (each from a different donor) with gamma-irradiated MSC from one donor. The results showed that MSC could elicit significant proliferation of responder PBMC with S.I. values ranging between 3.5 and 6.5.
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MSC have been reported to express genes for different growth factors (21, 34). Gamma-irradiated stimulator MSC could produce soluble factors in one-way MLR (Fig. 3) because the cells are metabolically activity. Thus, to determine whether cytokines produced by MSC were involved in the allogeneic responses of MSC (Fig. 3), we first studied the production of cytokines from MSC by microarray analysis. The microarray membranes, which comprised 43 different growth factors, were hybridized with supernatants from confluent MSC or fresh culture medium. Detectable spots were scanned by densitometer, and detection with fresh culture media was subtracted from the test samples. Positive controls, provided by the manufacturer, were normalized to 3-fold, and the unknown samples were calculated from the normalized spots. The results (Table II) are therefore presented as fold increase from the positive controls. High densities of growth factors belonging to the chemokine family and those associated with angiogenesis were detected. There was no obvious difference between pro- and anti-inflammatory cytokines. The results showed constitutive expression of growth factors that could be grouped into different categories based on hemopoietic functions, angiogenic properties, and immune regulation.
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The next set of studies determined whether the allogeneic responses shown in Fig. 3 could be due to soluble factors produced by MSC (Table II). This question was addressed by comparing MLR using paraformaldehyde-treated vs gamma-irradiated MSC as stimulators. The proliferative responses of MLR performed with paraformaldehyde-treated and gamma-irradiated stimulators were not significantly (p > 0.05) different (Fig. 4A). Furthermore, the addition of supernatants obtained from paraformaldehyde-treated cells cultured for up to 24 h showed no effect on cell proliferation in one-way MLR with PBMC from two different donors (not shown).
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Third-party effects of MSC in one-way MLR
An in vitro model of on-going immune responses was used to further explore the dichotomy in the functions of MSC, i.e., their ability to stimulate allogeneic MLR (Fig. 3) vs their reported immunosuppressive activities (27). MSC were used as third-party cells in one-way MLR containing PBMC as responders and allogeneic gamma-irradiated PBMC as stimulators. Fig. 5A shows the responses of PBMC from four different donor/responder combinations (A–D). Each bar represents cultures of one responder and four allogeneic PBMC stimulators in the presence or the absence of MSC from three different donors (gamma-irradiated or nonirradiated). Analyses of the results indicate an 3-fold decrease in cell proliferation when MSC were added as third-party cells. The effects were the same when irradiated or nonirradiated MSC were used (not shown). Furthermore, when supernatants from confluent MSC (in lieu of cells) were added to allogeneic MLR, they did not suppress the reaction (not shown).
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). Each bar in Fig. 5B represents the mean S.I. ± SD of one PBMC responder and four gamma-irradiated PBMC stimulators. Allogeneic differences between responders and stimulators were verified in one-way MLR (data not shown). Together the results indicate that while third-party MSC suppress one-way allogeneic MLR, similar suppression was not observed when BM fibroblasts from the same donor were added to the MLR. Veto-like functions of MSC
MSC as third-party cells in MLR mediated inhibitory effects on cell proliferation (Fig. 5A). To further clarify the effects of MSC as an immune suppressor, we added MSC to an assay in which PBMC were sensitized to recognize and produce cytotoxic effects or cells against stimulator cell alloantigens. Presensitized PBMC (effector) were established in 5-day cocultures with gamma-irradiated PBMC from an unrelated donor. Effectors from these MLR were analyzed for cytotoxicity against blast-activated targets, which were obtained from 3-day cultures with PHA.
Cytotoxic assays were performed with 104 target cells and variable number of effector cells necessary to obtain different E:T cell ratios. Cultures were established in the presence or the absence of constant numbers (103 cells) of MSC, and 51Cr released from targets was used as a readout of cytotoxicity. The percent 51Cr release at an E:T cell ratio of 25:1 (Fig. 6A) indicated significant suppression in wells containing MSC (p < 0.05). However, suppression was not observed in wells containing a similar number of BM fibroblasts that were cultured from the donor of MSC (Fig. 6A). The data in Fig. 6B show a typical cytotoxic response in the absence of MSC for one donor at different E:T cell ratios. As shown in Fig. 6C, the donor cells were verified for allogeneic differences in parallel studies with one-way MLR. Based on the S.I. shown, it could be deduced that the donors used to induce cytotoxicity (effectors and targets) were significantly different with respect to allogeneic variations. It should be noted that allogeneic variation was considered if the donors of effector/target pairs showed an S.I. >3 in one-way MLR. These results indicated that MSC could also suppress the cytotoxic effects of Ag-primed effector cells in a short term (4-h) assay.
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The effects of MSC on Ag-specific cytotoxic responses (Fig. 6) suggested a veto-like function of MSC. Since veto cells should not be able to alter stimulatory effects of recall Ags, the next set of studies was performed to determine whether MSC could affect cell proliferation induced by common recall Ags. PBMC were stimulated with three different recall Ags (Candida albicans, Bordetella pertusis, and tetanus toxoid) in the presence or the absence of allogeneic or autologous MSC. Dose-response curves determined the optimal concentrations of each Ag. Suppressive effects by MSC on 7-day stimulation with the recall Ags was not observed (Fig. 7). The increased proliferation following the addition of MSC to allogeneic PBMC (Fig. 7) was expected, since MSC were previously shown to stimulate MLR when added to naive allogeneic PBMC (Fig. 3). In contrast, MSC showed no effect on proliferation when they were added to recall Ag-stimulated autologous PBMC (not shown). The results indicated that compared with the suppressive effects by MSC on allogeneic MLR and alloantigen-induced cytotoxic responses, they had no effect on the response to recall Ags.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although there are several reports on the isolation of MSC (37), in our hands, we have standardized the most efficient method to obtain highly purified MSC. The study was pursued with the intent that MSC could trans-differentiate into cells of different tissues (26). The issue regarding the ability of MSC to elicit allogeneic responses is unclear and has been addressed in detail in this study. At present, it is unclear how MSC would be applied in repair medicine, e.g., should MSC be transplanted as unmanipulated cells or should cells be implanted as fully or partly differentiated? However, regardless of their stage of maturation, the allogeneic properties of MSC would be relevant.
The allo-suppressive effect of MSC is specific since similar suppressive effects were not observed for BM fibroblasts from the same donor (Fig. 5B). The BM fibroblasts used in the studies were pure, with no detectable contaminating cells such as macrophages (not shown). BM fibroblasts represent the major subset of stromal cells and can be differentiated from MSC (38, 39). Thus, our findings differ from other recent studies by Di Nicola et al. (40), which showed immunosuppressive effects of BM fibroblasts. The contrast between studies by Di Nicola (40) and those in our report could be explained by differences in the experimental design. Studies by Di Nicola et al. (40) used purified T cells from peripheral blood as their responding population, while we used the entire mononuclear cell fraction. Our goal was to establish a condition that could model a situation of graft-vs-host-like responses. Our results pertaining to cytokine production are consistent with those reported by Di Nicola (40), in which a role for TGF- was established (Table II). Further studies regarding the data on cytokines (Table II) are needed, and extrapolation of the findings would be speculation.
The immune-suppressive functions of MSC are supported by two independent reports (27, 40). Noort et al. (41) showed that MSC promote engraftment of human cells in SCID mice. This property of MSC in engraftment could not be explained by MSC forming their own stromal cells, since longitudinal studies with human transplant patients showed that the stromal cells were of host origin (42). The facilitating effects of MSC in BM transplantation might be explained by the veto-like functions of MSC. Thus, contrary to current thought that MSC could facilitate engraftment by forming stromal layers, the same end point might be explained by another mechanism, suppression of graft-vs-host disease during transplantation. The studies presented in this report do not discriminate concerning whether MSC exert suppressive functions in both direct and indirect pathways by alloantigen (43). Further experimental studies are warranted to address this pertinent question to understand the immunology of MSC. Further basic and clinical research into the biology of MSC in the BM and identification of a possible precursor of MSC will facilitate their use in repair medicine.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pranela Rameshwar, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, MSB, Room E-579, 185 South Orange Avenue, Newark, NJ 07103. E-mail address: rameshwa{at}umdnj.edu
3 Abbreviations used in this paper: BM, bone marrow; LHSC, lymphohemopoietic cells; MSC, mesenchymal stem cells; PFA, paraformaldehyde; S.I., stimulation index.
Received for publication March 14, 2003. Accepted for publication July 29, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signaling components by cooperative interaction between Sp1 and KLF6: their potential role in the response to vascular injury. Blood 100:4001. in the veto effect. Transplantation 67:372.[Medline]
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K. C. Tang, K. A. Trzaska, S. V. Smirnov, S. V. Kotenko, S. K. Schwander, J. J. Ellner, and P. Rameshwar Down-Regulation of MHC II in Mesenchymal Stem Cells at High IFN-{gamma} Can Be Partly Explained by Cytoplasmic Retention of CIITA J. Immunol., February 1, 2008; 180(3): 1826 - 1833. [Abstract] [Full Text] [PDF]
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A. J. Nauta and W. E. Fibbe Immunomodulatory properties of mesenchymal stromal cells Blood, November 15, 2007; 110(10): 3499 - 3506. [Abstract] [Full Text] [PDF]
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 S. J. Greco, S. V. Smirnov, R. G. Murthy, and P. Rameshwar Synergy between the RE-1 Silencer of Transcription and NF{kappa}B in the Repression of the Neurotransmitter Gene TAC1 in Human Mesenchymal Stem Cells J. Biol. Chem., October 12, 2007; 282(41): 30039 - 30050. [Abstract] [Full Text] [PDF]
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 I. Rasmusson, M. Uhlin, K. Le Blanc, and V. Levitsky Mesenchymal stem cells fail to trigger effector functions of cytotoxic T lymphocytes J. Leukoc. Biol., October 1, 2007; 82(4): 887 - 893. [Abstract] [Full Text] [PDF]
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 S. J. Greco and P. Rameshwar MicroRNAs regulate synthesis of the neurotransmitter substance P in human mesenchymal stem cell-derived neuronal cells PNAS, September 25, 2007; 104(39): 15484 - 15489. [Abstract] [Full Text] [PDF]
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S. J. Greco and P. Rameshwar Enhancing Effect of IL-1{alpha} on Neurogenesis from Adult Human Mesenchymal Stem Cells: Implication for Inflammatory Mediators in Regenerative Medicine J. Immunol., September 1, 2007; 179(5): 3342 - 3350. [Abstract] [Full Text] [PDF]
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 P. Batten, N. A Rosenthal, and M. H Yacoub Immune response to stem cells and strategies to induce tolerance Phil Trans R Soc B, August 29, 2007; 362(1484): 1343 - 1356. [Abstract] [Full Text] [PDF]
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R. Romieu-Mourez, M. Francois, M.-N. Boivin, J. Stagg, and J. Galipeau Regulation of MHC Class II Expression and Antigen Processing in Murine and Human Mesenchymal Stromal Cells by IFN-{gamma}, TGF-beta, and Cell Density J. Immunol., August 1, 2007; 179(3): 1549 - 1558. [Abstract] [Full Text] [PDF]
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M. A. Haniffa, X.-N. Wang, U. Holtick, M. Rae, J. D. Isaacs, A. M. Dickinson, C. M. U. Hilkens, and M. P. Collin Adult Human Fibroblasts Are Potent Immunoregulatory Cells and Functionally Equivalent to Mesenchymal Stem Cells J. Immunol., August 1, 2007; 179(3): 1595 - 1604. [Abstract] [Full Text] [PDF]
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 C. Prevosto, M. Zancolli, P. Canevali, M. R. Zocchi, and A. Poggi Generation of CD4+ or CD8+ regulatory T cells upon mesenchymal stem cell-lymphocyte interaction Haematologica, July 1, 2007; 92(7): 881 - 888. [Abstract] [Full Text] [PDF]
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C. P. Sevigny, L. Li, A. S. Awad, L. Huang, M. McDuffie, J. Linden, P. I. Lobo, and M. D. Okusa Activation of Adenosine 2A Receptors Attenuates Allograft Rejection and Alloantigen Recognition J. Immunol., April 1, 2007; 178(7): 4240 - 4249. [Abstract] [Full Text] [PDF]
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 S. H. Ramkissoon, P. S. Patel, M. Taborga, and P. Rameshwar Nuclear Factor-{kappa}B Is Central to the Expression of Truncated Neurokinin-1 Receptor in Breast Cancer: Implication for Breast Cancer Cell Quiescence within Bone Marrow Stroma Cancer Res., February 15, 2007; 67(4): 1653 - 1659. [Abstract] [Full Text] [PDF]
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A. J. Nauta, G. Westerhuis, A. B. Kruisselbrink, E. G. A. Lurvink, R. Willemze, and W. E. Fibbe Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting Blood, September 15, 2006; 108(6): 2114 - 2120. [Abstract] [Full Text] [PDF]
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A. J. Nauta, A. B. Kruisselbrink, E. Lurvink, R. Willemze, and W. E. Fibbe Mesenchymal Stem Cells Inhibit Generation and Function of Both CD34+-Derived and Monocyte-Derived Dendritic Cells J. Immunol., August 15, 2006; 177(4): 2080 - 2087. [Abstract] [Full Text] [PDF]
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J. L. Chan, K. C. Tang, A. P. Patel, L. M. Bonilla, N. Pierobon, N. M. Ponzio, and P. Rameshwar Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-{gamma} Blood, June 15, 2006; 107(12): 4817 - 4824. [Abstract] [Full Text] [PDF]
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J. Stagg, S. Pommey, N. Eliopoulos, and J. Galipeau Interferon-{gamma}-stimulated marrow stromal cells: a new type of nonhematopoietic antigen-presenting cell Blood, March 15, 2006; 107(6): 2570 - 2577. [Abstract] [Full Text] [PDF]
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G. M. Spaggiari, A. Capobianco, S. Becchetti, M. C. Mingari, and L. Moretta Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation Blood, February 15, 2006; 107(4): 1484 - 1490. [Abstract] [Full Text] [PDF]
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N. Eliopoulos, J. Stagg, L. Lejeune, S. Pommey, and J. Galipeau Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recipient mice Blood, December 15, 2005; 106(13): 4057 - 4065. [Abstract] [Full Text] [PDF]
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A. Poggi, C. Prevosto, A.-M. Massaro, S. Negrini, S. Urbani, I. Pierri, R. Saccardi, M. Gobbi, and M. R. Zocchi Interaction between Human NK Cells and Bone Marrow Stromal Cells Induces NK Cell Triggering: Role of NKp30 and NKG2D Receptors J. Immunol., November 15, 2005; 175(10): 6352 - 6360. [Abstract] [Full Text] [PDF]
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B. R Blazar and W. J Murphy Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) Phil Trans R Soc B, September 29, 2005; 360(1461): 1747 - 1767. [Abstract] [Full Text] [PDF]
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S. Glennie, I. Soeiro, P. J. Dyson, E. W.-F. Lam, and F. Dazzi Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells Blood, April 1, 2005; 105(7): 2821 - 2827. [Abstract] [Full Text] [PDF]
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S. Beyth, Z. Borovsky, D. Mevorach, M. Liebergall, Z. Gazit, H. Aslan, E. Galun, and J. Rachmilewitz Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness Blood, March 1, 2005; 105(5): 2214 - 2219. [Abstract] [Full Text] [PDF]
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S. Aggarwal and M. F. Pittenger Human mesenchymal stem cells modulate allogeneic immune cell responses Blood, February 15, 2005; 105(4): 1815 - 1822. [Abstract] [Full Text] [PDF]
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