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TISSUE-SPECIFIC STEM CELLS |
aStem Cell Research Center, National Health Research Institutes, Zhunan, Taiwan;
bDepartment of Primary Care Medicine and Department of Obstetrics/Gynecology, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan;
cDepartments of Laboratory Medicine and Forensic Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan;
dDepartment of Medicine, School of Medicine, Fu Jen Catholic University, Taipei, Taiwan;
eCathay General Hospital Neihu;
fCathay Medical Research Institute, Cathay General Hospital, Taipei, Taiwan;
gCentral Laboratory, Shin Kong WHS Memorial Hospital, Taipei, Taiwan
Key Words. Mesenchymal stem cell • Placenta • Multilineage differentiation • Immunosuppression • Mixed lymphocyte culture • Interferon-
• Human leukocyte antigen, class I, G • Indoleamine 2,3-dioxygenase
Correspondence: B. Linju Yen, M.D., Stem Cell Research Center, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan. Telephone: +886-2-2653-4401, ext. 27502; Fax: +886-2-2792-9679; e-mail: blyen{at}nhri.org.tw
Received on February 3, 2006;
accepted for publication on July 14, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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for 3 days only very minimally upregulated HLA-DR. On the contrary, PD-L1, a cell surface marker that plays an inhibitory role in T-cell activation, was upregulated and TGF-ß expression was seen. The immunosuppressive properties of PDMCs, along with their multilineage differentiation potential, ease of accessibility, and abundant cell numbers, may render these cells as good potential sources for future therapeutic applications.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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Recent research, however, is revealing that MSCs can suppress immune reactivity of allogeneic lymphocytes, both in vitro and in vivo. Allogeneic BMMSCs from humans, baboons, and mice have been shown to decrease the immune response of lymphocytes in vitro [10–14]. The inhibitory effects of MSCs appear to work via a number of mechanisms, only some of which have been elucidated. Most reports find that the immunosuppressive properties are broad, effective whether the stimulation is specific or nonspecific [10, 11, 13, 14], across species [10, 12, 15], and across different populations of lymphocytes [11, 15–17]. The data are more mixed regarding whether cell-cell contact and soluble factors are involved [11, 12, 14, 17–19] or not [13, 16, 20, 21]. In addition to in vitro data, skin engraftment in mice is improved with concurrent transplantation of MSCs [10].
Although the recent data regarding immunosuppressive effects of BMMSCs are promising, the fact remains that these cells are rare and numbers decrease with increasing donor age [22]. Recently, a number of laboratories have independently isolated multipotent cells from the human term placenta capable of osteogenic, adipocytic [23, 24], and neural [25] differentiation. Traditionally discarded after childbirth, the term placenta now appears to be an easily accessible and abundant source of stem cells. Moreover, because placenta-derived multipotent cells (PDMCs) are fetal in origin, they may generate less of an immune response than adult tissues, as is seen with the decrease in immune-related complications in transplantations using umbilical cord blood HSCs versus adult BM HSCs [26]. We therefore studied the immunological characteristics of PDMCs and found that these cells have potent inhibitory effects. Comparisons with BMMSCs were performed, and the mechanisms behind the immunosuppression by PDMCs were explored.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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Placenta-Derived Multipotent Cells. Term (38–40 weeks gestation) placentas from healthy donor mothers were obtained with informed consent approved according to the procedures of the institutional review board. The cells were isolated as previously reported [25]. Briefly, placental tissue was dissected after drainage of umbilical cord blood. After mechanical and enzymatic treatment, the homogenate was cultured in complete medium consisting of Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented by 10% fetal bovine serum (FBS) (selected lots; HyClone, Logan, UT, http://www.hyclone.com), 100 U/ml penicillin, and 100 g/ml streptomycin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Cell cultures were maintained at 37°C with a water-saturated atmosphere and 5% CO2. Medium was replaced one to two times every week. PDMCs between the passages of 5 and 8 were used. For control, BMMSCs were cultured according to Pittenger et al. [3]. For use as third-party cells in mixed lymphocyte cultures (MLCs), PDMCs and BMMSCs were inactivated with mitomycin C (Sigma-Aldrich).
Differentiation studies were carried out as previously described [25]. All reagents were from Sigma-Aldrich. Briefly, for adipogenic differentiation, cells were cultured in complete medium with the addition of 0.5 µM isobutyl-methylxanthine, 1 µM dexamethasone (Decadron; Merck & Co., Inc., Whitehouse Station, NJ, http://www.merck.com), 10 µM insulin, and 60 µM indomethacin. Osteogenic differentiation was achieved by culturing cells in complete medium along with 0.1 µM dexamethasone, 10 mM ß-glycerol phosphate, and 50 µm ascorbate. Neurogenic differentiation was induced by culturing cells in serum-free medium with the addition of 10–6 M retinoic acid. Cells were stained for oil red and alizarin red for visualization of adipogenic and osteogenic differentiations, respectively.
Generation of Human Mature Dendritic Cells. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (1.077 g/ml; Invitrogen) density gradient centrifugation. Monocytes were purified from PBMCs by using the MACS (magnetic cell sorting) CD14 Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com/nn.404,company.html). Briefly, PBMCs were incubated with anti-CD14 magnetic beads in 4°C for 20 minutes. After incubation, the PBMCs and magnetic beads complex were washed with phosphate-buffered saline (PBS) containing 1% FBS and isolated to CD14+ and CD14– fractions by AutoMACS (Miltenyi Biotec). The purity was determined to be more than 85% CD14+ by flow cytometric analysis. Cells (1 x 106 per milliliter) were cultured subsequently for 7 days in RPMI-1640 (Invitrogen) and 10% FBS (HyClone) with granulocyte-macrophage colony-stimulating factor (GM-CSF) (1,000 U/ml; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and interleukin-4 (IL-4) (500 U/ml; R&D Systems Inc.). Cytokines were replenished every other day. After 7 days, cells were washed by PBS, and the medium was changed. To induce maturation of monocyte-derived cells to mature dendritic cells (mDCs), lipopolysaccharide (1 µg/ml; Sigma-Aldrich) was added for another 48 hours of culture with GM-CSF and IL-4.
Isolation of Human CD4 and CD8 Cells. CD4 and CD8 cells were isolated from PBMCs by using the MACS CD4 and CD8 cell isolation kits (Miltenyi Biotec) in the same methodology as reported above. Briefly, PBMCs were incubated with the appropriate magnetic beads in 4°C for 20 minutes. After the incubation, the PBMCs and magnetic beads complex were washed with PBS containing 1% FBS and isolated to positive and negative fractions by AutoMACS (Miltenyi Biotec). The positive fraction was collected, and the purities of the CD4 and CD8 T lymphocytes were demonstrated to be greater than 98% by flow cytometric analysis. T cells were cultured in RPMI-1640 (Invitrogen) and 10% FBS (HyClone).
Immunophenotyping of Cells
Cells were detached with trypsin/EDTA, washed, and resuspended in PBS with 1% FBS. The cells (1 x 105 per milliliter) were then stained for 30 minutes on ice with saturating amounts of fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated monoclonal antibodies. Antibodies recognizing the following human antigens were used: CD4, CD8, CD14, CD25, CD34, CD40L, CD80, CD86, CD122, CD90/Thy-1, CD117/c-kit, CD166/ALCAM, PD-1, PD-L1, HLA-ABC, and HLA-DR (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com); CD13, CD29, and CD44 (Dako Denmark A/S, Glostrup, Denmark, http://www.dako.dk); CD133 (Miltenyi Biotec); Foxp3 (Serotec Ltd., Oxford, U.K., http://www.serotec.com); and CD105/endoglin/SH-2 and stage-specific embryonic antigen-4 (SSEA-4) (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/
dshbwww). Antibodies against the human antigen SH-3 were purified; the SH-3 hybridoma cell line was acquired from American Type Culture Collection (ATCC) (Manassas, VA, http://www.atcc.org). For analysis of T regulatory cells (Tregs), responder PBMCs were harvested before and after 3 days of mixed lymphocyte culture (MLCs) and were double-stained in the following combinations: CD4/CD25, CD4/CD122, CD4/Foxp3, CD8/CD25, and CD8/CD122. Each analysis included the appropriate FITC- and PE-conjugated isotype controls. All analyses were done on a Becton Dickinson FACScan laser flow cytometric system (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) equipped with a Macintosh PowerMac G3 personal computer (Apple Computer, Inc., Cupertino, CA, http://www.apple.com) using CellQuest software (BD Biosciences).
Immunofluorescence
Cultured cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes at room temperature and permeabilized with 0.1% Triton-X 100 (Sigma-Aldrich) for 10 minutes. Primary antibodies against indoleamine 2,3-dioxygenase (IDO) and HLA-G were purchased from Serotec Ltd. Samples were first incubated with the primary antibodies at 4°C overnight, rinsed three times with PBS, and incubated for 60 minutes at room temperature with FITC- or PE-conjugated secondary antibodies. All samples were stained with 4',6-diamidino-2-phenylindole (Molecular Probes Inc., Eugene, OR, http://www.invitrogen.com) for 5 minutes. Staining was visualized under a fluorescence microscope (Nikon Corporation, Tokyo, http://www.nikon.com).
MLCs and Cell Proliferation Assay
PDMCs were plated into 96-well plates (5 x 104 cells per milliliter) containing responder cells (CD4, CD8 T cells, or PBMCs) and stimulator cells (PBMCs, mDCs, or 4 µg/ml phytohemagglutinin [PHA]; Sigma-Aldrich). Stimulator cells were inactivated with mitomycin C (Sigma-Aldrich). Inactivated third-party cells (PDMCs or BMMSCs) were added directly to plates. For Transwell experiments, MLCs were carried out in 24-well plates with third-party cells plated on the transwell. After 72 hours of coculture, 100 µl of cells from each well was transferred to new 96-well plates with 10 µl of Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan, http://www.dojindo.co.jp) added to each well. After incubation for 1–4 hours at 37°C, the absorbance was measured at 450 µm with a microplate reader (Molecular Devices Corporation, Sunnyvale, CA, http://www.moleculardevices.com) using a reference wavelength of 600 nm.
Carboxyfluorescein Diacetate, Succinimidyl Ester Labeling and Analysis
PBMCs and CD4+ and CD8+ T cells were labeled with 2.5 µmol/l of carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes) for 10 minutes at 20°C in PBS with 0.1% bovine serum albumin (BSA). After washing twice with 1% BSA in PBS, the cells were resuspended in RPMI-1640 (Sigma-Aldrich) + 10% FBS (HyClone) and incubated at 20°C for another 10 minutes. MLCs were performed in the following manner: PDMCs were plated into 24-well plates (5 x 104 cells per milliliter) containing CFSE-labeled PBMCs, CD4, or CD8 T cells (5 x 105 cells per milliliter) with mDCs (5 x 104 cells per milliliter) or PHA (4 µg/ml) with or without IL-2 (50 U/ml; R&D Systems Inc.) in RPMI-1640 (Invitrogen) and 10% FBS (HyClone). After 72 and 96 hours, the T cells were harvested and washed twice with PBS containing 1% FBS. Analysis of cell division was performed by flow cytometry. To assess the effects of interferon- (IF-), PDMCs were pretreated with 100 ng/ml of IF- (R&D Systems Inc.) for 48 hours prior to MLCs. In experiments using neutralizing antibodies to IL-10 and transforming growth factor-ß (TGF-ß; R&D Systems Inc.), the antibodies were added to MLCs for 3 days of incubation after which T-cell proliferation was assessed by flow cytometry.
Apoptosis
The apoptosis assay for T cells was performed with the annexin V/propidium iodide (PI) staining kit (Roche Diagnostics GmbH, Mannheim, Germany, http://www.roche.de/diagnostics/index.htm). Briefly, cells were harvested by centrifugation (1,200 rpm, 5 minutes), and the medium was discarded. The lymphocytes were then recovered, stained with FITC-conjugated annexin V and PI, and analyzed by flow cytometry.
Natural Killer Cell Cytotoxicity Assay
Purified natural killer cells (NKCs) were obtained from PBMCs by negative selection with the MACS NKC Isolation Kit (Miltenyi Biotec). The degree of purity was measured with fluorescence-activated cell sorting analysis with labeled antibodies against CD56 (Becton, Dickinson and Company). K562 (ATCC), a well-characterized chronic myeloic lymphoma cell line known for its NKC sensitivity caused by a lack of major histocompatibility complex (MHC) class I molecules, was used as a positive target cell, and MCF-7 (ATCC), a breast cancer cell line known to be a poor NKC target [27], was used as negative control. NKCs were plated in quadriplicate in 96-well culture plates at a density of 2 x 105 cells per milliliter, and target cells (K562, MCF-7, and PDMCs) were plated at a density of 2 x 104 cells per milliliter and incubated at 37°C for 4 hours. Alamar blue (25 µl; Biosource International, Camarillo, CA, http://www.biosource.com) [28] was added, cells were further incubated for 1 hour, and fluorescence was measured in a fluorescence reader (Molecular Devices Corporation).
Reverse Transcription-Polymerase Chain Reaction
Total mRNA was extracted using Trizol (Invitrogen) and reverse-transcribed using the SuperScript III First-Strand (Invitrogen). Primers were as follows: IL-4, forward primer 5'-CAACTTTGTCCACGGACAC-3', reverse primer 5'-TCCAACGTACTCTGGTTGG-3'; IL-10, forward primer 5'-ATGCCCCAAGCTGAGAACCAAGACCCA-3', reverse primer 5'-AAGTCTCAAGGGGCTGGGTCAGCTATCCCA-3'; TGF-ß, forward primer 5'-CTATCCACCTGCAAGACTATCGAC-3', reverse primer 5'-GGAGCTGAAGCAATAGTTGGTGTC-3'; and ß-actin positive control, forward primer 5'-TGGCACCACCTTCTACAATGAGC-3', reverse primer 5'-GCACAGCTTCTCCTTAATGTCACGC-3'. Polymerase chain reaction (PCR) was carried out with 25-µl reaction volumes of Platinum PCR SuperMix (Invitrogen) and 60 to approximately 80 ng of cDNA template. The annealing temperature was 60°C, and amplification was set for 30 cycles. The PCR was performed in a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, CA, https://www2.appliedbiosystems.com).
Statistical Analysis
Statistical analysis was performed with the statistical SPSS 12.0 software (SPSS Inc., Chicago, http://www.spss.com). The t test (t distribution) was used to test the probability of significant differences between samples. Statistical significance was defined as p < .05. All experiments were performed at least in triplicate.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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Suppression of Lymphocyte Reactivity by PDMCs Is Not Due to Apoptosis but to Decreased Cell Proliferation and Increased Numbers of Tregs
The mechanism behind PDMC suppression of lymphocyte reactivity was further investigated. To rule out the possibility that the suppressive effect was due to lymphocyte cell death, cell apoptosis was assayed by flow cytometric analysis of PI and annexin V staining. No significant difference was seen between control MLCs lymphocytes and PDMC-cocultured lymphocytes (Fig. 3A). Next, we investigated whether cell division was affected. To assess this, PBMCs were stained with the cell surface dye CFSE prior to antigen stimulation with PHA and IL-2. Lymphocyte cell division was assessed after 3 days of MLCs. In MLCs cocultured with either PDMCs or BMMSCs, decreased lymphocyte cell division could be seen (Fig. 3B).
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PDMCs Have Similar Inhibitory Effects on CD4 and CD8 T Cells, and the Inhibitory Effects Are Seen with Either Nonspecific Mitogen or Alloantigen Stimulation
To further clarify the inhibitory effects of PDMCs, we investigated whether the immune suppression was directed at a particular T-cell population. We used AutoMACS magnetic beads to separate T cells into CD4 and CD8 populations. These separated populations of CD4 (Fig. 4A) and CD8 (Fig. 4B) T cells were then stimulated by both a nonspecific mitogen, PHA, or allogeneic mDCs. Analyzing cell proliferation by flow cytometry, we found that PDMCs can reduce proliferation in either T-cell population. Moreover, proliferation was inhibited whether mitogens or allogeneic mDCs, which are professional antigen-presenting cells (APCs), were used. When stimulated with mDCs, PDMCs appear to have a stronger inhibitory effect on CD4 than CD8 cells.
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Enhances the Inhibitory Effects of PDMCs, one of the most potent inflammatory cytokines known [34]. Thus, we investigated the effects of IF- on PDMC immune suppression. We initially investigated whether any phenotypic changes in immune-related cell surface markers would occur. Unlike BMMSCs, in which the HLA-DR expression before and after IF- treatment increased from 1.52% to 69.9% of the cells, PDMCs showed only minimal increases, from 0.80% to 1.75% of the cells (Fig. 6A). However, treatment with IF- did result in slight upregulation of PD-L1, which plays an inhibitory role in T-cell activation [35, 36]. This was seen with PDMCs but not BMMSCs (Fig. 6A).
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treatment of PDMCs resulted in any changes in expression of the anti-inflammatory cytokines TGF-ß, IL-4, and IL-10. Reverse transcription-PCR showed a strong increase in the expression of TGF-ß in PDMCs after 3 days of IF- treatment (Fig. 6B). In contrast, for BMMSCs, there appears to be constitutive expression of TGF-ß, levels of which did not change after IF- treatment. No expression of IL-4 or IL-10 was detected in PDMCs or BMMSCs, either with or without IF- treatment (data not shown).
Finally, we examined whether these phenotypic changes induced by IF- would enhance the immunosuppressive effects of PDMCs. Using CFSE-labeled CD4 and CD8 T cells, MLCs were performed with third-party PDMCs treated either with or without IF-, and with the addition of IL-10- and TGF-ß-neutralizing antibodies. IF--treated PDMCs are able to resuppress the lymphocyte proliferation restored by the neutralizing antibodies, whereas untreated PDMCs were not able to do so (Fig. 6C).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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stimulation for 3 days, unlike BMMSCs. On the contrary, PD-L1, a molecule involved in inhibition of T-cell activation, is upregulated [36, 37]. Moreover, based on our in vitro results, the immunosuppression seen with PDMCs is significantly stronger than that with BMMSCs (Fig. 2A). The fetal origin of PDMCs may be responsible for these differences with BMMSCs, which are of adult source. One of the greatest mysteries of science continues to be the state of mutual immune tolerance that exists between the mother and fetus during pregnancy, mediated by a number of mechanisms, including IDO expression, which we found to be expressed in PDMCs as well as BMMSCs [30, 42]. The expression of HLA-G, which is a placenta-specific MHC-I antigen critical to the immune-tolerant state of pregnancy [31], was found intracellularly in PDMCs but not BMMSCs (Fig. 1D); this may be a contributing factor in the stronger immunosuppression seen with PDMCs, as well as in making these cells a poor target for NKC lysis (Fig. 2B). Although based on in vitro data, our results would suggest that PDMCs (being of fetal origin) may harbor an "immunologic advantage," which would be of significant benefit regarding future clinical use of these cells. Further research, including in vivo studies, is necessary for the corroboration of these effects before therapeutic use can be considered. Host response to foreign antigen stimulation includes the production of proinflammatory cytokines and chemokines, recruitment of inflammatory cells to the site of infection, and activation of cytotoxic T lymphocytes and NKCs. Although these responses help to slow or eradicate the spread of the pathogen, such responses when uncontrolled can result in severe inflammation and host tissue damage. It is now clear that, concurrently, an immune-tolerant response is also induced that helps to maintain homeostasis of the immune system. One of the key players mediating tolerance is Tregs, a subpopulation of T cells that are CD4+CD25high and have immune-tolerant properties [32]. Our data show that, in MLCs cocultured with third-party PDMCs, a threefold increase in the number of Tregs can be seen. Although only a few reports show Treg involvement in BMMSC immunosuppression [43, 44], this may be due to species differences with the use of murine cells in negative studies [13]. Moreover, the presence of other populations of lymphocytes with immune-modulating properties cannot be excluded; one study on BMMSC immunosuppression found evidence of CD8+ cells with suppressive function [12]. We found that PDMCs inhibit DC-stimulated CD4 lymphocytes more effectively than CD8 lymphocytes (Fig. 4B), which may be indirect evidence for a CD8+ population of regulatory cells. Very recent data have revealed that, in mice, a population of CD8+ T cells that are also positive for CD122 have suppressive capabilities [45]. Although we did not find such a population of CD8+CD122+ cells (data not shown), this may be due to interspecies differences. More specific research is needed at this point to clarify these issues and show a mechanistic connection regarding Tregs and third-party stem/progenitor cells.
The immunosuppressive effects of PDMCs appear to be effective against mDCs, which are the most potent APCs and are crucially involved in allograft rejection. APCs are specialized immune cells capable of recognizing and processing antigens [46], initiating the inflammatory cascade. To test whether PDMCs are able to suppress DC-stimulated lymphocyte proliferation, we use monocyte-derived (CD14+) DC cells that were cultured to maturity with a standard protocol of IL-4 + GM-CSF stimulation [47]. Our data show that PDMCs are able to suppress mDC-induced lymphocyte proliferation. Recent studies have shown that BMMSCs inhibit the maturation of DCs [48] as well as affect the secreted cytokine profile of both mature and immature DCs, creating a more immunotolerant milieu [44]. Our data and those of others on the interactions of immune cells and mesenchymal tissues highlight the importance of microenvironments on immune cell development and phenotype, an area that has only begun to be explored [49].
The majority of studies to date show that cell-cell contact is not necessary for the inhibitory effects of BMMSCs [11, 12, 14, 18, 19], implicating a role for secreted soluble factors at work. We have also found that PDMCs did not require cell-cell contact and that TGF-ß, a potent anti-inflammatory cytokine, was expressed by PDMCs after IF- stimulation. This is in contrast to data from BMMSCs in which, although TGF-ß was detected in the supernatant of MLCs, it was determined that it was not produced by BMMSCs [14, 16, 17, 21]. In our hands, we did find baseline secretion of TGF-ß in BMMSCs but no upregulation after IF- treatment. Although we did not find gene expression of other anti-inflammatory molecules, including IL-4 and IL-10 in PDMCs or BMMSCs, this may be due to the fact that production of these molecules is not by the third-party stem cells but lymphocytes themselves [14, 17]. Indeed, the addition of neutralizing antibodies to IL-10 as well as TGF-ß partially reversed the inhibitory effects of PDMCs. The fact that only a partial reversal was seen may be indirect evidence that multiple mechanisms are responsible for the immunosuppression of PDMCs, some of which we have shown in this paper.
Although IF- is an important mediator in the host inflammatory response to allograft transplantation, we found that pretreatment with this cytokine actually enhanced the immunosuppressive effects of PDMCs. PDMCs increase the secretion of TFG-ß and upregulate the inhibitory cell surface marker PD-L1 after IF- treatment. PD-L1 is a ligand of the recently discovered PD-1 pathway, which when activated leads to inhibition of T-cell receptor-mediated lymphocyte proliferation and cytokine secretion [35, 36]. Although the mechanisms behind IF--related immunosuppression by third-party stem cells are just beginning to be unraveled [18, 42], the therapeutic implications of these results are significant: because increased levels of IF- are found in patients with acute GVHD, infusion of PDMCs and BMMSCs may decrease the incidence of this lethal disease. The report of the successful use of BMMSCs in treating a case of refractory acute GVHD will hopefully lead to more rapid consideration of PDMCs and other progenitor/stem cells for such lethal, immune-related diseases [50].
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CONCLUSION |
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stimulation of PDMCs actually enhanced the inhibitory effects rather than reduced it. PDMCs are an easily accessible and abundant supply of multipotent cells procured from a source without ethical controversy. The immunosuppressive properties now identified add further support for the consideration of these cells as a clinically viable source for cell therapy. |
DISCLOSURES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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