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First published online September 27, 2007
Stem Cells Vol. 26 No. 1 January 2008, pp. 182 -192
doi:10.1634/stemcells.2007-0491; www.StemCells.com
© 2008 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Human Amnion Mesenchyme Harbors Cells with Allogeneic T-Cell Suppression and Stimulation Capabilities

Marta Magatti, Silvia De Munari, Elsa Vertua, Lucia Gibelli, Georg S. Wengler, Ornella Parolini

Centro di Ricerca E. Menni, Fondazione Poliambulanza, Istituto Ospedaliero, Brescia, Italy

Key Words. Tolerance • Mesenchymal stromal cell • Immune escape • Immunosuppression • Human placenta • Amnion Mesenchymal stem cells

Correspondence: Correspondence: Ornella Parolini, Ph.D., Centro di Ricerca E. Menni, Fondazione Poliambulanza, Istituto Ospedaliero, Via Bissolati 57, I-25124 Brescia, Italy. Telephone: 390302455754; Fax: 390302455704; e-mail: ornella.parolini{at}tin.it

Received on June 21, 2007; accepted for publication on September 18, 2007.

First published online in STEM CELLS EXPRESS  September 27, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cells derived from the amniotic membrane of human placenta have been receiving particular attention because of their stem cell potentiality and immunomodulatory properties, which make them an attractive candidate source for cell therapy approaches. In this study, we isolated cells from the mesenchymal region of amnion and identified two subpopulations discordant for expression of the HLA-DR, CD45, CD14, and CD86 cellular markers. We therefore refer to the unfractionated cell population derived from this region as amniotic mesenchymal tissue cells (AMTC). We studied the suppressive and stimulatory characteristics of the unfractionated, HLA-DR-positive, and HLA-DR-negative AMTC populations and demonstrated that all three fail to induce an allogeneic T-cell response. However, unfractionated AMTC, which could inhibit T-cell allogeneic proliferation responses, induced proliferation of T cells stimulated via the T-cell receptor (TcR), in a cell-cell contact setting. We have shown that this stimulatory capacity can be attributed to the HLA-DR-positive AMTC subpopulation. Indeed, even though the HLA-DR-positive AMTC fraction surprisingly failed to induce proliferation of resting allogeneic T cells, they could cause strong proliferation of anti-CD3-primed allogeneic T cells. This stimulatory effect was not observed using the HLA-DR-negative AMTC fraction. The revelation that human amniotic mesenchyme possesses cell populations with both suppressive and stimulatory properties sheds additional light on the immunomodulatory functions of this tissue and may contribute to the clarification of some ongoing controversies associated with mesenchymal stromal cells of other sources, such as the presence of HLA-DR-positive cells and the suppressive versus stimulatory properties of these cells.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
The amniotic and chorionic mesenchymal layers from human term placenta harbor cells that present with fibroblast-like morphology, have clonogenic potential, and display multipotent differentiation capacity toward lineages including osteogenic, adipogenic, chondrogenic, vascular, endothelial, cardiomyocytic, and skeletal muscle [1–7]. These characteristics are reminiscent of the properties described for bone marrow-derived mesenchymal stem cells (BM-MSC), a much more extensively characterized cell type that is gaining increasing interest for clinical applications [8–10]. Besides bone marrow, where they have been first described [11], other sources of MSC that have been reported include adipose tissue [12–15], cord blood [16, 17], peripheral blood [18, 19], and amniotic fluid [20, 21]. One critical characteristic of MSC is their ability to suppress T-cell proliferation in mixed lymphocyte reaction (MLR) [22–26] in addition to other immunomodulatory properties, such as their induction of T helper (Th)2 responses, upregulation of T regulatory cells [27–29], and inhibitory effects on maturation of dendritic cells [27–32]. Whether or not MSC can induce tolerance in an allogeneic transplantation setting is still an area of debate [26, 33–35]; however, decreased graft-versus-host disease (GvHD) in allogeneic stem cell transplantation and treatment of acute GvHD in vivo have been demonstrated [9, 36, 37].

We and others have shown that cells isolated from the mesenchymal region of human amnion and chorion fail to induce allogeneic T-cell responses and actively suppress T-cell proliferation induced by alloantigens [1, 7]. Furthermore, we have shown that a heterogeneous population isolated from the human amniotic and chorionic fetal membranes demonstrated long-term chimerism in xenogenic animal transplantation models, suggesting their reduced immunogenicity and tolerogenic potential [1]. It is tempting to speculate that the immunomodulatory characteristics of the mesenchymal cells resident within the fetal membranes play a role in the fetal-maternal tolerance process; however, this theory remains to be proven.

Scientists have long been puzzled by the mechanisms involved in maternal tolerance to the fetus. Proposed explanations are the anatomical barrier between the mother and the fetus formed by the placenta, the immunological inertness of the mother, and the antigenic immaturity of the fetus [38]. However, several studies have indicated that the fetal placental barrier may be less inert or impervious than previously envisioned, with evidence presented for cellular trafficking in both directions across the fetal/maternal interface [39–41]. In addition, it is now clear that the maternal immune system is not anergic to all fetal tissues, since it can respond to and eliminate fetal cells that enter the maternal circulation [40, 42]. Finally, it is well-accepted that fetal trophoblast cells lack the major histocompatibility complex (MHC) class II (MHC-II) antigens, downregulate MHC class I proteins, and express high levels of HLA-G, an antigen known to protect against rejection [43]. However, fundamental questions still remain as to whether the fetus participates actively in suppressing maternal allogeneic immune responses and, if so, what fetal placental tissues play an immunomodulatory role. The mesodermal (stromal) layers of amnion and chorion are considered avascular and therefore inert in terms of immune presentation; however, macrophage-like populations in the chorion (Hofbauer cells) have been described in previous reports [44]. More recently, a defined population of HLA-DR-expressing cells with macrophage-monocyte phenotypic characteristics has also been described in the mesenchymal layer of the amnion [45–47], thus suggesting the presence of populations capable of active immune function within these tissues.

Because we have confirmed the presence of two distinct subpopulations in the mesenchymal region of amnion that differ in their expression of CD45, CD14, and HLA-DR, and considering that mesenchymal stromal cells from different origins are agreed to be negative for these markers [48, 49], we have adopted the designation of amniotic mesenchymal tissue cells (AMTC) when referring to the unfractionated population derived from the mesenchymal region of amnion, which includes both HLA-DR-positive and -negative cells. In this study, we investigated the immunomodulatory characteristics of unfractionated and fractionated cells derived from the mesenchymal layer of amnion, and we demonstrated that subfractions of cells isolated from the amniotic mesenchymal tissue can indeed induce either inhibitory or stimulatory effects on allogeneic T lymphocytes.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Isolation of Cells from the Amniotic Mesenchymal Tissue
Human term placentas were obtained from healthy women with informed consent after vaginal delivery or caesarean section and processed immediately. The amnion was manually separated from the chorion, washed extensively in phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) containing 100 U/ml penicillin and 100 µg/ml streptomycin (both from Euroclone, Whetherby, U.K., http://www.euroclone.net), and cut into small pieces. Amnion fragments were incubated at 37°C for 8 minutes in PBS containing 2.4 U/ml dispase (Roche Diagnostics, Mannheim, Germany, http://www.roche-applied-science.com) and then transferred at room temperature for 10 minutes into RPMI complete medium composed of RPMI 1640 medium (Cambrex, Verviers, Belgium, http://www.cambrex.com) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Cambrex). Afterward, the fragments were digested with collagenase (0.75 mg/ml) (Roche) and DNase (20 µg/ml) (Roche) for approximately 3 hours at 37°C. Resulting cell suspensions were gently centrifuged (150g for 3 minutes), and the supernatant was filtered through a 100-µm cell strainer (BD Falcon, Bedford, MA, http://www.bdbiosciences.com). Finally, cells were collected by centrifugation at 300g for 10 minutes. We refer to these freshly isolated cells from the mesenchymal tissue of the amnion as AMTC.

Isolation of HLA-DR-Positive and HLA-DR-Negative Cells from AMTC
The separation of HLA-DR-positive (HLA-DR+) cells from fresh preparations of AMTC was performed using the MACS Cell Separation (Miltenyi Biotec, Bergisch, Gladbach, Germany, http://www.miltenyibiotec.com). Cells were first incubated with anti-HLA-DR microbeads (Miltenyi Biotec) at 4°C for 20 minutes. After washing, separation of the HLA-DR+ and HLA-DR-negative (HLA-DR–) fractions was performed by two subsequent column purifications performed according to the manufacturer's specifications. The percentage of HLA-DR+ cells in the enriched and depleted fractions was determined by flow cytometry analysis.

Peripheral Blood and T-Cell Isolation
Human peripheral blood mononuclear cells (PBMC) were obtained from heparinized whole blood samples or buffy coats donated by healthy subjects, after informed consent was obtained, using density gradient centrifugation (Lymphoprep; Axis-Shield, Oslo, Norway, http://www.axis-shield.com). T lymphocytes were purified from PBMC after plastic adherence for 1.5–2 hours at 37°C, and the selection of T cells was performed through an indirect magnetic labeling system using the Pan T-cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer's instructions. Purity was assessed by flow cytometry analysis, and more than 95% of recovered cells were CD3-positive.

AMTC Cultures
Freshly isolated AMTC were plated in 75-cm2 flasks (Corning Enterprises, Corning, NY, http://www.corning.com) at a density of 4–5 x 106 cells per flask in 15 ml of RPMI complete medium. Confluent cells were washed in PBS and then detached with 0.25% trypsin (Sigma-Aldrich) before being replated in RPMI complete medium in 75-cm2 flasks at a density of 3 x 106 cells per flask.

For supernatant collection, AMTC were plated in 24-well plates at 1 x 106 cells per well, in a final volume of 1 ml of RPMI complete medium. Each day for 6 days, the supernatant was collected, centrifuged, filtered using a 0.2-µm sterile filter, and supplemented with 10% heat-inactivated FBS (Sigma-Aldrich) before being frozen at –80°C until use.

AMTC Coculture with PBMC or Purified T Cells
To study the effects of amniotic mesenchymal cells (AMTC) and their subpopulations (HLA-DR-negative and -positive AMTC) on T lymphocyte proliferation, 1.6 x 105 unfractionated, HLA-DR-negative, or HLA-DR-positive AMTC were plated in RPMI complete medium and left to adhere overnight. The next day, the cells were irradiated (3,000 cGy), and an equal number of PBMC or purified T cells was added. All cultures were carried out in triplicate, using round-bottomed 96-well tissue culture plates (Corning), in a final volume of 200 µl of RPMI complete medium. AMTC were irradiated to ensure that any proliferation observed could be attributed solely to the proliferation of responder lymphocytes. Proliferation of T cells and PBMC was assessed after 2–3 and 5 days by adding [3H]-thymidine (1 µCi per well; INC Biomedicals, Irvine, CA, http://www.mpbio.com) for 16–18 hours. Cells were then harvested with a Filtermate Harvester (PerkinElmer Life and Analytical Sciences, Zaventem, Belgium, http://www.perkinelmer.com), and thymidine incorporation was measured using a microplate scintillation and luminescence counter (Top Count NXT; PerkinElmer).

Effect of AMTC on Mixed Lymphocyte Reaction
For MLR with AMTC in cell-cell contact, 1 x 105 fresh or cultured AMTC were plated in RPMI complete medium and left to adhere overnight. The next day, AMTC were {gamma}-irradiated (3,000 cGy), and an equal number of "responder" PBMC or T cells was added, together with an equal number of {gamma}-irradiated (3,000 cGy) allogeneic "stimulator" PBMC. MLR without AMTC were used as controls. Experiments with different AMTC concentrations were performed by maintaining constant the number of PBMC (1 x 105) and decreasing the number of AMTC added, to obtain ratios of PBMC:AMTC of 1:1, 1:0.4, 1:0.2, 1:0.1, and 1:0. All cultures were carried out in triplicate, using round-bottomed 96-well tissue culture plates (Corning), in a final volume of 200 µl of RPMI complete medium.

For mixed lymphocyte reactions with segregated AMTC, transwell chambers with 0.4-µm pore size membranes (Corning) were used to physically separate the lymphocytes from the AMTC. PBMC (1.5 x 106) or T cells (1.5 x 106) were cocultured with equal numbers of {gamma}-irradiated (3,000 cGy) allogeneic PBMC in a 24-well tissue culture plate (Corning) in a final volume of 1 ml of RPMI complete medium. An equal number of fresh or cultured AMTC in a volume of 300 µl of RPMI complete medium was then added to the transwell chambers. Experiments were performed with different AMTC numbers and maintaining constant the number of PBMC, to obtain ratios of PBMC:AMTC of 1:1,1:0.4, 1:0.2, 1:0.1, and 1:0.

MLR were also performed in the presence of supernatants collected from AMTC cultured for various numbers of days. All cultures were carried out in triplicate using round-bottomed 96-well tissue culture plates (Corning), with the addition of 150 µl of AMTC-"conditioned" medium in a final volume of 200 µl.

In all cases, cell proliferation was assessed after 5 days of culture by adding 1 µCi per well (96-well tissue culture plates) or 5 µCi per well (24-well tissue culture plates) of [3H]-thymidine (INC Biomedicals) for 16–18 hours. Cells were then harvested with a Filtermate Harvester (PerkinElmer), and thymidine incorporation was measured using a microplate scintillation and luminescence counter (Top Count NXT; PerkinElmer).

Effect of AMTC on CD3/CD28-Stimulated PBMC or T Cells
To study the effect of AMTC and their subpopulations (HLA-DR-negative and -positive AMTC) on CD3/CD28-stimulated PBMC or T cells, fresh or cultured unfractionated, HLA-DR-negative, or HLA-DR-positive AMTC (1.6 x 105) were seeded in 96-well plates and left to adhere overnight. The next day, AMTC were {gamma}-irradiated (3,000 cGy), and an equal number of PBMC or purified T cells were added and activated with 1 µg/ml of soluble anti-CD3 monoclonal antibody (Orthoclone OKT3; Ortho Biotech, Bridgewater, NJ, http://www.orthobiotech.com) either alone or in combination with 7 µg/ml soluble anti-CD28 (clone CD28.2; Biolegend, San Diego, http://www.biolegend.com). Cultures were carried out in triplicate using round-bottomed 96-well tissue culture plates (Corning), in a final volume of 200 µl of RPMI complete medium.

For proliferation assays using transwell chambers, 1.5 x 106 PBMC or purified T cells were cultured in 24-well plates and stimulated by anti-CD3 and anti-CD28 monoclonal antibodies as described above. Cultures were carried out in a final volume of 1 ml of RPMI complete medium. AMTC (1.5 x 106) were seeded in the inner transwell chamber in a volume of 300 µl of RPMI complete medium. Cellular proliferation was assessed after 2–3 days of culture by adding [3H]-thymidine for 16–18 hours and assessing incorporation of radioactivity as described above.

Fixation of AMTC
As previously reported [50], AMTC were fixed with 0.5% paraformaldehyde (PFA) (Sigma-Aldrich) for 20 minutes at room temperature. Cells were then washed twice in PBS (Sigma-Aldrich) before being resuspended in RPMI complete medium and used in cell culture experiments.

Restimulation of Lymphocytes Following Culture with AMTC
PBMCs (1.5 x 106) were incubated with equal numbers of {gamma}-irradiated (3,000 cGy) allogeneic PBMCs in 24-well tissue culture plates (Corning) in a final volume of 1 ml of RPMI complete medium. Equal numbers of {gamma}-irradiated (3,000 cGy) AMTC were added in a volume of 300 µl of RPMI complete medium in the transwell chambers (Corning). After 5 days of culture, the transwell chambers containing AMTC were removed. Lymphocytes that had been cultured in the presence of AMTC via transwell were then collected, washed twice in phosphate buffered saline containing 100 U/ml penicillin and 100 µg/ml streptomycin, and recultured with the original or third-party PBMC stimulators. Lymphocyte proliferation was assessed after 5 days of culture as described above.

Flow Cytometry Analysis
For evaluation of cell phenotype, cell suspensions were incubated for 20 minutes at 4°C with fluorescein isothiocyanate or phycoerythrin-conjugated antibodies specific for human CD1a (clone HI149), CD3 (clone UCHT1), CD11b (clone ICRF44), CD14 (clone M{varphi}P9), CD16 (clone 3G8), CD45 (clone HI30), CD80 (clone L307.4), CD83 (clone HB15e), CD86 (clone 2331), and HLA-DR (clone TÜ36), or isotype controls IgG1 (clone X40), IgG2a (clone X39), and IgG2b (clone MG2b-57). All monoclonal antibodies were obtained from BD Biosciences (San Jose, CA, http://www.bdbiosciences.com) except for isotype control IgG2b, which was obtained from Biolegend. Samples were analyzed with a FACSCalibur instrument and the CellQuest software (BD Biosciences).

Immunohistochemistry Analysis
Immunohistochemical studies were performed on formalin-fixed and paraffin-embedded sections using the Super Sensitive IHC Detection System (BioGenex, San Ramon, CA, http://www.biogenex.com) with monoclonal antibodies specific for HLA-DR (clone LN-3; Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk) diluted 1:200, CD68 (clone KP1; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) diluted 1:100, and CD45RO (clone A6; DBS, Pleasanton, CA, http://www.dbiosys.com) diluted 1:100. The sections were deparaffinized in xylene and rehydrated in graded ethanol. The endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution.

The primary antibody was then applied for 1 hour at room temperature before incubation with the Super Enhancer Reagent (BioGenex) for 20 minutes, followed by application of Poly-HRP Reagent (BioGenex) for 30 minutes at room temperature. 3,3'-Diaminobenzidine (BioGenex) was used as the chromogen, and hematoxylin was used for counterstaining.

Restriction Fragment Length Polymorphism Analysis
DNA was extracted from placental deciduas, as well as unfractionated and HLA-DR-positive AMTC, using the Nucleospin Tissue Kit II (BD Biosciences) according to the manufacturer's instructions. Polymerase chain reaction (PCR) analysis of the minisatellite polymorphic locus D1S80 (forward primer, 5'-GAA ACT GGC CTC CAA ACA CTG CCC GCC G-3'; reverse primer, 5'-GTC TTG TTG GAG ATG CAC GTG CCC CTT GC-3') was then performed using an ABI 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) and GoTaq DNA Polymerase reagents (Promega, Madison, WI, http://www.promega.com) as previously described [51]. The PCR mixtures contained 200 µM dNTPs and 25 pmol of each primer in a total volume of 50 µl. The cycling conditions consisted of an initial denaturation step at 95°C for 10 minutes, followed by 35 cycles of 95°C for 15 seconds, 66°C for 45 seconds, and 72°C for 1 minute. PCR products were then separated by electrophoresis on 2.5% agarose gel (Bio-Rad, Hercules, CA, http://www.bio-rad.com), which was then stained with ethidium bromide.

Statistical Analysis
The Student's t test for paired data was used to test the probability of significant difference between paired samples for variables with normal distribution. Differences were considered statistically significant at p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
AMTC Inhibit Mixed Lymphocyte Reaction
We set out to study the effects of AMTC on classic MLR cultures as a model of allogeneic lymphocyte responses. We found that exposure of PBMC or isolated T cells to AMTC reproducibly inhibited MLR-induced cell proliferation both through cell-cell contact and in a transwell system (Fig. 1). The inhibitory effect was dependent on the amount of AMTC present in the cocultures, with the strongest effects observed at a ratio of responder cells: AMTC of 1:1, both in the cell-cell contact and transwell experiments (Fig. 2A).


Figure 1
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  		Figure 1. Suppression of allogeneic response by AMTC. Human PBMC (black bars) or human T cells (white bars) were used as responders and incubated with irradiated allogeneic PBMC (PBMC*), either alone or in the presence of AMTC. AMTC were added either in direct contact or in transwell chambers. Proliferation was assessed by [3H]-thymidine incorporation after 5 days of culture and expressed as a percentage of proliferation observed in the absence of AMTC. Data are mean and SD for more than 30 (PBMC+ PBMC*) or seven independent experiments (T cells+ PBMC*). ***, p < .001 versus corresponding control sample. Abbreviations: AMTC, amniotic mesenchymal tissue cells; PBMC, peripheral blood mononuclear cells.

 


Figure 2
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  		Figure 2. AMTC inhibitory conditions in MLR. (A): In MLR, R were incubated with irradiated allogeneic stimulator PBMC. Increasing concentrations of AMTC were added either in direct contact ({square}) or in transwell chambers (bullet). Data are mean and SD from three independent experiments. (B): MLR were performed alone (black column) or in the presence of SN from AMTC cultures (white columns). SN were collected from day 1 to day 6 of AMTC culture. Data are mean and SD from nine independent experiments. *, p < .05; ***, p < .001 versus corresponding control sample. (C): MLR were performed alone (black column) or in the presence of AMTC at P1, P2, or P3. AMTC were added either in direct contact or in transwell chambers. Data are mean and SD from three independent experiments. *, p < .05; ***, p < .001 versus corresponding control sample. (D): MLRs were set up between R and equal numbers of allogeneic S1*, with and without the addition of an equal number of AMTC in a transwell chamber. Re were then collected and restimulated with the original (Re + S1*) or third-party (Re + S2*) irradiated allogeneic PBMC, in the absence of AMTC. Data are mean and SD from three independent experiments. ***, p < .001 versus corresponding control sample. Abbreviations: AMTC, amniotic mesenchymal tissue cells; P, passage; PBMC, peripheral blood mononuclear cells; R, responder PBMC; Re, responders that had been exposed to AMTC; S1*, stimulator PBMC; SN, supernatant.

 
The inhibitory effect observed in the transwell system was suggestive of soluble inhibitory factor(s). This possibility was confirmed by the observation that MLR inhibition was also induced by the addition of FBS-supplemented supernatant collected from cultured AMTC. The inhibitory factor(s) seemed to accumulate with time in the AMTC conditioned medium, since the inhibitory effects were observed only in cultures with added supernatant obtained from AMTC cultured for more than 3 days, and increased gradually with the length of AMTC culture (Fig. 2B); similar results were obtained with essential and nonessential amino acid plus FBS supplementation of AMTC culture supernatants (data not shown). Surprisingly, the supernatant from AMTC cultured for 1 day increased MLR activation (Fig. 2B).

The inhibitory potential of AMTC was maintained for up to three AMTC culture passages in experiments using both cell-cell contact and transwell coculture, even though the inhibition was lower in the transwell setup when AMTC were used at passage 3 (Fig. 2C). Interestingly, responder cells previously exposed to AMTC in the transwell system were able to proliferate in new MLR cultures against either the original or new allogeneic PBMC stimulator cells (Fig. 2D), suggesting that the inhibitory effect is only transient and that AMTC do not induce T-cell death.

AMTC Inhibit Proliferation Induced by CD3 and CD28 Activation
To better define the observed inhibition of cell proliferation, we tested the effects of AMTC on PBMC and purified T cells stimulated with anti-CD3 with or without anti-CD28. In the absence of TcR stimulation, AMTC did not induce proliferation responses in either PBMC or purified T cells (Fig. 3A), thus indicating that these cells do not induce allogeneic responses. However, AMTC inhibited PBMC and T-cell proliferation induced by anti-CD3/anti-CD28 stimulation (Fig. 3B) either when cocultured in cell-cell contact or in a transwell system. In addition, AMTC inhibited PBMC proliferation induced by anti-CD3 stimulation both in transwell and in cell-cell contact culture (Fig. 3C). Surprisingly, however, the anergy of purified T cells to stimulation with anti-CD3 was overcome by the addition of AMTC. This proliferative effect was observed only in the cell-cell contact setting and not in transwell experiments (Fig. 3C). Stimulation of AMTC with anti-CD3 did not induce cell proliferation (data not shown).


Figure 3
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  		Figure 3. Effect of amniotic mesenchymal tissue cells (AMTC) on unstimulated and CD3- and CD3/CD28-stimulated PBMC and T cells. PBMC and T cells were either cultured alone (A) or stimulated with anti-CD3 plus anti-CD28 antibody (B) or with anti-CD3 antibody alone (C). All experiments were performed in the absence (black columns) or presence (white columns) of AMTC, both in cell contact and in transwell systems. Proliferation was assessed by [3H]-thymidine incorporation after culturing and expressed in cpm. Data are mean and SD from at least three independent experiments. **, p < .01; ***, p < .001 versus corresponding control sample. Abbreviation: PBMC, peripheral blood mononuclear cells.

 
Determination of AMTC Subpopulations
To investigate the basis of the costimulatory properties of AMTC in the presence of purified T cells, we performed flow cytometry analysis of freshly isolated AMTC. While performing routine exclusion of propidium iodide-positive cells from forward scatter/side scatter-gated AMTC (R1) using FLH-3 channel, we observed the presence of two distinct populations (R2 and R3) with different autofluorescence characteristics before addition of propidium iodide (Fig. 4Ai–4Aiii). We confirmed the presence of these two different populations in all placenta preparations analyzed. The percentage of the population with high autofluorescence (R2) ranged from 5% to 15% of AMTC.


Figure 4
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  		Figure 4. Representative fluorescence-activated cell sorting analysis of cells isolated from the amniotic mesenchymal region. (A): Gating strategies to characterize AMTC. R1 was defined based on side and forward scatter properties of AMTC (Ai). Analysis of R1 events using FLH-3 shows two distinct subpopulations, individuated by gates R2 and R3 (Aii) that can be back-gated to R1 (Aiii). AMTC acquired in presence of propidium iodide to stain dead cells (Aiv). (B): Surface expression of HLA-DR in total (gate R1) and R2- and R3-gated AMTC. (C): Surface expression of indicated hematopoietic markers on cells gated in R2 (HLA-DR-positive cells). Red histograms show positive cells, whereas black histograms show IgG isotype control stainings. Percentages of positive cells are indicated. AMTC were analyzed at passage 0. Abbreviation: M, histogram marker.

 
To begin to characterize these two different populations, we performed a series of immunophenotypic studies that showed that cells in gate R2 were more than 90% positive for HLA-DR, whereas the remaining subpopulation (R3) was HLA-DR-negative (Fig. 4B). Absence of propidium iodide positivity suggested that these cells were viable (Fig. 4Aiv).

Further analysis of R2 cells showed expression of the hematopoietic marker CD45 and the monocytic antigens CD14, CD11b, and CD86 in the absence of dendritic (CD1a, CD83), T (CD3) and natural killer (CD16) cell markers (Fig. 4C). Interestingly, the HLA-DR+ cells showed strong positivity for the costimulatory molecule CD86 and the absence of CD80. In addition, immunohistochemical staining of placenta sections confirmed the presence of CD45+, HLA-DR+, and CD68+ cells in the stromal region of amnion (Fig. 5A). Interestingly, these cells are located at the interface between the mesenchymal regions of amnion and chorion (Fig. 5A). Molecular analysis of DNA obtained from HLA-DR+ AMTC using highly polymorphic restriction fragment length polymorphisms allowed us to demonstrate the fetal origin of such cells (Fig. 5B).


Figure 5
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  		Figure 5. Determination of subpopulations present in the amniotic mesenchymal region. (A): Immunohistochemical staining of representative paraffin sections of term placental amniotic and chorionic membranes. Left panel: section stained with anti-human CD68 antibody; middle panel: section stained with anti-human HLA-DR antibody; right panel: section stained with anti-human CD45RO antibody. Original magnification, x40. (B): Restriction fragment length polymorphism analysis of DNA extracted from HLA-DR+ AMTC. Placental deciduas and total AMTC were used as maternal and fetal controls, respectively. Abbreviations: AETC, amniotic epithelial tissue cells; AMTC, amniotic mesenchymal tissue cells; CMTC, chorionic mesenchymal tissue cells; CTTC, chorionic trophoblastic tissue cells.

 
Effects of HLA-DR+ and HLA-DR– AMTC on T-Cell Proliferation
We hypothesized that HLA-DR+ amniotic cells played a role in the costimulation of T-cell proliferation observed in the presence of AMTC. To test this hypothesis, we obtained HLA-DR+-enriched (>90% HLA-DR-positive) and -depleted (<5% HLA-DR-positive) AMTC fractions. Both HLA-DR+ and HLA-DR– AMTC fractions did not induce allogeneic T-cell responses. However, in contrast to the HLA-DR– AMTC population, HLA-DR+ and unfractionated AMTC induced marked T-cell proliferation in the presence of anti-CD3 stimulation. Similar stimulatory capacity was also observed when unfractionated and HLA-DR+ AMTC were used after PFA fixation (Fig. 6A).


Figure 6
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  		Figure 6. Effect of cells isolated from mesenchymal amniotic region on CD3-stimulated T cells. (A): Left side: purified T cells were cultured alone or in direct contact with unfractionated, HLA-DR-negative or HLA-DR-positive AMTC. Right side: purified T cells in the presence of anti-CD3 were cultured alone or in direct contact with either paraformaldehyde-fixed or nonfixed, unfractionated, HLA-DR-negative or HLA-DR-positive AMTC. T-cell proliferation was assessed by [3H]-thymidine incorporation after 3 days of culture and expressed in cpm. Data are mean and SD from at least four independent experiments. ***, p < .001 versus corresponding control sample. (B): Purified T cells, stimulated with anti-CD3, were cultured alone or in direct contact with increasing concentrations of total AMTC (bullet), HLA-DR-negative AMTC ({blacksquare}), or HLA-DR-positive AMTC ({blacktriangleup}). T-cell proliferation was measured by [3H]-thymidine incorporation after 2 to 3 days of culture and expressed in cpm. Data are mean and SD from three independent experiments. (C): Purified T cells, stimulated with anti-CD3, were cultured in direct contact with total AMTC at different passage numbers. At each AMTC passage, the percentage of HLA-DR-positive cells present, measured by fluorescence-activated cell sorting analysis, is reported. T-cell proliferation was assessed by [3H]-thymidine incorporation after 3 days of culture and expressed in cpm. Representative results of three independent experiments are shown. Abbreviation: AMTC, amniotic mesenchymal tissue cells.

 
We also observed a dose-dependent effect on T-cell proliferation by both unfractionated and HLA-DR+ AMTC (Fig. 6B). These results suggest that the HLA-DR+ AMTC population is involved in T-cell proliferation in the presence of anti-CD3 stimulation.

We observed that HLA-DR+ AMTC decrease markedly in number during in vitro AMTC culture passages, with an approximate percentage of only 0.5%–2% remaining after three passages. Such a decline correlated with a reduction in the costimulatory effects of AMTC on T cells previously stimulated by anti-CD3 (Fig. 6C).

In transwell experiments with purified T cells activated with anti-CD3 and anti-CD28 antibodies, AMTC always inhibited T-cell proliferation in a dose-dependent manner, as shown in Figure 7A. In contrast, in cell-cell contact conditions, AMTC inhibited T-cell proliferation when added at higher concentrations (T cell:AMTC ratio of 1:1 or 1:1.3), whereas they induced T-cell activation when added at lower concentrations (Fig. 7B).


Figure 7
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  		Figure 7. Effect of amnion mesenchymal cells on purified T cells stimulated by CD3 plus CD28. Purified T cells, stimulated with anti-CD3 and anti-CD28, were cultured alone or in the presence of increasing concentrations of total AMTC in transwell chambers (A) or in direct contact (B). T-cell proliferation was assessed by [3H]-thymidine incorporation after 2 to 3 days of culture and expressed in cpm. Data are mean and SD from at least three independent experiments. (C): Purified T cells, stimulated with anti-CD3 and anti-CD28, were cultured alone or in direct contact with increasing concentrations of total AMTC (bullet), HLA-DR-negative AMTC ({blacksquare}), or HLA-DR-positive AMTC ({blacktriangleup}). T-cell proliferation was measured by [3H]-thymidine incorporation after 2 to 3 days of culture and expressed in cpm. Data are mean and SD from three independent experiments. Abbreviation: AMTC, amniotic mesenchymal tissue cells.

 
To characterize these findings, we performed cell-cell contact experiments with purified T cells activated with anti-CD3 and anti-CD28 and cocultured with irradiated unfractionated, HLA-DR+, and HLA-DR– AMTC. As shown in Figure 7C, the HLA-DR+ AMTC induced T-cell proliferation, whereas the HLA-DR– AMTC fraction had an inhibitory effect in a dose-dependent manner. Interestingly, unfractionated AMTC showed both activation and inhibition of cell proliferation, which was dependent on the dose of AMTC included in the culture.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
We and others have recently reported that the mesenchymal region of amnion from human term placenta contains cells with phenotypic, functional, and immunomodulatory characteristics similar to those described for MSC derived from other sources, such as BM, adipose tissue, and cord blood [1, 4–7, 52, 53]. In the present study, we report on the immunomodulatory properties of different cell subpopulations isolated from the mesenchymal tissue of the amniotic membrane, with a focus on their effect on the proliferation of T cells stimulated with allogeneic target cells (MLR) or via T-cell receptor engagement.

Unfractionated AMTC are capable of inhibiting MLR T-cell proliferation not only when cultured in cell-cell contact with responder cells but also when separated from them by a transwell membrane. The inhibitory effects were more pronounced when increasing numbers of AMTC were added to the cultures, suggesting a dose-dependent effect. The finding that inhibition of T-cell proliferation was induced by AMTC in the transwell system suggests that a soluble factor is implicated in this phenomenon. This hypothesis is also supported by our findings that T-cell proliferation was inhibited by the addition of AMTC culture supernatant.

The nature of these transferable inhibitory factor(s), as well as the identity of the factor(s) present in day 1 AMTC culture supernatant that resulted in the increase of MLR cell proliferation, remains unknown. However, it is of note that soluble factors, even though not unequivocally identified, have been implicated in the antiproliferative capabilities and inhibitory effects on maturation of dendritic cells by MSC of other origins, such as bone marrow and adipose tissue [22, 29–32, 54–59], although the need for cell-cell contact [23, 60] and/or additional cell types (monocytes, dendritic cells) [28, 61] remains an area of debate.

The inhibitory potential of AMTC in MLR is essentially maintained until passage 3, both in direct cell contact and in the transwell system. However, AMTC at passage 3 showed diminished ability to inhibit lymphocyte proliferation in the transwell system. Whether this phenomenon is associated with selection or loss of particular cells within the AMTC population during culture remains to be determined.

We were particularly intrigued by the identification of subpopulations expressing the leukocyte HLA-DR molecules. The mesodermal region of amnion is considered to be avascular, and therefore, the presence of hematopoietic cells is not expected. However, fluorescence-activated cell sorting analysis of freshly isolated AMTC revealed a defined HLA-DR-positive subpopulation coexpressing the monocyte-specific markers CD14, CD11b, and CD86. The presence of cells with monocytic immunophenotype was confirmed by immunohistochemistry of whole placenta samples, whereas PCR analysis proved the fetal origin of these cells. Although previous studies have reported on the presence of HLA-DR+ cells in the mesenchymal region of the amniotic membrane [45–47], expression of MHC II antigens has generally been reported to be low or absent on cells isolated from the amnion mesenchymal region [4, 7]. The discrepancy between these previous reports and our findings can be explained by the use of different cell isolation protocols. In addition, given the rapid reduction in the number of the HLA-DR+ cells that we observed in cultured AMTC, it is also possible that the timing of analysis of MHC II expression can account for some of the findings described in the literature. It is important to note that contrasting reports on the presence of HLA-DR+ and CD45+ cells exist also for the BM-MSC field [13, 23, 60], as well for adipose-derived MSC [13, 62].

We observed that unfractionated and purified HLA-DR+ and HLA-DR– AMTC failed to induce T-cell proliferation in the absence of additional stimuli. The lack of T-cell responses against HLA-DR+ allogeneic cells may appear surprising. However, previous studies have shown that exposure to interferon-{gamma} can induce a high level of expression of MHC II on BM-MSC, which remain unable to induce T-cell proliferation [55, 63].

Interestingly, AMTC induced strong proliferation in CD3-stimulated T cells when cocultured in direct cell contact. This effect was even stronger when the HLA-DR+ AMTC subpopulation was used, suggesting that these cells are capable of providing costimulatory signals. Of note, we observed that the prevalence of HLA-DR+ AMTC decreased with in vitro culture, which correlated with a reduction of the stimulatory properties of unfractionated AMTC. In contrast to their inhibitory function, which involves soluble factors, the "costimulatory" effect of AMTC on CD3-primed T cells is very likely not to be associated with the production of soluble factors, as demonstrated by the finding that CD3-primed T cells also proliferate when cultured with PFA-fixed AMTC. Cell-cell contact, however, is required for CD3-primed T-cell proliferation, which is prevented when AMTC are cultured in a transwell system.

The detection of the CD86 molecule on HLA-DR+ AMTC suggests that the CD86/CD28 costimulatory pathway may be involved in the activation of T-lymphocyte proliferation induced by AMTC in cells that received TcR stimulation. Furthermore, a significant AMTC dose-dependent phenomenon was observed in proliferation experiments using purified T responder cells and TcR engagement with CD3 and CD28 costimulus, with an increase in T-cell proliferation at low AMTC:T-cell ratios. The stimulatory effect was not observed when AMTC were cultured in a transwell system, indicating the need for cell-cell contact for this phenomenon to occur. Importantly, we demonstrated that activation of cell proliferation is associated with the presence of HLA-DR+ AMTC. These observations are reminiscent of previous puzzling findings indicating that low ratios of human MSC to T cells can augment responder cell proliferation rather than suppressing it [24, 64].

Furthermore, our findings may help to reconcile the current debate both on the presence of HLA-DR+ cells, which remains a controversial issue in MSC in general [13, 23, 60, 62], and also the paradoxical results that have been obtained after transplantation of BM-MSC. For example, allogeneic BM-MSC are used in clinical trials to control GvHD [36, 37]; however, in murine bone marrow transplantation (BMT) models, coinfusion of allogeneic BM and BM-MSC induces a memory T-cell response resulting in cell graft rejection [34]. If a population of HLA-DR+ cells exists within the BM-MSC population, these cells may account for these opposite immunomodulatory functions, as we have observed. Therefore, the testing of fractionated versus unfractionated cell populations for transplantation into animal models may yield interesting results that could become relevant in clinical settings by allowing tolerance of transplanted cells or organs.

Additional studies are warranted to understand whether immunomodulatory functions of AMTC and their possible counterpart in MSC from other tissues can be exploited therapeutically to modulate the outcome of tissue therapies or pregnancy pathologies caused by problems at the fetal-maternal interface. Reliable procedures to obtain such cells, including those we describe here, should facilitate these important tasks.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 Acknowledgments
 References
 
We have shown that unfractionated AMTC can inhibit T-cell proliferation with features that are very similar to those observed in MSC of various origins. In addition, we have identified an HLA-DR+ fraction of AMTC of fetal origin with immunophenotypic characteristics similar to those of human monocytes that is unable to induce proliferation unless T cells are primed by TcR engagement with anti-CD3. In addition to bringing new insights into the immunomodulatory functions of cells isolated from the mesenchymal region of the amniotic membrane and raising interest for similar investigations for MSC from other sources, the data obtained may be relevant for understanding the phenomenon of fetal-maternal tolerance. Indeed, the histological detection of these cells in the amniotic membrane and their peculiar distribution in "sentinel" arrangements leads us to speculate that they may play a role in fetal-maternal tolerance homeostasis. If correct, this hypothesis may implicate in this critical process a region of the placenta (the amnion) located internally to the trophoblast and the maternal deciduas, which have generally attracted investigators' attention to date.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
Acknowledgments
 References
 
A European patent application has been filed with the application no. 07011824.5.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank the physicians and midwives of the Department of Obstetrics and Gynecology of Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy, and all of the mothers who donated placenta as well as all the volunteers who donated blood. We also thank Dr. Verzeletti (Clinica St. Anna, Brescia, Italy) for assistance with cell irradiation. We are indebted to Dr. Fabio Candotti for support in data interpretation and critical reviewing of the manuscript. We sincerely thank Dr. Mark Larche for valuable technical advice and Dr. Marco Evangelista for help in editing the manuscript. This study was supported in part by grants from Fondazione Cariplo, Bando 2004, and Fondazione Cariplo Progetto Nobel 2006. Author contributions: M.M. performed research and analyzed data; S.D.M. performed research; E.V. provided technical support; L.G. performed immunohistochemistry analysis; G.S.W. analyzed data and provided technical advice; O.P. designed the research, analyzed data, and wrote the manuscript.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Bailo M, Soncini M, Vertua E et al. Engraftment potential of human amnion and chorion cells derived from term placenta. Transplantation 2004;78:1439–1448.[CrossRef][Medline]

  2. In't Anker PS, Scherjon SA, Kleijburg-van der Keur C et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. STEM CELLS 2004;22:1338–1345.[Abstract/Free Full Text]

  3. Zhao P, Ise H, Hongo M et al. Human amniotic mesenchymal cells have some characteristics of cardiomyocytes. Transplantation 2005;79:528–535.[CrossRef][Medline]

  4. Portmann-Lanz CB, Schoeberlein A, Huber A et al. Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration. Am J Obstet Gynecol 2006;194:664–673.[CrossRef][Medline]

  5. Parolini O, Soncini M. Human placenta: A source of progenitor/stem cells? J Reprod Med Endocrinol 2006;3:117–126.

  6. Alviano F, Fossati V, Marchionni C et al. Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro. BMC Dev Biol 2007;7:11.[CrossRef][Medline]

  7. Wolbank S, Peterbauer A, Fahrner M et al. Dose-dependent immunomodulatory effect of human stem cells from amniotic membrane: A comparison with human mesenchymal stem cells from adipose tissue. Tissue Eng 2007;13:1173–1183.[CrossRef][Medline]

  8. Dominici M, Hofmann TJ, Horwitz EM. Bone marrow mesenchymal cells: Biological properties and clinical applications. J Biol Regul Homeost Agents 2001;15:28–37.[Medline]

  9. Le Blanc K, Ringden O. Mesenchymal stem cells: Properties and role in clinical bone marrow transplantation. Curr Opin Immunol 2006;18:586–591.[CrossRef][Medline]

  10. Rasmusson I. Immune modulation by mesenchymal stem cells. Exp Cell Res 2006;312:2169–2179.[CrossRef][Medline]

  11. Friedenstein AJ, Petrakova KV, Kurolesova AI et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 1968;6:230–247.[Medline]

  12. Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 2001;7:211–228.[CrossRef][Medline]

  13. Kern S, Eichler H, Stoeve J et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. STEM CELLS 2006;24:1294–1301.[Abstract/Free Full Text]

  14. Lee RH, Kim B, Choi I et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004;14:311–324.[CrossRef][Medline]

  15. Mitchell JB, McIntosh K, Zvonic S et al. Immunophenotype of human adipose-derived cells: Temporal changes in stromal-associated and stem cell-associated markers. STEM CELLS 2006;24:376–385.[Abstract/Free Full Text]

  16. Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: Candidate MSC-like cells from umbilical cord. STEM CELLS 2003;21:105–110.[Abstract/Free Full Text]

  17. Lee OK, Kuo TK, Chen WM et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004;103:1669–1675.[Abstract/Free Full Text]

  18. Villaron EM, Almeida J, Lopez-Holgado N et al. Mesenchymal stem cells are present in peripheral blood and can engraft after allogeneic hematopoietic stem cell transplantation. Haematologica 2004;89:1421–1427.[Abstract/Free Full Text]

  19. Roufosse CA, Direkze NC, Otto WR et al. Circulating mesenchymal stem cells. Int J Biochem Cell Biol 2004;36:585–597.[CrossRef][Medline]

  20. In't Anker PS, Scherjon SA, Kleijburg-van der Keur C et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003;102:1548–1549.[Free Full Text]

  21. De Coppi P, Bartsch G Jr, Siddiqui MM et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100–106.[CrossRef][Medline]

  22. Di Nicola M, Carlo-Stella C, Magni M et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002;99:3838–3843.[Abstract/Free Full Text]

  23. Krampera M, Glennie S, Dyson J et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 2003;101:3722–3729.[Abstract/Free Full Text]

  24. Le Blanc K, Tammik L, Sundberg B et al. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003;57:11–20.[CrossRef][Medline]

  25. Ueta M, Kweon MN, Sano Y et al. Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction. Clin Exp Immunol 2002;129:464–470.[CrossRef][Medline]

  26. Barry FP, Murphy JM, English K et al. Immunogenicity of adult mesenchymal stem cells: Lessons from the fetal allograft. Stem Cells Dev 2005;14:252–265.[CrossRef][Medline]

  27. Maccario R, Podesta M, Moretta A et al. Interaction of human mesenchymal stem cells with cells involved in alloantigen-specific immune response favors the differentiation of CD4+ T-cell subsets expressing a regulatory/suppressive phenotype. Haematologica 2005;90:516–525.[Abstract/Free Full Text]

  28. Beyth S, Borovsky Z, Mevorach D et al. Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood 2005;105:2214–2219.[Abstract/Free Full Text]

  29. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005;105:1815–1822.[Abstract/Free Full Text]

  30. Zhang W, Ge W, Li C et al. Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells. Stem Cells Dev 2004;13:263–271.[CrossRef][Medline]

  31. Jiang XX, Zhang Y, Liu B et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005;105:4120–4126.[Abstract/Free Full Text]

  32. Nauta AJ, Kruisselbrink AB, Lurvink E et al. Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells. J Immunol 2006;177:2080–2087.[Abstract/Free Full Text]

  33. Zappia E, Casazza S, Pedemonte E et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 2005;106:1755–1761.[Abstract/Free Full Text]

  34. Nauta AJ, Westerhuis G, Kruisselbrink AB et al. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 2006;108:2114–2120.[Abstract/Free Full Text]

  35. Sudres M, Norol F, Trenado A et al. Bone marrow mesenchymal stem cells suppress lymphocyte proliferation in vitro but fail to prevent graft-versus-host disease in mice. J Immunol 2006;176:7761–7767.[Abstract/Free Full Text]

  36. Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363:1439–1441.[CrossRef][Medline]

  37. Ringden O, Uzunel M, Rasmusson I et al. Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation 2006;81:1390–1397.[CrossRef][Medline]

  38. Medawar PB. Some immunological and endocrinological problems raised by the evolution of viviparity in vertebrates. Symp Soc Exp Biol 1953;7:320–338.

  39. Collins GD, Chrest FJ, Adler WH. Maternal cell traffic in allogeneic embryos. J Reprod Immunol 1980;2:163–172.[CrossRef]

  40. Bonney EA, Matzinger P. The maternal immune system's interaction with circulating fetal cells. J Immunol 1997;158:40–47.[Abstract]

  41. Bianchi DW, Romero R. Biological implications of bi-directional fetomaternal cell traffic: A summary of a National Institute of Child Health and Human Development-sponsored conference. J Matern Fetal Neonatal Med 2003;14:123–129.[Medline]

  42. Bonney EA, Matzinger P. Much IDO about pregnancy. Nat Med 1998;4:1128–1129.[CrossRef][Medline]

  43. Koch CA, Platt JL. Natural mechanisms for evading graft rejection: The fetus as an allograft. Springer Semin Immunopathol 2003;25:95–117.[CrossRef][Medline]

  44. Enders AC, King BF. The cytology of Hofbauer cells. Anat Rec 1970;167:231–236.[CrossRef][Medline]

  45. Sutton L, Gadd M, Mason DY et al. Cells bearing class II MHC antigens in the human placenta and amniochorion. Immunology 1986;58:23–29.[Medline]

  46. Sutton L, Mason DY, Redman CW. HLA-DR positive cells in the human placenta. Immunology 1983;49:103–112.[Medline]

  47. Bulmer JN, Morrison L, Smith JC. Expression of class II MHC gene products by macrophages in human uteroplacental tissue. Immunology 1988;63:707–714.[Medline]

  48. Dominici M, Le Blanc K, Mueller I et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–317.[CrossRef][Medline]

  49. Parolini O, Alviano F, Bagnara GP et al. Isolation and characterization of cells from human term placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells. STEM CELLS 2007; in press.

  50. Bartholomew A, Sturgeon C, Siatskas M et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42–48.[CrossRef][Medline]

  51. Soncini M, Vertua E, Gibelli L et al. Isolation and characterization of mesenchymal cells from human fetal membranes. Tissue Eng Regen Med 2007;1:296–305.[CrossRef]

  52. Fukuchi Y, Nakajima H, Sugiyama D et al. Human placenta-derived cells have mesenchymal stem/progenitor cell potential. STEM CELLS 2004;22:649–658.[Abstract/Free Full Text]

  53. Chang CJ, Yen ML, Chen YC et al. Placenta-derived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-gamma. STEM CELLS 2006;24:2466–2477.[Abstract/Free Full Text]

  54. Djouad F, Plence P, Bony C et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 2003;102:3837–3844.[Abstract/Free Full Text]

  55. Tse WT, Pendleton JD, Beyer WM et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: Implications in transplantation. Transplantation 2003;75:389–397.[CrossRef][Medline]

  56. Meisel R, Zibert A, Laryea M et al. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood 2004;103:4619–4621.[Abstract/Free Full Text]

  57. Hainz U, Obexer P, Winkler C et al. Monocyte-mediated T-cell suppression and augmented monocyte tryptophan catabolism after human hematopoietic stem-cell transplantation. Blood 2005;105:4127–4134.[Abstract/Free Full Text]

  58. Rasmusson I, Ringden O, Sundberg B et al. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp Cell Res 2005;305:33–41.[CrossRef][Medline]

  59. Puissant B, Barreau C, Bourin P et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells: Comparison with bone marrow mesenchymal stem cells. Br J Haematol 2005;129:118–129.[CrossRef][Medline]

  60. Potian JA, Aviv H, Ponzio NM et al. Veto-like activity of mesenchymal stem cells: Functional discrimination between cellular responses to alloantigens and recall antigens. J Immunol 2003;171:3426–3434.[Abstract/Free Full Text]

  61. Groh ME, Maitra B, Szekely E et al. Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive T cells. Exp Hematol 2005;33:928–934.[CrossRef][Medline]

  62. McIntosh K, Zvonic S, Garrett S et al. The immunogenicity of human adipose-derived cells: Temporal changes in vitro. STEM CELLS 2006;24:1246–1253.[Abstract/Free Full Text]

  63. Klyushnenkova E, Mosca JD, Zernetkina V et al. T cell responses to allogeneic human mesenchymal stem cells: Immunogenicity, tolerance, and suppression. J Biomed Sci 2005;12:47–57.[CrossRef][Medline]

  64. Fang L, Lange C, Engel M et al. Sensitive balance of suppressing and activating effects of mesenchymal stem cells on T-cell proliferation. Transplantation 2006;82:1370–1373.[CrossRef][Medline]





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