Amniotic Membrane Mesenchymal Cells-Derived Factors Skew T Cell Polarization Toward Treg and Downregulate Th1 and Th17 Cells Subsets
- 2k Downloads
We previously demonstrated that cells derived from the mesenchymal layer of the human amniotic membrane (hAMSC) and their conditioned medium (CM-hAMSC) modulate lymphocyte proliferation in a dose-dependent manner. In order to understand the mechanisms involved in immune regulation exerted by hAMSC, we analyzed the effects of CM-hAMSC on T-cell polarization towards Th1, Th2, Th17, and T-regulatory (Treg) subsets. We show that CM-hAMSC equally suppresses the proliferation of both CD4+ T-helper (Th) and CD8+ cytotoxic T-lymphocytes. Moreover, we prove that the CM-hAMSC inhibitory ability affects both central (CD45RO+CD62L+) and effector memory (CD45RO+CD62L−) subsets. We evaluated the phenotype of CD4+ cells in the MLR setting and showed that CM-hAMSC significantly reduced the expression of markers associated to the Th1 (T-bet+CD119+) and Th17 (RORγt+CD161+) populations, while having no effect on the Th2 population (GATA3+CD193+/GATA3+CD294+cells). T-cell subset modulation was substantiated through the analysis of cytokine release for 6 days during co-culture with alloreactive T-cells, whereby we observed a decrease in specific subset-related cytokines, such as a decrease in pro-inflammatory, Th1-related (TNFα, IFNγ, IL-1β), Th2 (IL-5, IL-6), Th9 (IL-9), and Th17 (IL-17A, IL-22). Furthermore, CM-hAMSC significantly induced the Treg compartment, as shown by an induction of proliferating CD4+FoxP3+ cells, and an increase of CD25+FoxP3+ and CD39+FoxP3+ Treg in the CD4+ population. Induction of Treg cells was corroborated by the increased secretion of TGF-β. Taken together, these data strengthen the findings regarding the immunomodulatory properties of CM-hAMSC derived from human amniotic membrane MSC, and in particular provide insights into their effect on regulation of T cell polarization.
KeywordsMesenchymal stromal cells Human amniotic membrane mesenchymal cells Human placenta Conditioned medium Secretome Immunomodulation Cytokines T cells Th1 Th2 Treg Th17
Mesenchymal stromal cells
Human amniotic membrane
Human amniotic mesenchymal stromal cells
Bone marrow mesenchymal stromal cells
Conditioned Medium derived from the culture of hAMSC
Effector Memory RA
T regulatory cells
GATA-binding protein 3
glucocorticoid-induced TNFR-related protein
Retinoic acid-related orphan receptor gamma t
Forkhead box P3
Glycoprotein A Repetitions Predominant
T-box transcription factor TBX21
The human amniotic membrane, as well as the other perinatal tissues, have recently attracted much attention in regenerative medicine applications ; indeed they can be easily obtained in a non-invasive manner from tissues normally discarded after birth, and they also offer an abundant source for bank development. The therapeutic potential of the perinatal stem cells has been prevalently associated to their immunomodulatory capacities [2, 3, 4, 5, 6] and consequent paracrine effects, as observed in different animal models of disease [7, 8, 9].
Amongst perinatal tissues, the human amniotic membrane from term placenta has been recently recognized as a valuable source of mesenchymal stromal cells, referred to as hAMSC [10, 11, 12]. Interestingly, studies have shown the ability of hAMSC to interact with and modulate the functions of a wide variety of immune cells. For example, we and others have shown that hAMSC can inhibit T cell proliferation in vitro induced by alloantigens, T-cell receptor cross-linking, or mitogens [13, 14, 15, 16, 17]. Furthermore, we and others have previously shown that cells derived from the human amniotic membrane strongly inhibit the generation, maturation, and function of monocyte-derived dendritic cells (DCs) in vitro [18, 19]. The in-vitro anti-inflammatory potential of amniotic cells is in line with the in vivo findings showing reduction of inflammation and fibrosis in animal models of disease following the transplantation of cells derived from the amniotic membrane. For example, therapeutic effects have been observed in bleomycin-challenged mice as shown by a reduction in lung fibrosis following treatment with amniotic cells [5, 20]. Moreover, amniotic cells have been reported to ameliorate prognosis of autoimmune diseases such as rheumatoid arthritis, encephalomyelitis , and experimental autoimmune myocarditis . Furthermore, the use of amniotic membrane patches were also able to attenuate disease progression. The transplantation of non-cryopreserved amniotic patches , or even those after cryopreservation , were able to improve liver fibrosis in rats with bile-duct ligation and promote ischemic heart repair in rats with coronary artery ligation . Interestingly, in these studies therapeutic effects were observed despite absence or rare presence of transplanted cells in host tissues. These findings have reinforced their capacity to exert paracrine effects inducing tissue repair by immunomodulation rather than cell differentiation . Confirmation that the molecules released from cells are the key players comes from studies showing that the conditioned medium exerts the same anti-inflammatory effects as cells [25, 26]. Evidence suggests that the conditioned medium obtained from the culture of AM patches or hAMSC inhibits T cell proliferation , inhibits the differentiation of monocytes towards DCs, and induces a shift toward M2-like macrophages  as observed with MSC from other placental regions . The molecules and mechanisms involved are still unclear, but there are many hypotheses which also take into consideration what is known on mesenchymal stromal cells derived from bone marrow, which have been reported to act through IDO, NO, PGE2, TGF-β, IL-10, HGF and galectins [30, 31]. Moreover, we have provided evidence that this effect seems to be mediated by low molecular weight, non-protein, thermostable compounds present in conditioned medium, and that prostaglandins are one of the key effector molecules in the immunomodulatory activity . Arising from the need to identify key effector molecules is the desire to understand the cells on which they act, and in turn how they are impacted. Specifically, even though the anti-proliferative effects on T cells are now widely accepted, the effects of hAMSC on the different T cell subpopulations remain to be clearly addressed. Recent studies report the capacity of amniotic mesenchymal stromal cells to regulate T cell subsets in animal models. For example, systemic administration of hAMSC has been shown to ameliorate experimental autoimmune myocarditis (EAM) via the suppression of Th1/Th17 immunity . Similar mechanisms have been extensively described for mesenchymal stromal cells obtained from other sources. For example, treatment with bone marrow MSC was shown to attenuate cutaneous delayed-type hypersensitivity in mice and was found to be associated with reduced CD4+ and CD8+ T cell infiltration at the challenge site . Moreover, the treatment of colitic mice (model of inflammatory bowel disease) with MSC from adipose tissue reduced the Th1 cell responses and induced T regulatory cells , while treatment with MSC from bone marrow prevented Th1-mediated autoimmune diabetes mellitus in rats, and was associated with increased CD4+ and CD8+ FoxP3+ T cells . We have very recently demonstrated that treatment of mice with collagen-induced arthritis using cells from the amniotic membrane impaired antigen specific Th1/Th17 cell expansion in the lymph nodes, and generated peripheral antigen-specific T regulatory cells . Taken together, these studies indicate that amnion-derived cells and its conditioned medium do indeed act on T cells. Nevertheless, a basic lack of information regarding the effects that hAMSCs have on individual T-cell effector subsets remains. In this study, we set out to clarify the polarization of T cells by performing detailed in vitro studies on both CD4 and CD8 lineages and we contribute to the understanding of the time-dependent effects on the polarization of CD4+ T cells in terms of T cell activation, proliferation, and cytokine production.
Materials and Methods
Human term placentas were collected after obtaining written informed consent according to the guidelines of the Ethical Committee of the Catholic Hospital (CEIOC, Parere 16/2012) and of the Ethical Committee of the Hospital Fondazione Poliambulanza-Istituto Ospedaliero (Brescia, Italy). The research project was authorized by Centro di Ricerca E. Menni-Fondazione Poliambulanza.
Isolation of Human Amniotic Mesenchymal Stromal Cells (hAMSC) and Production of Conditioned Medium (CM-hAMSC)
Human term placentas were processed immediately after birth using a previously described protocol . Briefly, the amnion was manually separated from the chorion and washed extensively in PBS (Sigma, St Louis, MO, USA) containing 100U/ml penicillin and 100 mg/ml streptomycin (herein referred to as P/S, Euroclone, Whetherby, UK) and 2.5 mg/ml amphotericin B (Lonza, Basel, CH). Afterwards, the amnion was cut into small pieces (3x3 cm2). Amnion fragments were sterilized by a brief incubation in PBS + 2.5 % Eso Jod (Esoform, Italy) and 3 min in PBS containing 500U/ml penicillin, 500 mg/ml streptomycin, 12.5 mg/ml amphotericin B and 1.87 mg/ml Cefamezin (Pfizer, Italy). Sterilized amnion fragments were then incubated for 9 min at 37 °C in HBSS (Lonza, Basel, CH) containing 2.5U/ml dispase (Roche, Mannheim, Germany). The fragments were digested in complete RPMI 1640 medium (Cambrex, Verviers, Belgium) supplemented with 0.94 mg/ml collagenase (Roche) and 20 mg/ml DNase (Roche) for 2.5–3 hrs at 37 °C. Amnion epithelium fragments were then removed by low-g centrifugation, mobilized hAMSC were passed through a 100 μm cell strainer and collected by centrifugation. These cells are referred to as hAMSC, for human Amniotic Mesenchymal Stromal Cells, and at passage 0 (freshly isolated) are characterized by the expression of CD90 (80.5 ± 10.7 %), CD13 (84.5 ± 8.7 %), CD73 (66 ± 6 %), CD44 (57 ± 10 %), CD105 (6 ± 4 %), CD166 (9.3 ± 6.5 %), CD324 (10.4 ± 6.4 %), CD45 (7.4 ± 2.8 %), CD14 (6 ± 3 %), and negative for CD34 and CD3.
Conditioned Medium generated from freshly isolated hAMSC. hAMSC (obtained from amniotic membranes of at least 30 different donors) were re-suspended in an opportune volume of UltraCulture serum-free medium (Lonza, Basel, CH) supplemented with P/S, and plated in 24-well plates at 0.5×105 cells/well in a final volume of 0.5 ml (referred to as CM-hAMSC). After 5-days of culture at 37 °C with 5 % CO2, the CM-hAMSC were collected, centrifuged at 300 g, filtered through a 0.8 μm sterile filter (Sartorius) and frozen at −80 °C until use. In order to obtain results that were less influenced by single donor variability and more representative of bioactive molecules released by hAMSC, we pooled 8 to 10 different CM-hAMSC and used them for each specific analysis.
Purification of T-Lymphocytes and Proliferation Assays
Human peripheral blood mononuclear cells (PBMC) were obtained from heparinized peripheral blood (PB) or buffy coats (BC) of healthy donors after Ficoll–Hypaque gradient centrifugation (Sigma, St Louis, MO, USA). The purity of PBMC preparations was checked by FACS analysis to ensure low red blood cell (RBC) and polymorphonuclear (PMN) cell contaminations. T-cells were purified from PBMC by negative selection using the MACS® system (Pan T Cell Isolation Kit), (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Lymphocyte proliferation was induced either by stimulating T cells (105/well in 96-well-round bottom plate) by immobilized anti-CD3 (1 μg/ml OKT3) / anti-CD28 (2.5 μg/ml), or by the co-culture with irradiated allogeneic stimulator PBMC in mixed lymphocyte reactions (MLR). MLR were set up with 105 effector T-lymphocytes and 105 γ-irradiated (3000 cGy) allogeneic PBMC in round-bottom 96-well plates (Nunc, Roskilde, Denmark). MLR and T + anti-CD3/28 were cultured in UltraCulture medium. Responder T-cell/stimulator cell combinations were chosen on the basis of a minimum of three human leucocyte antigen (HLA) mismatches. T-cells were labeled with CFSE dye using the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, Molecular Probes, USA), according to manufacturer’s instructions. T cell proliferation was assessed by flow cytometry and is expressed as a percentage of CFSE diluting cells (Proliferative Fraction PF) or as Proliferation Index (PI). The PF represents the percent of proliferating cells and the PI is the sum of the cells in all generations divided by the number of original parent cells present at the start of the experiment. It measures the increase in cell number in culture over the course of the experiment and is calculated by using FCS express v4.07 (DeNovo Software) from a cell division model which predicts a cell doubling as a cell proliferated through each daughter generation. In order to perform FACS analysis only on the responder T-cells, γ-irradiated allogeneic stimulator PBMC were labeled using the CellVue® NIR780 Cell Labeling Kit (eBiosciences) in order to identify and exclude them from analysis. To assess the effect of CM-hAMSC on the T cell subsets, we co-cultured T cells in 50 % CM-hAMSC.
Phenotype Analysis of T-Cell Subsets
Markers used to identify T cell subpopulations
CD4, CD183, CD119
CD4, CD193, CD294
CD4, CD25, CD39, CD73, CD152, CD357
IL-1β, IL-2, TNF-α, IFN-α, IL-12p70
IL-4, IL-5, IL-6, IL-10, IL-13
Detection of Secreted Cytokines
The supernatant from MLR experiments was collected from day 1 to day 6 of culture, and the quantification of secreted cytokines was evaluated by using a multiple cytometric beads array system Human Th1/Th2/Th9/Th17/Th22 13plex FlowCytoMix kit (eBiosciences, San Diego, USA), according to the manufacturer’s instructions. The following cytokines were measured: IL-1-β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-22, TNF-α, and IFN-α. The levels of TGF-β and sIL2R were measured using the FlowCytoMix kit (eBiosciences, San Diego, USA), according to the manufacturer’s instructions. Samples were acquired with a FACSAria (Becton Dickinson, Franklin Lakes, New Jersey, USA) and analyzed with FlowCytomix Pro software (eBiosciences, San Diego, USA)
Statistical analyses were performed by means of unpaired, two-tailed t-tests using GraphPad Prism 6 Software (GraphPad Software, San Diego, CA, USA). Results are represented as mean ± standard deviation (SD) or standard error mean (SEM) as specified in the text. A P-value lower than 0.05 was considered statistically significant.
The Effects of CM-hAMSC on T-Cell Proliferation
The Effects of CM-hAMSC on Memory/Naïve Subsets
We then set out to clarify the effects of CM-hAMSC on CD4 and CD8 subsets in terms of proliferation and phenotype. To this end, we used CFSE-labeled T cells stimulated by allogeneic PBMC (MLR) and we evaluated T cell proliferation after 6 days of culture. T cell proliferation was expressed as a percentage of CFSE diluting cells (Proliferative Fraction, PF) and by the Proliferation Index (PI).
On the basis of the observed inhibitory ability, we also evaluated how the CM-hAMSC influenced the relative content/frequency of each T-cell subset described above. Regarding the CD4 positive cells, we observed a reduction in the Central Memory compartment, no variation in the Effector Memory compartment, and a relative increase of Naïve population (Fig. 2I). Similarly, within the CD8+ population, we observed a decrease of the Central Memory and a relative increase of the Naïve compartment (Fig. 2J). Finally, we observed that in the presence of CM-hAMSC, the percentage of CD8 Effector Memory did not change while that of CD8 Effector increased in the presence of CM-hAMSC (Fig. 2J).
The Effects of CM-hAMSC on T Helper (Th) Differentiation
The Effects of CM-hAMSC on T Regulatory (Treg) Cells
CM-hAMSC Modifies the Secretion of Th-Cytokines
Increasing evidence indicate that derivatives from the human amniotic membrane, such as patches, cells, and conditioned medium derived from these cells, exert therapeutical effects in diseases associated with altered inflammatory processes  or in autoimmune disorders . The lack or very low engraftment of transplanted cells, and the evidence that conditioned medium per se is effective, supports the notion that the therapeutic effect is due to bioactive molecules released from hAMSC that act through a paracrine/endocrine mechanism.
A likely explanation of the beneficial effects exerted by hAMSC is associated to the immunomodulatory potential of these cells, a characteristic identified previously in MSC from other sources, such as bone marrow [37, 38, 39, 40]. Indeed, it has been extensively reported that hAMSC can inhibit T cell proliferation induced by alloantigens, T-cell receptor cross-linking, or mitogens in vitro [13, 14, 15, 17, 19] and can inhibit the generation, maturation and function of monocyte-derived dendritic cells (DCs) [18, 41]. The confirmation that the molecules released from cells are the key players in their immunomodulatory effect comes from the observation that the conditioned medium obtained from the culture of both AM patches and hAMSC inhibit T cell proliferation , inhibit the differentiation of monocytes towards DCs and induce a shift toward M2-like macrophages [28, 29]. T cells have a prominent role in immune regulation, and polarization of the different T-cell subsets plays an important role in controlling the mechanisms of immune response in phenomena like acute and chronic inflammation and autoimmune responses. Since until now reports which provide evidence that hAMSC act on T cells are mainly based on the effects on total T cells and often limited to the proliferative parameters, we set out to perform a detailed study on the effects of conditioned medium from hAMSC on both CD4 and CD8 subsets and on different Th subsets. To this aim, we analyzed cell proliferation, alterations in phenotype, and cytokine production in a time-course response.
First, we observed that conditioned medium derived from hAMSC, when cultured without inflammatory stimuli, suppresses the proliferation of both CD4+ Th cells and CD8+ cytotoxic T lymphocytes (CTLs) stimulated by allogeneic PBMC, and also via T cell receptor (TCR) stimulation with anti-CD3/anti-CD28. This supports our previous observations that CM derived from amnion possess anti-proliferative effect in absence of stimulating culture conditions . This is in contrast to BM-MSC which possess an anti-proliferative ability only when cultured in the presence of activating stimuli, such as IL-1β, TNF-α or IFN-γ [30, 42, 43].
The differentiation of T cells into effector and memory subsets is one of the key aspects of T cell mediated immunity. We therefore characterized the effect of hAMSC on the T-cell response of Naïve and Memory T cells and demonstrate that CM-hAMSC inhibited the proliferation of both CD4/CD8 Effector Memory and CD4/CD8 Central Memory cells, while no variation was observed regarding the proliferation of the CD4/CD8 Naïve T-cell population. Others have shown that bone marrow MSC are able to equally inhibit the proliferation of Memory and Naïve T cells using HY peptide-stimulated splenocytes from transgenic HY-TCRhigh mice . The relative increase we observed in the percentage of Naïve cells (CD45RO−CD62L+) after CM-hAMSC co-culture can be justified by the decrease observed in the other subsets.
Within the CD8+ population (which drastically decreased in the presence of CM-hAMSC), we observed an increase of the CD8+CD28− population. This can be explained by the preferential survival of this population, as manifested by the fact that we observed a relative increase of CD8+CD28− within the Effector Memory, Central Memory, and EMRA compartments. Interestingly, CD8+CD28− T cells have been reported as T regulatory cells [45, 46]. Indeed, CD8+CD28− T cells have been shown to be accountable for regulatory functions associated to disease amelioration in an autoimmune mouse model . Moreover, they have been reported to be able to down-regulate the Th reactivity by suppression of antigen-presenting cells , and to be responsible for the inhibition of both T-cell proliferation and CTL function . The effect of MSC on the CD8+CD28− population is still a matter of debate. Indeed, it has been shown that MSC from adipose tissue induce an inhibition of CD8+CD28− cells , while others, in accordance with our data, have shown that MSC from bone marrow induce an increase of this population thus contributing to the attenuation of refractory dry eye secondary to chronic graft-versus-host-disease .
Within the CD4 population we were able to confirm the evident anti-inflammatory properties of hAMSC. Indeed our results showed that CM-hAMSC induced the inhibition of Th1 (Tbet+CD183+) and Th17 (RORγt+CD161+) subset proliferation, and down-regulated pro-inflammatory Th1 cytokines IFN-γ, TNFα, and IL-1β, and Th17 cytokines such as IL-17A and IL-22. Even though CM-hAMSC did not influence Th2 (GATA3+/CD193+ or GATA3+/CD294+) cell expansion, the release of Th2 cytokines, such as IL-5 and IL-6, was significantly reduced in the presence of CM-hAMSC.
Treg, a subpopulation of CD4+ T cells commonly identified by the expression of Forkhead box P3 (FoxP3) transcription factor, are key players in the mechanisms that are evoked to control the immune response. The two main subsets of Treg are natural Treg, which are thymus-derived and specific for self-antigens, and adaptive/induced Treg which can be generated from Naïve CD4+ T cells in peripheral lymphoid tissues following inflammatory stimuli [52, 53]. Both bone marrow [37, 54, 55] and adipose tissue [56, 57]-derived MSC have been extensively studied for their capacity to induce Treg induction.
The major cytokines responsible for inducing the differentiation of iTregs are IL-2 and TGF-β . In presence of CM-hAMSC, we observed an increase of IL-2 and soluble form of CD25 (sCD25 or sIL-2R), and also of TGF-β. These observations further support T cell differentiation toward the Treg phenotype in the presence of CM-hAMSC. Tregs are able to secrete anti-inflammatory cytokines such as TGF-β, IL-10, IL-13 [59, 60] and these cytokines are known to be critical factors involved in the suppression of the pro-inflammatory cytokine response. Indeed, we observed an increase of the production of TGF-β and IL-13 during the co-culture of allogeneic activated T cells with CM-hAMSC. In addition to CD4+CD25+FoxP3+ Tregs, we also observed an increase in Tregs expressing CD39, suggesting that the adenosynergic pathway, which has functional relevance for cellular immunoregulation [61, 62, 63], could also be involved in the immunomodulatory functions exerted by CM-hAMSC.
Furthermore, in co-cultures with CM-hAMSC, CD4+CD25+FoxP3+ and CD4+CD39+FoxP3+ Tregs showed an increase in percentage of cells positive for Cytolytic T lymphocyte-associated antigen (CTLA)-4 and Glycoprotein A Repetitions Predominant (GARP), which have been shown to selectively identify activated human FoxP3+ regulatory T cells . CTLA-4 has been shown to participate in Treg-mediated suppression by inhibition of dendritic cell (DC)-mediated T-cell stimulation [65, 66]. GITR appears to control DC and monocyte development and in its absence, mice develop aggravated chronic enterocolitis via an imbalance of colitogenic Th1 cells and Treg cells . Taken together, these data strongly demonstrate that CM-hAMSC not only induces upregulation of the Treg population, but also induces Treg functions as shown by the altered activation of specific surface molecules that could contribute to the control of the immune suppression.
Interestingly, we demonstrated up-regulation of Treg in the culture of allogeneic activated T cells in the presence of conditioned medium derived from unstimulated hAMSC culture. This is in line with what we previously observed for CM on total T cell populations , and our current observation regarding CD4 and CD8 subpopulations, as well as for Th1 or Th17 subsets. These findings also support the in vivo data showing upregulation of Treg in autoimmune disorders, such as that seen with PBMC from patients with rheumatoid arthritis after addition of either hAMSC or CM-hAMSC . This is in contrast to MSC derived from bone marrow, whereby stimulation with inflammatory cytokines, such as TNFα or IFNγ, are required in order to have immune regulatory effects and specifically induce Treg [68, 69].
In conclusion, this study provides new insights regarding the immune-modulating mechanism of hAMSC associated to the therapeutical effect observed in pre-clinical in vivo models and hypothesized to constitute the basis for their clinical application. Altogether, these results reinforce the potential use of these cells, and in particular their conditioned medium, which could constitute a cell-free treatment in diseases correlated to an altered inflammatory response.
The authors 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 and the volunteers who donated blood. The authors also wish to thank the personnel of Department of Radiation Oncology of Fondazione Poliambulanza-Istituto Ospedaliero (Brescia, Italy) for their assistance with cell irradiation.
This work was supported by Competitiveness ROP ERDF 2007–2013 of Lombardy Region (Regional Operational Programme of the European Regional Development Fund – Progetto NUTEC NUove TECnologie ID n. 30263049) and Fondazione Cariplo, Grant n° 2011–0495
Conflict of Interest
The authors declare no conflicts of interest.
- 1.Cetrulo, K. J., Cetrulo, C. L., Jr., & Taghizadeh, R. R. (Eds.). (2013). Perinatal stem cells. Hoboken: Wiley.Google Scholar
- 2.Anzalone, R., Iacono Lo, M., Loria, T., Di Stefano, A., Giannuzzi, P., Farina, F., & La Rocca, G. (2011). Wharton’s jelly mesenchymal stem cells as candidates for beta cells regeneration: extending the differentiative and immunomodulatory benefits of adult mesenchymal stem cells for the treatment of type 1 diabetes. Stem Cell Reviews and Reports, 7(2), 342–363. doi: 10.1007/s12015-010-9196-4.CrossRefPubMedGoogle Scholar
- 3.La Rocca, G., Corrao, S., Iacono Lo, M., Corsello, T., Farina, F., & Anzalone, R. (2012). Novel immunomodulatory markers expressed by human WJ-MSC: an updated review in regenerative and reparative medicine. The Open Tissue Engineering and Regenerative Medicine Journal, 5, 50–58.CrossRefGoogle Scholar
- 4.La Rocca, G., Iacono Lo, M., Corsello, T., Corrao, S., Farina, F., & Anzalone, R. (2013). Human Wharton’s jelly mesenchymal stem cells maintain the expression of key immunomodulatory molecules when subjected to osteogenic, adipogenic and chondrogenic differentiation in vitro: new perspectives for cellular therapy. Current Stem Cell Research & Therapy, 8(1), 100–113.CrossRefGoogle Scholar
- 6.Parolini, O., Souza-Moreira, L., O’Valle, F., Magatti, M., Hernandez-Cortes, P., Gonzalez-Rey, E., & Delgado, M. (2014). Therapeutic effect of human amniotic membrane-derived cells on experimental arthritis and other inflammatory disorders. Arthritis & Rheumatology, 66(2), 327–339. doi: 10.1002/art.38206.CrossRefGoogle Scholar
- 7.Parolini, O., Alviano, F., Bergwerf, I., Boraschi, D., De Bari, C., De Waele, P., et al. (2010, February). Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table. Stem Cells and Development, 19(2), 143–154. doi: 10.1089/scd.2009.0404.
- 10.Parolini, O., Alviano, F., Bagnara, G. P., Bilic, G., Bühring, H.-J., Evangelista, M., et al. (2008). Concise review: isolation and characterization of cells from human term placenta: outcome of the first international workshop on placenta derived stem cells. Stem Cells, 26(2), 300–311. doi: 10.1634/stemcells.2007-0594.CrossRefPubMedGoogle Scholar
- 15.Wolbank, S., Peterbauer, A., Fahrner, M., Hennerbichler, S., van Griensven, M., Stadler, G., et al. (2007). Dose-dependent immunomodulatory effect of human stem cells from amniotic membrane: a comparison with human mesenchymal stem cells from adipose tissue. Tissue Engineering, 13(6), 1173–1183. doi: 10.1089/ten.2006.0313.CrossRefPubMedGoogle Scholar
- 17.Manochantr, S., U-pratya, Y., Kheolamai, P., Rojphisan, S., Chayosumrit, M., Tantrawatpan, C., et al. (2013). Immunosuppressive properties of mesenchymal stromal cells derived from amnion, placenta, Wharton’s jelly and umbilical cord. Internal Medicine Journal, 43(4), 430–439. doi: 10.1111/imj.12044.CrossRefPubMedGoogle Scholar
- 18.Magatti, M., De Munari, S., Vertua, E., Nassauto, C., Albertini, A., Wengler, G. S., & Parolini, O. (2009). Amniotic mesenchymal tissue cells inhibit dendritic cell differentiation of peripheral blood and amnion resident monocytes. Cell Transplantation, 18(8), 899–914. doi: 10.3727/096368909X471314.CrossRefPubMedGoogle Scholar
- 20.Cargnoni, A., Gibelli, L., Tosini, A., Signoroni, P. B., Nassuato, C., Arienti, D., et al. (2009). Transplantation of allogeneic and xenogeneic placenta-derived cells reduces bleomycin-induced lung fibrosis. Cell Transplantation, 18(4), 405–422. doi: 10.3727/096368909788809857.CrossRefPubMedGoogle Scholar
- 21.Ohshima, M., Yamahara, K., Ishikane, S., Harada, K., Tsuda, H., Otani, K., et al. (2012). Systemic transplantation of allogenic fetal membrane-derived mesenchymal stem cells suppresses Th1 and Th17 T cell responses in experimental autoimmune myocarditis. Journal of Molecular and Cellular Cardiology, 53(3), 420–428. doi: 10.1016/j.yjmcc.2012.06.020.CrossRefPubMedGoogle Scholar
- 23.Ricci, E., Vanosi, G., Lindenmair, A., Hennerbichler, S., Peterbauer-Scherb, A., Wolbank, S., et al. (2013). Anti-fibrotic effects of fresh and cryopreserved human amniotic membrane in a rat liver fibrosis model. Cell and Tissue Banking, 14(3), 475–488. doi: 10.1007/s10561-012-9337-x.CrossRefPubMedGoogle Scholar
- 25.Cargnoni, A., Ressel, L., Rossi, D., Poli, A., Arienti, D., Lombardi, G., & Parolini, O. (2012). Conditioned medium from amniotic mesenchymal tissue cells reduces progression of bleomycin-induced lung fibrosis. Cytotherapy, 14(2), 153–161. doi: 10.3109/14653249.2011.613930.CrossRefPubMedCentralPubMedGoogle Scholar
- 26.Cargnoni, A., Piccinelli, E. C., Ressel, L., Rossi, D., Magatti, M., Toschi, I., et al. (2014). Conditioned medium from amniotic membrane-derived cells prevents lung fibrosis and preserves blood gas exchanges in bleomycin-injured mice-specificity of the effects and insights into possible mechanisms. Cytotherapy, 16(1), 17–32. doi: 10.1016/j.jcyt.2013.07.002.CrossRefPubMedGoogle Scholar
- 27.Rossi, D., Pianta, S., Magatti, M., Sedlmayr, P., & Parolini, O. (2012). Characterization of the conditioned medium from amniotic membrane cells: prostaglandins as Key effectors of its immunomodulatory activity. PLoS ONE, 7(10), e46956–14. doi: 10.1371/journal.pone.0046956.
- 28.Magatti, M., Caruso, M., De Munari, S., Vertua, E., De, D., Manuelpillai, U., & Parolini, O. (2014). Human amniotic membrane-derived mesenchymal and epithelial cells exert different effects on monocyte-derived dendritic cell differentiation and function. Cell Transplantation. doi: 10.3727/096368914X684033.
- 29.Abumaree, M. H., Jumah, M. A., Kalionis, B., Jawdat, D., Khaldi, A., Abomaray, F. M., et al. (2013). Human placental mesenchymal stem cells (pMSCs) play a role as immune suppressive cells by shifting macrophage differentiation from inflammatory M1 to anti-inflammatory M2 macrophages. Stem Cell Reviews and Reports, 9(5), 620–641. doi: 10.1007/s12015-013-9455-2.
- 30.Singer, N. G., & Caplan, A. I. (2011). Mesenchymal stem cells: mechanisms of inflammation. Annual Review of Pathology, 6(1), 457–478. doi: 10.1146/annurev-pathol-011110-130230.
- 31.Law, S., & Chaudhuri, S. (2013). Mesenchymal stem cell and regenerative medicine: regeneration versus immunomodulatory challenges. American Journal of Stem Cells, 2(1), 22–38.Google Scholar
- 32.Lim, J. H., Kim, J. S., Yoon, I. H., Shin, J. S., Nam, H. Y., Yang, S. H., et al. (2010). Immunomodulation of delayed-type hypersensitivity responses by mesenchymal stem cells is associated with bystander T cell apoptosis in the draining lymph node. The Journal of Immunology, 185(7), 4022–4029. doi: 10.4049/jimmunol.0902723.
- 33.González, M. A., Gonzalez-Rey, E., Rico, L., Büscher, D., & Delgado, M. (2009). Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology, 136(3), 978–989. doi: 10.1053/j.gastro.2008.11.041.CrossRefPubMedGoogle Scholar
- 34.Boumaza, I., Srinivasan, S., Witt, W. T., Feghali-Bostwick, C., Dai, Y., Garcia-Ocana, A., & Feili-Hariri, M. (2009). Autologous bone marrow-derived rat mesenchymal stem cells promote PDX-1 and insulin expression in the islets, alter T cell cytokine pattern and preserve regulatory T cells in the periphery and induce sustained normoglycemia. Journal of Autoimmunity, 32(1), 33–42. doi: 10.1016/j.jaut.2008.10.004.CrossRefPubMedGoogle Scholar
- 35.Sallusto, F., Geginat, J., & Lanzavecchia, A. (2004). Central memory and effector memory T cell subsets: function, generation, and maintenance. Annual Review of Immunology, 22, 745–763. doi: 10.1146/annurev.immunol.22.012703.104702
- 36.Filaci, G., Fenoglio, D., Fravega, M., Ansaldo, G., Borgonovo, G., Traverso, P., et al. (2007). CD8+CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers. Journal of Immunology, 179(7), 4323–4334. doi: 10.4049/jimmunol.179.7.4323.CrossRefGoogle Scholar
- 41.Kronsteiner, B., Peterbauer-Scherb, A., Grillari-Voglauer, R., Redl, H., Gabriel, C., van Griensven, M., & Wolbank, S. (2011). Human mesenchymal stem cells and renal tubular epithelial cells differentially influence monocyte-derived dendritic cell differentiation and maturation. Cellular Immunology, 267(1), 30–38. doi: 10.1016/j.cellimm.2010.11.001.CrossRefPubMedGoogle Scholar
- 43.Sivanathan, K. N., Gronthos, S., Rojas-Canales, D., Thierry, B., & Coates, P. T. (2014). Interferon-gamma modification of mesenchymal stem cells: implications of autologous and allogeneic mesenchymal stem cell therapy in allotransplantation. Stem Cell Reviews and Reports, 10(3), 351–375. doi: 10.1007/s12015-014-9495-2
- 44.Krampera, M., Glennie, S., Dyson, J., Scott, D., Laylor, R., Simpson, E., & Dazzi, F. (2003). Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood, 101(9), 3722–3729. doi: 10.1182/blood-2002-07-2104.
- 49.Filaci, G., Fravega, M., Negrini, S., Procopio, F., Fenoglio, D., Rizzi, M., et al. (2004). Nonantigen specific CD8+ T suppressor lymphocytes originate from CD8+CD28- T cells and inhibit both T-cell proliferation and CTL function. Human Immunology, 65(2), 142–156. doi: 10.1016/j.humimm.2003.12.001.CrossRefPubMedGoogle Scholar
- 50.Engela, A. U., Baan, C. C., Litjens, N. H. R., Franquesa, M., Betjes, M. G. H., Weimar, W., & Hoogduijn, M. J. (2013). Mesenchymal stem cells control alloreactive CD8 +CD28 −T cells. Clinical and Experimental Immunology, 174(3), 449–458. doi: 10.1111/cei.12199.CrossRefPubMedCentralPubMedGoogle Scholar
- 54.Ghannam, S., Pène, J., Moquet-Torcy, G., Torcy-Moquet, G., Jorgensen, C., & Yssel, H. (2010). Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. The Journal of Immunology, 185(1), 302–312. doi: 10.4049/jimmunol.0902007.CrossRefPubMedGoogle Scholar
- 55.Zuo, D., Liu, X., Shou, Z., Fan, H., Tang, Q., Duan, X., et al. (2013). Study on the interactions between transplanted bone marrow-derived mesenchymal stem cells and regulatory T cells for the treatment of experimental colitis. International Journal of Molecular Medicine, 32(6), 1337–1344. doi: 10.3892/ijmm.2013.1529.PubMedGoogle Scholar
- 56.Gonzalez-Rey, E., González, M. A., Varela, N., O’Valle, F., Hernandez-Cortes, P., Rico, L., et al. (2009). Human adipose-derived mesenchymal stem cells reduce inflammatory and T cell responses and induce regulatory T cells in vitro in rheumatoid arthritis. Annals of the Rheumatic Diseases, 69(01), 241–248. doi: 10.1136/ard.2008.101881.CrossRefGoogle Scholar
- 57.Ivanova-Todorova, E., Bochev, I., Dimitrov, R., Belemezova, K., Mourdjeva, M., Kyurkchiev, S., et al. (2012). Conditioned medium from adipose tissue-derived mesenchymal stem cells induces CD4+FOXP3+ cells and increases IL-10 secretion. Journal of Biomedicine and Biotechnology, 2012(1), 1–8. doi: 10.1111/j.1600-0897.2009.00707.x.CrossRefGoogle Scholar
- 59.Tiemessen, M. M., Jagger, A. L., Evans, H. G., van Herwijnen, M. J. C., John, S., & Taams, L. S. (2007). CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages. Proceedings of the National Academy of Sciences, 104(49), 19446–19451. doi: 10.1073/pnas.0706832104.CrossRefGoogle Scholar
- 60.Ochoa-Reparaz, J., Rynda, A., Ascon, M. A., Yang, X., Kochetkova, I., Riccardi, C., et al. (2008). IL-13 production by regulatory T cells protects against experimental autoimmune encephalomyelitis independently of autoantigen. Journal of Immunology, 181(2), 954–968. doi: 10.4049/jimmunol.181.2.954.CrossRefGoogle Scholar
- 61.Borsellino, G., Kleinewietfeld, M., Di Mitri, D., Sternjak, A., Diamantini, A., Giometto, R., et al. (2007). Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Blood, 110(4), 1225–1232. doi: 10.1182/blood-2006-12-064527.CrossRefPubMedGoogle Scholar
- 62.Moncrieffe, H., Nistala, K., Kamhieh, Y., Evans, J., Eddaoudi, A., Eaton, S., & Wedderburn, L. R. (2010). High expression of the ectonucleotidase CD39 on T cells from the inflamed site identifies two distinct populations, one regulatory and one memory T cell population. The Journal of Immunology, 185(1), 134–143. doi: 10.4049/jimmunol.0803474.CrossRefPubMedCentralPubMedGoogle Scholar
- 63.Dwyer, K. M., Hanidziar, D., Putheti, P., Hill, P. A., Pommey, S., McRae, J. L., et al. (2010). Expression of CD39 by human peripheral blood CD4+ CD25+ T cells denotes a regulatory memory phenotype. American Journal of Transplantation, 10(11), 2410–2420. doi: 10.1111/j.1600-6143.2010.03291.x.CrossRefPubMedCentralPubMedGoogle Scholar
- 65.Moseman, E. A., Liang, X., Dawson, A. J., Panoskaltsis-Mortari, A., Krieg, A. M., Liu, Y. J., et al. (2004). Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. Journal of Immunology, 173(7), 4433–4442. doi: 10.4049/jimmunol.173.7.4433.CrossRefGoogle Scholar
- 67.Liao, G., Detre, C., Berger, S. B., Engel, P., de Waal Malefyt, R., Herzog, R. W., et al. (2012). lucocorticoid-induced tumor necrosis factor receptor family-related protein regulates CD4(+)T cell-mediated colitis in mice. Gastroenterology, 142(3), 582–591. doi: 10.1053/j.gastro.2011.11.031.CrossRefPubMedCentralPubMedGoogle Scholar
- 69.Ren, G., Zhao, X., Zhang, L., Zhang, J., L’Huillier, A., Ling, W., et al. (2010). Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells Are critical for immunosuppression. Journal of Immunology, 184(5), 2321–2328. doi: 10.4049/jimmunol.0902023.CrossRefGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.