OBJECTIVETo investigate the immunological characteristics of human tonsil mesenchymal stem cells (TMSCs).

METHODSHuman tonsil tissues were obtained from the children patients with chronic tonsillitis. TMSCs were separated, cultured, and were detected the expression profiles of HLA-I, HLA-II, CD80, CD86 by flow cytometry. The measurement of immunogenicity, the effect on phytohemagglutinin (PHA) induced peripheral blood mononuclear cell (PBMCs) proliferation and mixed lymphocytes reaction (MLR) were performed to identify the immunological characteristics of TMSCs. The co-cultures of TMSCs + PBMCs + PHA and TMSCs + MLR were established, respectively, and the concentration of kynurenine, which is the metabolin of indoleamine 2, 3-dioxygenase, in the culture supernatant were examined. Then we added 1-methyl-L-tryptophan into the co-culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, and tested the proliferation of PBMCs. Each experiment was repeated three times, and there were six samples in each group. Statistical significance was assessed by analysis of variance (ANOVA), and a P value less than 0.05 was considered statistically significant.

RESULTSTMSCs expressed HLA-I, were negative for HLA-II and co-stimulatory molecules CD80 and CD86. The stimulation index in the group of TMSCs + allogeneic PBMCs was 1.38 ± 0.26, whereas the stimulation index in the group of allogeneic PBMCs was 1.22 ± 0.28, and there was no significant difference between the two groups (P > 0.05), indicating that TMSCs could not initiate the proliferation of allogeneic PBMCs. The stimulation indexes in the group of TMSCs + allogeneic PBMCs + PHA were 1.49 ± 0.29 and 1.23 ± 0.22, respectively, whereas the stimulation index in the group of allogeneic PBMCs + PHA was 4.60 ± 0.81, and the difference between the two groups had a statistical significance (P < 0.05) suggesting that TMSCs could inhibit PHA-induced PBMCs proliferation. The stimulation indexes in the group of TMSCs + MLR were 1.29 ± 0.23 and 1.26 ± 0.27, respectively, however, the stimulation index in the group of MLR was 3.04 ± 0.66, and the difference between the two groups had a statistical significance (P < 0.05), demonstrating that TMSCs could suppress MLR-induced PBMCs proliferation. The levels of kynurenine were (26.0 ± 2.3) μmol/L and (23.5 ± 4.5) μmol/L in the culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, thus elevating significantly. After adding of 1-methyl-L-tryptophan, TMSCs-mediated-proliferation suppression of PBMCs restored to normal levels.

CONCLUSIONTMSCs possess low immunogenecity and immunosuppressive function, may be used in allogeneic transplantation.

Cell Proliferation ; Cells, Cultured ; Child ; Coculture Techniques ; Flow Cytometry ; Humans ; Immunosuppression ; Kynurenine ; analysis ; Leukocytes, Mononuclear ; Lymphocyte Culture Test, Mixed ; methods ; Mesenchymal Stromal Cells ; cytology ; immunology ; Palatine Tonsil ; cytology ; Tryptophan ; administration & dosage ; analogs & derivatives

Mesenchymal stem cells (MSCs) have been widely studied for their applications in stem cell-based regeneration. During myocardial infarction (MI), infiltrated macrophages have pivotal roles in inflammation, angiogenesis and cardiac remodeling. We hypothesized that MSCs may modulate the immunologic environment to accelerate regeneration. This study was designed to assess the functional relationship between the macrophage phenotype and MSCs. MSCs isolated from bone marrow and bone marrow-derived macrophages (BMDMs) underwent differentiation induced by macrophage colony-stimulating factor. To determine the macrophage phenotype, classical M1 markers and alternative M2 markers were analyzed with or without co-culturing with MSCs in a transwell system. For animal studies, MI was induced by the ligation of the rat coronary artery. MSCs were injected within the infarct myocardium, and we analyzed the phenotype of the infiltrated macrophages by immunostaining. In the MSC-injected myocardium, the macrophages adjacent to the MSCs showed strong expression of arginase-1 (Arg1), an M2 marker. In BMDMs co-cultured with MSCs, the M1 markers such as interleukin-6 (IL-6), IL-1beta, monocyte chemoattractant protein-1 and inducible nitric oxide synthase (iNOS) were significantly reduced. In contrast, the M2 markers such as IL-10, IL-4, CD206 and Arg1 were markedly increased by co-culturing with MSCs. Specifically, the ratio of iNOS to Arg1 in BMDMs was notably downregulated by co-culturing with MSCs. These results suggest that the preferential shift of the macrophage phenotype from M1 to M2 may be related to the immune-modulating characteristics of MSCs that contribute to cardiac repair.
Animals ; Biomarkers/metabolism ; *Cell Differentiation ; Cells, Cultured ; Coculture Techniques ; Culture Media, Conditioned/pharmacology ; Humans ; *Macrophage Activation ; Macrophage Colony-Stimulating Factor/*pharmacology ; Macrophages/drug effects/*immunology/metabolism ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology/drug effects/metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Myocardial Infarction/surgery ; Rats

This study was objective to explore the effect of IFN-γ on immunosuppressive capability of mesenchymal stem cells (MSC) derived from umbilical cord. The immunomodulating capability of MSC was changed by stimulating cell surface receptors like Toll-like receptors (TLR). The inhibition of T-lymphocyte proliferation by MSC was tested via cell co-cultures. Further RT-PCR and ELISA were performed to examine the expression changes in gene and protein level. The results showed that the IFN-γ could promote the immunosuppressive effect of umbilical cord derived MSC. IFN-γ-stimulated MSC could suppress the proliferation of T cells more effectively. IFN-γ stimulation up-regulated the expression of immunosuppressive genes like IDO1, COX2, HLA-G, and soluble suppressive proteins such as HLA-G, KYN, IL10, PGE2 of MSC. And the immuno suppression capability of IFN-γ-stimulated MSC was 2-7 folds higher than control in MSC and lymphocyte co-culture tests. It is concluded that IFN-γ can effectively enhance the immunosuppressive capability of MSC.
Cells, Cultured ; Humans ; Immunosuppression ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology ; Umbilical Cord ; cytology

This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization.
Adult ; Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cells, Cultured ; Exosomes ; immunology ; metabolism ; Female ; Humans ; Interferon-gamma ; metabolism ; Leukocytes, Mononuclear ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice, Nude ; Middle Aged ; Neovascularization, Physiologic

This study was aimed to investigate the inhibitory mechanism of human amniotic mesenchymal stem cells (HAMSC) on lymphocyte proliferation and to validate the participation of the nonclassic human leukocyte antigen (HLA) class I molecule (HLA-G) in immunosuppressive action of HAMSC. HAMSC were isolated from fetal membranes of human placentas, and were cultured and expanded. The phenotypes of HAMSC were identified by flow cytometry, at same time the HLA-G levels on membrane surface and in cytoplasm were detected by flow cytometry. The soluble HLA-G (sHLA-G) level in HAMSC supernatants was determined by ELISA, MTT assay was used to examine the effect of mixed cultured HAMSC on proliferation of lymphocytes. The results showed that both surface and cytoplasm of HAMSC expressed HLA-G, the average rates of HLA-G expression on surface and in cytoplasm were (16.75 ± 3.871)% and (39.14 ± 4.274)%, respectively. The sHLA-G level in cell culture supernatant was 5.2 ng/ml. After HAMSC and culture supernatants were added in the MLR, the inhibitory rate on lymphocyte proliferation increased obviously, meanwhile the inhibitory rate on lymphocyte proliferation decreased when the HLA-G antibody was added in MLR. It is concluded that the surface and cytoplasm of HAMSC express HAL-G, at same time HAMSC secrete the HLA-G to supernatants of culture. The HLA-G is one of critical factors inhibiting immuno-function of HAMSC. This study contributes to improve the clinical therapeutic trails for using the HAMSC to prevent rejection.
Amnion ; cytology ; Cell Proliferation ; Cells, Cultured ; HLA-G Antigens ; immunology ; Humans ; Lymphocyte Activation ; Lymphocytes ; cytology ; immunology ; Mesenchymal Stromal Cells ; cytology ; immunology

OBJECTIVETo investigate the potential immunomodulatory properties of fetal bone marrow derived mesenchymal stem cells (FBM- MSCs).

METHODSMononuclear cells from the bone marrow of second trimester (14-22 wks) fetus were isolated and cultured for the derivation of MSCs. The derived FBM-MSC cells were characterized via morphology, immunophenotyping and the adipogenic and osteogenic differentiation assays. The immunomodulatory properties of FBM-MSC on lymphocytes were evaluated through the co- culture assay with PHA activated adult peripheral blood mononuclear cells (PBMCs).

RESULTSDerived FBM-MSCs were CD29⁺, CD44⁺, CD49e⁺, CD73⁺, CD90⁺, CD105⁺ and CD31⁻ , CD34⁻ , CD45⁻ , HLA-DR⁻ and can be differentiated into adipocytes and osteocytes. When co-cultured with PHA-activated PBMCs, FBM-MSCs inhibited the proliferation of lymphocytes up to 96% and down-regulated the secretion of inflammatory cytokines such as IFN-γ and TNF-α up to 90.9% and 58.4% respectively. When compared with FBM-MSCs cultured alone, the expression of MSCs derived immunomodulatory cytokines, such as IDO, TSG-6 and TGF-β, was up-regulated significantly in the co-culture system.

CONCLUSIONMSC derived from fetal bone marrow demonstrated immunosuppressive effects on adult PBMCs in vitro. MSC-derived cytokines like IDO, TSG-6 and TGF-β may be critical for FBM-MSCs mediated immunosuppressive function.

Adult ; Bone Marrow ; Bone Marrow Cells ; cytology ; immunology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Cytokines ; Hematopoietic Stem Cells ; Humans ; Immune Tolerance ; Immunophenotyping ; In Vitro Techniques ; Leukocytes, Mononuclear ; Lymphocytes ; Mesenchymal Stromal Cells ; cytology ; immunology ; Osteogenesis

This study was purposed to observe the influence of umbilical cord mesenchymal stem cells (UC-MSC) on the peripheral blood CD4(+)CD25(+)regulatory T cells (Treg), Th17 cells and neutrophils in rats with collagen type II-induced arthritis(CIA), and to explore the regulating effect of UC-MSC transplantation on immunocyte subgroup. The rats wee divided into 3 groups: CIA group (model group), UC-MSC treated group and blank control group. The CIA rats were injected with UC-MSC via tail vein. The percentage of CD4(+)CD25(+) cells in peripheral blood and the expression of NCD11b on neutrophil surface in CIA rates was detected by flow cytometry (FCM), and the serum interleukin-17 (IL-17) was observed by enzyme-linked immunosorbent assay (ELISA). The results showed that the mean fluorescence intensity(MFI) of NCD11b and the level of IL-17 in the model group were significantly higher than those in the blank control group, and the ratio of CD4(+)CD25(+) cells were significantly lower (P < 0.05). The MIF of NCD11b and the level of IL-17 in the UC-MSC treated group were significantly lower than that in the model group (P < 0.05), while the proportion of CD4(+)CD25(+) Treg increased (P < 0.05). Since the fifth week, the above indicators in the UC-MSC group have almostly approached the control group. It is concluded that the UC-MSC can increase peripheral blood Treg proportion in CIA rat, inhibit the secretion of Th17 and the activity of neutrophils, reduce the immune inflammation reaction, decrease the release of proinflammatory factor, and induce immune reconstruction.
Animals ; Arthritis, Experimental ; immunology ; therapy ; Female ; Interleukin-17 ; metabolism ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Neutrophils ; immunology ; Rats ; Rats, Sprague-Dawley ; Th17 Cells ; immunology ; Umbilical Cord ; cytology

OBJECTIVETo observe the effect of mesenchymal stem cells derived from umbilical cord (UC-MSCs) on natural killer (NK) cells-mediated cytotoxicity against dendritic cells (DCs) and explore the mechanism.

METHODSMSCs were isolated from human umbilical cord by collagen digestion and cultured in vitro. NK cells were separated from healthy human peripheral blood by magnetic bead sorting. Mononuclear cells from healthy human peripheral blood were cultured in the presence of granulocyte and macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to obtain the immature DCs. The DCs were then co-cultured with UC-MSCs in the presence of tumor necrosis factor α (TNFα) for 2 days, and the expressions of CD11c and CD86 on DCs and IL-12 level in the culture medium was detected using flow cytometry and ELISA, respectively. The cytotoxicity of NK cells against DCs was analyzed by LDH-releasing assay, and the expressions of ligands for killer activator receptor (MICA/B and ULBP1-3) on the DCs were detected with flow cytometry.

RESULTSCompared with the cytokine-induced DCs, the DCs induced by co-culture with UC-MSCs showed an identical CD11c expression but lowered CD86 expression and IL-12 secretion. The natural killer cells produced a stronger cytotoxicity against UC-MSCs-induced DCs than against cytokine-induced DCs. The UC-MSCs-induced DCs also showed increased expressions of MICA and MICB on the surface.

CONCLUSIONUC-MSCs can enhance NK cells-mediated cytotoxicity against DCs possibly by inhibiting DC maturation and up-regulating the ligands for killer activator receptor on the surface of the DCs.

Cells, Cultured ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; Humans ; Killer Cells, Natural ; cytology ; Mesenchymal Stromal Cells ; cytology ; Umbilical Cord ; cytology

OBJECTIVETo investigate mesenchymal stem cells (MSCs) immunosuppressive activity in the presence of interferon-gamma (IFN-γ) to reveal synergistic immunomodulatory effects of IFN-γ and MSCs.

METHODS① MSCs were cultured in the presence or absence of IFN-γ（100 ng/ml）, the supernatants were collected for measurements of PGE2、HGF and TGF-β1 by ELISA kits. ② MSCs were cultured in the presence or absence of IFN-γ （100 ng/ml）for 48 h. The cDNA was analysed for the expression of human indoleamine 2, 3-dioxygenase（IDO）mRNA by semiquantitative RT-PCR. ③ Mononuclear cells (MNCs) were extracted from peripheral blood of healthy donors. The T cell proliferation was tested in the co-culture system added with MSCs, recombinant human IFN-γ (100 ng/ml) and anti-IFN-γ mAb (5 μg/ml) by BrdU ELISA kit.

RESULTS①The immunosuppressive cytokines PGE2、HGF and TGF-β1 were detectable within 24-48 h in the supernatants. Their expressions were significantly up-regulated in the presence of IFN-γ. Concentrations of these cytokines were as of (1715.5±628.6) pg/ml vs (1344.5±709.4) pg/ml (P=0.001);(4031.8±1496.8) pg/ml vs (2452.4±1375.3) pg/ml（P=0.011）;(1753.5±413.8) pg/ml vs (1026.6±450.5) pg/ml（P<0.001）,respectively. ②The expression of IDO mRNA was undetectable when MSCs were cultured alone. In contrast, The IDO mRNA expression was remarkably enhanced in the presence of IFN-γ. ③Bone marrow-derived MSCs remarkably suppressed allogeneic T cell proliferation in vitro. Addition of exogenous IFN-γ had no significant effect on the inhibitory capacity of MSCs, the inhibitory ratios of T cell proliferation were （40.4±10.9）% vs（36.7±7.4）% (P=0.272). By contrast, the inhibitory ratio of T cell proliferation was significantly decreased in the presence of anti-IFN-γ mAb[(40.4±10.9)% vs (23.9±7.6)%,P=0.002].

CONCLUSION①Human MSCs constitutively expressed immunosuppressive concentrations of PGE2, HGF and TGF-β1, and their expressions were significantly up-regulated by IFN-γ. ②IFN-γ-induced expression of IDO on MSCs involved in tryptophan catabolism. ③MSCs notably suppressed allogeneic T cell proliferation in vitro. IFN-γ promoted the immunosuppressive capacity of human MSCs, indicating the synergistic immunomodulatory effect of IFN-γ and MSCs.

Bone Marrow Cells ; immunology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Cytokines ; immunology ; Humans ; Immune Tolerance ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; immunology ; T-Lymphocytes ; cytology

In the last 10 years, mesenchymal stem cells (MSCs) have emerged as a therapeutic approach to regenerative medicine, cancer, autoimmune diseases, and many more due to their potential to differentiate into various tissues, to repair damaged tissues and organs, and also for their immunomodulatory properties. Findings in vitro and in vivo have demonstrated immune regulatory function of MSCs and have facilitated their application in clinical trials, such as those of autoimmune diseases and chronic inflammatory diseases. There has been an increasing interest in the role of MSCs in allogeneic hematopoietic stem cell transplantation (HSCT), including hematopoietic stem cell engraftment and the prevention and treatment of graft-versus-host disease (GVHD), and their therapeutic potential has been reported in numerous clinical trials. Although the safety of clinical application of MSCs is established, further modifications to improve their efficacy are required. In this review, we summarize advances in the potential use of MSCs in HSCT. In addition, we discuss their use in clinical trials of the treatment of GVHD following HSCT, the immunomodulatory capacity of MSCs, and their regenerative and therapeutic potential in the field of HSCT.
Animals ; Chimerism ; Clinical Trials as Topic ; Graft vs Host Disease/immunology/therapy ; *Hematopoietic Stem Cell Transplantation ; Humans ; Immunomodulation ; Mesenchymal Stromal Cells/*cytology/immunology