The main aim of this study was to investigate the biological activities and immune modulation changes of chorionic villi mesenchymal stem cells (CV-MSC) after long term culture. The morphology of the CV-MSC of passage 3 and passage 9 were observed by microscopy, and their phenotypes were detected by flow cytometry. CV-MSC of passage 3 and 9 were co-cultured with PHA-stimulated PBMNC, and IFN-γ concentration in culture medium was detected by ELISA. The mRNA expression of COX-2, HGF and HLA-G in CV-MSC were detected by real-time PCR. The results showed that after long term culture, the CV-MSC kept the MSC morphology and most of the phenotypes including CD31, CD34, CD44, CD45, CD62L, CD73, CD90, CD105, CD117, CD151, CD235a, CD271 and HLA-DR, while the CD49d was significantly up-regulated. Immune modulation ability of CV-MSC was reduced and the mRNA expression of COX-2 and HGF was down regulated after long term culture, but the expression of HLA-G did not found to be obvious change. It is concluded that the long term in vitro expansion changes the expression of CD49d and reduces immune modulation of CV-MSC.
Cells, Cultured ; Chorionic Villi ; immunology ; Female ; Humans ; Integrin alpha4 ; metabolism ; Mesenchymal Stromal Cells ; cytology ; immunology ; Monocytes ; cytology ; Placenta ; cytology ; Pregnancy

Mesenchymal stem cells (MSCs) are partially defined by their ability to differentiate into tissues including bone, cartilage and adipose in vitro, but it is their trophic, paracrine and immunomodulatory functions that may have the greatest therapeutic impact in vivo. Unlike pharmaceutical treatments that deliver a single agent at a specific dose, MSCs are site regulated and secrete bioactive factors and signals at variable concentrations in response to local microenvironmental cues. Significant progress has been made in understanding the biochemical and metabolic mechanisms and feedback associated with MSC response. The anti-inflammatory and immunomodulatory capacity of MSC may be paramount in the restoration of localized or systemic conditions for normal healing and tissue regeneration. Allogeneic MSC treatments, categorized as a drug by regulatory agencies, have been widely pursued, but new studies demonstrate the efficacy of autologous MSC therapies, even for individuals affected by a disease state. Safety and regulatory concerns surrounding allogeneic cell preparations make autologous and minimally manipulated cell therapies an attractive option for many regenerative, anti-inflammatory and autoimmune applications.
Animals ; *Cellular Microenvironment ; Humans ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/cytology/immunology/*metabolism ; Regenerative Medicine/*methods

This study was purposed to investigate the immunoregulatory effect of endothelial cells derived from mesenchymal stem cells (MSC). The human MSC was induced to differentiate into endothelial cells for one week. The phenotypes were evaluated by flow cytometry, the cell morphologic feature was observed by invert phase-contrast microscope and analysis of capillary formation was performed by using the in vitro angiogenesis kit. The immunoregulatory effect was detected by lymphocyte transformation test. The result indicated that during the differentiation cells grew fast and there was no significant change in the phenotypes, i.e. CD73, CD105, HLA-ABC were positive and CD34, CD80, CD86, HLA-DR, CD31 were negative. Immunofluorescence analysis showed typical expression of the von Willebrand factor. Differentiated MSCs formed capillary-like structure. Endothelial cells derived from MSC also revealed immunosuppressive effect on T cell proliferation in a dose-dependent manner. It is concluded that endothelial cells derived from MSC also harbor immunoregulatory effect on T lymphocytes.
5'-Nucleotidase ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Child ; Endothelial Cells ; cytology ; immunology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; T-Lymphocytes ; immunology ; von Willebrand Factor ; metabolism

The objective of this study was to investigate the effect of human bone marrow mesenchymal stem cells (MSCs) on T lymphocyte killing K562 cells. MSCs were isolated from bone marrow and cultured, T cells were harvested by using nylon column method from peripheral blood. The T cells were co-cultured with MSCs, the phenotype expressions of T cell subsets were detected by flow cytometry. Killing effects of T cells (culture alone and co-culture with MSCs) on K562 cells were detected by LDH, expressions of IFN-gamma and IL-4 were detected by ELISA. The results showed that after T cells were co-cultured with MSCs for three days, the proportion of CD4+ and CD4+CD25+ T cells raised significantly (p<0.05) as compared with group of culture alone, but the proportion of CD8+ T cell were not significantly changed (p>0.05). In group of T cells co-cultured with MSCs, killing effects of T cells on K562 cells weakened, at the same time, expression of IFN-gamma decreased while expression of IL-4 increased. It is concluded that the MSCs weaken killing effects of T cells on K562 cells, which associates with increase of CD4+CD25+ T cell subsets and changes of IFN-gamma and IL-4 levels.
Bone Marrow Cells ; cytology ; CD4 Antigens ; immunology ; Coculture Techniques ; Humans ; Interferon-gamma ; metabolism ; Interleukin-2 Receptor alpha Subunit ; immunology ; Interleukin-4 ; metabolism ; K562 Cells ; Mesenchymal Stromal Cells ; cytology ; immunology ; T-Lymphocyte Subsets ; immunology ; metabolism ; T-Lymphocytes ; cytology ; immunology

This study was aimed to find a better method to isolate and enrich mesenchymal stem cells (MSCs)from bone marrow between CD271 (low affinity nerve growth factor receptor, LNGFR) and CD133 used for immunomagnetic positive selections through comparison of characteristics of MSCs isolated by these two agents. CD271+ and CD133+ cells were isolated from bone marrow and their colony forming unit-fibroblast (CFU-F) efficiency and proliferative capacity were assessed. Cell surface phenotype, adipogenic and osteogenic inductions were also assayed on the cells (after passage 3) isolated by both methods. The results showed that the purities of immunomagnetically selected CD271+ and CD133+ cells were (89.50+/-0.98)% and (88.03+/-3.06)% respectively. The CFU-F median frequency of CD271+ cells was 3 times as high as that of CD133+ cells, no CFU-F was observed in CD271- cells, while a few CFU-F was found in the CD133- cells. Phenotype of cells (after passage 3) isolated by the two methods was same, that is CD34-, CD14-, CD45-, CD90+, CD29+, CD44+, CD105+, CD73+. CD271+ cells possessed faster proliferation and stronger osteogenic and adipogenic differentiation potential than that of CD133+ cells. It is concluded that as compared with CD133 positive selection, CD271 positive selection is a better method for isolating and enriching mesenchymal stem cells from bone marrow.
AC133 Antigen ; Antigens, CD ; immunology ; metabolism ; Bone Marrow Cells ; cytology ; Glycoproteins ; immunology ; metabolism ; Humans ; Immunomagnetic Separation ; methods ; Mesenchymal Stromal Cells ; cytology ; Nerve Tissue Proteins ; immunology ; metabolism ; Peptides ; immunology ; metabolism ; Receptors, Nerve Growth Factor ; immunology ; metabolism ; Stem Cells

The aim of this study was to investigate the effects of interferon (IFN)-γ on biological characteristics and immunomodulatory property of human umbilical cord-derived mesenchymal stem cells (hUC-MSC). hUC-MSC were treated with IFN-γ 10 ng/ml (IFN-γ group) or without IFN-γ (control group). The phenotype of hUC-MSC was detected by flow cytometry. The proliferation status was detected by CCK-8 method, and its differentiation ability was assessed by oil red O and von Kossa staining. The production of PGE-2 was measured by ELISA, and the mRNA expression levels of COX-2, IDO-1 and IDO-2 in hUC-MSC were detected by real-time quantitative PCR. Furthermore, the proliferation of human peripheral blood mononuclear cells (hPBMNC) was evaluated after co-culture with hUC-MSC, IFN-γ pretreatment or not. The results showed that after IFN-γ stimulation, the expression of SSEA-4 on hUC-MSC decreased significantly [(8.15 ± 2.94) vs (16.42 ± 8.5), P < 0.05], and the expression of CD54 increased [(96.64 ± 3.29) vs (84.12 ± 10.73), P = 0.051]. The immunomodulatory property of hUC-MSC on the proliferation of hPBMNC was enhanced (P < 0.05). All the above mentioned effects were IFN-γ concentration-dependent. When hUC-MSC were stimulated by IFN-γ for 24 h, the production of PGE-2 secreted by hUC-MSC decreased significantly (P < 0.01). The mRNA expression level of COX-2 also decreased though the difference did not reach to statistically significant level. Compared with control group, IDO-1 expression level in IFN-γ group increased significantly (P < 0.01), and the mRNA expression level of IDO-2 remained unchanged. It is concluded that IFN-γ can influence the phenotype of hUC-MSC and enhance the immunomodulatory property of hUC-MSC.
Cells, Cultured ; Cyclooxygenase 2 ; metabolism ; Flow Cytometry ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology ; Umbilical Cord ; cytology

This study was purposed to explore the effect of bone marrow derived mesenchymal stem cells (MSCs) on the expression of CD69 on cytokine-induced killer (CIK)/natural killer (NK) cells derived from cord blood and on the quantity ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture system using Transwell non-contact cell culture system. The experiments were divided into two groups: Transwell non-contact culture and mixture culture. The ratio of MSC to CIK/ NK cells was 1:20, 1:50 and 1:100. In mixture culture groups, MSC and CIK/NK cells were co-cultured by together contact as the same ratio of Transwell non-contact culture groups. The expression of CD69 on CIK/NK cells, as well as the quantity ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture were evaluated by flow cytometry. The results showed that the expression of CD69 on CIK/NK cells in experimental groups were significantly lower than that in control group (p<0.001). As to Transwell groups, CD69 expression on the CIK/NK cells at 1:20 ratio of MSC and CIK/NK was significantly lower than that at 1:50 and 1:100 ratio. There were no differences in the expression of CD69 on CIK cells in mixture groups with various MSC ratios, whereas the expression of CD69 on NK cells at 1:20 ratio was significantly lower than that at 1:50 and 1:100. The quantity ratio of CD4+CD25+ cells in CIK/NK cell culture system of experimental groups with MSC co-culture was significantly higher than that in control. As to Transwell groups, the ratio of CD4+CD25+ cells in CIK/NK cell culture system at 1:20 and 1:50 was significantly higher than that at 1:100. The quantity ratio of CD4+CD25+ cells in CIK/NK cell culture system showed significant differences in various mixture groups. As to 1:20 ratio the amount of CD4+CD25+ cells in CIK/NK cell culture system of mixture groups was significantly higher than that in Transwell groups, while there were no differences of the quantity ratio of CD4+CD25+ cells in CIK/NK cell culture at 1:50 and 1:100. It is concluded that either by non-contact Transwell or mixed co-culture, the MSC can suppress the activation of allogeneic CB-CIK/NK cells, which maybe relate to up-regulating the ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture system in dose-dependent manner.
Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Cell Culture Techniques ; Cells, Cultured ; Cytokine-Induced Killer Cells ; immunology ; metabolism ; Fetal Blood ; cytology ; immunology ; Humans ; Lectins, C-Type ; metabolism ; Mesenchymal Stromal Cells ; cytology ; T-Lymphocytes, Regulatory ; cytology ; metabolism

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

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

The study was purposed to investigate the immune regulatory effects of human bone marrow mesenchymal stem cells (hMSCs) on Foxp3 expressing CD4(+)CD25(+) regulatory T cells and to explore the mechanism of immune modulation by hMSCs. Human MSCs were isolated and expanded from bone marrow cells, and identified with cell morphology, and the phenotypes were assessed by immunohistochemistry. Human peripheral blood mononuclear cells (hPBMNCs) were prepared by centrifugation on a Ficoll Hypaque density gradient. The hMSCs (1 x 10(3), 1 x 10(4), 1 x 10(5)) were added into wells containing hPBMNCs (1 x 10(6)) from an unrelated donor in the presence of rhIL-2. After 5 days of co-culture, the percentage of CD4(+)CD25(+) T cells was detected by flow cytometry. T cell proliferation was assessed by [(3)H] thymidine incorporation using a liquid scintillation counter. The expression of Foxp3 in CD4(+)CD25(+) T cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Cytokines (TGF-beta, IL-12, IFN-gamma, IL-10) concertrations of cultured supernatants were measured with ELISA. The results indicated that in all the experiments, the presence of hMSCs with hPBMNCs resulted in a statistically significant decrease in T cell proliferation, in dose-dependent manner. The increase of percentage of CD4(+)CD25(+) T cells in the peripheral CD4(+) T cell was observed after coculturing lymphocytes with hMSCs (p < 0.01). The expression of Foxp3-mRNA (Foxp3/beta-actin) in hMSCs groups was significantly higher than that in the control and was negatively associated with the value of CPM representing T proliferation. The levels of TGF-beta and IL-10 were higher in hMSCs groups than that in the control, and the levels of TGF-beta and IL-10 correlated positively with Foxp3-mRNA expression and the percentage of CD4(+)CD25(+) T cells. However, the secretion of IL-12 and IFN-gamma was significantly attenuated by hMSCs coculture, and there was no correlation with Foxp3-mRNA expression and the percentage of CD4(+)CD25(+) T cells. It is concluded that the Foxp3 expressing regulatory T cells may play an important role in the immune regulatory by hMSCs. Its mechanism is related to that the hMSCs-mediated TGF-beta and IL-10 convert CD4(+)CD25(-) T cells into CD4(+)CD25(+) T regulatory T cells, which specifically inhibits the proliferation of T cells.
Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Forkhead Transcription Factors ; metabolism ; Humans ; Immunization ; Interleukin-10 ; biosynthesis ; Interleukin-2 Receptor alpha Subunit ; immunology ; metabolism ; Mesenchymal Stromal Cells ; cytology ; T-Lymphocytes, Regulatory ; immunology ; metabolism ; Transforming Growth Factor beta ; biosynthesis