Immunosuppressive properties of mesenchymal stem cells

Mesenchymal stem cells (MSC) can be isolated from different adult tissues including bone marrow, adipose tissue, cord blood and placenta. MSCs modulate the immune function of the major immune cell populations involved in alloantigen recognition and elimination, including antigen presenting cells, T cells, B cells and natural killer cells. Many clinical trials are currently underway that employ MSCs to treat human immunological diseases. However, the molecular mechanism that mediates the immunosuppressive effect of MSCs is still unclear and the safety of using MSC in patient needs further confirmation. Here, we review the cytokines that activate MSCs and the soluble factors produced by MSCs, which allow them to exert their immunosuppressive effects. We review the mechanism responsible, at least in part, for the immune suppressive effects of MSCs and highlight areas of research required for a better understanding of MSC immune modulation.     

Murine mesenchymal stem cells suppress dendritic cell migration, maturation and antigen presentation

Mesenchymal stem cells (MSC) possess a wide range of immunosuppressive functions. Among these is the ability to inhibit CD4+ T cell proliferation. Dendritic cells (DC) play a role in initiating cell-mediated immunity; however, the immunosuppressive influence of MSC on professional antigen presenting cells remains unclear. DC exposed to TNF-alpha and cultured with murine MSC failed to show regular upregulation of maturation markers. Similarly, the presence of MSC abrogated the capacity of ovalbumin-pulsed DC to support antigen specific CD4+ T cell proliferation, or for DC to display an MHC class II- peptide complex recognizable by specific antibody. Interestingly, culture of MSC with DC resulted in reduced expression of CCR7 by DC following stimulation. Likewise, DC matured in the presence of MSC, showed significantly less migration to CCL19. In contrast, murine MSC prevented loss of expression of the tissue anchoring protein E-cadherin by DC. Modulation of DC maturation and function was not permanent and could be restored after removal of MSC. These data demonstrate that MSC modulate the three cardinal features of DC maturation, providing the first demonstration of MSC interference with DC migration.     

Identification of cord blood-derived mesenchymal stem/stromal cell populations with distinct growth kinetics, differentiation potentials, and gene expression profiles

Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB1 and UCB2). The use of isogenic populations eliminated differences contributed by genetic background. We characterized these UCB MSCs for cell morphology, growth kinetics, immunophenotype, and potential for differentiation. UCB1 displayed faster growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. However, osteogenic differentiation was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic differences, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB1, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming growth factor-beta (TGF-beta) and wnt genes, and markers of early embryonic stages and mesodermal differentiation displayed significant differences among the MSC populations. In undifferentiated UCB1 cells, multiple genes were significantly up-regulated (p < 0.0001): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic differentiation capacities, hepatocyte growth factor (HGF), and stromal-derived factor 1 (SDF1/CXCL12), which could both potentially contribute to the higher growth kinetics observed in UCB1 cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage differentiation potentials, and helped clarify the heterogeneity observed in these cells.     

Embryonic Stem Cell Marker Expression Pattern in Human Mesenchymal Stem Cells Derived from Bone Marrow, Adipose Tissue, Heart and Dermis

Mesenchymal stem cells (MSCs) have been isolated from a variety of human tissues, e.g., bone marrow, adipose tissue, dermis, hair follicles, heart, liver, spleen, dental pulp. Due to their immunomodulatory and regenerative potential MSCs have shown promising results in preclinical and clinical studies for a variety of conditions, such as graft versus host disease (GvHD), Crohn’s disease, osteogenesis imperfecta, cartilage damage and myocardial infarction. MSC cultures are composed of heterogeneous cell populations. Complications in defining MSC arise from the fact that different laboratories have employed different tissue sources, extraction, and cultivation methods. Although cell-surface antigens of MSCs have been extensively explored, there is no conclusive evidence that unique stem cells markers are associated with these adult cells. Therefore the aim of this study was to examine expression of embryonic stem cell markers Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4 in adult mesenchymal stem cell populations derived from bone marrow, adipose tissue, dermis and heart. Furthermore, we tested whether human mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions. We found that bone marrow MSCs express embryonic stem cell markers Oct4, Nanog, alkaline phosphatase and SSEA-4, adipose tissue and dermis MSCs express Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4, whereas heart MSCs express Oct4, Nanog, SOX2 and SSEA-4. Our results also indicate that human adult mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions during early passages, as shown by distinct germ layer and embryonic stem cell marker expression patterns. Studies are now needed to determine the functional role of embryonic stem cell markers Oct4, Nanog and SOX2 in adult human MSCs.     

Isolation and expansion of synovial CD34(-)CD44(+)CD90(+) mesenchymal stem cells: comparison of an enzymatic method and a direct explant technique

Synovium-derived mesenchymal stem cells (MSCs) offer a promising therapeutic option for cartilage regeneration. The conventional method of MSC isolation involves single-cell suspensions using collagenases. Recently, a nonenzymatic explant technique was developed to isolate MSCs. We compared these techniques in the isolation of functional MSCs. MSCs were isolated from human fibrous and adipose synovium of osteoarthritic patients using explants or enzymatic methods. Total cell number, percentage of MSCs, and surface marker expression of MSCs were measured following expansion. Multipotentiality was determined using a MSC functional identification kit. MSCs isolated from fibrous or adipose synovium using these two techniques expressed similar levels of the surface markers CD44, CD90, and CD105, and displayed similar multipotentiality in generating adipocytes, osteoblasts, and chondrocytes. Total cell number and number of CD34(-)CD44(+)CD90(+) MSCs after 10-day expansion were similar in each culture, regardless of the source and method used, although the percentage of MSCs was slightly higher in explant cultures. There were no correlations between MSC yield and patient age, Hospital for Special Surgery score, and degree of deformity under all culture conditions. Both the enzymatic and explant techniques yielded similar yields of MSCs with similar characteristics. Because the explant technique is simpler and less invasive, it may be preferred over enzymatic techniques for isolating MSCs from the synovium of osteoarthritic patients for cartilage regeneration.     

FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (MSCs) exhibit limited in vitro growth. Fibroblast growth factors (FGFs) elicit a variety of biological responses, such as cell proliferation, differentiation and migration. FGF-4 represents one of the FGFs with the highest cell mitogenic activity. We studied the effect of FGF-4 on MSCs growth and pluripotency. MSCs duplication time (Td) was significantly reduced with FGF-4 compared to controls (2.2 +/- 0.2 vs. 4.1 +/- 0.2 days, respectively; p = 0.03) while BMP-2 and SCF-1 did not exert a significant growth effect. MSC expression of surface markers, differentiation into adipogenic and osteogenic lineages, and baseline expression of cardiomyogenic genes were unaffected by FGF-4. In summary, exogenous FGF-4 increases the rate at which MSC proliferate and has no significant effect on MSC pluripotency.     

Immune-Related Antigens, Surface Molecules and Regulatory Factors in Human-Derived Mesenchymal Stromal Cells: The Expression and Impact of Inflammatory Priming

Based on their ability to regulate immune responses, MSCs are considered to be potential candidates for managing immune-mediated diseases in the context of immune therapy. AT and WJ are considered valuable alternatives for BM as a source of MSCs. A detailed and comparative characterization of the immunological profile of MSCs derived from different sources, as well as an understanding of their responsiveness under certain circumstances, such as inflammation, is required to facilitate efficient and well-designed clinical studies. Flow cytometric analyses revealed clear differences among MSC types concerning the expression of the endothelial (e.g., CD31, CD34, CD144 and CD309) and stromal (e.g., CD90 and CD105) associated markers. Regardless of their source, MSCs did not express any of the known hematopoietic markers. All MSCs were uniformly positive for HLA-ABC and lacked the expression of HLA-DR and the co-stimulatory molecules (e.g., CD40, CD80, CD86, CD134 and CD252) required for full T-cell activation. Tissue-specific MSCs presented a modulated expression of cell adhesion molecules that is important for their cellular interactions. MSCs exhibited several surface (e.g., CD73, HLA-G, HO-1 and CD274) and soluble (e.g., HGF, PGE2 and IGFBP-3) immunoregulatory molecules. According to these immunological profiles, the present work provides evidence that the source from which MSCs are derived is important for the design of MSC-based immunointervention approaches. In light of these observations, we may suggest that WJ-MSCs appear to be the most attractive cell population to use in immune cellular therapy when immunosuppressive action is required.     

Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells defined by multilineage potential, ease to gene modification, and immunosuppressive ability, thus holding promise for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which, however, does not compensate for the very low percentage in their niches given the vast numbers of cells required for the relative studies. Taking into consideration the lack of a uniform approach for MSC isolation and expansion, we attempted in this study, by comparing various culture conditions, to identify the optimal protocol for the large-scale production of MSCs while maintaining their multilineage and immunosuppressive capacities. Our data indicate that, apart from the quality of fetal calf serum, other culture parameters, including basal medium, glucose concentration, stable glutamine, bone marrow mononuclear cell plating density, MSC passaging density, and plastic surface quality, affect the final outcome. Furthermore, the use of basic fibroblast growth factor (bFGF), the most common growth supplement in MSC culture media, greatly increases the proliferation rate but also upregulates HLA-class I and induces low HLA-DR expression. However, not only does this upregulation not elicit significant in vitro allogeneic T cell responses, but also bFGF-cultured MSCs exhibit enhanced in vivo immunosuppressive potential. Besides, addition of bFGF affects MSC multilineage differentiation capacity, favoring differentiation toward the osteogenic lineage and limiting neurogenic potential. In conclusion, in this report we define the optimal culture conditions for the successful isolation and expansion of human MSCs in high numbers for subsequent cellular therapeutic approaches.     

Mesenchymal stem cell therapy induces glucocorticoid synthesis in colonic mucosa and suppresses radiation-activated T cells: new insights into MSC immunomodulation

Non-neoplastic tissues around an abdomino-pelvic tumor can be damaged by the radiotherapy protocol, leading to chronic gastrointestinal complications that affect the quality of life with substantial mortality. Stem cell-based approaches using immunosuppressive bone marrow mesenchymal stem cells (MSCs) are promising cell therapy tools. In a rat model of radiation proctitis, we evidenced that a single MSC injection reduces colonic mucosa damages induced by ionizing radiation with improvement of the re-epithelization process for up to 21 days. Immune cell infiltrate and inflammatory molecule expressions in the colonic mucosa were investigated. We report that MSC therapy specifically reduces T-cell infiltration and proliferation, and increases apoptosis of radiation-activated T cells. We assessed the underlying molecular mechanisms and found that interleukin-10 and regulatory T lymphocytes are not involved in the immunosuppressive process in this model. However, an increased level of corticosterone secretion and HSD11b1 (11β-hydroxysteroid dehydrogenase type 1)-steroidogenic enzyme expression was detected in colonic mucosa 21 days after MSC treatment. Moreover, blocking the glucocorticoid (GC) receptor using the RU486 molecule statistically enhances the allogenic lymphocyte proliferation inhibited by MSCs in vitro and abrogates the mucosal protection induced by MSC treatment in vivo. Using the irradiation model, we found evidence for a new MSC immunosuppressive mechanism involving GCs.     

The Origins of Mesenchymal Stromal Cell Heterogeneity

Cultured mesenchymal stromal cell (MSC) populations are best characterized by the capacity of some cells within this population to differentiate into mesodermal derivatives such as osteoblasts, chondrocytes and adipocytes. However, this progenitor property is not shared by all cells within the MSC population. Furthermore, MSCs exhibit variability in their phenotypes, including proliferation capacity, expression of cell surface markers and ability to secrete cytokines. These facts raise three major questions: (1) Does the in vitro observed variability reflect the existence of MSC subsets in vivo? (2) What is the molecular basis of the in vitro observed heterogeneity? and (3) What is the biological significance of this variability? This review considers the possibility that the variable nature of MSC populations contributes to the capacity of adult mammalian tissues to adapt to varying microenvironmental demands.     

Simplified protocol to isolate, purify, and culture expand mesenchymal stem cells

Mesenchymal stem cells (MSCs) are widely used for experimental regenerative strategies. Due to their differentiation capacity into mesenchymal lineages, they are a potential cellular source for tissue regeneration. Because there is no specific antigen that can be used to define MSCs directly, there is no consensus about how to isolate them. Here we describe a simple protocol to isolate, purify, and culture expand murine bone marrow MSCs using magnetic cell sorting and plastic adherence. We further show that cytokine supplementation enhances MSC proliferation without jeopardizing their pluripotency.     

Recent progress toward understanding the physiological function of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells that are being clinically explored as regenerative therapeutics. Cultured MSCs secrete various modulatory factors, which contribute to the immunosuppressive effects of transplanted MSCs as a therapy. Although the in vitro phenotype of MSCs has been well characterized, identification of MSCs in vivo is made difficult by the lack of specific markers. Current advances in murine MSC research provide valuable tools for studying the localization and function of MSCs in vivo. Recent findings suggest that MSCs exert diverse functions depending on tissue context and physiological conditions. This review focuses on bone marrow MSCs and their roles in haematopoiesis and immune responses.     

Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue

Human adult mesenchymal stem cells (MSCs) support the engineering of functional tissue constructs by secreting angiogenic and cytoprotective factors, which act in a paracrine fashion to influence cell survival and vascularization. MSCs have been isolated from many different tissue sources, but little is known about how paracrine factor secretion varies between different MSC populations. We evaluated paracrine factor expression patterns in MSCs isolated from adipose tissue (ASCs), bone marrow (BMSCs), and dermal tissues [dermal sheath cells (DSCs) and dermal papilla cells (DPCs)]. Specifically, mRNA expression analysis identified insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor-D (VEGF-D), and interleukin-8 (IL-8) to be expressed at higher levels in ASCs compared with other MSC populations whereas VEGF-A, angiogenin, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) were expressed at comparable levels among the MSC populations examined. Analysis of conditioned media (CM) protein confirmed the comparable level of angiogenin and VEGF-A secretion in all MSC populations and showed that DSCs and DPCs produced significantly higher concentrations of leptin. Functional assays examining in vitro angiogenic paracrine activity showed that incubation of endothelial cells in ASC(CM) resulted in increased tubulogenic efficiency compared with that observed in DPC(CM). Using neutralizing antibodies we concluded that VEGF-A and VEGF-D were 2 of the major growth factors secreted by ASCs that supported endothelial tubulogenesis. The variation in paracrine factors of different MSC populations contributes to different levels of angiogenic activity and ASCs maybe preferred over other MSC populations for augmenting therapeutic approaches dependent upon angiogenesis.     

Human mesenchymal stromal cells modulate T‐cell responses through TNF‐α‐mediated activation of NF‐κB

Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF‐κB) in human MSCs. This pathway is activated by TNF‐α that is generated following TCR stimulation of T cells. Inhibition of NF‐κB through silencing of IκB kinase β or the TNF‐α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC‐associated NF‐κB activation primarily leads to inhibition of T‐cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF‐α/NF‐κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF‐κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.     

Inhibition of platelet-derived growth factor receptor signaling regulates Oct4 and Nanog expression, cell shape, and mesenchymal stem cell potency

Defining the signaling mechanisms that regulate the fate of adult stem cells is an essential step toward their use in regenerative medicine. Platelet-derived growth factor receptor (PDGFR) signaling plays a crucial role in specifying mesenchymal stem cell (MSC) commitment to mesenchymal lineages. Based on the hypothesis that selective inhibition of signaling pathways involved in differentiation may increase stem cell potency, we examined the role of PDGFR signaling in controlling the fate of human MSCs. Using a small molecular PDGFR inhibitor that induced MSCs toward a more rounded shape, expression of Oct4 and Nanog were markedly upregulated. In these PDGFR inhibitor-treated MSCs, Oct4 and Nanog expression and cell shape were regulated by janus kinase (JAK), MAPK kinase (MEK), and epidermal growth factor receptor (EGFR) signaling. Under defined differentiation conditions, these PDGFR-inhibited MSCs expressed definitive endodermal, ectodermal, and mesodermal markers. We also confirmed that depletion of individual PDGF receptors upregulated expression of Oct4A and Nanog. This study identifies PDGFR signaling as a key regulator of Oct4 and Nanog expression and of MSC potency. Thus, inhibiting these specific receptor tyrosine kinases, which play essential roles in tissue formation, offers a novel approach to unlock the therapeutic capacity of MSCs.     

Mesenchymal stem cells might be used to induce tolerance in heart transplantation

Heart transplantation is the last treatment selection for end-stage heart diseases, but the side-effects of immunosuppressive agents and morbidity brought by lifelong immunosuppression still remains unavoidable. Mesenchymal stem cell (MSC) is one kind of adult pluripotent cell that not only can differentiate into cardiomyocytes but also maintain some unique immunomodulatory function. There were reports that MSCs could both in vitro and in vivo suppress alloreactive CD4(+) T cell which is mainly responsible for chronic rejection that leads to progressive graft vascular stenosis. We based our hypothesis on that MSC could escape recognition of alloreactive cells and would interact with important immune cells such as T cells, dendritic cells after cardiac transplantation to induce a cytokine profile shift in the Th1/Th2 balance toward the anti-inflammatory phenotype Th2. And then tolerance may be induced. Also it is conceivable that MSCs may differentiate toward cardiomyocyte in the specific microenvironment. Further work are necessary to verify if MSCs can protect heart graft from chronic rejection and its potential to be used as substitutes for classic immunosuppressants.     

Biophysical and chemical effects of fibrin on mesenchymal stromal cell gene expression

Mesenchymal stromal cells (MSCs) are multipotent cells that have high expansion yields and fibrin is a native extracellular matrix (ECM) material widely used for cell delivery and surgery. MSCs and fibrin have tremendous potential for tissue engineering applications, but the effect of fibrin on MSCs is not well characterized. The purpose of this study was to analyze the role of fibrin in modulating MSC phenotype by gene expression analysis. The results demonstrate that fibrin up-regulated MSC gene expression of vasculogenic (FLK1, ACTA2, VECAD, SM22 and CNN1), myogenic (MYF5 and MYH13), neurogenic (TH and GFAP) and chondrogenic (COL2A1) markers after 5 days incubation. These gene expression results were supported by induction of expression on the protein level for early lineage-specific markers such as ACTA2, FLK1 and MYF5. The ability of fibrin to modulate MSC gene expression was not affected by matrix pore size (80–110 μm diameter) or Young’s modulus (5–25 kPa) and the differential expression of some phenotypic markers could be partially mimicked by other ECM proteins, such as fibronectin and collagen I. In some cases the inductive effect of fibrin on gene expression could be further augmented by the treatment with growth factors such as nerve growth factor. However, the effect of fibrin appeared to be limited, as MSCs did not differentiate into fully mature cells based on immunofluorescence staining after 12 days. This body of work provides a rational approach for studying the interactions of MSC with fibrin, which has important therapeutic implications for the delivery of stem cells.     

Canine and Equine Mesenchymal Stem Cells Grown in Serum Free Media Have Altered Immunophenotype

Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE₂, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE₂. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions.     

Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA-stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA-stimulated lymphocytes dose-dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor-beta (TGF-beta). Stromal-cell-derived factor-1 (SDF-1) is not expressed on the cell surface. A recent report suggested that T-cell suppression by MSC is mediated by HGF and TGF-beta. MSC suppression was not restored by the addition of neutralizing antibodies against SDF-1, OPG, HGF or TGF-beta, alone or in combination. Addition of guanosine to PHA-stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA-activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.