Martin J. Hoogduijn

Effect of Arthritic Synovial Fluids on the Expression of Immunomodulatory Factors by Mesenchymal Stem Cells: An Explorative in vitro Study

Background: In diseased joints, the catabolic environment results in progressive joint damage. Mesenchymal stem cells (MSCs) can have immunomodulatory effects by secreting anti-inflammatory factors. To exert these effects, MSCs need to be triggered by pro-inflammatory cytokines. To explore the potential of MSCs as a treatment for diseased joints, we studied the effect of synovial fluid (SF) from donors with different joint diseases and donors without joint pathology on the immunomodulatory capacities of human MSCs in vitro. We hypothesized that SF of diseased joints influences the immunomodulatory effects of MSCs. Materials and Methods: MSCs were cultured in medium with SF of six osteoarthritis (OA) or six rheumatoid arthritis (RA) donors and three donors without joint pathology were used as control. Gene expressions of IL-6, HGF, TNFa, TGFb1, and indoleamine 2,3-dioxygenase (IDO) were analyzed. l-kynurenine concentration in conditioned medium (CM) by MSCs with SF was determined as a measure of IDO activity by MSCs. Furthermore, the effect of CM with SF on proliferation of activated lymphocytes was analyzed. Results: Addition of SF significantly up-regulated the mRNA expression of IL-6 and IDO in MSCs. SF(OA) induced significantly higher expression of IDO than SF(control), although no difference in IDO activity of the MSCs could be shown with a l-kynurenine assay. Medium conditioned by MSCs with SF(OA or RA) suppressed activated lymphocyte proliferation in vitro more than medium conditioned by MSCs without SF or with SF(control). Discussion: SF can influence the expression of genes involved in immunomodulation by MSCs and the effect on lymphocyte proliferation. We found indications for disease-specific differences between SFs but the variation between donors, even within one disease group was high. These data warrant further research to examine the potential application of MSC therapy in arthritic joints.

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.

Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion

Mesenchymal stem cells (MSC) are under investigation as a therapy for a variety of disorders. Although animal models show long term regenerative and immunomodulatory effects of MSC, the fate of MSC after infusion remains to be elucidated. In the present study the localization and viability of MSC was examined by isolation and re-culture of intravenously infused MSC. C57BL/6 MSC (500,000) constitutively expressing DsRed-fluorescent protein and radioactively labeled with Cr-51 were infused via the tail vein in wild-type C57BL/6 mice. After 5 min, 1, 24, or 72 h, mice were sacrificed and blood, lungs, liver, spleen, kidneys, and bone marrow removed. One hour after MSC infusion the majority of Cr-51 was found in the lungs, whereas after 24 h Cr-51 was mainly found in the liver. Tissue cultures demonstrated that viable donor MSC were present in the lungs up to 24 h after infusion, after which they disappeared. No viable MSC were found in the other organs examined at any time. The induction of ischemia-reperfusion injury in the liver did not trigger the migration of viable MSC to the liver. These results demonstrate that MSC are short-lived after i.v. infusion and that viable MSC do not pass the lungs. Cell debris may be transported to the liver. Long term immunomodulatory and regenerative effects of infused MSC must therefore be mediated via other cell types.

The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy.

There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohns disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti‐inflammatory macrophages

Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture‐expanded bone marrow‐derived MSC promote the generation of CD4+CD25hiFoxP3+ T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC‐conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF‐β1 mediates induction of Tregs. TGF‐β1 is constitutively secreted by MSC, suggesting that the MSC‐induced generation of Tregs by TGF‐β1 was independent of the interaction between MSC and PBMC. Monocyte‐depletion studies showed that monocytes are indispensable for MSC‐induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL‐10 and CCL‐18, which is mediated by as yet unidentified MSC‐derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF‐β1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC. Stem Cells 2013;31:1980‐1991

The Life and Fate of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) are present throughout the body and are thought to play a role in tissue regeneration and control of inflammation. MSC can be easily expanded in vitro and their potential as a therapeutic option for degenerative and inflammatory disease is therefore intensively investigated. Whilst it was initially thought that MSC would replace dysfunctional cells and migrate to sites of injury to interact with inflammatory cells, experimental evidence indicates that the majority of administered MSC get trapped in capillary networks and have a short life span. In this review, we discuss current knowledge on the migratory properties of endogenous and exogenous MSC and confer on how culture-induced modifications of MSC may affect these properties. Finally, we will discuss how, despite their limited survival, administered MSC can bring about their therapeutic effects.

Mesenchymal Stem Cells in Transplantation and Tissue Regeneration

Mesenchymal stem cells (MSC) were identified in the 1960s as bone marrow cells capable of osteogenic differentiation (Friedenstein and Petrakova, 1966). In the following decennia, these cells were further attributed with the capacity to differentiate into adipogenic, chondrogenic, and myogenic lineages (Pittenger et al., 1999), to secrete trophic factors that stimulate other cell types (Caplan and Dennis, 2006), and to possess immunomodulatory properties (Di Nicola et al., 2002). Cells with these properties were found not to be restricted to the bone marrow, but also to reside at other locations including adipose tissue (Zuk et al., 2002), skin (Toma et al., 2001), and in organs like liver, kidney, and brain (da Silva Meirelles et al., 2006). In particular adipose tissue has proven to be a valuable source of MSC due to its accessibility and its abundance. The characteristics of MSC initiated interest in their potential clinical use for tissue regenerative and immunomodulatory purposes. The first clinical applications were in the treatment of osteogenesis imperfecta (Horwitz et al., 1999) and graft versus host disease (Le Blanc et al., 2004). Since then, the use of MSC for the treatment of a variety of diseases has been investigated in clinical trials, including in Crohns disease (Duijvestein et al., 2010), myocardial infarction (Hare et al., 2009), rheumatoid arthritis (Liang et al., 2011), multiple sclerosis (Freedman et al., 2010), and organ transplantation (Dahlke et al., 2009). In organ transplantation, the use of MSC is aimed at the prolongation of allograft survival. Thus, MSC therapy may be used for the treatment of acute rejection, but also to prevent currently untreatable chronic rejection. Furthermore, there is evidence that MSC therapy has a tissue regenerative component that repairs organ injury caused by immunological or ischemic events and thereby prevents the loss of organ transplants in animal models (Morigi et al., 2008; Popp et al., 2008). This may offer another window of opportunity for MSC therapy in particular shortly after transplantation, when organ loss peaks partly due to ischemia–reperfusion injury of the graft. As with all therapies in development, the reasons for the use of MSC as an immunomodulatory and regenerative agent should be taken into careful consideration. Open questions are whether MSC therapy is effective and, if so, whether it is more efficient than existing drugs? Are there safety issues involved? Is MSC therapy cost-effective? Whether MSC can replace existing drugs is not clear at the moment as the efficacy of MSC therapy is difficult to determine. A one to one comparison of efficacy between MSC and conventional drugs is not easy to make. While pharmacological drugs target specific molecular pathways, MSC have a wide range of effects. Furthermore, while pharmaceuticals can be given to patients at a daily frequency, for safety, practical, and financial reasons there is a limit to the frequency at which cell therapy can be applied. However, at these early stages, a head on comparison with standard therapy may not be required as the use of MSC will primarily be aimed at applications where conventional therapies fail. As such, MSC will be applied as an adjuvant for current therapies. In the more distant future, MSC may be used to replace medication that has significant side effects, as may be the case with calcineurin inhibitors in organ transplantation. Although very effective in preventing organ rejection, calcineurin inhibitors are nephrotoxic, thereby limiting the life span of kidney transplants. Side effects of MSC therapy have not been reported yet, but certainly some will occur when MSC are used more widely. A high incidence of infections after MSC therapy in graft versus host disease patients was recently reported (von Bahr et al., 2011). Whether the risk for infection was significantly elevated in MSC treated patients compared to controls was however not demonstrated. To be able to map the side effects of MSC therapy, these effects should be investigated parallel to their clinical effects in placebo-controlled studies. Nevertheless, from where we stand today, we can conclude with considerable certainty that the infusion of MSC does not harbor serious health threats. While the in vitro properties of MSC suggest a beneficial effect of MSC in immunological and degenerative diseases and early clinical trials are triumphant about the feasibility and safety of MSC therapy, there is thus far little evidence that MSC are effective in curing disease. The effectiveness of MSC therapy needs to be established in follow up trials and knowledge of the mechanisms of action of MSC may help optimizing the therapy. The mechanisms of action of MSC after infusion may be very different to those observed in vitro. There is for instance accumulating evidence that MSC are short-lived after infusion (Popp et al., 2008). Even though, long-term effects are observed after infusion of MSC. These effects may be mediated by other cell types to which the effect of MSC is transferred. It has been demonstrated that MSC induce immunoregulatory capacity of T cells (Prevosto et al., 2007) and macrophages (Maggini et al., 2010). More knowledge on how MSC interact with these cell types could provide tools for optimizing MSC therapy. An alternative approach for MSC therapy is to design drugs or therapies that target tissue resident MSC. MSC respond to cytokines and growth factors by changing their immunoregulatory function and/or their differentiation status. Therefore, these factors could be the basis of such drugs, which should induce a specific response in MSC that reside in transplanted organs. In this way, MSC therapy can be more localized and more specific compared to the infusion of MSC. In order for a therapy to be successful, it needs to be cost-effective. MSC treatment is a costly therapy as MSC need to be cultured under GMP conditions. In particular the generation of custom-made, i.e., autologous, MSC of clinical grade is an expensive process. Centralization of MSC production at specialized laboratories can reduce costs. Furthermore, for some applications allogeneic MSC may be suitable and these can be generated in large batches, which further brings down costs. A real cost–effect analysis can however only be made once the efficacy of MSC therapy has become clear. Summarizing, MSC have the potential to be used as an immunomodulatory and regenerative therapy in organ transplantation and immune and degenerative diseases. Basic and clinical research will have to point toward the right directions on the effective use of MSC. In this special feature of Frontiers in Immunology, the most recent findings on the immunomodulatory capacity of MSC, such as their interaction with regulatory T cells, and on their potential to induce regeneration of liver, kidney, and heart after ischemia–reperfusion injury and of bone and cartilage damage in rheumatoid and osteoarthritis, will be presented. Furthermore, challenges on how to generate a high quality and effective cell product will receive attention. Finally, the use of MSC in transplantation and regenerative medicine and ideas on how to drive this field forward will be reviewed and discussed by leaders in the field. We are confident that this special topic will generate new directions to be followed in translational research and clinical trials.

Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells

There is emerging interest in the application of mesenchymal stem cells (MSC) for the prevention and treatment of autoimmune diseases, graft‐versus‐host disease and allograft rejection. It is, however, unknown how inflammatory conditions affect phenotype and function of MSC. Adipose tissue‐derived mesenchymal stem cells (ASC) were cultured with alloactivated peripheral blood mononuclear cells (PBMC) (mixed lymphocyte reaction: MLR), with proinflammatory cytokines [interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6] or under control conditions, and their full genome expression and function examined. Proinflammatory cytokines mainly increased indoleamine‐2,3‐dioxygenase expression, whereas ASC cultured with MLR showed increased expression of COX‐2, involved in prostaglandin E2 production. Both conditions had a stimulatory, but differential, effect on the expression of proinflammatory cytokines and chemokines, while the expression of fibrotic factors was decreased only in response to proinflammatory cytokines. Functional analysis demonstrated that inflammatory conditions affected morphology and proliferation of ASC, while their differentiation capacity and production of trophic factors was unaffected. The immunosuppressive capacity of ASC was enhanced strongly under inflammatory conditions. In conclusion, ASC showed enhanced immunosuppressive capacity under inflammatory conditions, while their differentiation capacity was preserved. Therefore, in vitro preconditioning provides ASC with improved properties for immediate clinical immune therapy.

Donor-derived mesenchymal stem cells suppress alloreactivity of kidney transplant patients.

Background. Human mesenchymal stem cells (MSC) have immunosuppressive capacities. Although their efficacy is currently studied in graft-versus-host disease, their effect on alloreactivity in solid organ transplant patients is unknown. In this study, the immunosuppressive effect of MSC on recipient anti-donor reactivity was examined before and after clinical kidney transplantation. Methods. Anti-donor reactivity was established in pretransplant and posttransplant mixed lymphocyte reactions (MLR) of 14 living-kidney donor-recipient pairs. MSC from donors and third-party controls were added to the MLR in a ratio of 1:5. Results. MSC were isolated from donor perirenal fat and showed multilineage differentiation potential and the capacity to inhibit lymphocyte proliferation. The immunosuppressive effect of MSC was dose dependent and mediated by cell-membrane contact and soluble factors, including interleukin-10 and indoleamine 2,3-dioxygenase. Donor-derived MSC significantly inhibited the recipient anti-donor reactivity before and 1 month after transplantation. This effect was independent of human leukocyte antigen background of MSC. Flow cytometric analysis showed that MSC inhibited the proliferation of CD4+ and CD8+ T-lymphocyte subsets in pretransplant and posttransplant donor-directed MLR, whereas MSC had no effect on B- or natural killer-cell proliferation. Conclusion. Donor MSC significantly inhibited the proliferation of alloactivated recipient T cells before and after kidney transplantation. We believe these findings should encourage MSC-based intervention in clinical organ transplantation.

Immunomodulatory effect of mesenchymal stem cells on B cells.

The research on T cell immunosuppression therapies has attracted most of the attention in clinical transplantation. However, B cells and humoral immune responses are increasingly acknowledged as crucial mediators of chronic allograft rejection. Indeed, humoral immune responses can lead to renal allograft rejection even in patients whose cell-mediated immune responses are well controlled. On the other hand, newly studied B cell subsets with regulatory effects have been linked to tolerance achievement in transplantation. Better understanding of the regulatory and effector B cell responses may therefore lead to new therapeutic approaches. Mesenchymal stem cells (MSC) are arising as a potent therapeutic tool in transplantation due to their regenerative and immunomodulatory properties. The research on MSCs has mainly focused on their effects on T cells and although data regarding the modulatory effects of MSCs on alloantigen-specific humoral response in humans is scarce, it has been demonstrated that MSCs significantly affect B cell functioning. In the present review we will analyze and discuss the results in this field.