Marc H. Dahlke

Immunomodulatory effects of mesenchymal stem cells in a rat organ transplant model

Background. Recent reports suggest that mesenchymal stem cells (MSCs) have immunomodulatory properties. Mesenchymal stem cells can suppress the immune response toward alloantigen in vitro by inhibiting T cell proliferation in mixed-lymphocyte reactions (MLRs). However, relatively little has been reported regarding the immunomodulative potential of MSCs in vivo. Herein the authors confirm the immunomodulatory effects of rat MSCs in vitro and tested for tolerogenic features in a model of allogeneic heart transplantation. Methods. Mesenchymal stem cells were obtained from bone marrow aspirates of male Lewis rats (major histocompatibility complex [MHC] haplotype RT1l) and ACI rats (RT1a). Lewis MSCs were cocultured with ACI spleen cells to reveal direct effects of MSCs on lymphocytes. In addition, MSCs were added to MLRs between ACI T cells as responders and irradiated Lewis spleen cells as stimulators. Finally, MSCs were administered in an allogeneic heart transplantation model at different doses (with and without cyclosporin A [CsA]). Results. Mesenchymal stem cells appeared with typical spindle-shaped morphology in culture and readily differentiated into adipocytes when exposed to differentiation media. Mesenchymal stem cells expressed MHC class I, but not class II or costimulatory molecules. In vitro, MSCs phagocytosed ACI spleen cells. When introduced into an MLR, donor MSCs suppressed the proliferation of stimulated T cells. However, in vivo, MSC injection did not prolong allograft survival. In addition, concurrent treatment with low-dose CsA and MSCs accelerated allograft rejection. Conclusions. The present data confirm previous reports suggesting that MSCs have immunomodulatory properties in vitro. However, their tolerogenic properties in vivo must be questioned, as MSC injections were not only ineffective at prolonging allograft survival, but tended to promote rejection.

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.

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.

Targeting Heat Shock Protein 90 in Pancreatic Cancer Impairs Insulin-like Growth Factor-I Receptor Signaling, Disrupts an Interleukin-6/Signal-Transducer and Activator of Transcription 3/Hypoxia-Inducible Factor-1α Autocrine Loop, and Reduces Orthotopic Tumor Growth

Purpose: Inhibitors of heat-shock protein 90 (Hsp90) may interfere with oncogenic signaling pathways, including Erk, Akt, and hypoxia-inducible factor-1α (HIF-1α). Because insulin-like growth factor-I receptor (IGF-IR) and signal transducer and activator of transcription 3 (STAT3) signaling pathways are implicated in the progression of pancreatic cancer, we hypothesized that blocking Hsp90 with geldanamycin derivates [17-allylamino-geldanamycin (17-AAG), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)] would impair IGF-I– and interleukin-6–mediated signaling and thus reduce pancreatic tumor growth and angiogenesis in vivo. Experimental Design: Human pancreatic cancer cells (HPAF-II, L3.6pl) were used for experiments. Changes in signaling pathway activation upon Hsp90 blockade were investigated by Western blotting. Effects of Hsp90 inhibition (17-AAG) on vascular endothelial growth factor were determined by ELISA and real-time PCR. Effects of 17-DMAG (25 mg/kg; thrice a week; i.p.) on tumor growth and vascularization were investigated in a s.c. xenograft model and in an orthotopic model of pancreatic cancer. Results: 17-AAG inhibited IGF-IR signaling by down-regulating IGF-IRβ and directly impairing IGF-IR phosphorylation. Hypoxia- and IL-6–mediated activation of HIF-1α or STAT3/STAT5 were substantially inhibited by 17-AAG. Moreover, a novel IL-6/STAT3/HIF-1α autocrine loop was effectively disrupted by Hsp90 blockade. In vivo, 17-DMAG significantly reduced s.c. tumor growth and diminished STAT3 phosphorylation and IGF-IRβ expression in tumor tissues. In an orthotopic model, pancreatic tumor growth and vascularization were both significantly reduced upon Hsp90 inhibition, as reflected by final tumor weights and CD31 staining, respectively. Conclusions: Blocking Hsp90 disrupts IGF-I and IL-6–induced proangiogenic signaling cascades by targeting IGF-IR and STAT3 in pancreatic cancer, leading to significant growth-inhibitory effects. Therefore, we suggest that Hsp90 inhibitors could prove to be valuable in the treatment of pancreatic cancer.

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.

Inhibition of heat shock protein 90 impairs epidermal growth factor-mediated signaling in gastric cancer cells and reduces tumor growth and vascularization in vivo

Oncogenic signaling through activation of epidermal growth factor receptor (EGFR), HER-2, and hypoxia inducible-factor-1α (HIF-1α) has been implicated in gastric cancer growth and angiogenesis through up-regulation of vascular endothelial growth factor (VEGF). Recently, heat shock protein 90 (Hsp90) has been identified as a critical regulator of oncogenic protein stability, including EGFR, HER-2, and HIF-1α. We hypothesized that inhibition of Hsp90 impairs EGF- and hypoxia-mediated angiogenic signaling in gastric cancer cells and consequently inhibits angiogenesis and tumor growth. In vitro, the geldanamycin derivate 17-allylamino-17-demethoxygeldanamycin (17-AAG) led to marked reduction in constitutive and inducible activation of extracellular signal-regulated kinase 1/2, Akt, and signal transducer and activator of transcription 3 and decreased nuclear HIF-1α protein. In addition, EGFR and HER-2 were down-regulated after Hsp90 inhibition. With respect to regulation of angiogenic molecules, 17-AAG significantly reduced EGF-mediated VEGF secretion. Phosphorylation of focal adhesion kinase and paxillin were both abrogated by 17-AAG, which resulted in significant impairment of cancer cell motility. Interestingly, cytotoxic effects of 17-AAG in vitro were higher on cancer cells and gastric fibroblasts than on pericytes. In vivo, the water-soluble compound 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; 25 mg/kg, thrice per week) significantly reduced s.c. xenografted tumor growth. By immunohistochemistry, 17-DMAG significantly reduced vessel area and numbers of proliferating tumor cells in sections. Furthermore, similar significant growth-inhibitory effects of 17-DMAG were achieved when administered as low-dose therapy (5 mg/kg, thrice per week). In conclusion, blocking Hsp90 disrupts multiple proangiogenic signaling pathways in gastric cancer cells and inhibits xenografted tumor growth in vivo. Hence, gastric cancer harbors attractive molecular targets for therapy with Hsp90 inhibitors, which could lead to improved efficacy of antineoplastic therapy regimens. [Mol Cancer Ther 2007;6(3):1123–32]

Conversion of Th17 into IL-17Aneg Regulatory T Cells: A Novel Mechanism in Prolonged Allograft Survival Promoted by Mesenchymal Stem Cell–Supported Minimized Immunosuppressive Therapy

The ultimate goal in transplantation medicine is the promotion of operational tolerance. Although Th cells of the Th17 type have been predominantly associated with rejection of allogeneic solid organ grafts, regulatory T (Treg) cells appear to foster operational tolerance. Induced Treg and Th17 cells have a higher lineage plasticity than has been recognized thus far. We found that when mesenchymal stem cells (MSCs) were used to induce long-term acceptance of allogeneic heart grafts in mice, the induction of Treg cells was preceded by development of a CD11bhiGr1int myeloid–derived immunosuppressive cell–mediated Th17 response. Substantial suppression of Foxp3+ Treg cell generation from retinoic acid receptor–related orphan receptor γ−/− T cells by MSCs revealed that retinoic acid receptor–related orphan receptor γ is a common factor in the differentiation of Treg and Th17 cells. Immunosuppressant mycophenolate mofetil treatment of enriched IL-17A+ cells from MSC-primed allograft mouse recipients resulted in a reduction of IL-17A production and an increase in the Foxp3+ Treg cell fraction. Furthermore, identification of IL-17A+ Foxp3+ double-positive and ex–IL-17–producing IL-17AnegFoxp3+ T cells strongly argues for direct conversion of Th17 cells into Treg cells as the underlying mechanism of immune regulation in MSC-mediated allograft survival. The Th17 into Treg conversion identified in this study constitutes an important immunological mechanism by which MSC-induced myeloid-derived immunosuppressive cells mediate operational transplant tolerance. The possibility to create Treg cell–regulated operational tolerance in the absence of complete immune suppression provides strong clinical implications for cell therapy–assisted minimization protocols.

Advancement of mesenchymal stem cell therapy in solid organ transplantation (MISOT).

There is evolving interest in the use of mesenchymal stem cells (MSC) in solid organ transplantation. Pre-clinical transplantation models show efficacy of MSC in prolonging graft survival and a number of clinical studies are planned or underway. At a recent meeting of the MISOT consortium (MSC In Solid Organ Transplantation) the advances of these studies were evaluated and mechanisms underlying the potential effects of MSC discussed. Continued discussion is required for definition of safety and eventually efficacy endpoints for MSC therapy in solid organ transplantation.

Toward MSC in Solid Organ Transplantation: 2008 Position Paper of the MISOT Study Group

The following position paper summarizes the recommendations for early clinical trials and ongoing basic research in the field of mesenchymal stem cell-induced solid organ graft acceptance—agreed upon on the first meeting of the Mesenchymal Stem Cells In Solid Organ Transplantation (MISOT) study group in late 2008.