Marina Gazdic

Mesenchymal stem cells: a friend or foe in immune-mediated diseases.

Mesenchymal stem cells (MSCs) are adult, self-renewable, multipotent cells that can be found in almost all postnatal tissues. Because of their capacity for self-renewal and differentiation into tissues of mesodermal origin and due to their immunomodulatory ability, MSCs are used in many preclinical and clinical studies as possible new therapeutic agents for the autoimmune or degenerative diseases treatment. In dependence of inflammatory environment to which they are exposed to, MSCs adopt immunosuppressive or pro-inflammatory phenotype. In the presence of high levels of pro-inflammatory cytokines or through activation of Toll-like receptor (TLR)-3, MSCs adopt an immune-suppressive phenotype and suppress the proliferation, activation and effector function of professional antigen presenting cells (dendritic cells, macrophages, B lymphocytes), T lymphocytes, NK cells, NKT cells, and neutrophils. During the early phase of inflammation, through TLR4 activation and in the presence of low levels of inflammatory cytokines, MSCs adopt a pro-inflammatory phenotype, promote neutrophil and T cell activation and enhance immune response. Here we review the current findings on the immunoregulatory plasticity of MSCs involved in regulation of immune response.

Ethical and Safety Issues of Stem Cell-Based Therapy

Results obtained from completed and on-going clinical studies indicate huge therapeutic potential of stem cell-based therapy in the treatment of degenerative, autoimmune and genetic disorders. However, clinical application of stem cells raises numerous ethical and safety concerns. In this review, we provide an overview of the most important ethical issues in stem cell therapy, as a contribution to the controversial debate about their clinical usage in regenerative and transplantation medicine. We describe ethical challenges regarding human embryonic stem cell (hESC) research, emphasizing that ethical dilemma involving the destruction of a human embryo is a major factor that may have limited the development of hESC-based clinical therapies. With previous derivation of induced pluripotent stem cells (iPSCs) this problem has been overcome, however current perspectives regarding clinical translation of iPSCs still remain. Unlimited differentiation potential of iPSCs which can be used in human reproductive cloning, as a risk for generation of genetically engineered human embryos and human-animal chimeras, is major ethical issue, while undesired differentiation and malignant transformation are major safety issues. Although clinical application of mesenchymal stem cells (MSCs) has shown beneficial effects in the therapy of autoimmune and chronic inflammatory diseases, the ability to promote tumor growth and metastasis and overestimated therapeutic potential of MSCs still provide concerns for the field of regenerative medicine. This review offers stem cell scientists, clinicians and patients useful information and could be used as a starting point for more in-depth analysis of ethical and safety issues related to clinical application of stem cells.

Galectin-3 Plays an Important Pro-inflammatory Role in the Induction Phase of Acute Colitis by Promoting Activation of NLRP3 Inflammasome and Production of IL-1β in Macrophages

BACKGROUND AND AIMS Galectin-3 [Gal-3] is an endogenous lectin with a broad spectrum of immunoregulatory effects: it plays an important role in autoimmune/inflammatory and malignant diseases, but the precise role of Gal-3 in pathogenesis of ulcerative colitis is still unknown. METHODS We used a model of dextran sulphate sodium [DSS]-induced acute colitis. The role of Gal-3 in pathogenesis of this disease was tested by evaluating disease development in Gal-3 deficient mice and administration of Gal-3 inhibitor. Disease was monitored by clinical, histological, histochemical, and immunophenotypic investigations. Adoptive transfer was used to detect cellular events in pathogenesis. RESULTS Genetic deletion or pharmacological inhibition of Gal-3 significantly attenuate DSS-induced colitis. Gal-3 deletion suppresses production of pro-inflammatory cytokines in colonic macrophages and favours their alternative activation, as well as significantly reducing activation of NOD-like receptor family, pyrin domain containing 3 [NLRP3] inflammasome in macrophages. Peritoneal macrophages isolated from untreated Gal-3(-/-) mice and treated in vitro with bacterial lipopolysaccharide or DSS produce lower amounts of tumour necrosis factor alpha [TNF-α] and interleukin beta [IL-1β] when compared with wild type [WT] cells. Genetic deletion of Gal-3 did not directly affect total neutrophils, inflammatory dendritic cells [DCs] or natural killer [NK] T cells. However, the total number of CD11c+ CD80+ DCs which produce pro-inflammatory cytokines, as well as TNF-α and IL-1β producing CD45+ CD11c- Ly6G+ neutrophils were significantly lower in colons of Gal-3(-/-) DSS-treated mice. Adoptive transfer of WT macrophages significantly enhanced the severity of disease in Gal-3(-/-) mice. CONCLUSIONS Gal-3 expression promotes acute DSS-induced colitis and plays an important pro-inflammatory role in the induction phase of colitis by promoting the activation of NLRP3 inflammasome and production of IL-1β in macrophages.

Mesenchymal stem cells protect from acute liver injury by attenuating hepatotoxicity of liver natural killer T cells in an inducible nitric oxide synthase- and indoleamine 2,3-dioxygenase-dependent manner.

The effects of mesenchymal stem cells (MSCs) on the phenotype and function of natural killer T (NKT) cells is not understood. We used concanavalin A (Con A) and α‐galactosylceramide (α‐GalCer)‐induced liver injury to evaluate the effects of MSCs on NKT‐dependent hepatotoxicity. Mouse MSCs (mMSCs) significantly reduced Con A‐ and α‐GalCer‐mediated hepatitis in C57Bl/6 mice, as demonstrated by histopathological and biochemical analysis, attenuated the influx of inflammatory [T‐bet+, tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ)‐producing and GATA3+, interleukin‐4 (IL‐4)‐producing] liver NKT cells and downregulated TNF‐α, IFN‐γ and IL‐4 levels in the sera. The liver NKT cells cultured in vitro with mMSCs produced lower amounts of inflammatory cytokines (TNF‐α, IFN‐γ, IL‐4) and higher amounts of immunosuppressive IL‐10 upon α‐GalCer stimulation. mMSC treatment attenuated expression of apoptosis‐inducing ligands on liver NKT cells and suppressed the expression of pro‐apoptotic genes in the livers of α‐GalCer‐treated mice. mMSCs reduced the cytotoxicity of liver NKT cells against hepatocytes in vitro. The presence of 1‐methyl‐dl‐tryptophan, a specific inhibitor of indoleamine 2,3‐dioxygenase (IDO), or l‐NG‐monomethyl arginine citrate, a specific inhibitor of inducible nitric oxide synthase (iNOS), in mMSC‐conditioned medium injected into α‐GalCer‐treated mice, counteracted the hepatoprotective effect of mMSCs in vivo and restored pro‐inflammatory cytokine production and cytotoxicity of NKT cells in vitro. Human MSCs attenuated the production of inflammatory cytokines in α‐GalCer‐stimulated human peripheral blood mononuclear cells in an iNOS‐ and IDO‐dependent manner and reduced their cytotoxicity against HepG2 cells. In conclusion, MSCs protect from acute liver injury by attenuating the cytotoxicity and capacity of liver NKT cells to produce inflammatory cytokines in an iNOS‐ and IDO‐dependent manner.

Mesenchymal stem cells attenuate acute liver injury by altering ratio between interleukin 17 producing and regulatory natural killer T cells

Mesenchymal stem cells (MSCs) are, due to immunomodulatory characteristics, considered as novel agents in the treatment of immune‐mediated acute liver failure. Although it is known that MSCs can regulate activation of T lymphocytes, their capacity to modulate function of neutrophils and natural killer T (NKT) cells, major interleukin (IL) 17–producing cells in acute liver injury, is still unknown. By using 2 well‐established murine models of neutrophil and NKT cell–mediated acute liver failure (induced by carbon tetrachloride and α‐galactoceramide), we investigated molecular and cellular mechanisms involved in MSC‐mediated modulation of IL17 signaling during acute liver injury. Single intravenous injection of MSCs attenuate acute hepatitis and hepatotoxicity of NKT cells in a paracrine, indoleamine 2,3‐dioxygenase (IDO)–dependent manner. Decreased levels of inflammatory IL17 and increased levels of immunosuppressive IL10 in serum, reduced number of interleukin 17–producing natural killer T (NKT17) cells, and increased presence of forkhead box P3 + IL10–producing natural killer T regulatory cells (NKTregs) were noticed in the injured livers of MSC‐treated mice. MSCs did not significantly alter the total number of IL17‐producing neutrophils, CD4+, and CD8 + T lymphocytes in the injured livers. Injection of mesenchymal stem cell–conditioned medium (MSC‐CM) resulted with an increased NKTreg/NKT17 ratio in the liver and attenuated hepatitis in vivo and significantly reduced hepatotoxicity of NKT cells in vitro. This phenomenon was completely abrogated in the presence of IDO inhibitor, 1‐methyltryptophan. In conclusion, the capacity of MSCs to alter NKT17/NKTreg ratio and suppress hepatotoxicity of NKT cells in an IDO‐dependent manner may be used as a new therapeutic approach in IL17‐driven liver inflammation. Liver Transplantation 23 1040–1050 2017 AASLD.

Mesenchymal stem cell‐derived factors: Immuno‐modulatory effects and therapeutic potential

Stem cell‐based therapy is considered to be a new hope in transplantation medicine. Among stem cells, mesenchymal stem cells (MSCs) are, due to their differentiation and immuno‐modulatory characteristics, the most commonly used as therapeutic agents in the treatment of immune‐mediated diseases. MSCs migrate to the site of inflammation and modulate immune response. The capacity of MSC to alter phenotype and function of immune cells are largely due to the production of soluble factors which expression varies depending on the pathologic condition to which MSCs are exposed. Under inflammatory conditions, MSCs‐derived factors suppress both innate and adaptive immunity by attenuating maturation and capacity for antigen presentation of dendritic cells, by inducing polarization of macrophages towards alternative phenotype, by inhibiting activation and proliferation of T and B lymphocytes and by reducing cytotoxicity of NK and NKT cells. In this review, we emphasized current findings regarding immuno‐modulatory effects of MSC‐derived factors and emphasize their potential in the therapy of immune‐mediated diseases.

Pharmacological Inhibition of Gal-3 in Mesenchymal Stem Cells Enhances Their Capacity to Promote Alternative Activation of Macrophages in Dextran Sulphate Sodium-Induced Colitis.

Transplantation of mesenchymal stem cells (MSCs) reduces the severity of dextran sulphate sodium- (DSS-) induced colitis. MSCs are able to secrete Galectin-3 (Gal-3), a protein known to affect proliferation, adhesion, and migration of immune cells. We investigate whether newly synthetized inhibitor of Gal-3 (Davanat) will affect production of Gal-3 in MSCs and enhance their potential to attenuate DSS-induced colitis. Pharmacological inhibition of Gal-3 in MSCs enhances their capacity to promote alternative activation of peritoneal macrophages in vitro and in vivo. Injection of MSCs cultured in the presence of Davanat increased concentration of IL-10 in sera of DSS-treated animals and markedly enhanced presence of alternatively activated and IL-10 producing macrophages in the colons of DSS-treated mice. Pharmacological inhibition of Gal-3 in MSCs significantly attenuates concentration of Gal-3 in sera of DSS-treated animals, indicating that MSCs produce Gal-3 in this disease. In conclusion, our findings indicate that Davanat could be used for improvement of MSC-mediated polarization towards immunosuppressive M2 phenotype of macrophages.

Mesenchymal stem cells attenuate liver fibrosis by suppressing Th17 cells – an experimental study

This study investigates molecular and cellular mechanisms involved in mesenchymal stem cell (MSC)‐mediated modulation of IL‐17 signaling during liver fibrosis. Mice received CCl4 (1 μl/g intraperitoneally) twice/week for 1 month. MSCs (1 × 106), or MSC‐conditioned medium (MSC‐CM), were intravenously injected 24 h after CCl4 and on every 7th day. Liver fibrosis was determined by macroscopic examination, histological analysis, Sirius red staining, and RT‐PCR. Serum levels of cytokines, indoleamine 2,3‐dioxygenase (IDO), and kynurenine were determined by ELISA. Flow cytometry was performed to identify liver‐infiltrated cells. In vitro, CD4+ T cells were stimulated and cultured with MSCs. 1‐methyltryptophan was used for inhibition of IDO. MSCs significantly attenuated CCl4‐induced liver fibrosis by decreasing serum levels of inflammatory IL‐17, increasing immunosuppressive IL‐10, IDO, and kynurenine, reducing number of IL‐17 producing Th17 cells, and increasing percentage of CD4+IL‐10+ T cells. Injection of MSC‐CM resulted with attenuated fibrosis accompanied with the reduced number of Th17 cells in the liver and decreased serum levels of IL‐17. MSC‐CM promoted expansion of CD4+FoxP3+IL‐10+ T regulatory cells and suppressed proliferation of Th17 cells. This phenomenon was completely abrogated in the presence of IDO inhibitor. MSCs, in IDO‐dependent manner, suppress liver Th17 cells which lead to the attenuation of liver fibrosis.

Mesenchymal Stem Cells Promote Metastasis of Lung Cancer Cells by Downregulating Systemic Antitumor Immune Response

Since majority of systemically administered mesenchymal stem cells (MSCs) become entrapped within the lungs, we used metastatic model of lung cancer, induced by intravenous injection of Lewis lung cancer 1 (LLC1) cells, to investigate the molecular mechanisms involved in MSC-mediated modulation of metastasis. MSCs significantly augmented lung cancer metastasis, attenuate concentrations of proinflammatory cytokines (TNF-α, IL-17), and increase levels of immunosuppressive IL-10, nitric oxide, and kynurenine in sera of LLC1-treated mice. MSCs profoundly reduced infiltration of macrophages, TNF-α-producing dendritic cells (DCs), TNF-α-, and IL-17-producing CD4+ T cells but increased IL-10-producing CD4+ T lymphocytes in the lungs of tumor-bearing animals. The total number of lung-infiltrated, cytotoxic FasL, perforin-expressing, TNF-α-, and IL-17-producing CD8+ T lymphocytes, and NKG2D-expressing natural killer (NK) cells was significantly reduced in LLC1 + MSC-treated mice. Cytotoxicity of NK cells was suppressed by MSC-conditioned medium. This phenomenon was abrogated by the inhibitors of inducible nitric oxide synthase (iNOS) and indoleamine 2,3-dioxygenase (IDO), suggesting the importance of iNOS and IDO for MSC-mediated suppression of antitumor cytotoxicity of NK cells. This study provides the evidence that MSCs promote lung cancer metastasis by suppressing antitumor immune response raising concerns regarding safety of MSC-based therapy in patients who have genetic susceptibility for malignant diseases.

Stem Cells and Labeling for Spinal Cord Injury

Spinal cord injury (SCI) is a devastating condition that usually results in sudden and long-lasting locomotor and sensory neuron degeneration below the lesion site. During the last two decades, the search for new therapies has been revolutionized with the improved knowledge of stem cell (SC) biology. SCs therapy offers several attractive strategies for spinal cord repair. The transplantation of SCs promotes remyelination, neurite outgrowth and axonal elongation, and activates resident or transplanted progenitor cells across the lesion cavity. However, optimized growth and differentiation protocols along with reliable safety assays should be established prior to the clinical application of SCs. Additionally, the ideal method of SCs labeling for efficient cell tracking after SCI remains a challenging issue that requires further investigation. This review summarizes the current findings on the SCs-based therapeutic strategies, and compares different SCs labeling approaches for SCI.