The aryl hydrocarbon receptor controls mesenchymal stromal cell-mediated immunomodulation via ubiquitination of eukaryotic elongation factor-2 kinase.

Mesenchymal stem cells (MSCs) have great therapeutic advantages due to their immunosuppressive properties. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose signaling plays an important role in the immune system. AHR may be involved in the regulation of MSC-associated immunomodulatory functions. However, the mechanisms by which AHR controls the immunosuppressive functions of MSCs are not well understood. Here, we report that Ahr-deficient MSCs show decreased therapeutic efficacy against graft-versus-host disease (GVHD) compared to wild-type (WT)-MSCs. This was probably due to decreased iNOS protein expression, which is a key regulatory enzyme in MSC immunomodulation. The expression of eukaryotic elongation factor 2 kinase (eEF2K), which inhibits the elongation stage of protein synthesis, is significantly increased in the Ahr-deficient MSCs. Inhibition of eEF2K restored iNOS protein expression. AHR is known to act as an E3 ligase together with CUL4B. We observed constitutive binding of AHR to eEF2K. Consequently, ubiquitination and degradation of eEF2K were inhibited in Ahr-deficient MSCs and by the AHR antagonist CH223191 in WT-MSCs. In summary, AHR regulates the immunomodulatory functions of MSCs through ubiquitination of eEF2K, thereby controlling iNOS protein synthesis and its product, nitric oxide levels.

Long-term efficacy and safety of intravenous injection of clonal mesenchymal stem cells derived from bone marrow in five adults with moderate to severe atopic dermatitis.

Atopic dermatitis is a chronic and relapsing inflammatory skin disease that is treated with immunosuppressants. However, long-term use of immunosuppressants may cause toxicity and severe side-effects. To confirm the long-term efficacy and safety of clonal mesenchymal stem cell therapy, we performed investigator-initiated clinical trials and long-term observation in five adult patients with moderate to severe atopic dermatitis that was refractory to conventional treatments. The clinical response assessment values such as Eczema Area and Severity Index (EASI) improved significantly at 16 weeks, and 80% (4/5) of the patients achieved EASI-50 after one or two treatment cycles. Patients were observed for long-term efficacy and safety for an average of 38 weeks (range, 16-86) and showed no serious side-effects. Among the cytokines tested, CCL-17, interleukin (IL)-13, and IL-22 significantly decreased at the end-point of the five participants, two patients who maintained good clinical response over 84 weeks showed increased IL-17 cytokine levels in the blood.

Dynamics of T Lymphocyte between the Periphery and the Brain from the Acute to the Chronic Phase Following Ischemic Stroke in Mice.

Stroke causes systemic immunosuppression. T lymphocytes are involved in infarct size in the early stages of stroke. However, the phenotypes of T lymphocytes and their functions in peripheral immune organs and the brain have not been well analyzed in the acute and chronic phases of stroke. Here, we investigated pathological phenotypic alterations in the systemic immune response, especially changes in T lymphocytes, from one day to six months after ischemic stroke in mice. Impairment in thymocyte numbers, development, proliferation, and apoptosis were observed for up to two weeks. The number of mature T cells in the spleen and blood decreased and showed reduced interferon-γ production. Increased numbers of CD4-CD8-CD3+ double-negative T cells were observed in the mouse brain during the early stages of stroke, whereas interleukin (IL)-10+Foxp3+ regulatory T lymphocytes increased from two weeks during the chronic phase. These phenotypes correlated with body weight and neurological severity scores. The recovery of T lymphocyte numbers and increases in IL-10+Foxp3+ regulatory T lymphocytes may be important for long-term neurological outcomes. Dynamic changes in T lymphocytes between the acute and chronic phases may play different roles in pathogenesis and recovery. This study provides fundamental information regarding the T lymphocyte alterations from the brain to the peripheral immune organs following stroke.

Treatment with phosphodiester CpG-ODN ameliorates atopic dermatitis by enhancing TGF-ß signaling.

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG phosphorothioate (PS CpG-ODN) are known to decrease IgE synthesis in Th2 allergy responses. Nonetheless, the therapeutic role of PS CpG-ODN is limited due to cytotoxicity. Therefore, we developed a phosphodiester (PO) form of CpG-ODN (46O) with reduced toxicity but effective against allergies. In this study, we first compared the toxicity of 46O with CpG-ODNs containing a PS backbone (1826S). We also investigated the therapeutic efficacy and mechanism of 46O injected intravenously in a mouse model of ovalbumin (OVA)-induced atopic dermatitis (AD). To elucidate the mechanism of 46O underlying the inhibition of IgE production, IgE- and TGF-ô€…-associated molecules were evaluated in CD40/IL-4- or LPS/IL-4-stimulated B cells. Our data showed that the treatment with 46O was associated with a lower hematological toxicity compared with 1826S. In addition, injection with 46O reduced erythema, epidermal thickness, and suppressed IgE and IL-4 synthesis in mice with OVA-induced AD. Additionally, 46O induced TGF-ß production in LPS/IL-4-stimulated B cells via inhibition of Smad7, which suppressed IgE synthesis via interaction between Id2 and E2A. These findings suggest that enhanced TGF-ß signaling is an effective treatment for IgE-mediated allergic conditions, and 46O may be safe and effective for treating allergic diseases such as AD and asthma. [BMB Reports 2021; 54(2): 142-147].

Prunus cerasoides Extract and Its Component Compounds Upregulate Neuronal Neuroglobin Levels, Mediate Antioxidant Effects, and Ameliorate Functional Losses in the Mouse Model of Cerebral Ischemia.

Prunus cerasoides (PC) has been reported to have antimicrobial and anti-inflammatory properties, but its potential as a neuroprotective agent in a mouse model of cerebral ischemia has not been explored. Considering neuroglobin (Ngb), an endogenous neuroprotective factor, as a novel approach to neuroprotection, in this study, Ngb promoter activity, Ngb expression changes, and antioxidant protection by PC extract (PCE) and PC component compounds (PCCs) were analyzed in oxygen-glucose deprivation (OGD)-treated neurons. In vivo analysis involved transient middle cerebral artery occlusion (tMCAO) in mice with pre- and post-treatment exposure to PCE. Following ischemic stroke induction, neurological behavior scores were obtained, and cellular function-related signals were evaluated in the ischemic infarct areas. In addition to PCE, certain component compounds from PCE also significantly increased Ngb levels and attenuated the intracellular ROS production and cytotoxicity seen with OGD in primary neurons. Administration of PCE reduced the infarct volume and improved neurological deficit scores in ischemic stroke mice compared with the vehicle treatment. Increased Ngb levels in infarct penumbra with PCE treatment were also accompanied by decreased markers of apoptosis (activated p38 and cleaved caspase-3). Our findings point to the benefits of Ngb-mediated neuroprotection via PCE and its antioxidant activity in an ischemic stroke model.

Oral treatment with Aloe polysaccharide ameliorates ovalbumin-induced atopic dermatitis by restoring tight junctions in skin.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. A hallmark of AD is dry itchy skin that results from defects in the epidermal barrier function. Aloe vera is used widely to promote general health and is administered topically to treat skin conditions such as eczema, burns and wounds. However, effects of A vera on AD were not fully elucidated. In this study, we investigated the oral administration of processed A vera gel (PAG) containing low molecular weight Aloe polysaccharides to treat ovalbumin (OVA)-induced AD in mice. Oral administration of PAG suppressed total and OVA-specific IgE production in sera and decreased the epidermal thickness of skin. Numbers of Ki-67-positive cells were reduced by PAG treatment. Expression levels of tight junction genes, including those that encode ZO-1, Claudin-1 and Claudin-8, were decreased in AD skin lesions, whereas oral administration of PAG partially restored the expression levels of tight junction genes. In addition, IL-4 and IL-17A mRNA transcript levels were reduced in skin lesions after PAG treatment. Taken together, our findings suggest that oral administration of PAG ameliorated AD, normalized tight junction gene expression and suppressed inflammatory cytokines in AD skin.

A Novel Immunomodulatory Mechanism Dependent on Acetylcholine Secreted by Human Bone Marrow-derived Mesenchymal Stem Cells.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are used to treat autoimmune or inflammatory diseases. Our aim was to determine the immunomodulatory mechanisms elicited by MSCs during inflammation. METHODS AND RESULTS: We cocultured MSCs with peripheral blood mononuclear cells for a mixed lymphocyte reaction or stimulated them by phytohemagglutinin. Morphological changes of MSCs and secretion of acetylcholine (ACh) from MSCs were measured. The effects of an ACh antagonist and ACh agonist on lymphocyte proliferation and proinflammatory-cytokine production were determined. The inflammatory milieu created by immune-cell activation caused MSCs to adopt a neuronlike phenotype and induced them to release ACh. Additionally, nicotinic acetylcholine receptors (nAChRs) were upregulated in activated peripheral blood mononuclear cells. We observed that ACh bound to nAChR on activated immune cells and led to the inhibition of lymphocyte proliferation and of proinflammatory-cytokine production. MSC-mediated immunosuppression through ACh activity was reversed by an ACh antagonist called α-bungarotoxin, and lymphocyte proliferation was inhibited by an ACh agonist, ACh chloride. CONCLUSIONS: Our findings point to a novel immunomodulatory mechanism in which ACh secreted by MSCs under inflammatory conditions might modulate immune cells. This study may provide a novel method for the treatment of autoimmune diseases by means of MSCs.

Comparable Immune Function Inhibition by the Infliximab Biosimilar CT-P13: Implications for Treatment of Inflammatory Bowel Disease.

BACKGROUND AND AIMS: CT-P13 is the first biosimilar monoclonal antibody to infliximab, and was recently approved in the European Union, Japan, Korea, and USA for all six indications of infliximab. However, studies directly assessing the biologic activity of CT-P13 versus inflximab in the context of inflammatory bowel disease [IBD] are still scanty. In the present study, we aimed to compare the biological activities of CT-P13 and infliximab with specific focus on intestinal cells so as to gain insight into the potential biosimilarity of these two agents for treatment of IBD. METHODS: CT-P13 and infliximab were investigated and compared by in vitro experiments for their neutralisation ability of soluble tumour necrosis factor alpha [sTNFα] and membrane-bound tumour necrosis factor alpha [mTNFα], suppression of cytokine release by reverse signalling, induction of regulatory macrophages and wound healing, and antibody-dependent cell cytotoxicity [ADCC]. RESULTS: CT-P13 showed similar biological activities to infliximab as gauged by neutralisation of soluble TNFα, as well as blockade of apoptosis and suppression of pro-inflammatory cytokines in intestinal Caco-2 cells. Infliximab and CT-P13 equally induced apoptosis and outside-to-inside signals through transmembrane TNFα [tmTNFα]. Moreover, regulatory macrophage induction and ensuing wound healing were similarly exerted by CT-P13 and infliximab. However, neither CT-P13 nor infliximab exerted any significant ADCC of ex vivo-stimulated peripheral blood monocytes or lamina propria mononuclear cells from IBD patients. CONCLUSIONS: These findings indicate that CT-P13 and infliximab exert highly similar biological activities in intestinal cells, and further support a mechanistic comparability of these two drugs in the treatment of IBD.

ICOSL expression in human bone marrow-derived mesenchymal stem cells promotes induction of regulatory T cells.

Mesenchymal stem cells (MSCs) can modulate lymphocyte proliferation and function. One of the immunomodulatory functions of MSCs involves CD4+CD25+FoxP3+ regulatory T cells (Tregs), which negatively regulate inflammatory responses. MSC-mediated Treg induction is supposed to be regulated by mechanisms requiring both soluble and cell contact-dependent factors. Although the involvement of soluble factors has been revealed, the contact-dependent mechanisms in MSC-mediated Treg induction remain unclear. We attempted to identify molecule(s) other than secreted factors that are responsible for MSC-mediated Treg induction and to uncover the underlying mechanisms. Under in vitro Treg-inducing conditions, ICOSL expression in MSCs coincided with Treg induction in co-cultures of MSCs with CD4+ T cells. When cultured in a transwell plate, MSCs failed to induce Tregs. Neutralization or knockdown of ICOSL significantly reduced Tregs and their IL-10 release. ICOSL overexpression in MSCs promoted induction of functional Tregs. ICOSL-ICOS signaling promoted Treg differentiation from CD4+ T cells through activation of the phosphoinositide 3-kinase-Akt pathway. MSCs primed with Interleukin-1ß significantly induced Tregs through ICOSL upregulation. We demonstrated that the Treg-inducing activity of MSCs is proportionate to their basal ICOSL expression. This study provides evidence that ICOSL expression in human MSCs plays an important role in contact-dependent regulation of MSC-mediated Treg induction.

Mesenchymal stromal cells inhibit CD25 expression via the mTOR pathway to potentiate T-cell suppression.

Mesenchymal stromal cells (MSCs) are known to suppress T-cell activation and proliferation. Several studies have reported that MSCs suppress CD25 expression in T cells. However, the molecular mechanism underlying MSC-mediated suppression of CD25 expression has not been fully examined. Here, we investigated the mTOR pathway, which is involved in CD25 expression in T cells. We showed that MSCs inhibited CD25 expression, which was restored in the presence of an inducible nitric oxide synthase (iNOS) inhibitor. Since CD25 mRNA expression was not inhibited, we focused on determining whether MSCs modulated components of the mTOR pathway in T cells. MSCs increased the phosphorylation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK) and decreased the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, the expression of 4E-BP1 increased dramatically in the presence of MSCs. An m7GTP pull-down assay showed increased binding of 4E-BP1 to the 5' cap-binding eukaryotic translation initiation factor 4E (eIF4E) complex in the presence of MSCs, which resulted in inhibition of mRNA translation. Treatment with 4EGI-1, a synthetic inhibitor of mRNA translation, also reduced CD25 expression in T cells. Polysome analysis confirmed decreased CD25 mRNA in the polysome-rich fraction in the presence of MSCs. Taken together, our results showed that nitric oxide, produced by MSCs, inhibits CD25 translation through regulation of the LKB1-AMPK-mTOR pathway to suppress T cells.

Neuroprotective effect of mesenchymal stem cell through complement component 3 downregulation after transient focal cerebral ischemia in mice.

Bone marrow-derived mesenchymal stem cells (MSCs) are used in stroke treatment despite the poor understanding of its mode of action. The immune suppressive and anti-inflammatory properties of MSCs possibly play important roles in regulating neuroinflammation after stroke. We investigated whether MSCs reduce the inflammatory complement component 3 (C3) levels, thus, providing neuroprotection during stroke. Mice were subjected to transient focal cerebral ischemia (tFCI), after which MSCs were intravenously injected. The infarct volume of the brain was reduced in MSC-injected tFCI mice, and C3 expression was significantly reduced in both the brain and the blood. Additionally, the profiles of other inflammatory mediators demonstrated neuroprotective changes in the MSCs-treated group. In order to analyze the effect of MSCs on neurons during cerebral ischemia, primary cortical neurons were co-cultured with MSCs under oxygen-glucose deprivation (OGD). Primary neurons co-cultured with MSCs exhibited reduced levels of C3 expression and increased protection against OGD, indicating that treatment with MSCs reduces excessive C3 expression and rescues ischemia-induced neuronal damage. Our finding suggests that reduction of C3 expression by MSCs can help to ameliorate ischemic brain damage, offering a new neuroprotective strategy in stroke therapy.

Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the ß-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

Therapeutic effects of mouse bone marrow-derived clonal mesenchymal stem cells in a mouse model of inflammatory bowel disease.

Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-α, IFN-γ, IL-1ß, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-κB activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy.

Manufacture of Clinical-Grade Human Clonal Mesenchymal Stem Cell Products from Single Colony Forming Unit-Derived Colonies Based on the Subfractionation Culturing Method.

Stem cell products derived from mesenchymal stem cells (MSCs) have been widely used in clinical trials, and a few products have been already commercialized. However, the therapeutic effects of clinical-grade MSCs are still controversial owing to mixed results from recent clinical trials. A potential solution to overcome this hurdle may be to use clonal stem cells as the starting cell material to increase the homogeneity of the final stem cell products. We have previously developed an alternative isolation and culture protocol for establishing a population of clonal MSCs (cMSCs) from single colony forming unit (CFU)-derived colonies. In this study, we established a good manufacturing practice (GMP)-compatible procedure for the clinical-grade production of human bone marrow-derived cMSCs based on the subfractionation culturing method. We optimized the culture procedures to expand and obtain a clonal population of final MSC products from single CFU-derived colonies in a GMP facility. The characterization results of the final cMSC products met our preset criteria. Animal toxicity tests were performed in a good laboratory practice facility, and showed no toxicity or tumor formation in vivo. These tests include single injection toxicity, multiple injection toxicity, biodistribution analysis, and tumorigenicity tests in vivo. No chromosomal abnormalities were detected by in situ karyotyping using oligo-fluorescence in situ hydridization (oligo-FISH), providing evidence of genetic stability of the clinical-grade cMSC products. The manufacture and quality control results indicated that our GMP methodology could produce sufficient clonal population of MSC products from a small amount of bone marrow aspirate to treat a number of patients.

Molecular Characterization of Neurally Differentiated Human Bone Marrow-derived Clonal Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, with the ability to differentiate into different cell types. Additionally, the immunomodulatory activity of MSCs can downregulate inflammatory responses. The use of MSCs to repair injured tissues and treat inflammation, including in neuroimmune diseases, has been extensively explored. Although MSCs have emerged as a promising resource for the treatment of neuroimmune diseases, attempts to define the molecular properties of MSCs have been limited by the heterogeneity of MSC populations. We recently developed a new method, the subfractionation culturing method, to isolate homogeneous human clonal MSCs (hcMSCs). The hcMSCs were able to differentiate into fat, cartilage, bone, neuroglia, and liver cell types. In this study, to better understand the properties of neurally differentiated MSCs, gene expression in highly homogeneous hcMSCs was analyzed. Neural differentiation of hcMSCs was induced for 14 days. Thereafter, RNA and genomic DNA was isolated and subjected to microarray analysis and DNA methylation array analysis, respectively. We correlated the transcriptome of hcMSCs during neural differentiation with the DNA methylation status. Here, we describe and discuss the gene expression profile of neurally differentiated hcMSCs. These findings will expand our understanding of the molecular properties of MSCs and contribute to the development of cell therapy for neuroimmune diseases.

Hypoxia induces adipocyte differentiation of adipose-derived stem cells by triggering reactive oxygen species generation.

Generation of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4) induces the proliferation and migration of adipose-derived stem cells (ASCs). However, the functional role of mitochondrial ROS (mtROS) generation in ASCs is unknown. Therefore, we have investigated whether hypoxia induces the differentiation of ASCs via ROS generation. We also have tried to identify the cellular mechanisms of ROS generation underlying adipocyte differentiation. Hypoxia (2%) and ROS generators, such as antimycin and rotenone, induced adipocyte differentiation, which was attenuated by an ROS scavenger. Although Nox4 generates ROS and regulates proliferation of ASCs, Nox4 inhibition or Nox4 silencing did not inhibit adipocyte differentiation; indeed fluorescence intensity of mito-SOX increased in hypoxia, and treatment with mito-CP, a mtROS scavenger, significantly reduced hypoxia-induced adipocyte differentiation. Phosphorylation of Akt and mTOR was induced by hypoxia, while inhibition of these molecules prevented adipocyte differentiation. Thus hypoxia induces adipocyte differentiation by mtROS generation, and the PI3K/Akt/mTOR pathway is involved.

Additive anti-allergic effects of anti-interleukin-33 and anti-Siglec-F treatments in a murine model of allergic asthma.

BACKGROUND: Anti-interleukin-33 (anti-IL-33) and anti-Siglec-F antibodies have potent anti-allergic effects on murine allergic asthma and rhinitis and induce eosinophil apoptosis. OBJECTIVE: We aimed to determine whether post-sensitization with anti-IL-33/anti-Siglec-F treatments exhibited more potent effects compared to individual treatments in a murine allergic asthma model. MATERIAL AND METHODS: Twenty-five BALB/c mice were separated into five groups (n = 5): Group A (control), Group B (ovalbumin [OVA] challenge), Group C (OVA + anti-IL-33), Group D (OVA + anti-Siglec-F), and Group E (OVA + anti-IL-33 + anti-Siglec-F). Serum total/ OVA-specific IgE, bronchoalveolar lavage (BAL) inflammatory cells and cytokines (IL-4 and IL-5), histopathological lung properties, and airway hyperreactivity were compared. RESULTS: Ovalbumin challenge induced strong immune and inflammatory responses with > 6-fold IgE level increases; 10- to 25-fold BAL eosinophil, neutrophil, and lymphocyte count increases; and > 1.5-fold IL-4 and IL-5 level increases (p < 0.05). Whereas anti-IL-33 reduced neutrophil counts, anti-Siglec-F and anti-IL-33/anti-Siglec-F reduced both eosinophil and neutrophil counts (p < 0.05). Individual treatments reduced OVA-mediated bronchiolar infiltration by 50% (p <0.05). Ovalbumin challenge increased airway hyperreactivity by 4-fold (Group B; 2000.0 ±671.8% increase in Penh) compared to controls (Group A; 445.7 ±33.5% increase in Penh) (p = 0.016). The anti-IL-33 (Group C: 1579.4 ±973.6% increase in Penh) and anti-Siglec-F (Group D: 930.4 ±236.5%) groups demonstrated significantly reduced hyperreactivity (p = 0.029). Anti-IL-33/anti-Siglec-F therapy showed synergism towards neutrophil counts, IL-5 concentrations, bronchial infiltration, and hyperreactivity (p < 0.05). CONCLUSIONS: Combination treatment with anti-IL-33/anti-Siglec-F had more potent anti-allergic effects, reducing eosinophilic infiltration through their additive effects in a murine allergic asthma model.

Mesenchymal Stem Cell Lines Isolated by Different Isolation Methods Show Variations in the Regulation of Graft-versus-host Disease.

Since the discovery of the immunomodulation property of mesenchymal stem cells (MSCs) about a decade ago, it has been extensively investigated whether MSCs can be used for the treatment of immune-related diseases, such as graft-versus-host disease (GvHD). However, how to evaluate the efficacy of human MSCs for the clinical trial is still unclear. We used an MHC-mismatched model of GvHD (B6 into BALB/c). Surprisingly, the administration of the human MSCs (hMSCs) could reduce the GvHD-related mortality of the mouse recipients and xenogeneically inhibit mouse T-cell proliferation and IFN-γ production in vitro. We recently established a new protocol for the isolation of a homogeneous population of MSCs called subfractionation culturing methods (SCM), and established a library of clonal MSC lines. Therefore, we also investigated whether MSCs isolated by the conventional gradient centrifugation method (GCM) and SCM show different efficacy in vivo. Intriguingly, clonal hMSCs (hcMSCs) isolated by SCM showed better efficacy than hMSCs isolated by GCM. Based on these results, the MHC-mismatched model of GvHD may be useful for evaluating the efficacy of human MSCs before the clinical trial. The results of this study suggest that different MSC lines may show different efficacy in vivo and in vitro.

Gene expression profile reveals that STAT2 is involved in the immunosuppressive function of human bone marrow-derived mesenchymal stem cells.

Emerging evidence of the potent immunosuppressive activity of mesenchymal stem cells (MSCs) by modulation of both innate and adaptive immune responses enables MSCs to be developed as a promising therapeutic modality for immune-related or inflammatory diseases. However, it is not clearly understood how MSCs exert their immunosuppressive effects on immune cells under inflammatory conditions. Using human bone marrow (BM)-derived clonal MSCs (hcMSCs), we obtained and analyzed a differentially expressed gene profile when stimulated with the inflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) to find novel candidate factors responsible for MSC immunomodulation. Microarray analysis showed that 5650 genes were upregulated and 5862 genes were downregulated with the cutoff of 2-fold expression change. Among these, the ICOSLG and STAT2 genes were drastically upregulated 173-fold and 154-fold, respectively. Reverse transcription-polymerase chain reaction analysis confirmed the microarray data. To evaluate whether their increased expression is related to MSC-mediated immunosuppression,siRNA-induced ICOSLG- or STAT2-knockdown hcMSCs were assessed for their T cell suppressive activity. We demonstrated that STAT2 but not ICOSLG is functionally involved in the immunosuppressive activity of hcMSCs as a novel regulator under inflammatory conditions. Gene ontology and pathway analyses further support the immunomodulatory function of hcMSCs when inflammatory stimulation was provided.Taken together, this study provides an informative genome-wide gene expression profile and molecular evidence for understanding the mechanisms underlying the modulation of immune cells by human BM-derived MSCs under inflammatory conditions.