Previous reports suggested that culture as 3D aggregates or as spheroids can increase the therapeutic potential of the adult stem/progenitor cells referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). Here we used a hanging drop protocol to prepare human MSCs (hMSCs) as spheroids that maximally expressed TNFα stimulated gene/protein 6 (TSG-6), the antiinflammatory protein that was expressed at high levels by hMSCs trapped in the lung after i.v. infusion and that largely explained the beneficial effects of hMSCs in mice with myocardial infarcts. The properties of spheroid hMSCs were found to depend critically on the culture conditions. Under optimal conditions for expression of TSG-6, the hMSCs also expressed high levels of stanniocalcin-1, a protein with both antiinflammatory and antiapoptotic properties. In addition, they expressed high levels of three anticancer proteins: IL-24, TNFα-related apoptosis inducing ligand, and CD82. The spheroid hMSCs were more effective than hMSCs from adherent monolayer cultures in suppressing inflammatory responses in a coculture system with LPS-activated macrophages and in a mouse model for peritonitis. In addition, the spheroid hMSCs were about one-fourth the volume of hMSCs from adherent cultures. Apparently as a result, larger numbers of the cells trafficked through the lung after i.v. infusion and were recovered in spleen, liver, kidney, and heart. The data suggest that spheroid hMSCs may be more effective than hMSCs from adherent cultures in therapies for diseases characterized by sterile tissue injury and unresolved inflammation and for some cancers that are sensitive to antiinflammatory agents.

Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing β-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of β-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and β-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote β-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.

IntroductionConsiderable efforts are currently being made to explore the therapeutic potentials of the stem/progenitor cells from bone marrow referred to initially as colony forming units-fibroblastic, then as marrow stromal cells, subsequently as mesenchymal stem cells, and most recently as multipotent mesenchymal stromal cells (MSCs). [1][2][3][4][5][6] The cells are readily isolated from small aspirates of bone marrow from normal human donors or patients, they expand rapidly for 30 or more population doublings in culture, and they can differentiate into several cellular phenotypes in culture and in vivo. The therapeutic potentials of the cells have been tested in animal models and in clinical trials for a large number of diseases (see www.clinicaltrials.gov). Initially, it was assumed that the cells repaired tissues by engrafting and differentiating to replace injured cells. Engraftment with differentiation was observed in some animal models such as those with severe injuries to tissues, in embryos, or with local infusions of high concentrations of the cells. In most experimental situations, however, repair with functional improvements was observed without evidence of long-term engraftment. Therefore, most of the beneficial effects were explained by paracrine secretions or cell-to-cell contacts that had multiple effects including modulation of inflammatory or immune reactions. [6][7][8][9][10] Of special importance were the observations that although MSCs in culture secreted a large number of cytokines, 11,12 they were activated by cross-talk with injured cells to express high levels of additional therapeutic factors. 10,13 Previously we observed 14 that intravenously infused human MSCs (hMSCs) improved a mouse model for myocardial infarction in part because the hMSCs were trapped in the lung as microemboli and the injury produced by the microemboli activated the cells to secrete the anti-inflammatory protein TNF-␣-stimulated gene 6 protein (TSG-6). TSG-6 suppressed inflammatory reactions triggered by ischemia in the heart and thereby limited destruction of cardiomyocytes by invading neutrophils and monocytes/macrophages. TSG-6 is a 30 kD glycoprotein that was shown to produce anti-inflammatory effects in several animal models. 15,16 In transgenic mice, inactivation of the gene increased inflammatory responses, 17 and over-expression of the gene decreased inflammatory responses. 18 In addition, administration of the recombinant protein improved arthritis and decreased inflammation in several murine models. 19,20 To better understand the anti-inflammatory effects of hMSCs, we used the model of zymosan-induced peritonitis in mice. 21,22 The results indicated that hMSCs decreased inflammation in the model in part because the hMSCs were activated by the initial inflammatory microenvironment of the peritoneal cavity to secrete TSG-6. TSG-6 then produced a CD44-dependent decrease in the zymosan/ TLR2-mediated stimulation of NF-B signaling in resident macrophages. In effect, the hMSCs and TSG-6 generated a negativef...

Previous reports demonstrated that adult stem/progenitor cells from bone marrow (multipotent mesenchymal stem cells; MSCs) can repair injured tissues with little evidence of engraftment or differentiation. In exploring this phenomenon, our group has recently discovered that the therapeutic benefits of MSCs are in part explained by the cells being activated by signals from injured tissues to express an anti-inflammatory protein TNF-α-stimulated gene/protein 6 (TSG-6). Therefore, we elected to test the hypothesis that TSG-6 would have therapeutic effects in inflammatory but noninfectious diseases of the corneal surface. We produced a chemical and mechanical injury of the cornea in rats by brief application of 100% ethanol followed by mechanical debridement of corneal and limbal epithelium. Recombinant human TSG-6 or PBS solution was then injected into the anterior chamber of the eye. TSG-6 markedly decreased corneal opacity, neovascularization, and neutrophil infiltration. The levels of proinflammatory cytokines, chemokines, and matrix metalloproteinases were also decreased. The data indicated that TSG-6, a therapeutic protein produced by MSCs in response to injury signals, can protect the corneal surface from the excessive inflammatory response following injury.inflammation | TNF-α-stimulated protein 6 | mesenchymal stem cells | neutrophil | matrix metalloproteinase

Objective Meniscal regeneration was previously shown to be enhanced by injection of mesenchymal stem/stromal cells (MSCs) but the mode of action of the MSCs was not established. The aim of this study was to define how injection of MSCs enhances meniscal regeneration. Design A hemi-meniscectomy model in rats was used. Rat-MSCs (rMSCs) or human-MSCs (hMSCs) were injected into the right knee joint after the surgery, and PBS was injected into the left. The groups were compared macroscopically and histologically at 2, 4, and 8 w. The changes in transcription in both human and rat genes were assayed by species-specific microarrays and real-time RT-PCRs. Results Although the number of hMSCs decreased with time, hMSCs enhanced meniscal regeneration in a manner similar to rMSCs. hMSCs injection increased expression of rat type II collagen (rat-Col II), and inhibited osteoarthritis progression. The small fraction of hMSCs were activated to express high levels of a series of genes including Indian hedgehog (Ihh), parathyroid hormone-like hormone (PTHLH), and bone morphogenetic protein 2 (BMP2). The presence of hMSCs triggered the subsequent expression of rat-Col II. An antagonist of hedgehog signaling inhibited the expression of rat-Col II and an agonist increased expression of rat-Col II in the absence of hMSCs. Conclusions Despite rapid reduction in cell numbers, intra-articular injected hMSCs were activated to express Ihh, PTHLH, and BMP2 and contributed to meniscal regeneration. The hedgehog signaling was essential in enhancing the expression of rat-Col II, but several other factors provided by the hMSCs probably contributed to the repair.

ObjectivesThe outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection in the Republic of Korea started from the index case who developed fever after returning from the Middle East. He infected 26 cases in Hospital C, and consecutive nosocomial transmission proceeded throughout the nation. We provide an epidemiologic description of the outbreak, as of July 2015.MethodsEpidemiological research was performed by direct interview of the confirmed patients and reviewing medical records. We also analyzed the incubation period, serial interval, the characteristics of superspreaders, and factors associated with mortality. Full genome sequence was obtained from sputum specimens of the index patient.ResultsA total of 186 confirmed patients with MERS-CoV infection across 16 hospitals were identified in the Republic of Korea. Some 44.1% of the cases were patients exposed in hospitals, 32.8% were caregivers, and 13.4% were healthcare personnel. The most common presenting symptom was fever and chills. The estimated incubation period was 6.83 days and the serial interval was 12.5 days. A total of 83.2% of the transmission events were epidemiologically linked to five superspreaders, all of whom had pneumonia at presentation and contacted hundreds of people. Older age [odds ratio (OR) = 4.86, 95% confidence interval (CI) 1.90–12.45] and underlying respiratory disease (OR = 4.90, 95% CI 1.64–14.65) were significantly associated with mortality. Phylogenetic analysis showed that the MERS-CoV of the index case clustered closest with a recent virus from Riyadh, Saudi Arabia.ConclusionA single imported MERS-CoV infection case imposed a huge threat to public health and safety. This highlights the importance of robust preparedness and optimal infection prevention control. The lessons learned from the current outbreak will contribute to more up-to-date guidelines and global health security.

Intravenously administered mesenchymal stem/stromal cells (MSCs) engraft only transiently in recipients, but confer long-term therapeutic benefits in patients with immune disorders. This suggests that MSCs induce immune tolerance by long-lasting effects on the recipient immune regulatory system. Here, we demonstrate that i.v. infusion of MSCs preconditioned lung monocytes/macrophages toward an immune regulatory phenotype in a TNF-α-stimulated gene/protein (TSG)-6-dependent manner. As a result, mice were protected against subsequent immune challenge in two models of alloand autoimmune ocular inflammation: corneal allotransplantation and experimental autoimmune uveitis (EAU). The monocytes/macrophages primed by MSCs expressed high levels of MHC class II, B220, CD11b, and IL-10, and exhibited T-cell-suppressive activities independently of FoxP3 + regulatory T cells. Adoptive transfer of MSCinduced B220 + CD11b + monocytes/macrophages prevented corneal allograft rejection and EAU. Deletion of monocytes/macrophages abrogated the MSC-induced tolerance. However, MSCs with TSG-6 knockdown did not induce MHC II + B220 + CD11b + cells, and failed to attenuate EAU. Therefore, the results demonstrate a mechanism of the MSC-mediated immune modulation through induction of innate immune tolerance that involves monocytes/macrophages. corneal allotransplantation | experimental autoimmune uveitis | immune tolerance | mesenchymal stem/stromal cell | monocyte/macrophage

Mesenchymal stem/stromal cells (MSCs) control excessive inflammatory responses by modulating a variety of immune cells including monocytes/macrophages. However, the mechanisms by which MSCs regulate monocytes/macrophages are unclear. Inflammasomes in macrophages are activated upon cellular "danger" signals and initiate inflammatory responses through the maturation and secretion of proinflammatory cytokines such as interleukin 1b (IL-1b). Here we demonstrate that human MSCs (hMSCs) negatively regulate NLRP3 inflammasome activation in human or mouse macrophages stimulated with LPS and ATP. Caspase-1 activation and subsequent IL-1b release were decreased in macrophages by direct or transwell coculture with hMSCs. Addition of hMSCs to macrophages either at a LPS priming or at a subsequent ATP step similarly inhibited the inflammasome activation. The hMSCs had no effect on NLRP3 and IL-1b expression at mRNA levels during LPS priming. However, MSCs markedly suppressed the generation of mitochondrial reactive oxygen species (ROS) in macrophages. Further analysis showed that NLRP3-activated macrophages stimulated hMSCs to increase the expression and secretion of stanniocalcin (STC)-1, an antiapoptotic protein. Addition of recombinant protein STC-1 reproduced the effects of hMSCs in inhibiting NLRP3 inflammasome activation and ROS production in macrophages. Conversely, the effects of hMSCs on macrophages were largely abrogated by an small interfering RNA (siRNA) knockdown of STC-1. Together, our results reveal that hMSCs inhibit NLRP3 inflammasome activation in macrophages primarily by secreting STC-1 in response to activated macrophages and thus by decreasing mitochondrial ROS. STEM CELLS 2014;32:1553-1563