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Significance Clearance of apoptotic neutrophils by alveolar macrophages (AMФs) is critical for the resolution of acute lung injury (ALI). Here, we demonstrated that induction of the M1 phenotype in MФs activates the transcription factor STAT6 and thereby promotes resolution of lung injury by the increased expression of Gas6, the ligand for efferocytosis. Therefore, targeting the STAT6 activation pathway in AMФs and Gas6 may be a potential therapeutic strategy for resolution of inflammatory lung injury., Efferocytosis of apoptotic neutrophils (PMNs) by alveolar macrophages (AMФs) is vital for resolution of inflammation and tissue injury. Here, we investigated the role of AMФ polarization and expression of the efferocytic ligand Gas6 in restoring homeostasis. In the murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI), we observed augmented temporal generation of cytokines IL-4 and TSG6 in bronchoalveolar fluid (BALF). Interestingly, we also observed increased expression of antiinflammatory markers consistent with a phenotype shift in AMФs. In particular, AMФs expressed the efferocytic ligand Gas6. In vitro priming of bone marrow–derived macrophages (BMMФs) with IL-4 or TSG6 also induced MФ transition and expression of Gas6. TSG6- or IL-4–primed BMMФs induced efferocytosis of apoptotic PMNs compared with control BMMФs. Adoptive transfer of TSG6- or IL-4–primed BMMФs i.t. into LPS-challenged mice more rapidly and effectively cleared PMNs in lungs compared with control BMMФs. We demonstrated that expression of Gas6 during AMФ transition was due to activation of the transcription factor signal transducer and activator of transcription-6 (STAT6) downstream of IL-4 or TSG6 signaling. Adoptive transfer of Gas6-depleted BMMФs failed to clear PMNs in lungs following LPS challenge and mice showed severely defective resolution of lung injury. Thus, activation of STAT6-mediated Gas6 expression during macrophage phenotype transition resulting in efferocytosis of PMNs plays a crucial role in the resolution of inflammatory lung injury.

Purpose: To investigate the therapeutic efficacy of tumor necrosis factor (TNF)-α stimulated gene/protein 6 (TSG-6) in a rat model of corneal alkali injury. Methods: Corneal alkali injury was produced by placing an NaOH-soaked filter paper disk on the central cornea of the right eye of an anesthetized male Lewis (LEW/Crl) rat. Recombinant human TSG-6, or an equal volume of phosphate-buffered saline (PBS), was administered intravenously (IV), by anterior chamber (AC) injection, or as a topical drop. The affected eyes were photographed daily using a dissecting microscope and documented for clinical time course analysis of corneal opacification. Corneal tissue was excised at pre-determined therapeutic endpoints, with subsequent qRT-PCR or histological analyses. Results: The continuous monitoring of corneal alkali injury progression revealed TSG-6 treatments do not show sufficient effectiveness in vivo regardless of IV injection, AC injection, or topical application. Corneal opacification and neovascularization were not diminished, and gene expression was not impacted by these treatments. However, both IV and AC administration of TSG-6 significantly suppressed pro-inflammatory cytokines compared to PBS-treated eyes. Conclusion: We conclude that the therapeutic potential of TSG-6 is insufficient in a rat corneal alkali injury model.

Significance This study provides evidence that human induced pluripotent stem cell (hiPSC)-derived medial ganglionic eminence (MGE) cell grafting into the hippocampus after status epilepticus can greatly reduce the frequency of spontaneous seizures in the chronic phase through both antiepileptogenic and antiepileptic effects. The antiepileptogenic changes comprised reductions in host interneuron loss, abnormal neurogenesis, and aberrant mossy fiber sprouting, whereas the antiepileptic effects were evident from an increased occurrence of seizures after silencing of graft-derived interneurons. Additional curative impacts of grafting comprised improved cognitive and mood function. The results support the application of autologous human MGE cell therapy for temporal lobe epilepsy. Autologous cell therapy is advantageous as such a paradigm can avoid immune suppression and promote enduring graft–host integration., Medial ganglionic eminence (MGE)-like interneuron precursors derived from human induced pluripotent stem cells (hiPSCs) are ideal for developing patient-specific cell therapy in temporal lobe epilepsy (TLE). However, their efficacy for alleviating spontaneous recurrent seizures (SRS) or cognitive, memory, and mood impairments has never been tested in models of TLE. Through comprehensive video- electroencephalographic recordings and a battery of behavioral tests in a rat model, we demonstrate that grafting of hiPSC-derived MGE-like interneuron precursors into the hippocampus after status epilepticus (SE) greatly restrained SRS and alleviated cognitive, memory, and mood dysfunction in the chronic phase of TLE. Graft-derived cells survived well, extensively migrated into different subfields of the hippocampus, and differentiated into distinct subclasses of inhibitory interneurons expressing various calcium-binding proteins and neuropeptides. Moreover, grafting of hiPSC-MGE cells after SE mediated several neuroprotective and antiepileptogenic effects in the host hippocampus, as evidenced by reductions in host interneuron loss, abnormal neurogenesis, and aberrant mossy fiber sprouting in the dentate gyrus (DG). Furthermore, axons from graft-derived interneurons made synapses on the dendrites of host excitatory neurons in the DG and the CA1 subfield of the hippocampus, implying an excellent graft–host synaptic integration. Remarkably, seizure-suppressing effects of grafts were significantly reduced when the activity of graft-derived interneurons was silenced by a designer drug while using donor hiPSC-MGE cells expressing designer receptors exclusively activated by designer drugs (DREADDs). These results implied the direct involvement of graft-derived interneurons in seizure control likely through enhanced inhibitory synaptic transmission. Collectively, the results support a patient-specific MGE cell grafting approach for treating TLE.

The cornea is a transparent tissue devoid of blood and lymphatic vessels. However, various inflammatory conditions can cause hemangiogenesis and lymphangiogenesis in the cornea, compromising transparency and visual acuity. Mesenchymal stem/stromal cells (MSCs) have therapeutic potentials in a variety of diseases because of anti-inflammatory properties. Herein, we investigated the effects of MSCs on corneal angiogenesis using a model of suture-induced inflammatory corneal neovascularization. Data demonstrated that an intravenous administration of MSCs suppressed corneal inflammation and neovascularization, inhibiting both hemangiogenesis and lymphangiogenesis. MSCs reduced the levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, Tek, MRC1, and MRC2 in the cornea, which are expressed by pro-angiogenic macrophages. Moreover, the number of CD11b+ monocytes/macrophages in the cornea, spleen, peripheral blood, and draining lymph nodes was decreased by MSCs. Depletion of circulating CD11b+ monocytes by blocking antibodies replicated the effects of MSCs. Importantly, knockdown of tumor necrosis factor alpha (TNF-α)-stimulated gene/protein 6 (TSG-6) in MSCs abrogated the effects of MSCs in inhibiting corneal hemangiogenesis and lymphangiogenesis and monocyte/macrophage infiltration. Together, the results suggest that MSCs inhibit inflammatory neovascularization in the cornea by suppressing pro-angiogenic monocyte/macrophage recruitment in a TSG-6-dependent manner.

Extracellular vesicles (EVs) play key roles in cell biology and may provide new clinical diagnostics and therapies. However, it has proven difficult to develop protocols for their purification and characterization. One of the major barriers in the field has been a lack of convenient assays for their bioactivity. Developing assays has not been a trivial matter, because of the heterogeneity of EVs, the multiple activities they demonstrate, and the uncertainty about their modes of action. Therefore, it is likely that multiple assays for their activities are needed. One important assay will be for the anti-inflammatory activity observed in mice after administration of the small EVs commonly referred to as exosomes. We developed an assay for the anti-inflammatory activity of exosomes with a line of mouse macrophages. The assay makes it possible to rank different preparations of exosomes by their anti-inflammatory activity, and their ranking predicts their efficacy in suppressing LPS-stimulated inflammation in mice. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols for the purification and characterization of anti-inflammatory exosomes., Graphical Abstract

Fibrosis and scarring are the end stage of many disease processes. In effect, the collagen fibers that initially provide a necessary strength during the repair of injured tissues are frequently synthesized in excessive amounts and become irreversible fibrotic deposits that limit regeneration of the endogenous cells of a tissue. This review will focus on the potential of mesenchymal stem/stromal cells for treatment of fibrotic diseases, with emphasis on the role of TSG-6 as a mediator of anti-inflammatory effects.

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (hMSCs) have great promise as biologics to treat neurological and neurodegenerative conditions due to their robust antiinflammatory and neuroprotective properties. Besides, intranasal (IN) administration of EVs has caught much attention because the procedure is noninvasive, amenable for repetitive dispensation, and leads to a quick penetration of EVs into multiple regions of the forebrain. Nonetheless, it is unknown whether brain injury-induced signals are essential for the entry of IN-administered EVs into different brain regions. Therefore, in this study, we investigated the distribution of IN-administered hMSC-derived EVs into neurons and microglia in the intact and status epilepticus (SE) injured rat forebrain. Ten billion EVs labeled with PKH26 were dispensed unilaterally into the left nostril of na&iuml, ve rats, and rats that experienced two hours of kainate-induced SE. Six hours later, PKH26 + EVs were quantified from multiple forebrain regions using serial brain sections processed for different neural cell markers and confocal microscopy. Remarkably, EVs were seen bilaterally in virtually all regions of intact and SE-injured forebrain. The percentage of neurons incorporating EVs were comparable for most forebrain regions. However, in animals that underwent SE, a higher percentage of neurons incorporated EVs in the hippocampal CA1 subfield and the entorhinal cortex, the regions that typically display neurodegeneration after SE. In contrast, the incorporation of EVs by microglia was highly comparable in every region of the forebrain measured. Thus, unilateral IN administration of EVs is efficient for delivering EVs bilaterally into neurons and microglia in multiple regions in the intact or injured forebrain. Furthermore, incorporation of EVs by neurons is higher in areas of brain injury, implying that injury-related signals likely play a role in targeting of EVs into neurons, which may be beneficial for EV therapy in various neurodegenerative conditions including traumatic brain injury, stroke, multiple sclerosis, and Alzheimer&rsquo, s disease.

The University of Vermont College of Medicine and the Vermont Lung Center, in collaboration with the NHLBI, Alpha-1 Foundation, American Thoracic Society, European Respiratory Society, International Society for Cell Therapy, and the Pulmonary Fibrosis Foundation, convened a workshop, “Stem Cells and Cell Therapies in Lung Biology and Lung Diseases,” held July 29 to August 1, 2013 at the University of Vermont. The conference objectives were to review the current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy and ex vivo bioengineering approaches for lung diseases. These are all rapidly expanding areas of study that both provide further insight into and challenge traditional views of mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, discuss and debate current controversies, and identify future research directions and opportunities for both basic and translational research in cell-based therapies for lung diseases. This conference was a follow-up to four previous biennial conferences held at the University of Vermont in 2005, 2007, 2009, and 2011. Each of those conferences, also sponsored by the National Institutes of Health, American Thoracic Society, and Respiratory Disease Foundations, has been important in helping guide research and funding priorities. The major conference recommendations are summarized at the end of the report and highlight both the significant progress and major challenges in these rapidly progressing fields.

Objective: Bone marrow-derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose-derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. Methods: C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA-transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony-forming unit assays. Results: In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS-induced myelosuppression. This effect was specifically dependent on the anti-inflammatory factor TSG-6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. Conclusion: Our results suggest that systemic administration of hASC or TSG-6 may be novel approaches to reverse CS-induced myelosuppression. Stem Cells 2015;33:468–478

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications.

Much of what we know about immunology suggests that little is to be gained from experiments in which human cells are administered to immunocompetent mice. Multiple reports have demonstrated that this common assumption does not hold for experiments with human mesenchymal stem/stromal cells (hMSCs). The data demonstrate that hMSCs can suppress immune responses to a variety of stimuli in immunocompetent mice by a range of different mechanisms that are similar to those employed by mouse MSCs. Therefore, further experiments with hMSCs in mice will make it possible to generate preclinical data that will improve both the efficacy and safety of the clinical trials with the cells that are now in progress.

Status epilepticus (SE), a medical emergency that is typically terminated through antiepileptic drug treatment, leads to hippocampus dysfunction typified by neurodegeneration, inflammation, altered neurogenesis, as well as cognitive and memory deficits. Here, we examined the effects of intranasal (IN) administration of extracellular vesicles (EVs) secreted from human bone marrow-derived mesenchymal stem cells (MSCs) on SE-induced adverse changes. The EVs used in this study are referred to as A1-exosomes because of their robust antiinflammatory properties. We subjected young mice to pilocarpine-induced SE for 2 h and then administered A1-exosomes or vehicle IN twice over 24 h. The A1-exosomes reached the hippocampus within 6 h of administration, and animals receiving them exhibited diminished loss of glutamatergic and GABAergic neurons and greatly reduced inflammation in the hippocampus. Moreover, the neuroprotective and antiinflammatory effects of A1-exosomes were coupled with long-term preservation of normal hippocampal neurogenesis and cognitive and memory function, in contrast to waned and abnormal neurogenesis, persistent inflammation, and functional deficits in animals receiving vehicle. These results provide evidence that IN administration of A1-exosomes is efficient for minimizing the adverse effects of SE in the hippocampus and preventing SE-induced cognitive and memory impairments.

One attractive strategy to treat cancers is to deliver an exogenous enzyme that will convert a non-toxic compound to a highly toxic derivative. The strategy was tested with viral vectors but was disappointing because the efficiency of transduction into tumor cells was too low. Recent reports demonstrated that the limitation can be addressed by using tissue-derived mesenchymal stromal cells (MSCs) to deliver enzyme/prodrug systems that kill adjacent cancer cells through bystander effects. Here we addressed the limitation that tissue-derived MSCs vary in their properties and are difficult to generate in the large numbers needed for clinical applications. We prepared a Feeder Stock of MSCs from induced pluripotent stem cells (iPSs) that provided an extensively expandable source of standardized cells. We then transduced the iPS-derived MSCs to express cytosine deaminase and injected them locally into a mouse xenogeneic model of human breast cancer. After administration of the prodrug (5-fluorocytosine), the transduced iPS-MSCs both limited growth of preformed tumors and decreased lung metastases.

Background aims Human mesenchymal stromal cells (MSCs) are being used in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3 dimensions (3D). We compared the activation of MSCs in 3D in fetal bovine serum containing medium and in multiple xeno-free media formulations. Methods MSC aggregation and sphere formation was studied with the use of hanging drop cultures with medium containing fetal bovine serum or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with the use of gene expression and protein secretion measurements and with functional studies with the use of macrophages and cancer cells. Results MSCs did not condense into tight spheroids and express a full complement of therapeutic genes in α-minimum essential medium or several commercial stem-cell media. However, we identified a chemically defined xeno-free media, which, when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (prostaglandin E2, TSG-6 TNF-alpha induced gene/protein 6) and anti-cancer molecules (TRAIL TNF-related apoptosis-inducing ligand, interleukin-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage system and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death. Conclusions We demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed in the present study for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications.

There has been great interest in research and clinical trials with the adult stem/progenitor cells referred to as mesenchymal stem/stromal cells (MSCs). However, there are no definitive markers for the cells and no assays that would reflect the therapeutic efficacy of the cells in vivo. There are in effect no adequate release criteria that define the quality or efficacy of the cells. The problems are compounded by the fact that a variety of different protocols has been used to isolate the cells and expand them in culture. The result is that many publications have used MSCs with different properties, frequently without the investigators being aware of the differences. As a partial solution to these problems, we have devised a simple table to record in-process data on the preparation of MSCs. We suggest that comparisons of data generated by different laboratories would be facilitated if similar in-process data, probably as supplemental materials, were included in publications using MSCs.

Traumatic brain injury (TBI) causes multiple long-term defects including a loss of working memory that is frequently incapacitating. Administrations of mesenchymal stem/stromal cells (MSCs) previously produced beneficial effects in models of TBI as well as other disease models. In several models, the beneficial effects were explained by the MSCs being activated to express TSG-6, a multifunctional protein that modulates inflammation. In a mouse model of TBI, we found the initial mild phase of the inflammatory response persisted for at least 24 h and was followed by secondary severe response that peaked at 3 days. Intravenous human MSCs or TSG-6 during initial mild phase decreased neutrophil extravasation, expression of matrix metalloproteinase 9 by endothelial cells and neutrophils, and the subsequent blood brain barrier leakage in secondary phase. Administration of TSG-6 also decreased the lesion size at 2 weeks. Importantly, the acute administration of TSG-6 within 24 h of TBI was followed 6 to 10 weeks later by improvements in memory, depressive-like behavior and the number of newly born-neurons. The data suggested that acute administration of TSG-6 may be an effective therapy for decreasing some of the long-term consequences of TBI.

TNFα-stimulated gene-6 (TSG6), a 30-kDa protein generated by activated macrophages, modulates inflammation; however, its mechanism of action and role in the activation of macrophages are not fully understood. Here we observed markedly augmented LPS-induced inflammatory lung injury and mortality in TSG6-/- mice compared with WT (TSG6+/+) mice. Treatment of mice with intratracheal instillation of TSG6 prevented LPS-induced lung injury and neutrophil sequestration, and increased survival in mice. We found that TSG6 inhibited the association of TLR4 with MyD88, thereby suppressing NF-κB activation. TSG6 also prevented the expression of proinflammatory proteins (iNOS, IL-6, TNFα, IL-1β, and CXCL1) while increasing the expression of anti-inflammatory proteins (CD206, Chi3l3, IL-4, and IL-10) in macrophages. This shift was associated with suppressed activation of proinflammatory transcription factors STAT1 and STAT3. In addition, we observed that LPS itself up-regulated the expression of TSG6 in TSG6+/+ mice, suggesting an autocrine role for TSG6 in transitioning macrophages. Thus, TSG6 functions by converting macrophages from a proinflammatory to an anti-inflammatory phenotype secondary to suppression of TLR4/NF-κB signaling and STAT1 and STAT3 activation.

Patients with breast cancer often develop malignant regrowth of residual drug-resistant dormant tumor cells years after primary treatment, a process defined as cancer relapse. Deciphering the causal basis of tumor dormancy therefore has obvious therapeutic significance. Because cancer cell behavior is strongly influenced by stromal cells, particularly the mesenchymal stem/stromal cells (MSCs) that are actively recruited into tumor-associated stroma, we assessed the impact of MSCs on breast cancer cell (BCC) dormancy. Using 3D cocultures to mimic the cellular interactions of an emerging tumor niche, we observed that MSCs sequentially surrounded the BCCs, promoted formation of cancer spheroids, and then were internalized/degraded through a process resembling the well-documented yet ill-defined clinical phenomenon of cancer cell cannibalism. This suspected feeding behavior was less appreciable in the presence of a rho kinase inhibitor and in 2D monolayer cocultures. Notably, cannibalism of MSCs enhanced survival of BCCs deprived of nutrients but suppressed their tumorigenicity, together suggesting the cancer cells entered dormancy. Transcriptome profiles revealed that the resulting BCCs acquired a unique molecular signature enriched in prosurvival factors and tumor suppressors, as well as inflammatory mediators that demarcate the secretome of senescent cells, also referred to as the senescence-associated secretory phenotype. Overall, our results provide intriguing evidence that cancer cells under duress enter dormancy after cannibalizing MSCs. Importantly, our practical 3D coculture model could provide a valuable tool to understand the antitumor activity of MSCs and cell cannibalism further, and therefore open new therapeutic avenues for the prevention of cancer recurrence.

Background aims Mesenchymal stromal cells (MSCs) offer tremendous potential for therapeutic applications for inflammatory diseases. However, tissue-derived MSCs, such as bone marrow–derived MSCs (BM-MSCs), have considerable donor variations and limited expandability. It was recently demonstrated that MSCs derived from induced pluripotent stem cells (iPSC-MSCs) have less pro-tumor potential and greater expandability of homogenous cell population. In this study, we investigated the anti-inflammatory effects and mechanism of iPSC-MSCs in a murine model of chemical and mechanical injury to the cornea and compared the effects with those of BM-MSCs. Methods To create an injury, ethanol was applied to the corneal surface in mice, and the corneal epithelium was removed with a blade. Immediately after injury, mice received an intravenous injection of (i) iPSC-MSCs, (ii) BM-MSCs or (iii) vehicle. Clinical, histological and molecular assays were performed in the cornea to evaluate inflammation. Results We found that corneal opacity was significantly reduced by iPSC-MSCs or BM-MSCs. Histological examination revealed that the swelling and inflammatory infiltration in the cornea were markedly decreased in mice treated with iPSC-MSCs or BM-MSCs. Corneal levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were lower in iPSC-MSC- and BM-MSC-treated mice, compared with vehicle-treated controls. In contrast, iPSC-MSCs with a knockdown of the TNF-α stimulating gene (TSG)-6 did not suppress the levels of inflammatory cytokines and failed to reduce corneal opacity. Conclusions Together these data demonstrate that iPSC-MSCs exert therapeutic effects in the cornea by reducing inflammation in part through the expression of TSG-6, and the effects are similar to those seen with BM-MSCs.

From the outset, it was apparent that developing new therapies with mesenchymal stem/stromal cells (MSCs) was not a simple or easy task. Among the earliest experiments was administration of MSCs from normal mice to transgenic mice that developed brittle bones because they expressed a mutated gene for type 1 collagen isolated from a patient with osteogenesis imperfecta. The results prompted a clinical trial of MSCs in patients with severe osteogenesis imperfecta. Subsequent work by large numbers of scientists and clinicians has established that, with minor exceptions, MSCs do not engraft or differentiate to a large extent in vivo. Instead the cells produce beneficial effects in a large number of animal models and some clinical trials by secreting paracrine factors and extracellular vesicles in a “hit and run” scenario. The field faces a number of challenges, but the results indicate that we are on the way to effective therapies for millions of patients who suffer from devastating diseases. This report was presented by the Author on receipt of the Career Achievement Award in Cell Therapy from the International Society for Cellular Therapy. Singapore, May 25, 2016.

Stanniocalcin-1 (STC-1) is a multifunctional glycoprotein with antioxidant and anti-inflammatory properties. Ischemic myocardial necrosis generates "danger" signals that perpetuate detrimental inflammatory reactions often involving monocyte recruitment and their subsequent differentiation into proinflammatory macrophages. Therefore, we evaluated the effects of recombinant STC-1 (rSTC-1) on monocyte phenotype and in a mouse model of myocardial infarction. Using an established protocol to differentiate human monocytes into macrophages, we demonstrated that rSTC-1 did not alter morphology of the differentiated cells, toll-like receptor (TLR) 4 expression, or expression of the myeloid cell marker CD11b. However, rSTC-1 treatment before differentiation attenuated the rise in the expression of CD14, a TLR4 coreceptor and pathogen sensor that propagates innate immune responses, and suppressed levels of inflammatory cytokines produced by the differentiated cells in response to the CD14-TLR4 ligand lipopolysaccharide. Moreover, rSTC-1 treatment reduced CD14 expression in monocytes stimulated with endogenous danger signals. Interestingly, the effects of rSTC-1 on CD14 expression were not reproduced by a superoxide dismutase mimetic. In mice with induced myocardial infarcts, intravenous administration of rSTC-1 decreased CD14 expression in the heart as well as levels of tumor necrosis factor alpha, C-X-C motif ligand 2, interleukin 1 beta, and myeloperoxidase. It also suppressed the formation of scar tissue while enhancing cardiac function. The data suggests that one of the beneficial effects of STC-1 might be attributed to suppression of CD14 on recruited monocytes and macrophages that limits their inflammatory response. STC-1 may be a promising therapy to protect the heart and other tissues from ischemic injury.

TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional protein that has a number of potential therapeutic applications. Experiments and clinical trials with TSG-6, however, have been limited by the technical difficulties of producing the recombinant protein. We prepared stable clones of CHO cells that expressed recombinant human TSG-6 (rhTSG-6) as a secreted glycoprotein. Paradoxically, both cell number and protein production decreased dramatically when the clones were expanded. The decreases occurred because the protein aggregated the synthesizing CHO cells by binding to the brush border of hyaluronan that is found around many cultured cells. In addition, the rhTSG-6 readily self-aggregated. To address these problems, we added to the medium an inhibitor of hyaluronan synthesis and heparin to compete with the binding of TSG-6 to hyaluronan. Also, we optimized the composition of the culture medium, and transferred the CHO cells from a spinner culture system to a bioreactor that controlled pH and thereby decreased pH-dependent binding properties of the protein. With these and other improvements in the culture conditions, we obtained 57.0 mg ± 9.16 S.D. of rhTSG-6 in 5 or 6 liter of medium. The rhTSG-6 accounted for 18.0% ± 3.76 S.D. of the total protein in the medium. We then purified the protein with a Ni-chelate column that bound the His tag engineered into the C-terminus of the protein followed by an anion exchange column. The yield of the purified monomeric rhTSG-6 was 4.1 mg to 5.6 mg per liter of culture medium. After intravenous injection into mice, the protein had a longer plasma half-life than commercially available rhTSG-6 isolated from a mammalian cell lysate, apparently because it was recovered as a secreted glycoprotein. The bioactivity of the rhTSG-6 in suppressing inflammation was demonstrated in a murine model.

Previous studies have demonstrated that mesenchymal stromal cells (MSCs) enhance cell survival through upregulation and secretion of stanniocalcin-1 (STC1). This study shows that MSC-derived STC1 promotes survival of lung cancer cells by uncoupling oxidative phosphorylation, reducing intracellular reactive oxygen species (ROS), and shifting metabolism towards a more glycolytic metabolic profile. MSC-derived STC1 upregulated uncoupling protein 2 (UCP2) in injured A549 cells in an STC1-dependent manner. Knockdown of UCP2 reduced the ability of MSCs and recombinant STC1 (rSTC1) to reduce cell death in the A549 population. rSTC1-treated A549 cells displayed decreased levels of ROS, mitochondrial membrane potential (MMP), and increased lactate production, all of which were dependent on the upregulation of UCP2. Our data suggest that MSCs can promote cell survival by regulating mitochondrial respiration via STC1.

Recent observations have demonstrated that one of the functions of mesenchymal stem/stromal cells (MSCs) is to serve as guardians against excessive inflammatory responses. One mode of action of the cells is that they are activated to express the interleukin (IL)-1 receptor antagonist. A second mode of action is to create a negative feedback loop in which tumor necrosis factor-α (TNF-α) and other proinflammatory cytokines from resident macrophages activate MSCs to secrete the multifunctional anti-inflammatory protein TNF-α stimulated gene/protein 6 (TSG-6). The TSG-6 then reduces nuclear factor-κB (NF-κB) signaling in the resident macrophages and thereby modulates the cascade of proinflammatory cytokines. A third mode of action is to create a second negative feedback loop whereby lipopolysaccharide, TNF-α, nitric oxide, and perhaps other damage-associated molecular patterns (DAMPs) from injured tissues and macrophages activate MSCs to secrete prostaglandin E(2) (PGE(2)). The PGE(2) converts macrophages to the phenotype that secretes IL-10. There are also suggestions that MSCs may produce anti-inflammatory effects through additional modes of action including activation to express the antireactive oxygen species protein stanniocalcin-1.

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 allo- and 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 MSC-induced 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.

Extracellular vesicles (EVs) secreted by cells present an attractive strategy for developing new therapies, but progress in the field is limited by several issues: The quality of the EVs varies with the type and physiological status of the producer cells; protocols used to isolate the EVs are difficult to scale up; and assays for efficacy are difficult to develop. In the present report, we have addressed these issues by using human mesenchymal stem/stromal cells (MSCs) that produce EVs when incubated in a protein-free medium, preselecting the preparations of MSCs with a biomarker for their potency in modulating inflammation, incubating the cells in a chemically defined protein-free medium that provided a stable environment, isolating the EVs with a scalable chromatographic procedure, and developing an in vivo assay for efficacy of the cells in suppressing neuroinflammation after traumatic brain injury (TBI) in mice. In addition, we demonstrate that i.v. infusion of the isolated EVs shortly after induction of TBI rescued pattern separation and spatial learning impairments 1 mo later.

Recently we demonstrated that the miRNA regulates human Mesenchymal Stem Cells (hMSCs) differentiation. To determine the role of the miRNA pathway in hMSCs proliferation, Drosha and Dicer knockdown hMSCs were generated using a lentiviral based tetracycline inducible shRNA. hMSCs with reduced Drosha expression had a significantly reduced proliferation rate, while hMSCs with reduced Dicer expression displayed a proliferation rate similar to untransduced cells. Cell cycle analysis identified that unlike Dicer knockdown, Drosha knockdown hMSCs contained an increased number of G1 phase cells, with a reduced level of cells in S phase, compared to controls. ELISAs of hMSCs revealed decreased levels of pRB and stable levels of total RB with Drosha knockdown. Two key regulators of the G1/S phase transition, cyclin dependent kinase inhibitor 2A (p16) and cyclin dependent kinase inhibitor 2B (p15), were increased in Drosha knockdown cells but not in Dicer knockdown. Transcripts of 28S and 18S rRNA were significantly reduced in Drosha knockdown hMSCs, with no change in rRNA levels in Dicer knockdown hMSCs. 45S pre-rRNA transcripts were not significantly different in either knockdown model. The above results indicate that Drosha modifies hMSCs proliferation through a miRNA independent mechanism, potentially by regulating rRNA processing.

Mesenchymal stem/progenitor cells (MSCs) improve functional outcome in a number of disease models through suppression of inflammation. However, their effects on neuroinflammation are unknown. In this study, we show that MSCs suppress endotoxin-induced glial activation in organotypic hippocampal slice cultures (OHSCs). Lipopolysaccharide-stimulated OHSCs activated MSCs to increase the expression of cyclo-oxygenase-2 and produce prostaglandin E2. MSC-derived prostaglandin E2, then suppressed pro-inflammatory cytokine production by the OHSCs. Together, the results suggest the potential anti-inflammatory mechanism of MSCs in models of disease and support earlier observations that MSCs may offer a therapy for neuroinflammation produced by trauma or disease.

Multipotent stem/progenitor cells from bone marrow stroma (mesenchymal stromal cells or MSCs) were previously shown to enhance proliferation and differentiation of neural stem cells (NSCs) in vivo, but the molecular basis of the effect was not defined. Here coculturing human MSCs (hMSCs) with rat NSCs (rNSCs) was found to stimulate astrocyte and oligodendrocyte differentiation of the rNSCs. To survey the signaling pathways involved, RNA from the cocultures was analyzed by species-specific microarrays. In the hMSCs, there was an upregulation of transcripts for several secreted factors linked to differentiation: bone morphogenetic protein 1 (BMP1), hepatocyte growth factor (HGF), and transforming growth factor isoforms (TGFβ1 and TGFβ3). In both the hMSCs and the rNSCs, there was an upregulation of transcripts for Notch signaling. The role of TGFβ1 was verified by the demonstration that hMSCs in coculture increased secretion of TGFβ1, the rNSCs expressed the receptor, and an inhibitor of TGFβ signaling blocked differentiation. The role of Notch signaling was verified by the demonstration that in the cocultures hMSCs expressed a Notch ligand at sites of cell contact with rNSCs, and the rNSCs expressed the receptor, Notch 1. Increased Notch signaling in both cell types was then demonstrated by assays of transcript expression and by a reporter construct for downstream targets of Notch signaling. The results demonstrated that glial differentiation of the rNSCs in the cocultures was driven by increased secretion of soluble factors such as TGFβ1 by the hMSCs and probably through increased cell contact signaling between the hMSCs and rNSCs through the Notch pathway.

AIM Mesenchymal stromal cells from human bone marrow (hMSCs) were observed to produce therapeutic benefits in some models for cardiac and vascular injuries but their mode of action was not defined. We tested the effects of hMSCs in models for restricted vascular flow. METHODS We made model for restricted vascular flow produced by permanent ligation of a carotid artery and injected hMSCs to clarify the effects of hMSCs to vascular lesions. RESULTS Seven, 14, and 28 days after infusion of hMSCs into the cardiac left ventricle of the mice, there was a significant reduction in neointimal hyperplasia (p

There has been considerable interest in developing new therapies with adult multipotent progenitor stromal cells or mesenchymal stem cells (MSCs) in organ replacement and repair. To be effectively seeded into scaffolds for therapy, large numbers of cells are needed, but concerns remain regarding their chromatin stability in long-term culture. We therefore expanded four donors of human MSCs (hMSCs) from bone marrow aspirates with a protocol that maintains the cells at low density. MSCs initially proliferated at average doubling times of 24 h and then gradually reached senescence after 8-15 passages (33-55 population doublings) without evidence of immortalization. Comparative genomic hybridization assays of two preparations revealed no abnormalities through 33 population doublings. One preparation had a small amplification of unknown significance in chromosome 7 (7q21:11) after 55 population doublings. Microarray assays demonstrated progressive changes in the transcriptome of the cells. However, the transcriptomes clustered more closely over time within a single passage, rather than with passage number, indicating a partial reversibility of the patterns of gene expression. One of the largest changes was a decrease in mRNA for Sox11, a transcription factor previously identified in neural progenitor cells. Knockdown of Sox11 with siRNA decreased the proliferation and osteogenic differentiation potential of hMSCs. The results suggested that assays for Sox11 may provide a biomarker for early progenitor hMSCs.

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.

Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits beta1, beta2, and alpha3, 20%-55% expressed alpha1, alpha2, alpha4, alpha5, alpha6, and alphaV, and about 10% expressed beta3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: beta5, alpha7, alpha8, alpha9, alpha11, alphaX, and alphaD. Expression of some integrins changed with MSC confluency: integrins beta3, alpha1, alpha3, alpha5, and alphaV increased, and alpha6 decreased. Furthermore, alpha4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-beta5 reduced MSC adhesion to all types of ECs, anti-beta1 to both HUVEC and HPAEC, anti-beta3 to HUVEC, and anti-beta2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-alphaX reduced adhesion to HPAEC and HMVEC-C, anti-alphaV to HPAEC, and both anti-alpha7 and anti-alphaD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro.

SummaryQuantitative assays for human DNA and mRNA were used to examine the paradox that intravenously (i.v.) infused human multipotent stromal cells (hMSCs) can enhance tissue repair without significant engraftment. After 2 × 106 hMSCs were i.v. infused into mice, most of the cells were trapped as emboli in lung. The cells in lung disappeared with a half-life of about 24 hr, but

Research on stem cells has progressed at a rapid pace and, as might be anticipated, the results have generated several controversies, a few myths and a change in a major paradigm. Some of these issues will be reviewed in this study with special emphasis on how they can be applied to the adult stem/progenitor cells from bone marrow, referred to as MSCs.

Multipotent stromal cells (MSCs) have been shown to reduce apoptosis in injured cells by secretion of paracrine factors, but these factors were not fully defined. We observed that coculture of MSCs with previously UV-irradiated fibroblasts reduced apoptosis of the irradiated cells, but fresh MSC conditioned medium was unable reproduce the effect. Comparative microarray analysis of MSCs grown in the presence or absence of UV-irradiated fibroblasts demonstrated that the MSCs were activated by the apoptotic cells to increase synthesis and secretion of stanniocalcin-1 (STC-1), a peptide hormone that modulates mineral metabolism and has pleiotrophic effects that have not been fully characterized. We showed that STC-1 was required but not sufficient for reduction of apoptosis of UV-irradiated fibroblasts. In contrast, we demonstrated that MSC-derived STC-1 was both required and sufficient for reduction of apoptosis of lung cancer epithelial cells made apoptotic by incubation at low pH in hypoxia. Our data demonstrate that STC-1 mediates the antiapoptotic effects of MSCs in two distinct models of apoptosis in vitro. STEM CELLS2009;27:670–681

We screened for surface proteins expressed only by the early progenitor cells present in low-passage, low-density cultures of the adult stem/progenitor cells from bone marrow referred to as mesenchymal stem cells or multipotent stromal cells (MSCs). Six proteins were identified that were selectively expressed in the early progenitors: podocalyxin-like protein (PODXL), α6-integrin (CD49f), α4-integrin (CD49d), c-Met, CXCR4, and CX3CR1. All were previously shown to be involved in cell trafficking or tumor progression. Antibodies to CD49f and PODXL, a sialomucin in the CD34 family, were the most robust for FACScan assays. PODXLhi/CD49fhi MSCs were more clonogenic and differentiated more efficiently than PODXLlo/CD49flo cells. Inhibition of expression of PODXL with RNA interference caused aggregation of the cells. Furthermore, PODXLhi/CD49fhi MSCs were less prone to produce lethal pulmonary emboli, and larger numbers were recovered in heart and kidney after intravenous infusion into mice with myocardial infarcts.

Objective Human multipotent stromal cells readily form single-cell–derived colonies when plated at clonal densities. However, the colonies are heterogeneous because cells from a colony form new colonies that vary in size and differentiation potential when replated at clonal densities. The experiments here tested the hypothesis that cells in the inner regions of colonies are partially differentiated, but the differentiation is reversible. Materials and Methods Cells were separately isolated from the dense inner (IN) regions and less-dense outer regions (OUT) of single-cell–derived colonies. Cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation. Results IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when replated at clonal densities and assayed for rates of propagation and clonogenicity. Also, colonies formed by IN cells were similar to colonies formed by OUT cells because they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30to75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded. Conclusions The results demonstrated that cells in the IN region of single-cell–derived colonies are partially differentiated, but the differentiation can be reversed by replating the cells at clonal densities.

The University of Vermont College of Medicine and the Vermont Lung Center, with support of the National Heart, Lung, and Blood Institute (NHLBI), the Alpha-1 Foundation, the American Thoracic Society, the Emory Center for Respiratory Health,the Lymphangioleiomyomatosis (LAM) Treatment Alliance,and the Pulmonary Fibrosis Foundation, convened a workshop,‘‘Stem Cells and Cell Therapies in Lung Biology and Lung Diseases,’’ held July 26-29, 2009 at the University of Vermont,to review the current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy approaches for lung diseases. These are rapidly expanding areas of study that provide further insight into and challenge traditional views of the mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, discuss and debate current controversies, and identify future research directions and opportunities for both basic and translational research in cell-based therapies for lung diseases.

Canonical Wnt signaling is an osteo-inductive signal that promotes bone repair through acceleration of osteogenic differentiation by progenitors. Dkk-1 is a secreted inhibitor of canonical Wnt signaling and thus inhibits osteogenesis. To examine a potential osteo-inhibitory role of Dkk-1 in osteosarcoma (OS), we measured serum Dkk-1 in pediatric patients with OS (median age, 13.4 years) and found it to be significantly elevated. We also found that Dkk-1 was maximally expressed by the OS cells at the tumor periphery and in vitro Dkk-1 and RANKL are co-expressed by rapidly proliferating OS cells. Both Dkk-1 and conditioned media from OS cells reduces osteogenesis by human mesenchymal cells and by immuno-depletion of Dkk-1, or by adding a GSK3[beta] inhibitor, the effects of Dkk-1 were attenuated. In mice, we found that the expression of Dkk-1 from implanted tumors was similar to the human tumor biopsies in that human Dkk-1 was present in the serum of recipient animals. These data demonstrate that systemic levels of Dkk-1 are elevated in osteosarcoma. Furthermore, the expression of Dkk-1 by the OS cells at the periphery of the tumor probably contributes to its expansion by inhibiting repair of the surrounding bone. These data demonstrate that Dkk-1 may serve as a prognostic or diagnostic marker for evaluation of OS and furthermore, immuno-depletion of Dkk-1 or administration of GSK3[beta] inhibitors could represent an adjunct therapy for this disease.

Human mesenchymal stem cells, or multipotent stromal cells (MSCs), are of interest for clinical therapy, in part because of their capacity for proliferation and differentiation. However, results from clinical trials and in vitro models have been variable, possibly because of MSC heterogeneity and a lack of standardization between MSC in vitro expansion protocols. Here we defined changes in MSCs during expansion in vitro. In low-density cultures, MSCs expand through distinct lag, exponential growth, and stationary phases. We assayed cultures of passage 2 human MSCs from three donors at low density (50 cells per cm2) at approximately 5% confluence on day 2 and after the cultures had expanded to approximately 70% confluence on day 7. On day 2, genes involved in cell division were upregulated. On day 7, genes for cell development were upregulated. The variations among three donors were less than the variation within the expansion of MSCs from a single donor. The microarray data for selected genes were confirmed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and FACScan analysis. Approximately 50% of cells at day 2 were in S-phase compared with 10% at day 7. The results demonstrated major differences in early and late stage cultures of MSCs that should be considered in using the cells in experiments and clinical applications. Disclosure of potential conflicts of interest is found at the end of this article.

Mesenchymal stem cells or multipotent stromal cells (MSCs) isolated from the bone marrow of adult organisms were initially characterized as plastic adherent, fibroblastoid cells with the capacity to generate heterotopic osseous tissue when transplanted in vivo. In recent years, MSCs or MSC-like cells have been shown to reside within the connective tissue of most organs, and their surface phenotype has been well described. A large number of reports have also indicated that the cells possess the capacity to transdifferentiate into epithelial cells and lineages derived from the neuroectoderm. The broad developmental plasticity of MSCs was originally thought to contribute to their demonstrated efficacy in a wide variety of experimental animal models of disease as well as in human clinical trials. However, new findings suggest that the ability of MSCs to alter the tissue microenvironment via secretion of soluble factors may contribute more significantly than their capacity for transdifferentiation in tissue repair. Herein, we critically evaluate the literature describing the plasticity of MSCs and offer insight into how the molecular and functional heterogeneity of this cell population, which reflects the complexity of marrow stroma as an organ system, may confound interpretation of their transdifferentiation potential. Additionally, we argue that this heterogeneity also provides a basis for the broad therapeutic efficacy of MSCs. Disclosure of potential conflicts of interest is found at the end of this article.