Aline M. Betancourt

A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype.

Background Our laboratory and others reported that the stimulation of specific Toll-like receptors (TLRs) affects the immune modulating responses of human multipotent mesenchymal stromal cells (hMSCs). Toll-like receptors recognize “danger” signals, and their activation leads to profound cellular and systemic responses that mobilize innate and adaptive host immune cells. The danger signals that trigger TLRs are released following most tissue pathologies. Since danger signals recruit immune cells to sites of injury, we reasoned that hMSCs might be recruited in a similar way. Indeed, we found that hMSCs express several TLRs (e.g., TLR3 and TLR4), and that their migration, invasion, and secretion of immune modulating factors is drastically affected by specific TLR-agonist engagement. In particular, we noted diverse consequences on the hMSCs following stimulation of TLR3 when compared to TLR4 by our low-level, short-term TLR-priming protocol. Principal Findings Here we extend our studies on the effect on immune modulation by specific TLR-priming of hMSCs, and based on our findings, propose a new paradigm for hMSCs that takes its cue from the monocyte literature. Specifically, that hMSCs can be polarized by downstream TLR signaling into two homogenously acting phenotypes we classify here as MSC1 and MSC2. This concept came from our observations that TLR4-primed hMSCs, or MSC1, mostly elaborate pro-inflammatory mediators, while TLR3-primed hMSCs, or MSC2, express mostly immunosuppressive ones. Additionally, allogeneic co-cultures of TLR-primed MSCs with peripheral blood mononuclear cells (PBMCs) predictably lead to suppressed T-lymphocyte activation following MSC2 co-culture, and permissive T-lymphocyte activation in co-culture with MSC1. Significance Our study provides an explanation to some of the conflicting reports on the net effect of TLR stimulation and its downstream consequences on the immune modulating properties of stem cells. We further suggest that MSC polarization provides a convenient way to render these heterogeneous preparations of cells more uniform while introducing a new facet to study, as well as provides an important aspect to consider for the improvement of current stem cell-based therapies.

Mesenchymal Stem Cell 1 (MSC1)-Based Therapy Attenuates Tumor Growth Whereas MSC2-Treatment Promotes Tumor Growth and Metastasis

Background Currently, there are many promising clinical trials using mesenchymal stem cells (MSCs) in cell-based therapies of numerous diseases. Increasingly, however, there is a concern over the use of MSCs because they home to tumors and can support tumor growth and metastasis. For instance, we established that MSCs in the ovarian tumor microenvironment promoted tumor growth and favored angiogenesis. In parallel studies, we also developed a new approach to induce the conventional mixed pool of MSCs into two uniform but distinct phenotypes we termed MSC1 and MSC2. Methodology/Principal Findings Here we tested the in vitro and in vivo stability of MSC1 and MSC2 phenotypes as well as their effects on tumor growth and spread. In vitro co-culture of MSC1 with various cancer cells diminished growth in colony forming units and tumor spheroid assays, while conventional MSCs or MSC2 co-culture had the opposite effect in these assays. Co-culture of MSC1 and cancer cells also distinctly affected their migration and invasion potential when compared to MSCs or MSC2 treated samples. The expression of bioactive molecules also differed dramatically among these samples. MSC1-based treatment of established tumors in an immune competent model attenuated tumor growth and metastasis in contrast to MSCs- and MSC2-treated animals in which tumor growth and spread was increased. Also, in contrast to these groups, MSC1-therapy led to less ascites accumulation, increased CD45+leukocytes, decreased collagen deposition, and mast cell degranulation. Conclusion/Significance These observations indicate that the MSC1 and MSC2 phenotypes may be convenient tools for the discovery of critical components of the tumor stroma. The continued investigation of these cells may help ensure that cell based-therapy is used safely and effectively in human disease.

New concepts on the immune modulation mediated by mesenchymal stem cells

Mesenchymal stem cells (MSCs) are the nonhematopoietic multipotent progenitor cells found in various adult tissues. They are characterized by their ease of isolation and their rapid growth in vitro while maintaining their differentiation potential, allowing for extensive expansion in culture that yields large quantities suitable for therapeutic use. This article reviews the immunomodulatory activities associated with MSCs. Numerous studies have demonstrated that MSCs are potently immunosuppressive in vitro and in vivo. However, this article presents a new paradigm in MSC biology, in which MSCs, at least in vitro, can undergo polarization into either a pro-inflammatory or an immunosuppressive phenotype.

Emerging roles of the host defense peptide LL-37 in human cancer and its potential therapeutic applications

Human cathelicidin LL‐37, a host defense peptide derived from leukocytes and epithelial cells, plays a crucial role in innate and adaptive immunity. Not only does LL‐37 eliminate pathogenic microbes directly but also modulates host immune responses. Emerging evidence from tumor biology studies indicates that LL‐37 plays a prominent and complex role in carcinogenesis. Although overexpression of LL‐37 has been implicated in the development or progression of many human malignancies, including breast, ovarian and lung cancers, LL‐37 suppresses tumorigenesis in gastric cancer. These data are beginning to unveil the intricate and contradictory functions of LL‐37. The reasons for the tissue‐specific function of LL‐37 in carcinogenesis remain to be elucidated. Here, we review the relationship between LL‐37, its fragments and cancer progression as well as discuss the potential therapeutic implications of targeting this peptide.

Comparison of the therapeutic effects of human and mouse adipose-derived stem cells in a murine model of lipopolysaccharide-induced acute lung injury.

IntroductionAdipose-derived stem cells (ASCs) have emerged as important regulators of inflammatory/immune responses in vitro and in vivo and represent attractive candidates for cell-based therapies for diseases that involve excessive inflammation. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive due to lack of effective therapies. In this study, the therapeutic effects of ASC-based therapy were assessed in vivo by comparison of the anti-inflammatory properties of both human and murine ASCs in a mouse model of lipopolysaccharide (LPS)-induced ALI.MethodsHuman ASCs (hASCs) or mouse ASCs (mASCs) were delivered to C57Bl/6 mice (7.5 × 105 total cells/mouse) by oropharyngeal aspiration (OA) four hours after the animals were challenged with lipopolysaccharide (15 mg/kg). Mice were sacrificed 24 and 72 hours after LPS exposure, and lung histology examined for evaluation of inflammation and injury. Bronchoalveolar lavage fluid (BALF) was analyzed to determine total and differential cell counts, total protein and albumin concentrations, and myeloperoxidase (MPO) activity. Cytokine expression in the injured lungs was measured at the steady-state mRNA levels and protein levels for assessment of the degree of lung inflammation.ResultsBoth human and mouse ASC treatments provided protective anti-inflammatory responses. There were decreased levels of leukocyte (for example neutrophil) migration into the alveoli, total protein and albumin concentrations in BALF, and MPO activity after the induction of ALI following both therapies. Additionally, cell therapy with both cell types effectively suppressed the expression of proinflammatory cytokines and increased the anti-inflammatory cytokine interleukin 10 (IL-10). Overall, the syngeneic mASC therapy had a more potent therapeutic effect than the xenogeneic hASC therapy in this model.ConclusionsTreatment with hASCs or mASCs significantly attenuated LPS-induced acute lung injury in mice. These results suggest a potential benefit for using an ASC-based therapy to treat clinical ALI and may possibly prevent the development of acute respiratory distress syndrome (ARDS).

Anti-Inflammatory Mesenchymal Stem Cells (MSC2) Attenuate Symptoms of Painful Diabetic Peripheral Neuropathy

Mesenchymal stem cells (MSCs) are very attractive candidates in cell‐based strategies that target inflammatory diseases. Preclinical animal studies and many clinical trials have demonstrated that human MSCs can be safely administered and that they modify the inflammatory process in the targeted injured tissue. Our laboratory developed a novel method that optimizes the anti‐inflammatory effects of MSCs. We termed the cells prepared by this method MSC2. In this study, we determined the effects of MSC2‐based therapies on an inflammation‐linked painful diabetic peripheral neuropathy (pDPN) mouse model. Streptozotocin‐induced diabetic mice were treated with conventionally prepared MSCs, MSC2, or vehicle at three specific time points. Prior to each treatment, responses to radiant heat (Hargreaves) and mechanical stimuli (von Frey) were measured. Blood serum from each animal was collected at the end of the study to compare levels of inflammatory markers between the treatment groups. We observed that MSC2‐treated mice had significant improvement in behavioral assays compared with the vehicle and MSC groups, and moreover these responses did not differ from the observations seen in the healthy wild‐type control group. Mice treated with conventional MSCs showed significant improvement in the radiant heat assay, but not in the von Frey test. Additionally, mice treated with MSC2 had decreased serum levels in many proinflammatory cytokines compared with the values measured in the MSC‐ or vehicle‐treated groups. These findings indicate that MSC2‐based therapy is a new anti‐inflammatory treatment to consider in the management of pDPN.

Interleukin 6 Mediates the Therapeutic Effects of Adipose-Derived Stromal/Stem Cells in Lipopolysaccharide-Induced Acute Lung Injury

Adipose‐derived stromal/stem cells (ASCs) have anti‐inflammatory as well as immunosuppressive activities and are currently the focus of clinical trials for a number of inflammatory diseases. Acute lung injury (ALI) is an inflammatory condition of the lung for which standard treatment is mainly supportive due to lack of effective therapies. Our recent studies have demonstrated the ability of both human ASCs (hASCs) and mouse ASCs (mASCs) to attenuate lung damage and inflammation in a rodent model of lipopolysaccharide‐induced ALI, suggesting that ASCs may also be beneficial in treating ALI. To better understand how ASCs may act in ALI and to elucidate the mechanism(s) involved in ASC modulation of lung inflammation, gene expression analysis was performed in ASC‐treated (hASCs or mASCs) and control sham‐treated lungs. The results revealed a dramatic difference between the expression of anti‐inflammatory molecules by hASCs and mASCs. These data show that the beneficial effects of hASCs and mASCs in ALI may result from the production of different paracrine factors. Interleukin 6 (IL‐6) expression in the mASC‐treated lungs was significantly elevated as compared to sham‐treated controls 20 hours after delivery of the cells by oropharyngeal aspiration. Knockdown of IL‐6 expression in mASCs by RNA interference abrogated most of their therapeutic effects, suggesting that the anti‐inflammatory properties of mASCs in ALI are explained, at least in part, by activation of IL‐6 secretion. Stem Cells 2014;32:1616–1628

Nonhuman primate lung decellularization and recellularization using a specialized large-organ bioreactor.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organs natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use. The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Treating Chronic Pain with Mesenchymal Stem Cells: A TherapeuticApproach Worthy of Continued Investigation

Though there are various etiologies to chronic pain, one common feature is that painful states are associated with increased inflammation. This is verified by studies in both animal models and humans that have found increased expression of inflammatory proteins in muscle tissue, and increased inflammatory cytokines in cerebral spinal fluid, synovial fluid, and serum. Over the last decade we have become aware of the anti-inflammatory effects that multipotent mesenchymal stromal cells frequently referred to as mesenchymal stem cells [MSC], elicit. This ability of MSC to affect the inflammatory milieu has led researchers to consider MSC as a treatment for various painful states such as degenerative disc disease and osteoarthritis. In this article we present relevant animal and human studies, which indicate that MSC are worthy of further study as a valuable therapy in the treatment of chronic pain.

Toll-like receptor 3 and suppressor of cytokine signaling proteins regulate CXCR4 and CXCR7 expression in bone marrow-derived human multipotent stromal cells.

Background The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to “danger” signals – by-products of damaged, infected or inflamed tissues – via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC. Methodology/Principal Findings We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization. Conclusion/Significance These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals.