Ruth S. Waterman

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.

Toll‐Like Receptors on Human Mesenchymal Stem Cells Drive Their Migration and Immunomodulating Responses

Adult human bone marrow‐derived mesenchymal stem cells (hMSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. To achieve the desired clinical outcomes for this strategy requires a better understanding of the mechanisms that drive the recruitment, migration, and engraftment of hMSCs to the targeted tissues. It is known that hMSCs are recruited to sites of stress or inflammation to fulfill their repair function. It is recognized that toll‐like receptors (TLRs) mediate stress responses of other bone marrow‐derived cells. This study explored the role of TLRs in mediating stress responses of hMSCs. Accordingly, the presence of TLRs in hMSCs was initially established by reverse transcription‐polymerase chain reaction assays. Flow cytometry and fluorescence immunocytochemical analyses confirmed these findings. The stimulation of hMSCs with TLR agonists led to the activation of downstream signaling pathways, including nuclear factor κB, AKT, and MAPK. Consequently, activation of these pathways triggered the induction and secretion of cytokines, chemokines, and related TLR gene products as established from cDNA array, immunoassay, and cytokine antibody array analyses. Interestingly, the unique patterns of affected genes, cytokines, and chemokines measured identify these receptors as critical players in the clinically established immunomodulation observed for hMSCs. Lastly, hMSC migration was promoted by TLR ligand exposure as demonstrated by transwell migration assays. Conversely, disruption of TLRs by neutralizing TLR antibodies compromised hMSC migration. This study defines a novel TLR‐driven stress and immune modulating response for hMSCs that is critical to consider in the design of 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.

Ovarian cancers overexpress the antimicrobial protein hCAP-18 and its derivative LL-37 increases ovarian cancer cell proliferation and invasion

The role of the pro‐inflammatory peptide, LL‐37, and its pro‐form, human cationic antimicrobial protein 18 (hCAP‐18), in cancer development and progression is poorly understood. In damaged and inflamed tissue, LL‐37 functions as a chemoattractant, mitogen and pro‐angiogenic factor suggesting that the peptide may potentiate tumor progression. The aim of this study was to characterize the distribution of hCAP‐18/LL‐37 in normal and cancerous ovarian tissue and to examine the effects of LL‐37 on ovarian cancer cells. Expression of hCAP‐18/LL‐37 was localized to immune and granulosa cells of normal ovarian tissue. By contrast, ovarian tumors displayed significantly higher levels of hCAP‐18/LL‐37 where expression was observed in tumor and stromal cells. Protein expression was statistically compared to the degree of immune cell infiltration and microvessel density in epithelial‐derived ovarian tumors and a significant correlation was observed for both. It was demonstrated that ovarian tumor tissue lysates and ovarian cancer cell lines express hCAP‐18/LL‐37. Treatment of ovarian cancer cell lines with recombinant LL‐37 stimulated proliferation, chemotaxis, invasion and matrix metalloproteinase expression. These data demonstrate for the first time that hCAP‐18/LL‐37 is significantly overexpressed in ovarian tumors and suggest LL‐37 may contribute to ovarian tumorigenesis through direct stimulation of tumor cells, initiation of angiogenesis and recruitment of immune cells. These data provide further evidence of the existing relationship between pro‐inflammatory molecules and ovarian cancer progression.

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.

Evaluation of the protective value of hospital gowns against blood strike-through and methicillin-resistant Staphylococcus aureus penetration

BACKGROUND Hospital gowns protect patients and health care workers from exposure to blood and other infectious materials. Previous studies have shown that certain gowns do allow blood strike-through. Because of worldwide increases in the incidence of Staphylococcus aureus infections, especially with methicillin-resistant strains, there is now increased concern regarding bacterial transmission through gowns. METHODS This study evaluated six gown types used in hospitals (one disposable cover or isolation gown, three disposable operating room gowns, and new and washed reusable operating room gowns). Gowns were evaluated for dry spore and S. aureus filtration efficiencies and were subjected to 20 time-pressure combinations with methicillin-resistant S. aureus-spiked blood (10(4)/ml) to evaluate blood strike-through and passage of methicillin-resistant S. aureus. RESULTS Blood strike-through was lowest with disposable operating room gowns 1 and 2 (polypropylene). Disposable operating room gown 3 (polyester-wood pulp) showed the greatest strike-through and overall passage of methicillin-resistant S. aureus. Operating room gowns 1 and 2 showed minimal bacterial passage, whereas the disposable cover (polypropylene) only allowed passage at pressures greater than 1 psi. Bacterial filtration efficiency testing showed operating room gowns 1 and 2 to be the most protective; operating room gown 3 and both reusable (cotton) gowns were the least protective. Dry spore passage was greatest for reusable gowns. CONCLUSION Different hospital gowns offer varying degrees of protection against fluid strike-through or bacterial passage. Gowns therefore should be chosen according to the task performed and conditions encountered.

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.

Dexmedetomidine: Clinical Application as an Adjunct for Intravenous Regional Anesthesia

The selective α-2 adrenoceptor agonist, dexmedetomidine, has been shown to be a useful, safe adjunct in perioperative medicine. Intravenous regional anesthesia is one of the simplest forms of regional anesthesia and has a high degree of success. However, intravenous regional anesthesia is limited by the development of tourniquet pain and its inability to provide postoperative analgesia. To improve block quality, prolong postdeflation analgesia, and decrease tourniquet pain, various chemical additives have been combined with local anesthetics, although with limited success. The antinociceptive effects of α-2 adrenoceptor agonists have been shown in animals and in humans. However, less is known about the clinical effects of dexmedetomidine when coadministered with local anesthetics in patients undergoing intravenous regional anesthesia. This review examines what is currently known to improve our understanding of the properties and application of dexmedetomidine when used as an adjunct in intravenous regional anesthesia.

The Role of Mesenchymal Stem Cells in the Tumor Microenvironment

Currently, there are many promising clinical trials using mesenchymal stem cells (MSCs) in cell-based therapies of diseases ranging widely from graft-versus-host to joint and cartilage disorders (Salem and Thiemermann 2010; Tolar, Le Blanc et al.). Increasingly, however, there is a concern over the clinical use of MSCs because they are also known to home to tumors and once resident in the tumor microenvironment (TME) to support tumor growth and spread (Karnoub, Dash et al. 2007; Kidd, Spaeth et al. 2008; Coffelt, Marini et al. 2009; Klopp, Gupta et al. 2010; Klopp, Gupta et al. 2011). Conversely, other studies have reported that MSCs found in the TME diminish tumor growth, which has further generated some controversy in this field (reviewed in (Klopp, Gupta et al. 2010; Klopp, Gupta et al. 2011). Either way as a result of the MSC propensity for the TME, genetically modified MSCs that can act as “Trojan horses” and deliver anti-cancer therapeutics into the tumor stroma are being evaluated as a promising new specific cell-based therapy for cancer.