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1.  Mesenchymal Stem Cell 1 (MSC1)-Based Therapy Attenuates Tumor Growth Whereas MSC2-Treatment Promotes Tumor Growth and Metastasis 
PLoS ONE  2012;7(9):e45590.
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.
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.
PMCID: PMC3447765  PMID: 23029122
2.  Interleukin 6 Mediates the Therapeutic Effects of Adipose-Derived Stromal/Stem Cells in Lipopolysaccharide-Induced Acute Lung Injury 
Stem cells (Dayton, Ohio)  2014;32(6):1616-1628.
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.
PMCID: PMC4365913  PMID: 24449042
Adipose-derived stromal/stem cell; Acute lung injury; Interleukin 6; Mouse
3.  Pericyte Dynamics during Angiogenesis: New Insights from New Identities 
Journal of vascular research  2014;51(3):163-174.
Therapies aimed at manipulating the microcirculation require the ability to control angiogenesis, defined as the sprouting of new capillaries from existing vessels. In multiple pathologies (cancer, retinopathies, rheumatoid arthritis), blocking angiogenesis would be beneficial. In others (myocardial infarction, stroke, hypertension), promoting angiogenesis would be desirable. We know vascular pericytes elongate around endothelial cells and are functionally associated with regulating vessel stabilization, vessel diameter, and endothelial cell proliferation. During angiogenesis, bidirectional pericyte-endothelial cell signaling is critical for capillary sprout formation. Observations of pericytes leading capillary sprouts also implicate their role in endothelial cell guidance. As such, pericytes have recently emerged as a therapeutic target to promote or inhibit angiogenesis. Advancing our basic understanding of pericytes and the ability to develop pericyte-related therapies are challenged, like in many other fields, by questions regarding cell identity. This review article discusses what we know about pericyte phenotypes and the opportunity to advance our understanding of pericytes by defining specific pericyte cell populations involved in capillary sprouting.
PMCID: PMC4149862  PMID: 24853910
pericyte; angiogenesis; capillary; sprouting; phenotype
4.  Hypertensive Rat Lungs Retain Hallmarks of Vascular Disease upon Decellularization but Support the Growth of Mesenchymal Stem Cells 
Tissue Engineering. Part A  2014;20(9-10):1426-1443.
There are an insufficient number of donor organs available to meet the demand for lung transplantation. This issue could be addressed by regenerating functional tissue from diseased or damaged lungs that would otherwise be deemed unsuitable for transplant. Detergent-mediated whole-lung decellularization produces a three-dimensional natural scaffold that can be repopulated with various cell types. In this study, we investigated the decellularization and initial recellularization of diseased lungs using a rat model of monocrotaline-induced pulmonary hypertension (MCT-PHT). Decellularization of control and MCT-PHT Sprague-Dawley rat lungs was accomplished by treating the lungs with a combination of Triton X-100, sodium deoxycholate, NaCl, and DNase. The resulting acellular matrices were characterized by DNA quantification, Western blotting, immunohistochemistry, and proteomic analyses revealing that decellularization was able to remove cells while leaving the extracellular matrix (ECM) components and lung ultrastructure intact. Decellularization significantly reduced DNA content (∼30-fold in MCT-PHT lungs and ∼50-fold in the control lungs) and enriched ECM components (>60-fold in both the control and MCT-PHT lungs) while depleting cellular proteins. MicroCT visualization of MCT-PHT rat lungs indicated that the vasculature was narrowed as a result of MCT treatment, and this characteristic was unchanged by decellularization. Mean arterial vessel diameter of representative decellularized MCT-PHT and control scaffolds was estimated to be 0.152±0.134 mm and 0.247±0.160 mm, respectively. Decellularized MCT-PHT lung scaffolds supported attachment and survival of rat adipose-derived stem cells (rASCs), seeded into the airspace or the vasculature, for at least 2 weeks. The cells seeded in MCT-PHT lung scaffolds proliferated and underwent apoptosis similar to control scaffolds; however, the initial percentage of apoptotic cells was slightly higher in MCT-PHT lungs (2.79±2.03% vs. 1.05±1.02% of airway-seeded rASCs, and 4.47±1.21% vs. 2.66±0.10% of vascular seeded rASCs). The ECM of cell-seeded scaffolds showed no signs of degradation by the cells after 14 days in culture. These data suggest that diseased hypertensive lungs can be efficiently decellularized similar to control lungs and have the potential to be recellularized with mesenchymal stem cells with the ultimate goal of generating healthy, functional pulmonary tissue.
PMCID: PMC4011420  PMID: 24378017
5.  Impact of primary influenza infection on the immune response to secondary bacterial infection in aged mice 
Influenza and other respiratory viruses  2011;5(Suppl 1):198-201.
Increased susceptibility of older populations to secondary bacterial pneumonia-like infections following influenza infection has been well documented.1 Recent evidence in mouse models suggests that this increased risk from secondary bacterial infection occurs through a desensitization of the innate immune response.2 This recent finding, however, does not account for potential differences in immune responsiveness due to age.
Materials and methods
To address this parameter, we used three age groups (aged, adult, and young mice) to evaluate the role of age in influenza-mediated vulnerability to secondary bacterial challenge with Pseudomonas aeruginosa. All mice were evaluated for multiple parameters including: (i) survival; (ii) lung bacterial load; (iii) total lung protein content; (iv) immune cell infiltration; (v) cytokine/chemokine expression; and (vi) toll-like receptor (TLR) RNA expression profiles.
Prior challenge with influenza contributed to aberrant cytokine/chemokine profiles and increased lung cellular infiltrate in response to secondary bacterial infection across all age groups, supporting a critical role for influenza infection in the alteration of immune responses to other pathogens. Also similar to human influenza, these changes were exacerbated by age in mice as demonstrated by increased bacterial load, mortality, and total lung protein content (an indicator of lung damage) after P. aeruginosa challenge.
These data support a potential role for virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and the increased susceptibility of influenza virus infected mice to secondary bacterial infection. The understanding of the complex interaction of host and pathogen – and the role of age – in human influenza is critical in the development of novel therapeutics and improved vaccine approaches for influenza. Our results support further examination of influenza-mediated alterations in innate immune responses in aged and non-aged animals to allow elucidation of the molecular mechanisms of influenza pathogenesis in humans.
PMCID: PMC4172348  PMID: 21477139
Aging; bacteria; innate immunity; secondary infection; secondary infection; toll-like receptors
6.  A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells 
Tissue Engineering. Part A  2012;18(23-24):2437-2452.
Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (∼92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.
PMCID: PMC3501118  PMID: 22764775
7.  Anti-Inflammatory Mesenchymal Stem Cells (MSC2) Attenuate Symptoms of Painful Diabetic Peripheral Neuropathy 
This study determined the effects of MSC2-based therapies on an inflammation-linked painful diabetic peripheral neuropathy (pDPN) mouse model. Findings indicate that MSC2-based therapy is a new anti-inflammatory treatment to consider in the management of pDPN.
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.
PMCID: PMC3659725  PMID: 23197860
Adult stem cells; Bone marrow stromal cells; Cellular therapy; Diabetes; Neuropathy; Mesenchymal stem cells; Marrow stromal stem cells; Immunotherapy
8.  Comparison of the therapeutic effects of human and mouse adipose-derived stem cells in a murine model of lipopolysaccharide-induced acute lung injury 
Adipose-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.
Human 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.
Both 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.
Treatment 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).
PMCID: PMC3706907  PMID: 23360775
9.  Toll-Like Receptor 3 and Suppressor of Cytokine Signaling Proteins Regulate CXCR4 and CXCR7 Expression in Bone Marrow-Derived Human Multipotent Stromal Cells 
PLoS ONE  2012;7(6):e39592.
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.
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.
PMCID: PMC3382127  PMID: 22745793
10.  Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis 
Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.
PMCID: PMC3466932  PMID: 22643964
Cellular Biology;  Issue 63;  mesentery;  rat;  angiogenesis;  microcirculation;  microvascular;  remodeling
11.  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.
PMCID: PMC3025436  PMID: 21092149
12.  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 it eliminate pathogenic microbes directly, LL-37 also modulates host immune responses. Emerging evidence from tumor biology studies indicates that LL-37 plays a prominent and complex role in carcinogenesis. While 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.
PMCID: PMC2930073  PMID: 20521250
13.  A New Mesenchymal Stem Cell (MSC) Paradigm: Polarization into a Pro-Inflammatory MSC1 or an Immunosuppressive MSC2 Phenotype 
PLoS ONE  2010;5(4):e10088.
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.
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.
PMCID: PMC2859930  PMID: 20436665

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