Aideen E. Ryan

Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications

Mesenchymal stem (stromal) cells (MSCs) are multipotent cells with the ability to differentiate into several cell types, thus serving as a cell reservoir for regenerative medicine. Much of the current interest in therapeutic application of MSCs to various disease settings can be linked to their immunosuppressive and anti-inflammatory properties. One of the key mechanisms of MSC anti-inflammatory effects is the secretion of soluble factors with paracrine actions. Recently it has emerged that the paracrine functions of MSCs could, at least in part, be mediated by extracellular vesicles (EVs). EVs are predominantly released from the endosomal compartment and contain a cargo that includes miRNA, mRNA, and proteins from their cells of origin. Recent animal model-based studies suggest that EVs have significant potential as a novel alternative to whole cell therapies. Compared to their parent cells, EVs may have a superior safety profile and can be safely stored without losing function. In this article, we review current knowledge related to the potential use of MSC-derived EVs in various diseases and discuss the promising future for EVs as an alternative, cell-free therapy.

Mesenchymal stem cells enhance recovery and repair following ventilator-induced lung injury in the rat

Background Bone-marrow derived mesenchymal stem cells (MSCs) reduce the severity of evolving acute lung injury (ALI), but their ability to repair the injured lung is not clear. A study was undertaken to determine the potential for MSCs to enhance repair after ventilator-induced lung injury (VILI) and elucidate the mechanisms underlying these effects. Methods Anaesthetised rats underwent injurious ventilation which produced severe ALI. Following recovery, they were given an intravenous injection of MSCs (2×106 cells) or vehicle immediately and a second dose 24 h later. The extent of recovery following VILI was assessed after 48 h. Subsequent experiments examined the potential for non-stem cells and for the MSC secretome to enhance VILI repair. The contribution of specific MSC-secreted mediators was then examined in a wound healing model. Results MSC therapy enhanced repair following VILI. MSCs enhanced restoration of systemic oxygenation and lung compliance, reduced total lung water, decreased lung inflammation and histological lung injury and restored lung structure. They attenuated alveolar tumour necrosis factor α concentrations while increasing concentrations of interleukin 10. These effects were not seen with non-stem cells (ie, rat fibroblasts). MSC-secreted products also enhanced lung repair and attenuated the inflammatory response following VILI. The beneficial effect of the MSC secretome on repair of pulmonary epithelial wounds was attenuated by prior depletion of keratinocyte growth factor. Conclusion MSC therapy enhances lung repair following VILI via a paracrine mechanism that may be keratinocyte growth factor-dependent.

Anti-donor immune responses elicited by allogeneic mesenchymal stem cells: what have we learned so far?

Mesenchymal stem (stromal) cells (MSCs) have potent anti‐inflammatory/immunosuppressive properties which underlie much of their therapeutic potential. This fact has led to the widely accepted belief that MSCs from genetically unrelated individuals (allogeneic (allo)‐MSCs) can be used therapeutically with equal efficacy to autologous MSCs and without triggering the donor‐specific immune responses that are typically associated with allo‐transplants. In this article, we critically review available experimental data to determine whether good in vivo evidence exists in support of the ‘immune privileged’ status of allo‐MSCs. We also examine published studies regarding the immunogenicity of allo‐MSCs following activation (‘licensing’) by inflammatory stimuli or following differentiation. Among the identified studies which have addressed in vivo immunogenicity of allo‐MSCs, there was substantial variability as regards experimental species, disease model, route of MSC administration, cell dose and stringency of the immunological assays employed. Nonetheless, the majority of these studies has documented specific cellular (T‐cell) and humoral (B‐cell/antibody) immune responses against donor antigens following administration of non‐manipulated, interferon‐γ‐activated and differentiated allo‐MSCs. The consequences of such anti‐donor immune responses were also variable and ranged from reduced in vivo survival of allo‐MSCs with accelerated rejection of subsequent allogeneic transplants to apparent promotion of donor‐specific tolerance. On the basis of these findings and on existing knowledge of allo‐antigen recognition from the field of transplant immunology, we propose that the concept of the immune privileged nature of allo‐MSCs should be reconsidered and that the range and clinical implications of anti‐donor immune responses elicited by allo‐MSCs be more precisely studied in human and animal recipients.

Effects of Intratracheal Mesenchymal Stromal Cell Therapy during Recovery and Resolution after Ventilator-induced Lung Injury

Background:Mesenchymal stromal cells (MSCs) have been demonstrated to attenuate acute lung injury when delivered by intravenous or intratracheal routes. The authors aimed to determine the efficacy of and mechanism of action of intratracheal MSC therapy and to compare their efficacy in enhancing lung repair after ventilation-induced lung injury with intravenous MSC therapy. Methods:After induction of anesthesia, rats were orotracheally intubated and subjected to ventilation-induced lung injury (respiratory rate 18 min−1, Pinsp 35 cm H2O,) to produce severe lung injury. After recovery, animals were randomized to receive: (1) no therapy, n = 4; (2) intratracheal vehicle (phosphate-buffered saline, 300 µl, n = 8); (3) intratracheal fibroblasts (4 × 106 cells, n = 8); (4) intratracheal MSCs (4 × 106 cells, n = 8); (5) intratracheal conditioned medium (300 µl, n = 8); or (6) intravenous MSCs (4 × 106 cells, n = 4). The extent of recovery after acute lung injury and the inflammatory response was assessed after 48 h. Results:Intratracheal MSC therapy enhanced repair after ventilation-induced lung injury, improving arterial oxygenation (mean ± SD, 146 ± 3.9 vs. 110.8 ± 21.5 mmHg), restoring lung compliance (1.04 ± 0.11 vs. 0.83 ± 0.06 ml·cm H2O−1), reducing total lung water, and decreasing lung inflammation and histologic injury compared with control. Intratracheal MSC therapy attenuated alveolar tumor necrosis factor-&agr; (130 ± 43 vs. 488 ± 211 pg·ml−1) and interleukin-6 concentrations (138 ± 18 vs. 260 ± 82 pg·ml−1). The efficacy of intratracheal MSCs was comparable with intravenous MSC therapy. Intratracheal MSCs seemed to act via a paracine mechanism, with conditioned MSC medium also enhancing lung repair after injury. Conclusions:Intratracheal MSC therapy enhanced recovery after ventilation-induced lung injury via a paracrine mechanism, and was as effective as intravenous MSC therapy.

Chondrogenic Differentiation Increases Antidonor Immune Response to Allogeneic Mesenchymal Stem Cell Transplantation

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3(+) and CD68(+) infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use.

Changes in immunological profile of allogeneic mesenchymal stem cells after differentiation: should we be concerned?

Mesenchymal stem cells (MSCs) are an adult stromal cell population possessing potent differentiation capacity and a potential for use across major histocompatibility complex barriers. Although allogeneic MSCs have potent immunosuppressive properties, evidence also suggests that they elicit a weak allogeneic immune response. However, the effect of induced differentiation on the immunosuppressive ability and immunogenicity of allogeneic MSCs is a potential obstacle when applying MSCs in tissue replacement therapies. These concerns will be explored in this review, with particular emphasis on changes in the cell surface expression of immunogenic markers, changes in the secretion of immunosuppressive molecules and in vivo functional benefits of the cell therapy. We review the literature from a translational point of view, focusing on pre-clinical studies that have utilised and analysed the effects of allogeneic immune responses on the ability of allogeneic MSCs to regenerate damaged tissue in models of bone, heart and cartilage defects.

Autophagosomal IκBα Degradation Plays a Role in the Long Term Control of Tumor Necrosis Factor-α-induced Nuclear Factor-κB (NF-κB) Activity

Transcription factor NF-κB is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-κB is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-α on NF-κB activity for up to 48 h in intestinal epithelial cells. Results show that NF-κB remained persistently activated up to 48 h after TNF-α and that the long term activation of NF-κB was accompanied by a biphasic degradation of IκBα. The first phase of IκBα degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-α stimulated formation of autophagosomes in intestinal epithelial cells and that IκBα co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-α-induced degradation of IκBα and lowered NF-κB target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-κB activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-κB may mediate this effect.

Fas Ligand Promotes Tumor Immune Evasion of Colon Cancer In Vivo

The study of the role of Fas ligand (FasL/CD95L) in tumor immune evasion has been complicated by the discovery that FasL may trigger cytokine secretion and induce inflammation. Antisense suppression of FasL expression by colon tumor cells was used to investigate if a reduction in endogenously expressed FasL in tumors resulted in reduced tumor development and improved anti-tumor immune challenge in vivo. Downregulation of FasL expression had no effect on tumor growth in vitro but significantly reduced tumor development in syngeneic immune-competent mice in vivo. Tumor size was also significantly decreased. Reduced FasL expression by tumor cells was associated with increased lymphocyte infiltration. Moreover, constitutively expressed FasL was not pro-inflammatory. This study indicates that upregulation of FasL expression by colon tumor cells results in an improved anti-tumor immune challenge in vivo, providing functional evidence in favor of the ‘Fas counterattack’ as a mechanism of tumor immune evasion.

Autophagosomal IκBα degradation plays a role in the long term control of tumor necrosis factor-α-induced NF-κB activity

Transcription factor NF-κB is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-κB is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-α on NF-κB activity for up to 48 h in intestinal epithelial cells. Results show that NF-κB remained persistently activated up to 48 h after TNF-α and that the long term activation of NF-κB was accompanied by a biphasic degradation of IκBα. The first phase of IκBα degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-α stimulated formation of autophagosomes in intestinal epithelial cells and that IκBα co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-α-induced degradation of IκBα and lowered NF-κB target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-κB activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-κB may mediate this effect.

Adenoviral Transduction of Mesenchymal Stem Cells: In Vitro Responses and In Vivo Immune Responses after Cell Transplantation

Adult mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential which makes them attractive targets for regenerative medicine applications. However, to date, therapeutic success of MSC-therapy is limited and the genetic modification of MSCs using viral vectors is one option to improve their therapeutic potential. Ex-vivo genetic modification of MSCs using recombinant adenovirus (Ad) could be promising to reduce undesired immune responses as Ad will be removed before cell/tissue transplantation. In this regard, we investigated whether Ad-modification of MSCs alters their immunological properties in vitro and in vivo. We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86. Moreover, Ad-transduction caused no significant changes in terms of pro-inflammatory cytokine expression, chemokine and chemokine receptor and Toll-like receptor expression. In addition, Ad-modification of MSCs had no affect on their ability to suppress T cell proliferation in vitro. In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues. Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.