Therapeutic Effects of Hypoxic and Pro-Inflammatory Priming of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Inflammatory Arthritis

Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O₂, 5% CO₂), hypoxically (2% O₂, 5% CO₂) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.     

Regenerative Potential of Mesenchymal Stem Cells’ (MSCs) Secretome for Liver Fibrosis Therapies

Chronic liver injuries lead to liver fibrosis and then to end-stage liver cirrhosis. Liver transplantation is often needed as a course of treatment for patients in critical conditions, but limitations associated with transplantation prompted the continuous search for alternative therapeutic strategies. Cell therapy with stem cells has emerged as an attractive option in order to stimulate tissue regeneration and liver repair. Transplanted mesenchymal stem cells (MSCs) could trans-differentiate into hepatocyte-like cells and, moreover, show anti-fibrotic and immunomodulatory effects. However, cell transplantation may lead to some uncontrolled side effects, risks associated with tumorigenesis, and cell rejection. MSCs’ secretome includes a large number of soluble factors and extracellular vesicles (EVs), through which they exert their therapeutic role. This could represent a cell-free strategy, which is safer and more effective than MSC transplantation. In this review, we focus on cell therapies based on MSCs and how the MSCs’ secretome impacts the mechanisms associated with liver diseases. Moreover, we discuss the important therapeutic role of EVs and how their properties could be further used in liver regeneration.     

Small Extracellular Vesicles Derived from Human Chorionic MSCs as Modern Perspective towards Cell-Free Therapy

Mesenchymal stem cells (MSCs) are of great interest to scientists due to their application in cell therapy of many diseases, as well as regenerative medicine and tissue engineering. Recently, there has been growing evidence surrounding the research based on extracellular vesicles (EVs), especially small EVs (sEVs)/exosomes derived from MSCs. EVs/exosomes can be secreted by almost all cell types and various types of EVs show multiple functions. In addition, MSCs-derived exosomes have similar characteristics and biological activities to MSCs and their therapeutic applications are considered as a safe strategy in cell-free therapy. The aim of this study was the characterization of MSCs isolated from the chorion (CHo-MSCs) of human full-term placenta, as well as the isolation and analysis of small EVs obtained from these cells. Accordingly, in this study, the ability of small EVs’ uptake is indicated by synovial fibroblasts, osteoblasts and periosteum-derived MSCs. Improvement in the understanding of the structure, characteristics, mechanism of action and potential application of MSCs-derived small EVs can provide new insight into improved therapeutic strategies.     

Comparisons of Extracellular Vesicles from Human Epidural Fat-Derived Mesenchymal Stem Cells and Fibroblast Cells

Extracellular vesicles (EVs) are generated and secreted by cells into the circulatory system. Stem cell-derived EVs have a therapeutic effect similar to that of stem cells and are considered an alternative method for cell therapy. Accordingly, research on the characteristics of EVs is emerging. EVs were isolated from human epidural fat-derived mesenchymal stem cells (MSCs) and human fibroblast culture media by ultracentrifugation. The characterization of EVs involved the typical evaluation of cluster of differentiation (CD antigens) marker expression by fluorescence-activated cell sorting, size analysis with dynamic laser scattering, and morphology analysis with transmission electron microscopy. Lastly, the secreted levels of cytokines and chemokines in EVs were determined by a cytokine assay. The isolated EVs had a typical size of approximately 30–200 nm, and the surface proteins CD9 and CD81 were expressed on human epidural fat MSCs and human fibroblast cells. The secreted levels of cytokines and chemokines were compared between human epidural fat MSC-derived EVs and human fibroblast-derived EVs. Human epidural fat MSC-derived EVs showed anti-inflammatory effects and promoted macrophage polarization. In this study, we demonstrated for the first time that human epidural fat MSC-derived EVs exhibit inflammatory suppressive potency relative to human fibroblast-derived EVs, which may be useful for the treatment of inflammation-related diseases.     

Mesenchymal Stem Cells and Extracellular Vesicles in Osteosarcoma Pathogenesis and Therapy

Osteosarcoma (OS) is an aggressive bone tumor that mainly affects children and adolescents. OS has a strong tendency to relapse and metastasize, resulting in poor prognosis and survival. The high heterogeneity and genetic complexity of OS make it challenging to identify new therapeutic targets. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into adipocytes, osteoblasts, or chondroblasts. OS is thought to originate at some stage in the differentiation process of MSC to pre-osteoblast or from osteoblast precursors. MSCs contribute to OS progression by interacting with tumor cells via paracrine signaling and affect tumor cell proliferation, invasion, angiogenesis, immune response, and metastasis. Extracellular vesicles (EVs), secreted by OS cells and MSCs in the tumor microenvironment, are crucial mediators of intercellular communication, driving OS progression by transferring miRNAs/RNA and proteins to other cells. MSC-derived EVs have both pro-tumor and anti-tumor effects on OS progression. MSC-EVs can be also engineered to deliver anti-tumor cargo to the tumor site, which offers potential applications in MSC-EV-based OS treatment. In this review, we highlight the role of MSCs in OS, with a focus on EV-mediated communication between OS cells and MSCs and their role in OS pathogenesis and therapy.     

The Role of MSCs and Cell Fusion in Tissue Regeneration

Regenerative medicine is concerned with the investigation of therapeutic agents that can be used to promote the process of regeneration after injury or in different diseases. Mesenchymal stem/stromal cells (MSCs) and their secretome—including extracellular vesicles (EVs) are of great interest, due to their role in tissue regeneration, immunomodulatory capacity and low immunogenicity. So far, clinical studies are not very conclusive as they show conflicting efficacies regarding the use of MSCs. An additional process possibly involved in regeneration might be cell fusion. This process occurs in both a physiological and a pathophysiological context and can be affected by immune response due to inflammation. In this review the role of MSCs and cell fusion in tissue regeneration is discussed.     

Molecular Mechanisms of Mesenchymal Stem Cell-Based Therapy in Acute Kidney Injury

Acute kidney injury (AKI) causes a lot of harm to human health but is treated by only supportive therapy in most cases. Recent evidence shows that mesenchymal stem cells (MSCs) benefit kidney regeneration through releasing paracrine factors and extracellular vesicles (EVs) to the recipient kidney cells and are considered to be promising cellular therapy for AKI. To develop more efficient, precise therapies for AKI, we review the therapeutic mechanism of MSCs and MSC-derived EVs in AKI and look for a better understanding of molecular signaling and cellular communication between donor MSCs and recipient kidney cells. We also review recent clinical trials of MSC-EVs in AKI. This review summarizes the molecular mechanisms of MSCs’ therapeutic effects on kidney regeneration, expecting to comprehensively facilitate future clinical application for treating AKI.     

Therapeutic Potential of Mesenchymal Stem Cells (MSCs) and MSC-Derived Extracellular Vesicles for the Treatment of Spinal Cord Injury

Spinal cord injury (SCI) is a life-threatening condition that leads to permanent disability with partial or complete loss of motor, sensory, and autonomic functions. SCI is usually caused by initial mechanical insult, followed by a cascade of several neuroinflammation and structural changes. For ameliorating the neuroinflammatory cascades, MSC has been regarded as a therapeutic agent. The animal SCI research has demonstrated that MSC can be a valuable therapeutic agent with several growth factors and cytokines that may induce anti-inflammatory and regenerative effects. However, the therapeutic efficacy of MSCs in animal SCI models is inconsistent, and the optimal method of MSCs remains debatable. Moreover, there are several limitations to developing these therapeutic agents for humans. Therefore, identifying novel agents for regenerative medicine is necessary. Extracellular vesicles are a novel source for regenerative medicine; they possess nucleic acids, functional proteins, and bioactive lipids and perform various functions, including damaged tissue repair, immune response regulation, and reduction of inflammation. MSC-derived exosomes have advantages over MSCs, including small dimensions, low immunogenicity, and no need for additional procedures for culture expansion or delivery. Certain studies have demonstrated that MSC-derived extracellular vesicles (EVs), including exosomes, exhibit outstanding chondroprotective and anti-inflammatory effects. Therefore, we reviewed the principles and patho-mechanisms and summarized the research outcomes of MSCs and MSC-derived EVs for SCI, reported to date.     

Comparative Proteomic Analysis of the Mesenchymal Stem Cells Secretome from Adipose,Bone Marrow,Placenta and Wharton’s Jelly

Mesenchymal stem cells (MSCs) have the potential to be a viable therapy against various diseases due to their paracrine effects, such as secretion of immunomodulatory, trophic and protective factors. These cells are known to be distributed within various organs and tissues. Although they possess the same characteristics, MSCs from different sources are believed to have different secretion potentials and patterns, which may influence their therapeutic effects in disease environments. We characterized the protein secretome of adipose (AD), bone marrow (BM), placenta (PL), and Wharton’s jelly (WJ)-derived human MSCs by using conditioned media and analyzing the secretome by mass spectrometry and follow-up bioinformatics. Each MSC secretome profile had distinct characteristics depending on the source. However, the functional analyses of the secretome from different sources showed that they share similar characteristics, such as cell migration and negative regulation of programmed cell death, even though differences in the composition of the secretome exist. This study shows that the secretome of fetal-derived MSCs, such as PL and WJ, had a more diverse composition than that of AD and BM-derived MSCs, and it was assumed that their therapeutic potential was greater because of these properties.     

Enhanced Suppression of Immune Cells In Vitro by MSC Overexpressing FasL

Mesenchymal stromal cells (MSC) display several mechanisms of action that may be harnessed for therapeutic purposes. One of their most attractive features is their immunomodulatory activity that has been extensively characterized both in vitro and in vivo. While this activity has proven to be very efficient, it is transient. We aimed to enhance it by transforming MSC to overexpress a first apoptosis signal (Fas) ligand (FasL). In this study, our goal was to induce FasL overexpression through adenoviral transduction in MSC to improve their immunomodulatory activity. We characterized the impact of FasL overexpression on the morphology, proliferation, viability, phenotype, multilineage differentiation potential and immunomodulation of MSC. Moreover, we determined their suppressive properties in mixed reactions with A20 cells, as well as with stimulated splenocytes. Our findings demonstrate that FasL-overexpressing MSC exhibit improved immunosuppressive properties, while maintaining their MSC-characteristic features. In conclusion, we establish, in a proof-of-concept set-up, that FasL-overexpressing MSC represent good candidates for therapeutic intervention targeted at autoimmune disorders.     

Administration of Human MSC-Derived Extracellular Vesicles for the Treatment of Primary Sclerosing Cholangitis: Preclinical Data in MDR2 Knockout Mice

Primary Sclerosing Cholangitis (PSC) is a progressive liver disease for which there is no effective medical therapy. PSC belongs to the family of immune-mediated biliary disorders and it is characterized by persistent biliary inflammation and fibrosis. Here, we explored the possibility of using extracellular vesicles (EVs) derived from human, bone marrow mesenchymal stromal cells (MSCs) to target liver inflammation and reduce fibrosis in a mouse model of PSC. Five-week-old male FVB.129P2-Abcb^4tm1Bor mice were intraperitoneally injected with either 100 µL of EVs (± 9.1 × 10⁹ particles/mL) or PBS, once a week, for three consecutive weeks. One week after the last injection, mice were sacrificed and liver and blood collected for flow cytometry analysis and transaminase quantification. In FVB.129P2-Abcb4^tm1Bor mice, EV administration resulted in reduced serum levels of alkaline phosphatase (ALP), bile acid (BA), and alanine aminotransferase (ALT), as well as in decreased liver fibrosis. Mechanistically, we observed that EVs reduce liver accumulation of both granulocytes and T cells and dampen VCAM-1 expression. Further analysis revealed that the therapeutic effect of EVs is accompanied by the inhibition of NFkB activation in proximity of the portal triad. Our pre-clinical experiments suggest that EVs isolated from MSCs may represent an effective therapeutic strategy to treat patients suffering from PSC.     

Stem Cells-Derived Extracellular Vesicles: Potential Therapeutics for Wound Healing in Chronic Inflammatory Skin Diseases

Endosome-derived small extracellular vesicles (EVs), often referred to as exosomes, are produced by almost all, if not all, cell types, and are critical for intercellular communication. They are composed of a lipid bilayer associated with membrane proteins and contain a payload of lipids, proteins and regulatory RNAs that depends on the parental cell physiological condition. By transferring their “cargo”, exosomes can modulate the phenotype of neighboring and distant cells. Stem cells (SC) were widely studied for therapeutic applications regarding their regenerative/reparative potential as well as their immunomodulatory properties. Whether from autologous or allogeneic source, SC beneficial effects in terms of repair and regeneration are largely attributed to their paracrine signaling notably through secreted EVs. Subsequently, SC-derived EVs have been investigated for the treatment of various diseases, including inflammatory skin disorders, and are today fast-track cell-free tools for regenerative/reparative strategies. Yet, their clinical application is still facing considerable challenges, including production and isolation procedures, and optimal cell source. Within the emerging concept of “allogeneic-driven benefit” for SC-based therapies, the use of EVs from allogeneic sources becomes the pragmatic choice although a universal allogeneic cell source is still needed. As a unique temporary organ that ensures the mutual coexistence of two allogeneic organisms, mother and fetus, the human placenta offers a persuasive allogeneic stem cell source for development of therapeutic EVs. Advancing cell-free therapeutics nurtures great hope and provides new perspectives for the development of safe and effective treatment in regenerative/reparative medicine and beyond. We will outline the current state of the art in regard of EVs, summarize their therapeutic potential in the context of skin inflammatory disorders, and discuss their translational advantages and hurdles.     

Extracellular Vesicles from Mesenchymal Stromal Cells for the Treatment of Inflammation-Related Conditions

Over the past two decades, mesenchymal stromal cells (MSCs) have demonstrated great potential in the treatment of inflammation-related conditions. Numerous early stage clinical trials have suggested that this treatment strategy has potential to lead to significant improvements in clinical outcomes. While promising, there remain substantial regulatory hurdles, safety concerns, and logistical issues that need to be addressed before cell-based treatments can have widespread clinical impact. These drawbacks, along with research aimed at elucidating the mechanisms by which MSCs exert their therapeutic effects, have inspired the development of extracellular vesicles (EVs) as anti-inflammatory therapeutic agents. The use of MSC-derived EVs for treating inflammation-related conditions has shown therapeutic potential in both in vitro and small animal studies. This review will explore the current research landscape pertaining to the use of MSC-derived EVs as anti-inflammatory and pro-regenerative agents in a range of inflammation-related conditions: osteoarthritis, rheumatoid arthritis, Alzheimer’s disease, cardiovascular disease, and preeclampsia. Along with this, the mechanisms by which MSC-derived EVs exert their beneficial effects on the damaged or degenerative tissues will be reviewed, giving insight into their therapeutic potential. Challenges and future perspectives on the use of MSC-derived EVs for the treatment of inflammation-related conditions will be discussed.     

Mesenchymal Stem Cells Can Both Enhance and Inhibit the Cellular Response to DNA Immunization by Genes of Nonstructural Proteins of the Hepatitis C Virus

Despite extensive research, there is still no vaccine against the hepatitis C virus (HCV). The aim of this study was to investigate whether MSCs can exhibit adjuvant properties during DNA vaccination against hepatitis C. We used the pcNS3-NS5B plasmid encoding five nonstructural HCV proteins and MSCs derived from mice bone marrow. Five groups of DBA mice were immunized with the plasmid and/or MSCs in a different order. Group 1 was injected with the plasmid twice at intervals of 3 weeks; Group 2 with the plasmid, and after 24 h with MSCs; Group 3 with MSCs followed by the plasmid the next day; Group 4 with only MSCs; and Group 5 with saline. When the MSCs were injected prior to DNA immunization, the cell immune response to HCV proteins assessed by the level of IFN-γ synthesis was markedly increased compared to DNA alone. In contrast, MSCs injected after DNA suppressed the immune response. Apparently, the high level of proinflammatory cytokines detected after DNA injection promotes the conversion of MSCs introduced later into the immunosuppressive MSC2. The low level of cytokines in mice before MSC administration promotes the high immunostimulatory activity of MSC1 in response to a DNA vaccine. Thus, when administered before DNA, MSCs are capable of exhibiting promising adjuvant properties.     

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer.     

Dental Mesenchymal Stem Cell Secretome: An Intriguing Approach for Neuroprotection and Neuroregeneration

Mesenchymal stem cells (MSCs) are known for their beneficial effects and regenerative potential. In particular, dental-derived MSCs have the advantage of easier accessibility and a non-invasive isolation method. Moreover, thanks to their neural crest origin, dental MSCs seem to have a more prominent neuroregenerative potential. Indeed, in basal conditions they also express neuronal markers. However, it is now well known that the beneficial actions of MSCs depend, at least in part, on their secretome, referring to all the bioactive molecules released in the conditioned medium (CM) or in extracellular vesicles (EVs). In this review we focus on the applications of the secretome derived from dental MSCs for neuroregeneration and neuroprotection. The secretomes of different dental MSCs have been tested for their effects for neuroregenerative purposes, and the secretomes of dental pulp stem cells and stem cells from human exfoliated deciduous teeth are the most studied. Both the CM and EVs obtained from dental MSCs showed that they are able to promote neurite outgrowth and neuroprotective effects. Interestingly, dental-derived MSC secretome showed stronger neuroregenerative and neuroprotective effects compared to that obtained from other MSC sources. For these reasons, the secretome obtained from dental MSCs may represent a promising approach for neuroprotective treatments.     

Fresh and Cryopreserved Human Umbilical-Cord-Derived Mesenchymal Stromal Cells Attenuate Injury and Enhance Resolution and Repair following Ventilation-Induced Lung Injury

Background: Ventilator-induced lung injury (VILI) frequently worsens acute respiratory distress syndrome (ARDS) severity. Human mesenchymal stem/stromal cells (MSCs) offer considerable therapeutic promise, but the key impediments of clinical translation stem from limitations due to cell source and availability, and concerns regarding the loss of efficacy following cryopreservation. These experiments compared the efficacy of umbilical-cord-derived MSCs (UC-MSCs), a readily available and homogenous tissue source, to the previously more widely utilised bone-marrow-derived MSCs (BM-MSCs). We assessed their capacity to limit inflammation, resolve injury and enhance repair in relevant lung mechanical stretch models, and the impact of cryopreservation on therapeutic efficacy. Methods: In series 1, confluent alveolar epithelial layers were subjected to cyclic mechanical stretch (22% equibiaxial strain) and wound injury, and the potential of the secretome from BM- and UC-derived MSCs to attenuate epithelial inflammation and cell death, and enhance wound repair was determined. In series 2, anesthetized rats underwent VILI, and later received, in a randomised manner, 1 × 10⁷ MSCs/kg intravenously, that were: (i) fresh BM-MSCs, (ii) fresh UC-MSCs or (iii) cryopreserved UC-MSCs. Control animals received a vehicle (PBS). The extent of the resolution of inflammation and injury, and repair was measured at 24 h. Results: Conditioned medium from BM-MSCs and UC-MSCs comparably decreased stretch-induced pulmonary epithelial inflammation and cell death. BM-MSCs and UC-MSCs comparably enhanced wound resolution. In animals subjected to VILI, both fresh BM-MSCs and UC-MSCs enhanced injury resolution and repair, while cryopreserved UC-MSCs comparably retained their efficacy. Conclusions: Cryopreserved UC-MSCs can reduce stretch-induced inflammation and cell death, enhance wound resolution, and enhance injury resolution and repair following VILI. Cryopreserved UC-MSCs represent a more abundant, cost-efficient, less variable and equally efficacious source of therapeutic MSC product.     

Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles

The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs) could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials.