Mesenchymal stem cell-derived extracellular vesicles as a new therapeutic strategy for ocular diseases

Mesenchymal stem cells (MSCs) have attracted considerable attention for their activity in the treatment of refractory visual disorders. Since MSCs were found to possess the beneficial effects by secreting paracrine factors rather than direct differentiation, MSC-derived extracellular vesicles (EVs) were widely studied in various disease models. MSCs generate abundant EVs, which act as important mediators by exchanging protein and genetic information between MSCs and target cells. It has been confirmed that MSC-derived EVs possess unique anti-inflammatory, anti-apoptotic, tissue repairing, neuroprotective, and immunomodulatory properties, similar to their parent cells. Upon intravitreal injection, MSC-derived EVs rapidly diffuse through the retina to alleviate retinal injury or inflammation. Due to possible risks associated with MSC transplantation, such as vitreous opacity and pathological proliferation, EVs appear to be a better choice for intravitreal injection. Small size EVs can pass through biological barriers easily and their contents can be modified genetically for optimal therapeutic effect. Hence, currently, they are also explored for the possibility of serving as drug delivery vehicles. In the current review, we describe the characteristics of MSC-derived EVs briefly, comprehensively summarize their biological functions in ocular diseases, and discuss their potential applications in clinical settings.     

Melatonin preconditioning is an effective strategy for mesenchymal stem cell‐based therapy for kidney disease

Based on multiple studies in animal models, mesenchymal stem cell (MSC)‐based therapy appears to be an innovative intervention approach with tremendous potential for the management of kidney disease. However, the clinical therapeutic effects of MSCs in either acute kidney injury (AKI) or chronic kidney disease (CKD) are still under debate. Hurdles originate from the harsh microenvironment in vivo that decreases the cell survival rate, paracrine activity and migratory capacity of MSCs after transplantation, which are believed to be the main reasons for their limited effects in clinical applications. Melatonin is traditionally regarded as a circadian rhythm‐regulated neurohormone but in recent years has been found to exhibit antioxidant and anti‐inflammatory properties. Because inflammation, oxidative stress, thermal injury, and hypoxia are abnormally activated in kidney disease, application of melatonin preconditioning to optimize the MSC response to the hostile in vivo microenvironment before transplantation is of great importance. In this review, we discuss current knowledge concerning the beneficial effects of melatonin preconditioning in MSC‐based therapy for kidney disease. By summarizing the available information and discussing the underlying mechanisms, we aim to improve the therapeutic effects of MSC‐based therapy for kidney disease and accelerate translation to clinical application.     

Mesenchymal stromal cell‐derived extracellular vesicles as cell‐free biologics for the ex vivo expansion of hematopoietic stem cells

Hematopoietic stem cell transplantation (HSCT) is the ultimate choice of treatment for patients with hematological diseases and cancer. The success of HSCT is critically dependent on the number and engraftment efficiency of the transplanted donor hematopoietic stem cells (HSCs). Various studies show that bone marrow‐derived mesenchymal stromal cells (MSCs) support hematopoiesis and also promote ex vivo expansion of HSCs. MSCs exert their therapeutic effect through paracrine activity, partially mediated through extracellular vesicles (EVs). Although the physiological function of EVs is not fully understood, inspiring findings indicate that MSC‐derived EVs can reiterate the hematopoiesis, supporting the ability of MSCs by transferring their cargo containing proteins, lipids, and nucleic acids to the HSCs. The activation state of the MSCs or the signaling mechanism that prevails in them also defines the composition of their EVs, thereby influencing the fate of HSCs. Modulating or preconditioning MSCs to achieve a specific composition of the EV cargo for the ex vivo expansion of HSCs is, therefore, a promising strategy that can overcome several challenges associated with the use of naïve/unprimed MSCs. This review aims to speculate upon the potential role of preconditioned/primed MSC‐derived EVs as “cell‐free biologics,” as a novel strategy for expanding HSCs in vitro.     

Prodigious therapeutic effects of combining mesenchymal stem cells with magnetic nanoparticles

Mesenchymal stem cells (MSCs) have gained wide-ranging reputation in the medical research community due to their promising regenerative abilities. MSCs can be isolated from various resources mostly bone marrow, Adipose tissues and Umbilical cord. Huge advances have been achieved in comprehending the possible mechanisms underlying the therapeutic functions of MSCs. Despite the proven role of MSCs in repairing and healing of many disease modalities, many hurdles hinder the transferring of these cells in the clinical settings. Among the most reported problems encountering MSCs therapy in vivo are loss of tracking signal post-transplantation, insufficient migration, homing and engraftment post-infusion, and undesirable differentiation at the site of injury. Magnetic nanoparticles (MNPs) have been used widely for various biomedical applications. MNPs have a metallic core stabilized by an outer coating material and their magnetic properties can be modulated by an external magnetic field. These magnetic properties of MNPs were found to enhance the quality of diagnostic imaging procedures and can be used to create a carrying system for targeted delivery of therapeutic substances mainly drug, genes and stem cells. Several studies highlighted the advantageous outcomes of combining MSCs with MNPs in potentiating their tracking, monitoring, homing, engraftment and differentiation. In this review, we will discuss the role of MNPs in promoting the therapeutic profile of MSCs which may improve the success rate of MSCs transplantation and solve many challenges that delay their clinical applicability.     

Generation of mesenchymal stem-like cells for producing extracellular vesicles

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with therapeutic potential against autoimmune diseases, inflammation, ischemia, and metabolic disorders. Contrary to the previous conceptions, recent studies have revealed that the tissue repair and immunomodulatory functions of MSCs are largely attributed to their secretome, rather than their potential to differentiate into desired cell types. The composition of MSC secretome encompasses cytokines and growth factors, in addition to the cell-derived structures known as extracellular vesicles (EVs). EVs are membrane-enclosed nanoparticles that are capable of delivering biomolecules, and it is now believed that MSC-derived EVs are the major players that induce biological changes in the target tissues. Based on these EVs’ characteristics, the potential of EVs derived from MSC (MSC-EV) in terms of tissue regeneration and immune modulation has grown during the last decade. However, the use of MSCs for producing sufficient amount of EVs has not been satisfactory due to limitations in the cell growth and large variations among the donor cell types. In this regard, pluripotent stem cells (PSCs)-derived MSC-like cells, which can be robustly induced and expanded in vitro, have emerged as more accessible cell source that can overcome current limitations of using MSCs for EV production. In this review, we have highlighted the methods of generating MSC-like cells from PSCs and their therapeutic outcome in preclinical studies. Finally, we have also discussed future requirements for making this cell-free therapy clinically feasible.     

Extracellular micro/nanovesicles rescue kidney from ischemia-reperfusion injury

Acute renal failure (ARF) is a clinical challenge that is highly resistant to treatment, and its high rate of mortality is alarming. Ischemia–reperfusion injury (IRI) is the most common cause of ARF. Especially IRI is implicated in kidney transplantation and can determine graft survival. Although the exact pathophysiology of renal IRI is unknown, the role of inflammatory responses has been elucidated. Because mesenchymal stromal cells (MSCs) have strong immunomodulatory properties, they are under extensive investigation as a therapeutic modality for renal IRI. Extracellular vesicles (EVs) play an integral role in cell-to-cell communication. Because the regenerative potential of the MSCs can be recapitulated by their EVs, the therapeutic appeal of MSC-derived EVs has dramatically increased in the past decade. Higher safety profile and ease of preservation without losing function are other advantages of EVs compared with their producing cells. In the current review, the preliminary results and potential of MSC-derived EVs to alleviate kidney IRI are summarized. We might be heading toward a cell-free approach to treat renal IRI.     

Proteomic Signature of Mesenchymal Stromal Cell‐Derived Small Extracellular Vesicles

Small extracellular vesicles (EVs) are 50–200 nm vesicles secreted by most cells. They are considered as mediators of intercellular communication, and EVs from specific cell types, in particular mesenchymal stem/stromal cells (MSCs), offer powerful therapeutic potential, and can provide a novel therapeutic strategy. They appear promising and safe (as EVs are non‐self‐replicating), and eventually MSC‐derived EVs (MSC‐EVs) may be developed to standardized, off‐the‐shelf allogeneic regenerative and immunomodulatory therapeutics. Promising pre‐clinical data have been achieved using MSCs from different sources as EV‐producing cells. Similarly, a variety EV isolation and characterization methods have been applied. Interestingly, MSC‐EVs obtained from different sources and prepared with different methods show in vitro and in vivo therapeutic effects, indicating that isolated EVs share a common potential. Here, well‐characterized and controlled, publicly available proteome profiles of MSC‐EVs are compared to identify a common MSC‐EV protein signature that might be coupled to the MSC‐EVs’ common therapeutic potential. This protein signature may be helpful in developing MSC‐EV quality control platforms required to confirm the identity and test for the purity of potential therapeutic MSC‐EVs.     

Preconditioning strategies for improving the survival rate and paracrine ability of mesenchymal stem cells in acute kidney injury

Acute kidney injury (AKI) is a common, severe emergency case in clinics, with high incidence, significant mortality and increased costs. Despite development in the understanding of its pathophysiology, the therapeutic choices are still confined to dialysis and renal transplantation. Considering their antiapoptotic, immunomodulatory, antioxidative and pro‐angiogenic effects, mesenchymal stem cells (MSCs) may be a promising candidate for AKI management. Based on these findings, some clinical trials have been performed, but the results are contradictory (NCT00733876, NCT01602328). The low engraftment, poor survival rate, impaired paracrine ability and delayed administration of MSCs are the four main reasons for the limited clinical efficacy. Investigators have developed a series of preconditioning strategies to improve MSC survival rates and paracrine ability. In this review, by summarizing these encouraging studies, we intend to provide a comprehensive understanding of various preconditioning strategies on AKI therapy and improve the prognosis of AKI patients by regenerative medicine.     

Preconditioning influences mesenchymal stem cell properties in vitro and in vivo

Various diseases and toxic factors easily impair cellular and organic functions in mammals. Organ transplantation is used to rescue organ function, but is limited by scarce resources. Mesenchymal stem cell (MSC)‐based therapy carries promising potential in regenerative medicine because of the self‐renewal and multilineage potency of MSCs; however, MSCs may lose biological functions after isolation and cultivation for a long time in vitro. Moreover, after they are injected in vivo and migrate into the damaged tissues or organs, they encounter a harsh environment coupled with death signals due to the inadequate tensegrity structure between the cells and matrix. Preconditioning, genetic modification and optimization of MSC culture conditions are key strategies to improve MSC functions in vitro and in vivo, and all of these procedures will contribute to improving MSC transplantation efficacy in tissue engineering and regenerative medicine. Preconditioning with various physical, chemical and biological factors is possible to preserve the stemness of MSCs for further application in studies and clinical tests. In this review, we mainly focus on preconditioning and the corresponding mechanisms for improving MSC activities in vitro and in vivo; we provide a glimpse into the promotion of MSC‐based cell therapy development for regenerative medicine. As a promising consequence, MSC transplantation can be applied for the treatment of some terminal diseases and can prolong the survival time of patients in the near future.     

Strategies to improve the efficiency of mesenchymal stem cell transplantation for reversal of liver fibrosis

End‐stage liver fibrosis frequently progresses to portal vein thrombosis, formation of oesophageal varices, hepatic encephalopathy, ascites, hepatocellular carcinoma and liver failure. Mesenchymal stem cells (MSCs), when transplanted in vivo, migrate into fibrogenic livers and then differentiate into hepatocyte‐like cells or fuse with hepatocytes to protect liver function. Moreover, they can produce various growth factors and cytokines with anti‐inflammatory effects to reverse the fibrotic state of the liver. In addition, only a small number of MSCs migrate to the injured tissue after cell transplantation; consequently, multiple studies have investigated effective strategies to improve the survival rate and activity of MSCs for the treatment of liver fibrosis. In this review, we intend to arrange and analyse the current evidence related to MSC transplantation in liver fibrosis, to summarize the detailed mechanisms of MSC transplantation for the reversal of liver fibrosis and to discuss new strategies for this treatment. Finally, and most importantly, we will identify the current problems with MSC‐based therapies to repair liver fibrosis that must be addressed in order to develop safer and more effective routes for MSC transplantation. In this way, it will soon be possible to significantly improve the therapeutic effects of MSC transplantation for liver regeneration, as well as enhance the quality of life and prolong the survival time of patients with liver fibrosis.     

Small extracellular vesicles from mesenchymal stromal cells: the next therapeutic paradigm for musculoskeletal disorders

Musculoskeletal disorders are one of the biggest contributors to morbidity and place an enormous burden on the health care system in an aging population. Owing to their immunomodulatory and regenerative properties, mesenchymal stromal/stem cells (MSCs) have demonstrated therapeutic efficacy for treatment of a wide variety of conditions, including musculoskeletal disorders. Although MSCs were originally thought to differentiate and replace injured/diseased tissues, it is now accepted that MSCs mediate tissue repair through secretion of trophic factors, particularly extracellular vesicles (EVs). Endowed with a diverse cargo of bioactive lipids, proteins, nucleic acids and metabolites, MSC-EVs have been shown to elicit diverse cellular responses and interact with many cell types needed in tissue repair. The present review aims to summarize the latest advances in the use of native MSC-EVs for musculoskeletal regeneration, examine the cargo molecules and mechanisms underlying their therapeutic effects, and discuss the progress and challenges in their translation to the clinic.     

Bone marrow mesenchymal stem cells reduce ureteral stricture formation in a rat model via the paracrine effect of extracellular vesicles

With no effective therapy to prevent or treat ureteral stricture (US), a multifactorial fibrotic disease after iatrogenic injury of the ureter, the need for new therapies is urgent. Mesenchymal stem cells (MSCs) have been widely studied for treating tissue defects and excessive fibrosis, and recent studies established that one of the main therapeutic vectors of MSCs is comprised in their secretome and represented by extracellular vesicles (EVs). Thus, we have determined to explore the specific role of MSCs‐derived EVs (MSC‐EVs) treatment in a pre‐clinical model of US. The results firstly showed that either a bolus dose of MSCs or a bolus dose of MSC‐EVs (administration via renal‐arterial) significantly ameliorated ureteral fibrosis and recuperated ureter morphological development in a US rat model. We confirmed our observations through MSCs or MSC‐EVs treatment alleviated hydronephrosis, less renal dysfunction and blunted transforming growth factor‐β1 induced fibration. Due to MSC‐EVs are the equivalent dose of MSCs, and similar curative effects of transplantation of MSCs and MSC‐EVs were observed, we speculated the curative effect of MSCs in treating US might on account of the release of EVs through paracrine mechanisms. Our study demonstrated an innovative strategy to counteract ureteral stricture formation in a rat model of US.     

Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro

Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.     

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.     

Extracellular Vesicles in chondrogenesis and Cartilage regeneration

Extracellular vesicles (EVs), mainly exosomes and microvesicles, are bilayer lipids containing biologically active information, including nucleic acids and proteins. They are involved in cell communication and signalling, mediating many biological functions including cell growth, migration and proliferation. Recently, EVs have received great attention in the field of tissue engineering and regenerative medicine. Many in vivo and in vitro studies have attempted to evaluate the chondrogenesis potential of these microstructures and their roles in cartilage regeneration. EVs derived from mesenchymal stem cells (MSCs) or chondrocytes have been found to induce chondrocyte proliferation and chondrogenic differentiation of stem cells in vitro. Preclinical studies have shown that exosomes derived from MSCs have promising results in cartilage repair and in cell-free therapy of osteoarthritis. This review will focus on the in vitro and in vivo chondrogenesis and cartilage regeneration of EVs as well as their potential in the treatment of osteoarthritis.     

Effect of mesenchymal stem cells-derived exosomes on tumor microenvironment: Tumor progression versus tumor suppression

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into different cell types. Owing to their immunosuppressive and anti-inflammatory properties, they are widely used in regenerative medicine, but they have a dual effect on cancer progression and exert both growth-stimulatory or -inhibitory effects on different cancer types. It has been proposed that these controversial effects of MSC in tumor microenvironment (TME) are mediated by their polarization to proinflammatory or anti-inflammatory phenotype. In addition, they can polarize the immune system cells that in turn influence tumor progression. One of the mechanisms involved in the TME communications is extracellular vesicles (EVs). MSCs, as one of cell populations in TME, produce a large amount of EVs that can influence tumor development. Similar to MSC, MSC-EVs can exert both anti- or protumorigenic effects. In the current study, we will investigate the current knowledge related to MSC role in cancer progression with a focus on the MSC-EV content in limiting tumor growth, angiogenesis, and metastasis. We suppose MSC-EVs can be used as safe vehicles for delivering antitumor agents to TME.     

Mesenchymal stem cells in arthritic diseases

Mesenchymal stem cells (MSCs), the nonhematopoietic progenitor cells found in various adult tissues, are characterized by their ease of isolation and their rapid growth in vitro while maintaining their differentiation potential, allowing for extensive culture expansion to obtain large quantities suitable for therapeutic use. These properties make MSCs an ideal candidate cell type as building blocks for tissue engineering efforts to regenerate replacement tissues and repair damaged structures as encountered in various arthritic conditions. Osteoarthritis (OA) is the most common arthritic condition and, like rheumatoid arthritis (RA), presents an inflammatory environment with immunological involvement and this has been an enduring obstacle that can potentially limit the use of cartilage tissue engineering. Recent advances in our understanding of the functions of MSCs have shown that MSCs also possess potent immunosuppression and anti-inflammation effects. In addition, through secretion of various soluble factors, MSCs can influence the local tissue environment and exert protective effects with an end result of effectively stimulating regeneration in situ. This function of MSCs can be exploited for their therapeutic application in degenerative joint diseases such as RA and OA. This review surveys the advances made in the past decade which have led to our current understanding of stem cell biology as relevant to diseases of the joint. The potential involvement of MSCs in the pathophysiology of degenerative joint diseases will also be discussed. Specifically, we will explore the potential of MSC-based cell therapy of OA and RA by means of functional replacement of damaged cartilage via tissue engineering as well as their anti-inflammatory and immunosuppressive activities.     

Mesenchymal stem cell–derived extracellular vesicles: a new impetus of promoting angiogenesis in tissue regeneration

Over the past few decades, extracellular vesicles (EVs) have emerged as crucial mediators of intercellular communication. EVs encapsulate and convey information to surrounding cells or distant cells, where they mediate cellular biological responses. Among their multifaceted roles in the modulation of biological responses, the involvement of EVs in vascular development, growth and maturation has been widely documented and their potential therapeutic application in regenerative medicine or in the treatment of angiogenesis-related diseases is drawing increasing interest. In this review, we have summarized the details about the current knowledge on biogenesis of EVs and conventional isolation methods. Evidence supporting the use of EVs derived from mesenchymal stromal cells (MSCs) to enhance angiogenesis in the development of insufficient angiogenesis, such as chronic wounds, stroke and myocardial infarction, will also be discussed critically. Finally, the main challenges and prerequisites for their therapeutic applications will be evaluated.     

Immunomodulatory effects of mesenchymal stem cells for the treatment of cardiac allograft rejection

Heart transplantation continues to be the gold standard clinical intervention to treat patients with end-stage heart failure. However, there are major complications associated with this surgical procedure that reduce the survival prognosis of heart transplant patients, including allograft rejection, malignancies, infections, and other complications that arise from the use of broad-spectrum immunosuppression drugs. Recent studies have demonstrated the use of mesenchymal stem cells (MSCs) against allotransplantation rejection in both in vitro and in vivo settings due to their immunomodulatory properties. Therefore, utilization of MSCs provides new and exciting strategies to improve heart transplantation and potentially reduce the use of broad-spectrum immunosuppression drugs while alleviating allograft rejection. In this review, we will discuss the current research on the mechanisms of cardiac allograft rejection, the physiological and immunological characteristics of MSCs, the effects of MSCs on the immune system, and immunomodulation of heart transplantation by MSCs.     

Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell-derived extracellular vesicles in stirred-tank bioreactors

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) act as signaling mediators of cellular responses. However, despite representing a promising alternative to cell-based therapies, clinical translation of EVs is currently limited by their lack of scalability and standardized bioprocessing. Herein, we integrated scalable downstream processing protocols with standardized expansion of large numbers of viable cells in stirred-tank bioreactors to improve EV production. Higher EV yields were linked to EV isolation by tangential flow filtration followed by size exclusion chromatography, rendering 5 times higher number of EVs comparatively to density gradient ultracentrifugation protocols. Additionally, when compared to static culture, EV manufacture in bioreactors resulted in 2.2 higher yields. Highlighting the role of operating under optimal cell culture conditions to maximize the number of EVs secreted per cell, MSCs cultured at lower glucose concentration favored EV secretion. While offline measurements of metabolites concentration can be performed, in this work, Raman spectroscopy was also applied to continuously track glucose levels in stirred-tank bioreactors, contributing to streamline the selection of optimal EV collection timepoints. Importantly, MSC-derived EVs retained their quality attributes and were able to stimulate angiogenesis in vitro, therefore highlighting their promising therapeutic potential.