Cell-free Therapeutics Research Articles

Exosomes from TNF-Α-Treated Human Gingiva-Derived MSCs Inhibit Periodontal Bone Loss Via CD73 and MiR-1260b-Mediated Attenuation of Inflammation

Mesenchymal stem cell (MSC)–derived exosome plays a central role in the cell-free therapeutics involving MSCs and the contents can be customized under disease-associated microenvironments. However, optimal MSC-preconditioning to enhance its therapeutic potential is largely unknown. Here, we show that preconditioning of gingival tissue-derived MSC (GMSC) with tumor necrosis factor-alpha (TNF-α) is ideal for the treatment of periodontitis. TNF-α stimulation not only increased the amount of exosome secreted from GMSC, but also enhanced the exosomal expression of CD73, thereby inducing anti-inflammatory M2 macrophage polarization. The effect of GMSC-derived exosomes on inflammatory bone loss were examined by ligature-induced periodontitis model in mice. Local injection of GMSCs-derived exosomes significantly reduced periodontal bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and these effects were further enhanced by preconditioning of GMSCs with TNF-α. Thus, GMSCs-derived exosomes also exhibited anti-osteoclastogenic activity. Receptor activator of NF-κB ligand (RANKL) expression was regulated by Wnt5a in periodontal ligament cells (PDLCs), and exosomal miR-1260b was found to target Wnt5a-mediated RANKL pathway and inhibit its osteoclastogenic activity. These results indicate that significant ability of the TNF-α-preconditioned GMSC-derived exosomes to regulate inflammation and osteoclastogenesis paves the way for establishment of a new therapeutic approach for periodontitis.

Cell-Free Extracellular Vesicles Derived from Human Bone Marrow Endothelial Progenitor Cells as Potential Therapeutics for Microvascular Endothelium Restoration in ALS.

Repairing the damaged blood-CNS-barrier in amyotrophic lateral sclerosis (ALS) is necessary to prevent entry of detrimental blood-borne factors contributing to motor neuron dysfunction. Recently, we showed benefits of human bone marrow endothelial progenitor cell (hBM-EPC) transplantation into symptomatic ALS mice on barrier restoration by replacing damaged endothelial cells (ECs). Additionally, transplanted cells may endogenously repair ECs by secreting angiogenic factors as our subsequent in vitro study demonstrated. Based on these study results, hBM-EPCs may secrete extracellular vesicles, which may contain and transfer diverse vesicular biomolecules towards maintenance of EC functionality. The study aimed to characterize extracellular vesicles (EVs) derived from hBM-EPCs as potential cell-free therapeutics for endothelium repair in ALS. EVs were isolated from hBM-EPC media at different culture times and vesicle properties were evaluated. The protective effects of EVs on mouse brain endothelial cells (mBECs) exposed to ALS mouse plasma were investigated. Uptake and blockage of EVs from GFP-transfected hBM-EPCs in ECs were determined in vitro. Results showed that EVs isolated from hBM-EPCs as nanosized vesicles significantly reduced mBEC damage from the pathological environment and these EVs were taken up by cells. Blockage of β1 integrin on EVs prevented internalization of vesicles in mBECs. Together, these results provide evidence for potential of hBM-EPC-derived EVs as novel cell-free therapeutics for repair of endothelium in ALS. Although determining translational potential of hBM-EPC-derived EVs will require evaluation in vivo, this in vitro study represents a step towards an extracellular vesicle-based approach for repair of the damaged microvascular endothelium in ALS.

The Hippo Pathway in Cardiac Regeneration and Homeostasis: New Perspectives for Cell-Free Therapy in the Injured Heart

Intractable cardiovascular diseases are leading causes of mortality around the world. Adult mammalian hearts have poor regenerative capacity and are not capable of self-repair after injury. Recent studies of cell-free therapeutics such as those designed to stimulate endogenous cardiac regeneration have uncovered new feasible therapeutic avenues for cardiac repair. The Hippo pathway, a fundamental pathway with pivotal roles in cell proliferation, survival and differentiation, has tremendous potential for therapeutic manipulation in cardiac regeneration. In this review, we summarize the most recent studies that have revealed the function of the Hippo pathway in heart regeneration and homeostasis. In particular, we discuss the molecular mechanisms of how the Hippo pathway maintains cardiac homeostasis by directing cardiomyocyte chromatin remodeling and regulating the cell-cell communication between cardiomyocytes and non-cardiomyocytes in the heart.

Three-dimensional culture of dental pulp pluripotent-like stem cells (DPPSCs) enhances Nanog expression and provides a serum-free condition for exosome isolation.

Stem cell‐derived exosomes have been identified as novel cell‐free therapeutics for regenerative medicine. Three‐dimensional (3D) culture of stem cells were reported to improve their “stemness” and therapeutic efficacy. This work focused on establishing serum‐free 3D culture of dental pulp pluripotent‐like stem cells (DPPSCs)—a newly characterized pluripotent‐like stem cell for exosome production. DPPSCs were expanded in regular 2D culture in human serum‐supplemented (HS)‐medium and transferred to a micropatterned culture plate for 3D culture in HS‐medium (default) and medium supplemented with KnockOut™ serum replacement (KO‐medium). Bright‐field microscopy observation throughout the culture period (24 days) revealed that DPPSCs in KO‐medium formed spheroids of similar morphology and size to that in HS‐medium. qRT‐PCR analysis showed similar Oct4A gene expression in DPPSC spheroids in both HS‐medium and KO‐medium, but Nanog expression significantly increased in the latter. Vesicles isolated from DPPSC spheroids in KO‐medium in the first 12 days of culture showed sizes that fall within the exosomal size range by nanoparticle tracking analysis (NTA) and express the canonical exosomal markers. It is concluded that 3D culture of DPPSCs in KO‐medium provided an optimal serum‐free condition for successful isolation of DPPSC‐derived exosomes for subsequent applications in regenerative medicine.

Toxicological evaluation of exosomes derived from human adipose tissue-derived mesenchymal stem/stromal cells

Several studies report that the therapeutic mechanism of action of mesenchymal stem/stromal cells (MSCs) is mainly mediated by paracrine factors that are released from MSCs such as exosomes. Exosomes are nano-sized extracellular vesicles that are transferred to target cells for cell-to-cell communication. Although MSC-derived exosomes (MSC-exosomes) are suggested as novel cell-free therapeutics for various human diseases, evaluation studies for the safety and toxicity of MSC-exosomes are limited. The purpose of our study was to evaluate the toxicological profile, including skin sensitization, photosensitization, eye and skin irritation, and acute oral toxicity using exosomes derived from human adipose tissue-derived MSCs (ASC-exosomes) in accordance with the OECD guidelines and the principles of Good Laboratory Practice. The ASC-exosomes were classified as a potential non-sensitizer in the skin sensitization test, UN GHS no category in the eye irritation test, and as a skin non-irritant in the skin irritation test, and did not induce any toxicity in the phototoxicity test or in acute oral toxicity testing. Our findings are the first to suggest that ASC-exosomes are safe for use as a topical treatment, with no adverse effects in toxicological testing, and have potential application as a therapeutic agent, cosmetic ingredient, or for other biological uses.

Mesenchymal Stem/Stromal Cell-Derived Exosomes for Immunomodulatory Therapeutics and Skin Regeneration.

Exosomes are nano-sized vesicles that serve as mediators for cell-to-cell communication. With their unique nucleic acids, proteins, and lipids cargo compositions that reflect the characteristics of producer cells, exosomes can be utilized as cell-free therapeutics. Among exosomes derived from various cellular origins, mesenchymal stem cell-derived exosomes (MSC-exosomes) have gained great attention due to their immunomodulatory and regenerative functions. Indeed, many studies have shown anti-inflammatory, anti-aging and wound healing effects of MSC-exosomes in various in vitro and in vivo models. In addition, recent advances in the field of exosome biology have enabled development of specific guidelines and quality control methods, which will ultimately lead to clinical application of exosomes. This review highlights recent studies that investigate therapeutic potential of MSC-exosomes and relevant mode of actions for skin diseases, as well as quality control measures required for development of exosome-derived therapeutics.

A comparison between long non-coding RNA repertoires of exosomes derived from naïve and cytokine-activated human mesenchymal stem cells

Background & Aim Mesenchymal stem cells (MSCs) are being used in the fields of regenerative medicine, especially in the treatment of musculoskeletal disorders, diabetes, autoimmune diseases, neuronal injury, and neurodegeneration. The therapeutic effects of MSCs are largely mediated by paracrine factors including exosomes, which are nanometer-sized membrane-bound vesicles with functions as mediators of cell-cell communication. Interestingly, the therapeutic potential of MSCs could be prompted by pro-inflammatory cytokines stimulation. The alternation of MSC-secreted immunomodulatory factors composition after cytokine treatment strengthen the modulation of exosomal regenerative nature. MSC-derived exosomes bypass the concern of pro-fibrogenic potential and the possibility of tumorigenicity upon in vivo transplantation of MSCs and could represent a novel cell-free therapeutics. However, the composition of exosomes, in particular long non-coding RNAs (lncRNAs), are not comprehensively investigated. In this study, we compared the expression level of long non-coding RNAs in exosomes derived from naive and cytokine-activated human adipose-derived MSCs. Methods, Results & Conclusion Human adipose-derived MSCs were incubated with or without TNF-α and IFN-γ in serum-free medium for 24 hrs, and the condition medium were harvested for exosome isolation and small RNA sequencing. Exosome isolation protocol is as previously described (Wu and Lee, Stem Cell Research & Therapy (2017) 8:117). Small RNAs in exosomes were sequenced by the Illumina HiSeq 2500 System (50SE). Twenty upregulated lncRNAs after cytokine treatment are selected for lncRNA-RNA and lncRNA-protein interaction prediction. Our results provide a reference dataset that can help to identify the therapeutic targets for future translational studies.

Human adipose stem cell-derived extracellular nanovesicles for treatment of chronic liver fibrosis

Liver fibrosis is an excessive wound healing process that occurs in response to liver damage depending on underlying aetiologies. Currently, there are no effective therapies and FDA-approved therapeutics for the treatment of liver fibrosis except liver transplantation. Multipotent adipose-derived stem cells (ADSCs) have received significant attention as regenerative medicine for liver fibrosis owing to their advantages over stem cells with other origins. However, intrinsic limitations of stem cell therapies, such as cellular rejection and tumor formation, have impeded clinical applications of the ADSC-based liver therapeutics. To overcome these problems, the extracellular nanovesicles (ENVs) responsible for the therapeutic effect of ADSCs (A-ENVs) have shown considerable promise as cell-free therapeutics for liver diseases. However, A-ENVs have not been used for the treatment of intractable chronic liver diseases including liver fibrosis and cirrhosis. Therefore, in this study, we investigated the in vitro and in vivo antifibrotic efficacy of A-ENVs in thioacetamide-induced liver fibrosis models. A-ENVs significantly downregulated the expression of fibrogenic markers, such as matrix metalloproteinase-2, collagen-1, and alpha-smooth muscle actin. The systemic administration of A-ENVs led to high accumulation in fibrotic liver tissue and the restoration of liver functionality in liver fibrosis models through a marked reduction in α-SMA and collagen deposition. These results demonstrate the significant potential of A-ENVs for use as extracellular nanovesicles-based therapeutics in the treatment of liver fibrosis and possibly other intractable chronic liver diseases.

Reprogramming Exosomes for Immunotherapy.

Exosomes are nanosized vesicles secreted by nearly all types of cells and play important roles in intercellular communication. Given their unique and important pharmacological properties, exosomes have been emerging as a new class of cell-free therapeutics. Herein, we describe exosomes developed against epidermal growth factor receptor (EGFR), a key factor in epithelial malignancies, involved in enhanced tumor growth, invasion, and metastasis. The exosomes are genetically modified for displaying two distinct types of monoclonal antibodies on the exosome surface, resulting in novel synthetic multivalent antibodies retargeted exosomes (SMART-Exos) that can simultaneously target tumor-associated human EGFR and T-cell surface CD3 receptor. By redirecting and activating T cells toward attacking EGFR-expressing cancer cells, the designed SMART-Exos exhibit highly potent and specific antitumor activity. In this chapter, the methodologies are outlined for generating and using SMART-Exos for cancer immunotherapy.

Injectable Supramolecular Ureidopyrimidinone Hydrogels Provide Sustained Release of Extracellular Vesicle Therapeutics.

Extracellular vesicles (EVs) are small vesicles secreted by cells and have gained increasing interest as both drug delivery vehicles or as cell-free therapeutics for regenerative medicine. To achieve optimal therapeutic effects, strategies are being developed to prolong EV exposure to target organs. One promising approach to achieve this is through EV-loaded injectable hydrogels. In this study, the use of a hydrogel based on ureido-pyrimidinone (UPy) units coupled to poly(ethylene glycol) chains (UPy-hydrogel) is examined as potential delivery platform for EVs. The UPy-hydrogel undergoes a solution-to-gel transition upon switching from a high to neutral pH, allowing immediate gelation upon administration into physiological systems. Here, sustained EV release from the UPy-hydrogel measured over a period of 4 d is shown. Importantly, EVs retain their functional capacity after release. Upon local administration of fluorescently labeled EVs incorporated in a UPy-hydrogel in vivo, EVs are still detected in the UPy-hydrogel after 3 d, whereas in the absence of a hydrogel, EVs are internalized by fat and skin tissue near the injection site. Together, these data demonstrate that UPy-hydrogels provide sustained EV release over time and enhance local EV retention in vivo, which could contribute to improved therapeutic efficacy upon local delivery and translation toward new applications.

Hydrogel-Mediated Sustained Systemic Delivery of Mesenchymal Stem Cell-Derived Extracellular Vesicles Improves Hepatic Regeneration in Chronic Liver Failure.

Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have been widely reported as promising cell-free products that show therapeutic effects of the parental cells but not their limitations. Due to the intrinsic liver tropism of MSC-EVs, they have been widely used as therapeutics or drug carriers for treatment of liver diseases. However, rapid clearance from the target site may attenuate the efficiency of systemically administered MSC-EVs. Herein, sustained release into the peritoneum has been proposed as a new strategy to prolong the bioavailability of the MSC-EVs in the target liver. During intraperitoneal injection, clickable polyethylene glycol (PEG) macromeres were mixed with MSC-EVs to form EV-encapsulated PEG hydrogels via a fast, biocompatible click reaction. Upon biodegradation, the EV-laden hydrogels were swollen gradually to release EVs in a sustained manner over 1 month. In vivo tracking of the labeled EVs revealed that the accumulation of EVs in the liver was extended by hydrogel-mediated delivery for 1 month. Four weeks after injection in a rat model of chronic liver fibrosis, the physical and histopathological investigations of the harvested liver showed superior antifibrosis, anti-apoptosis, and regenerative effects of the EVs when delivered by the sustained systemic release (Gel-EV) to the conventional bolus injection (Free-EV). Specifically, the Gel-EV system improved the antifibrosis, anti-inflammation, anti-apoptosis, and regenerative effects of the EVs to nearly 40, 50, 40, and 50% compared to Free-EV, respectively, as was specified by quantification of the fibrotic area, α-SMA density, and caspase-3 density in the harvested tissues and ALT enzyme in serum. This study may potentiate the use of MSC-EVs as cell-free therapeutics for chronic liver failure. The sustained systemic delivery strategy may open a new paradigm to extend the effects of disease-targeting EVs over time.

Polyunsaturated fatty acids modify the extracellular vesicle membranes and increase the production of proresolving lipid mediators of human mesenchymal stromal cells

Human mesenchymal stromal/stem cells (hMSCs) are used in experimental cell therapy to treat various immunological disorders, and the extracellular vesicles (hMSC-EVs) they produce have emerged as an option for cell-free therapeutics. The immunomodulatory function of hMSCs resembles the resolution of inflammation, in which proresolving lipid mediators (LMs) play key roles. Multiple mechanisms underlying the hMSC immunosuppressive effect has been elucidated; however, the impact of LMs and EVs in the resolution is poorly understood. In this study, we supplemented hMSCs with polyunsaturated fatty acids (PUFAs); arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which serve as precursors for multiple LMs. We then determined the consequent compositional modifications in the fatty acid, phospholipid, and LM profiles. Mass spectrometric analyses revealed that the supplemented PUFAs were incorporated into the main membrane phospholipid classes with different dynamics, with phosphatidylcholine serving as the first acceptor. Most importantly, the PUFA modifications were transferred into hMSC-EVs, which are known to mediate hMSC immunomodulation. Furthermore, the membrane-incorporated PUFAs influenced the LM profile by increasing the production of downstream prostaglandin E2 and proresolving LMs, including Resolvin E2 and Resolvin D6. The production of LMs was further enhanced by a highly proinflammatory stimulus, which resulted in an increase in a number of mediators, most notably prostaglandins, while other stimulatory conditions had less a pronounced impact after a 48-h incubation. The current findings suggest that PUFA manipulations of hMSCs exert significant immunomodulatory effects via EVs and proresolving LMs, the composition of which can be modified to potentiate the therapeutic impact of hMSCs.

Bioprocessing of Mesenchymal Stem Cells and Their Derivatives: Toward Cell-Free Therapeutics.

Mesenchymal stem cells (MSCs) have attracted tremendous research interest due to their ability to repair tissues and reduce inflammation when implanted into a damaged or diseased site. These therapeutic effects have been largely attributed to the collection of biomolecules they secrete (i.e., their secretome). Recent studies have provided evidence that similar effects may be produced by utilizing only the secretome fraction containing extracellular vesicles (EVs). EVs are cell-derived, membrane-bound vesicles that contain various biomolecules. Due to their small size and relative mobility, they provide a stable mechanism to deliver biomolecules (i.e., biological signals) throughout an organism. The use of the MSC secretome, or its components, has advantages over the implantation of the MSCs themselves: (i) signals can be bioengineered and scaled to specific dosages, and (ii) the nonliving nature of the secretome enables it to be efficiently stored and transported. However, since the composition and therapeutic benefit of the secretome can be influenced by cell source, culture conditions, isolation methods, and storage conditions, there is a need for standardization of bioprocessing parameters. This review focuses on key parameters within the MSC culture environment that affect the nature and functionality of the secretome. This information is pertinent to the development of bioprocesses aimed at scaling up the production of secretome-derived products for their use as therapeutics.

Abstract 550: Exosomes Released by Human Induced Pluripotent Stem Cell-Cardiomyocytes Induce In-Vitro Angiogenesis: A New Strategy for Cell-Free Therapeutics

Due to the poor regenerative properties of cardiac tissue, acute ischemic events can lead to progressive terminal heart failure unless cardiac regeneration can be stimulated. Recent evidence using stem cell therapy in the heart demonstrates a greater role of their paracrine signaling potential, as cell survival post-transplantation is very low. The main objective of this study was to characterize exosomes secreted by human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and evaluate their in vitro angiogenic potential. HiPSC-CMs were cultured under normal conditions with exosome-free serum. Isolated exosomes were characterized by TEM, NTA, and immunoblotting. Angiogenic potential was assessed by wound-healing, proliferation, and tube formation assays using bovine aortic endothelial cells (BAECs). TEM imaging showed secretion of exosomes for hiPSC-CMs, as confirmed by the size and shape of the isolated particles. The size of exosomes was determined to be 71.4 nm (mode), within the characteristic exosomal range. Immunoblot analysis of exosome lysate demonstrated enrichment of known exosome-specific protein markers (CD63 and Hsp70). Most importantly, BAECs treated with exosomes showed significant (p < 0.05) increases in migration, proliferation, and tube formation, as compared to the no exosome control. Overall, our results demonstrate that treating BAECs with exosomes isolated from hiPSC-CMs enhances angiogenesis. Overall, this study demonstrates the angiogenic potential of human iPSC-CM- derived exosomes. The cell-free therapeutics using exosomes constitutes a novel innovative approach for future clinical therapy to induce angiogenesis in the post-ischemic heart.

Mesenchymal Stromal Cell Microparticles Enhance Lung Endothelial Barrier Through CD44 and the S1P/ceramide Rheostat

IntroductionAcute respiratory distress syndrome (ARDS) is a fatal condition characterized by hyperinflammation and pulmonary microvascular leak. Treatment is only supportive with no pharmacological cure. Recently, mesenchymal stromal/stem cells (MSCs) have emerged as potential therapy, but greater knowledge on their mechanism(s) of action could yield development of cell‐free therapeutics.ObjectiveTo determine if MSC‐produced microparticles (MPs) ameliorate endothelial barrier disruption and identify their biomolecular contents responsible for such therapeutic effect.MethodsTo re‐capitulate lung capillary leak in ARDS, primary human pulmonary microvascular endothelial cell (HPMEC) monolayers grown on Transwell inserts were injured with thrombin to induce barrier disruption as measured by transendothelial electrical resistance (TEER). MPs were purified out of conditioned medium produced by MSCs either stimulated with Ca2+ ionophore or unstimulated. MPs were administered to treat thrombin‐injured HPMEC monolayers; surface markers were compared by flow cytometry and lipid content analyzed by HPLC‐MS.ResultsMPs from unstimulated MSCs enhanced HPMEC barrier function and TEER recovery after thrombin injury, while MPs from stimulated MSCs were injurious. Stimulated MSCs produced MPs with significantly less surface CD44. Antibody blockade of CD44 on unstimulated MPs eliminated their therapeutic effect on HPMEC monolayers. Moreover, stimulated MPs had significantly more total ceramides and less sphingosine‐1‐phosphate (S1P) than unstimulated MPs. Ceramide degradation by neutral ceramidase blunted the injurious effect of stimulated MPs. The therapeutic function of unstimulated MPs was eliminated with ceramide over‐production by acid sphingomyelinase or S1P degradation by S1P lyase.ConclusionThese results show MSC‐produced MPs were therapeutic after lung capillary endothelial barrier disruption. Beneficial effects were associated with surface CD44 and greater ratio of S1P:ceramide content. This knowledge provides a mechanistic basis for development of MSC‐based cell‐free therapeutics.Support or Funding InformationThis work was supported by Ontario Research Fund and Canadian Institutes of Health Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles

Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in) EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.

Mesenchymal stem cell-derived secretome and microvesicles as a cell-free therapeutics for neurodegenerative disorders

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that can be isolated from various fetal and adult tissues. Over the fast decades, their multipotency, tissue reparative action, homing ability and immunoregulatory properties raised a great expectation in the field of stem cell and regenerative medicine that is being actively translated in a number of clinical trials worldwide. While MSC-based therapies have proven effectiveness in a numerous experimental models of human diseases and clinical studies including neurodegenerative disorders, there are accumulating evidences suggesting that the observed therapeutic effects in tissue regeneration is likely due to the paracrine actions of MSCs. Indeed, MSCs secrete trophic factors and cytokines (secretome) that have therapeutic relevance for the neurogenic, neuroprotective, angiogenic and anti-inflammatory/immunoregulatory activities. In addition, extracellular vesicles, such as microvesicles and exosomes, are proposed as key mediators of information transfer between different cells for tissue repair and regeneration. Although the underlying mechanism(s) of reparative action is not clear, evidences suggest that MSC secretome and microvesicles can recapitulate the beneficial effects as their cellular counterparts in tissue regeneration. Thus, secretome and microvesicles represent a promising candidate for a novel cell-free therapy for neurodegenerative diseases that has many advantages in overcoming the limitations and risks associated with the cell-based therapeutics.