Cell-free Therapy Research Articles

A Novel, Cell-Free Therapy to Enter Our Hearts: The Potential Role of Small EVs in Prevention and Treatment of CVD.

Heart disease constitutes one of the leading causes of morbidity and mortality worldwide. Current therapeutic techniques, such as interventional revascularization, although lifesaving, come along with myocardial injury related to the reperfusion itself, called ischemia-reperfusion injury, which is an added factor for increased morbidity. For that reason, there is an imperative need for novel therapies to be developed that would either prevent or treat myocardial injury. Extracellular vesicles (EVs), specifically small EVs (sEVs), have proven to be important mediators of intercellular communication. The fact that they carry information reflecting that of the parental cell makes them an ideal candidate for diagnostic purposes. sEVs derived from immunoregulatory cells, such as mesenchymal stem cells or cardiac progenitor cells, could also be used therapeutically to exert the primary immunomodulatory function but without carrying the side effects related to cell therapy. Furthermore, as a natural product, they have the added advantage of low immunogenicity, offering the potential for safe drug delivery. In the field of cardiology, there has been great interest in the therapeutic and diagnostic potential of sEVs with significant translational potential. Here, we review the potential use of sEVs in the context of myocardial ischemia and ischemia-reperfusion injury.

TNF-α stimulation enhances the neuroprotective effects of gingival MSCs derived exosomes in retinal ischemia-reperfusion injury via the MEG3/miR-21a-5p axis

Retinal ischemia-reperfusion injury (IRI) is one of the main pathogenic mechanisms of glaucoma, which are largely unknown, including neuroinflammation and neuronal death in the pathological process. In our previous studies, mesenchymal stem cells (MSCs) have been reported to play anti-inflammatory and neuroprotective roles. Additionally, conditioned culture medium (CM) of MSCs stimulated by TNF-α have achieved better antiallergic effects in an experimental allergic conjunctivitis mouse model. However, there is an urgent need for cell-free therapy approaches, like exosomes, to reduce the side effects of autoimmunity. The present study aimed to elucidate the pathways involving TNF-α-stimulated gingival MSC (GMSC)-exosomes (TG-exos), in modulating inflammatory microglia and alleviating apoptosis. In this study, exosomes from the CM of GMSCs were isolated by ultracentrifugation and were injected into the vitreous of mice. The results showed that intraocular injection of TG-exos into mice with IRI notably reduced inflammation and cell loss than that with G-exos (GMSC-exosomes). Similar results were observed in vitro. Additionally, with the microRNA (miR) arrays, it was found that miR-21-5p acted as a crucial factor in TG-exos for neuroprotection and anti-inflammation. Following target prediction and dual-luciferase assay suggested that miR-21-5p played a role by combining with programmed cell death 4 (PDCD4), which was regulated by the long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) as a competing endogenous RNA (ceRNA). This study demonstrates a new therapeutic pathway for neuroprotection against IRI by delivering miR-21-5p-enriched exosomes through MEG3/miR-21-5p/PDCD4 axis and paves the way for the establishment of a cell-free therapeutic approach for glaucoma.

Development of a Chemical Cocktail That Rescues Mouse Brain Demyelination in a Cuprizone-Induced Model.

Oligodendrocytes are glial cells located in the central nervous system (CNS) that play essential roles in the transmission of nerve signals and in the neuroprotection of myelinated neurons. The dysfunction or loss of oligodendrocytes leads to demyelinating diseases such as multiple sclerosis (MS). To treat demyelinating diseases, the development of a therapy that promotes remyelination is required. In the present study, we established an in vitro method to convert human fibroblasts into induced oligodendrocyte-like cells (iOLCs) in 3 days. The induced cells displayed morphologies and molecular signatures similar to oligodendrocytes after treatment with valproic acid and exposure to the small molecules Y27632, SU9516, and forskolin (FSK). To pursue the development of a cell-free remyelination therapy in vivo, we used a cuprizone-induced demyelinated mouse model. The small molecules (Y27632, SU9516, and FSK) were directly injected into the demyelinated corpus callosum of the mouse brain. This combination of small molecules rescued the demyelination phenotype within two weeks as observed by light and electron microscopy. These results provide a foundation for exploring the development of a treatment for demyelinating diseases via regenerative medicine.

Bone Cell Exosomes and Emerging Strategies in Bone Engineering.

Bone tissue remodeling is a highly regulated process balancing bone formation and resorption through complex cellular crosstalk between resident bone and microenvironment cells. This cellular communication is mediated by direct cell and cell–matrix contact, autocrine, endocrine, and paracrine receptor mediated mechanisms such as local soluble signaling molecules and extracellular vesicles including nanometer sized exosomes. An impairment in this balanced process leads to development of pathological conditions. Bone tissue engineering is an emerging interdisciplinary field with potential to address bone defects and disorders by synthesizing three-dimensional bone substitutes embedded with cells for clinical implantation. However, current cell-based therapeutic approaches have faced hurdles due to safety and ethical concerns, challenging their clinical translation. Recent studies on exosome-regulated bone homeostasis and regeneration have gained interest as prospective cell free therapy in conjugation with tissue engineered bone grafts. However, exosome research is still in its nascent stages of bone tissue engineering. In this review, we specifically describe the role of exosomes secreted by cells within bone microenvironment such as osteoblasts, osteocytes, osteoclasts, mesenchymal stem cell cells, immune cells, endothelial cells, and even tumor cells during bone homeostasis and crosstalk. We also review exosome-based osteoinductive functionalization strategies for various bone-based biomaterials such as ceramics, polymers, and metals in bone tissue engineering. We further highlight biomaterials as carrier agents for exosome delivery to bone defect sites and, finally, the influence of various biomaterials in modulation of cell exosome secretome.

Melatonin Engineered Adipose-Derived Biomimetic Nanovesicles Regulate Mitochondrial Functions and Promote Myocardial Repair in Myocardial Infarction.

Myocardial infarction (MI), one type of ischemic heart disease, is a major cause of disability and mortality worldwide. Currently, extracellular vesicles (EVs) derived from adipose-derived stem cells (ADSC) have been proven to be a potentially promising therapeutic treatment for MI. However, the inconvenience of isolation, the low productivity, and the high cost of EVs greatly limits their application in clinic. In this study, we constructed novel biomimetic ADSC-derived nanovesicles (ADSC NVs) to achieve cell-free therapy for MI. Here, we firstly developed a novel Mel@NVs delivery system consisting of engineered ADSC NVs with melatonin (Mel). Then, the characterization and properties of Mel@NVs were performed. The effect of Mel@NVs on cellular oxidative stress and myocardial infarction repair was conducted. The results showed that Mel@NVs treatment under ischemia mimic condition reduced cell apoptosis from 42.59 ± 2.69% to 13.88 ± 1.77%. Moreover, this novel engineered Mel@NVs could ameliorate excessive ROS generation, promote microvessel formation, and attenuate cardiac fibrosis, which further alleviates mitochondrial dysfunction and finally enhance myocardial repair. Hence, the engineered NVs show a potential strategy for MI therapy.

Mesenchymal Stem Cell-Derived Exosomes: Toward Cell-Free Therapeutic Strategies in Chronic Kidney Disease.

Chronic kidney disease (CKD) is rising in global prevalence and has become a worldwide public health problem, with adverse outcomes of kidney failure, cardiovascular disease, and premature death. However, current treatments are limited to slowing rather than reversing disease progression or restoring functional nephrons. Hence, innovative strategies aimed at kidney tissue recovery hold promise for CKD therapy. Mesenchymal stem cells (MSCs) are commonly used for regenerative therapy due to their potential for proliferation, differentiation, and immunomodulation. Accumulating evidence suggests that the therapeutic effects of MSCs are largely mediated by paracrine secretion of extracellular vesicles (EVs), predominantly exosomes. MSC-derived exosomes (MSC-Exos) replicate the functions of their originator MSCs via delivery of various genetic and protein cargos to target cells. More recently, MSC-Exos have also been utilized as natural carriers for targeted drug delivery. Therapeutics can be effectively incorporated into exosomes and then delivered to diseased tissue. Thus, MSC-Exos have emerged as a promising cell-free therapy in CKD. In this paper, we describe the characteristics of MSC-Exos and summarize their therapeutic efficacy in preclinical animal models of CKD. We also discuss the potential challenges and strategies in the use of MSC-Exos-based therapies for CKD in the future.

Adverse effects of cell-free and concentrated ascites reinfusion therapy for malignant ascites: a single-institute experience

BackgroundCell-free and concentrated ascites reinfusion therapy (CART) is a strategy for improving various intractable symptoms due to refractory ascites, including hypoalbuminemia. CART has recently been applied in the treatment of cancer patients. This study was performed to assess the safety of CART in a single cancer institute.MethodsWe retrospectively reviewed 233 CART procedures that were performed for 132 cancer patients in our institute.ResultsThe median weight of ascites before and after concentration was 4,720 g and 490 g (median concentration rate, 10.0-fold), The median amounts of total protein and albumin were 64.0 g and 32.6 g (median recovery rates, 44.9% and 49.0%), respectively. Thirty-three adverse events (AEs) were observed in 22 (9.4%) of 233 procedures; 30 of these events occurred after reinfusion. The most common reinfusion-related AEs were fever (13 events) and chills (10 events). Univariate analyses revealed no significant relationships between the frequency of AEs and age, sex, appearance of ascites, weight of harvested and concentrated ascites, the ascites processing rate (filtration and concentration), weight of saline used for membrane cleaning, amount of calculated total protein for infusion, or prophylaxis against AEs; the reinfusion rate of ≥ 125 mL/h or ≥ 10.9 g/h of total protein affected the frequency of AEs, regardless of the prophylactic use of steroids.ConclusionsThe observed AEs were mainly mild reactions after reinfusion, which were related to a reinfusion rate of volume ≥ 125 mL/h, a simple indicator in practice, or total protein ≥ 10.9 g/h. Although our study was retrospective in nature and undertaken in a single institute, this information may be helpful for the management of cancer patients with refractory malignant ascites using CART.

Mesenchymal Stem Cell-Derived Extracellular Vesicles in Liver Immunity and Therapy.

Mesenchymal stem cells (MSCs), as the most common cell source for stem cell therapy, play an important role in the modulation of innate and adaptive immune responses and have been widely used in clinical trials to treat autoimmune and inflammatory diseases. Recent experimental and clinical studies have shown that MSC-derived extracellular vesicles (MSC-EVs) can inhibit the activation and proliferation of a variety of proinflammatory cells, such as Th1, Th17 and M1 macrophages, reducing the secretion of proinflammatory cytokines, while promoting the proliferation of anti-inflammatory cells, such as M2 macrophages and Tregs, and increasing the secretion of anti-inflammatory cytokines, thus playing a role in immune regulation and exhibiting immunomodulatory functions. Besides MSC-EVs are more convenient and less immunogenic than MSCs. There is growing interest in the role of MSC-EVs in liver diseases owing to the intrinsic liver tropism of MSC-EVs. In this review, we focus on the immunomodulatory effects of MSC-EVs and summarize the pivotal roles of MSC-EVs as a cell-free therapy in liver diseases, including NAFLD, AIH, acute liver failure, liver fibrosis and hepatic ischemia–reperfusion injury. Moreover, we provide a concise overview of the potential use and limits of MSC-EVs in clinical application.

Comparison of two cell-free therapeutics derived from adipose tissue: small extracellular vesicles versus conditioned medium

BackgroundCell-free therapy has been inspired as a promising approach to overcome the limitations of traditional stem cell therapy. However, the therapeutic effect between extracellular vesicles and conditioned medium with the same source had not been compared. Our previous studies have shown that both the conditioned medium of adipose tissue (adipose tissue extract, ATE) and its further purification product small extracellular vesicles (sEV-AT) contributed to adipose tissue regeneration. In this study, we aimed to compare the ATE and sEV-AT in composition, inductivity on cells and de novo adipose regenerative potential.MethodsThe characteristics of sEV-AT and ATE were compared through protein and particle yield, particle size distribution and composition. The inductivity of sEV-AT and ATE on cells were compared through co-culture of sEV-AT or ATE with ASC, HUVEC and RAW264.7 in vitro. The capacity of promoting de novo adipogenesis was compared by implanting the silicone tube containing sEV-AT or ATE subcutaneously in vivo.ResultsMore particles and concentrated particle size distribution were detected in sEV-AT. In turn, more soluble factors and multiple peaks in particle size distribution were detected in ATE. In 1662 common proteins of sEV-AT and ATE, there were 984 (59.2%) proteins enriched twice more in sEV-AT than in ATE. With the prerequisite of equivalent protein concentration, sEV-AT outperformed ATE in promoting proliferation, migration and regeneration potential of cells those contributing adipose tissue regeneration in vitro. Furthermore, sEV-AT expedited the de novo adipose tissue regeneration and angiogenesis at the early stage than ATE in vivo, but sEV-AT and ATE group formed similar neoadipose tissue and new vessels at week 12.ConclusionsOur results provided a direct comparison between EV and conditioned medium as cell-free therapeutic strategy. Both sEV and ATE had specific biological signature to facilitate tissue repair. Considering the convenience of extraction and acceptable effect, ATE represented a feasible product of cell-free therapy, providing another option for different situations in clinical application. Furthermore, the complex contents of both sEV-AT and ATE should be studied comprehensively to avoid possible negative effects and to ensure sufficient safety for clinical applications.

Feasibility of a fast-track randomized controlled trial of cell-free and concentrated ascites reinfusion therapy for patients with refractory malignant ascites

BackgroundMalignant ascites often causes discomfort in advanced cancer patients. Paracentesis is the most common treatment modality, but it requires frequently repeated treatment. Cell-free and concentrated ascites reinfusion therapy (CART) may prolong the paracentesis interval, but controlled trials are lacking. We assessed the feasibility of a randomized controlled trial of CART vs. paracentesis alone for patients with refractory malignant ascites.MethodsThis study was an open-label, fast-track, randomized controlled, feasibility trial. Patients admitted to four designated cancer hospitals who received no further anticancer treatments were eligible. Patients were randomly assigned 1:1 to a CART arm or control (simple paracentesis) arm. The feasibility endpoint was the percentage of patients who completed the study intervention. Secondary endpoints included paracentesis-free survival, patient’s request on the questionnaire for paracentesis (PRO-paracentesis)-free survival (the period until the patients first reported that they would want paracentesis if indicated), and adverse events.ResultsWe screened 953 patients for eligibility. Of 61 patients with refractory malignant ascites, 21 patients were determined as eligible. Finally, 20 patients consented and were allocated; 18 patients (90%, 95% CI: 68.3–98.8) completed the study intervention. All patients had an ECOG performance status of 3 or 4. The median drained ascites volume was 3,200 mL in the CART arm and 2,500 mL in the control arm. In the CART arm, the median reinfused albumin volume was 12.6 g. Median paracentesis-free survivals were 5 days (95% CI: 2–6) in the CART arm, and 6 days (3–9) in the control arm. Median PRO-paracentesis-free survivals were 4 days (2–5) and 5 days (1–9), respectively. A total of 73% of patients received paracentesis within 2 days from their first request for the next paracentesis. One patient in the CART arm developed Grade 1 fever.ConclusionsA fast-track randomized controlled trial of CART for patients with malignant ascites is feasible. The efficacy and safety of CART should be assessed in future trials. PRO-paracentesis-free survival may be a complementary outcome measure with paracentesis-free survival in future trials.Trial registrationRegistered at University Hospital Medical Information Network Clinical Trial Registry as UMIN000031029. Registered on 28/01/2018.

Multipotency and Immunomodulatory Benefits of Stem Cells From Human Exfoliated Deciduous Teeth

Stem cells derived from human exfoliated deciduous teeth (SHEDs) are considered a promising cell population for cell-based or cell-free therapy and tissue engineering because of their proliferative, multipotency and immunomodulator. Based on recent studies, we find that SHEDs show the superior ability of nerve regeneration in addition to the potential of osteogenesis, odontogenesis owing to their derivation from the neural crest. Besides, much evidence suggests that SHEDs have a paracrine effect and can function as immunomodulatory regents attributing to their capability of secreting cytokines and extracellular vesicles. Here, we review the characteristic of SHEDs, their multipotency to regenerate damaged tissues, specifically concentrating on bones or nerves, following the paracrine activity or immunomodulatory benefits of their potential for clinical application in regenerative medicine.

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies.

Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

Stem Cell-Derived Exosome as Potential Therapeutics for Microbial Diseases.

Exosomes, as the smallest extracellular vesicles that carry a cargo of nucleic acids, lipids, and proteins and mediate intercellular communication, have attracted much attention in diagnosis and treatment in the field of medicine. The contents of exosomes vary depending on the cell type and physiological conditions. Among exosomes derived from several cell types, stem cell-derived exosomes (stem cell-Exo) are increasingly being explored due to their immunomodulatory properties, regenerative capacity, anti-inflammatory and anti-microbial functions. Administration of stem cell-Exo, as a cell-free therapy for various diseases, has gained great promise. Indeed, the advantages of exosomes secreted from stem cells outweigh those of their parent cells owing to their small size, high stability, less immunogenicity, no risk of tumorigenesis, and easier condition for storage. Recently, the use of stem cell-Exo has been proposed in the field of microbial diseases. Pathogens including bacteria, viruses, fungi, and parasites can cause various diseases in humans with acute and chronic complications, sometimes resulting in mortality. On the other hand, treatments based on antibiotics and other chemical compounds have many side effects and the strains become resistant to drugs in some cases. Hence, this review aimed to highlight the effect of stem cell-derived extracellular vesicles including stem cell-Exo on microbial diseases. Although most published studies are preclinical, the avenue of clinical application of stem cell-Exo is under way to reach clinical applications. The challenges ahead of this cell-free treatment that might be applied as a therapeutic alternative to stem cells for translation from bench to bed were emphasized, as well.

Mesenchymal Stem Cell-Derived Extracellular Vesicles: The Novel Therapeutic Option for Regenerative Dentistry.

Dental mesenchymal stem cells (MSCs) are characterized by unlimited self-renewal ability and high multidirectional differentiation potential. Since dental MSCs can be easily isolated and exhibit a high capability to differentiate into odontogenic cells, they are considered as attractive therapeutic agents in regenerative dentistry. Recently, MSC-derived extracellular vesicles (MSC-EVs) have attracted widespread attention as carriers for cell-free therapy due to their potential functions. Many studies have shown that MSC-EVs can mediate microenvironment at tissue damage site, and coordinate the regeneration process. Additionally, MSC-EVs can mediate intercellular communication, thus affecting the phenotypes and functions of recipient cells. In this review, we mainly summarized the types of MSCs that could be potentially applied in regenerative dentistry, the possible molecular cargos of MSC-EVs, and the major effects of MSC-EVs on the therapeutic induction of osteogenic differentiation.

Extracellular vesicle and soluble fractions of adipose tissue-derived mesenchymal stem cells secretome induce inflammatory cytokines modulation in an in vitro model of discogenic pain

Mesenchymal stem cells (MSCs) secretome or conditioned medium (CM) is a complex cocktail of different molecules, some of which, particularly those contained in extracellular vesicles, already have proven therapeutic applications. CM may well represent promising therapy for discogenic pain and the intention of this work is to assess its therapeutic potential using an in vitro model of this condition. This is an experimental study. Our in vitro model comprised nucleus pulposus (NP) and annulus fibrosus (AF) cells inflamed with TNF. To assess the potential therapeutic value of CM and its components, extracellular vesicles (EVs) and soluble culture fraction (SF), cell inflammation took place under 3 different conditions: either in the presence of whole CM, isolated EVs or SF, and concentrations of pro-inflammatory cytokines, metalloproteinases (MMPs) and neurotrophic factors produced in all 3 cases were compared. In the presence of whole CM, both in vitro gene expression by the NP and AF test cells and analysis of their protein content showed high modulatory effects on inflammation and MMP inhibition. The presence of EVs and SF showed similar but much smaller effects, and this was particularly marked in the case of NP cells. Our results show that, compared to EVs and SF, the presence of whole CM has the greatest positive effect on the modulation of pro-inflammatory and catabolic factors. These observations suggest that CM could protect against inflammation and the resulting intervertebral disc (IVD) degeneration that leads to discogenic pain. Many patients' expectations are not met by current non-operative and surgical treatments for discogenic low back pain. We propose the use of the MSCs secretome for assessing its potential as cell-free therapy to treat degenerative disc disease modulating the inflammatory response.

Extracellular Vesicles as a Cell-free Therapy for Cardiac Repair: a Systematic Review and Meta-analysis of Randomized Controlled Preclinical Trials in Animal Myocardial Infarction Models.

Stem cell therapy for cardiac regeneration has been gaining traction as a possible intervention for the reduction of the burden associated with MI and heart failure. However, stem cell therapies have several shortcomings, including poor engraftment, limited improvements in cardiac function, and possible teratogenicity. Recently, extracellular vesicles (EVs) from stem cell sources have been explored as a novel therapy to regenerate the injured myocardium in several animal MI trials. In this systematic review and meta-analysis, we investigate the use of stem cell-derived EVs for cardiac repair preclinical trials in animal MI models. Cochrane Library, Medline, Embase, PubMed, Scopus and Web of Science and grey literature (Canadian Agency for Drugs, Technologies in Health, and Google Scholar) were searched through August 20, 2020 and 37 articles were included in the final analysis. The overall effect size observed in EV-treated small animals after MI for ejection fraction (EF) was 10.85 [95%CI: 8.79, 12.90] and for fractional shortening (FS) was 7.19 [95%CI: 5.43, 8.96] compared to control-treated animals. The most abundant stem cell source used were mesenchymal stem cells which showed robust improvements in EF and FS (MD = 11.89 [95 % CI: 9.44, 14.34] and MD = 6.96 [95 % CI: 4.97, 8.96], respectively). Significant publication bias was detected for EF and FS outcomes. This study supports the use of EVs derived from stem cells as a novel therapy for cardiac repair after MI. Further investigation in larger animal studies may be necessary before clinical trials.PROSPERO registration number: CRD42019142218.

Gene-Directed Enzyme/Prodrug Therapy of Rat Brain Tumor Mediated by Human Mesenchymal Stem Cell Suicide Gene Extracellular Vesicles In Vitro and In Vivo.

Simple SummaryExtracellular vesicles— exosomes—secreted by human mesenchymal stem/stromal cells are able to cross the blood–brain barrier and internalize glioblastoma cells. We prepared exosomes possessing a gene message, the product of which is able to convert nontoxic 5-fluorocytosine to cytotoxic drug 5-fluorouracil. Such therapeutic exosomes administered intranasally, intraperitoneally, or subcutaneously to rats bearing intracerebral glioblastoma cells inhibited their growth. The treatment cured a significant number of animals.MSC-driven, gene-directed enzyme prodrug therapy (GDEPT) mediated by extracellular vesicles (EV) represents a new paradigm—cell-free GDEPT tumor therapy. In this study, we tested the efficacy of yeast cytosine deaminase::uracilphosphoribosyl transferase (yCD::UPRT-MSC)-exosomes, in the form of conditioned medium (CM) to inhibit the growth of C6 glioblastoma cells both in vitro and in vivo. MSCs isolated from human adipose tissue, umbilical cord, or dental pulp engineered to express the yCD::UPRT gene secreted yCD::UPRT-MSC-exosomes that in the presence of the prodrug 5-fluorocytosine (5-FC), inhibited the growth of rat C6 glioblastoma cells and human primary glioblastoma cells in vitro in a dose-dependent manner. CM from these cells injected repeatedly either intraperitoneally (i.p.) or subcutaneously (s.c.), applied intranasally (i.n.), or infused continuously by an ALZET osmotic pump, inhibited the growth of cerebral C6 glioblastomas in rats. A significant number of rats were cured when CM containing yCD::UPRT-MSC-exosomes conjugated with 5-FC was repeatedly injected i.p. or applied i.n. Cured rats were subsequently resistant to challenges with higher doses of C6 cells. Our data have shown that cell-free GDEPT tumor therapy mediated by the yCD::UPRT-MSC suicide gene EVs for high-grade glioblastomas represents a safer and more practical approach that is worthy of further investigation.

Therapeutic exosomes for various human diseases: Special issue of BMB Reports in 2022

Extracellular vesicles (EVs), especially exosomes, are cell-derived nanoparticles harboring various cellular components such as RNAs, lipids, and proteins for intercellular communication. Roles of EVs as intercellular communicators have been extensively studied in the last few decades, especially under various pathological conditions. Deciphering the message in EVs isolated from biological fluids of patients can provide valuable information not only for disease diagnosis, but also for disease monitoring or treatment responses. EVs are also attractive treatment modality and drug delivery system with favorable properties of biocompatibility, selective tropism, and stability. Stem cell-derived naïve EVs have been tested for their regenerative or immunomodulatory effects in numerous preclinical and clinical studies. This so-called "cell-free cell therapy" is supported by the idea that most therapeutic actions of conventional cell therapy are mediated by paracrine action of EVs released from stem cells. In that sense, immune cell-derived EVs are regarded as a reasonable option for cancer immunotherapy. Such therapeutic effect of EVs can be dramatically augmented by incorporating active pharmaceutical ingredients (APIs) to make engineered exosomes as "Trojan Horses". Biomimetic EVs or cell-derived nanovesicles can be generated through various physicochemical methos such as serial extrusion. They provide alternative options due to their high productivity and relatively easy purification. In this special issue, therapeutic applications of naïve or engineered EVs are discussed in various human diseases including cardiovascular diseases, renal disorders, neurological diseases, cancers, and infectious diseases focusing on COVID-19. [BMB Reports 2022; 55(1): 1-2].

Matrix vesicles from dental follicle cells improve alveolar bone regeneration via activation of the PLC/PKC/MAPK pathway

BackgroundThe regeneration of bone loss that occurs after periodontal diseases is a significant challenge in clinical dentistry. Extracellular vesicles (EVs)-based cell-free regenerative therapies represent a promising alternative for traditional treatments. Developmental biology suggests matrix vesicles (MVs), a subtype of EVs, contain mineralizing-related biomolecules and play an important role in osteogenesis. Thus, we explore the therapeutic benefits and expect to find an optimized strategy for MV application.MethodsHealthy human dental follicle cells (DFCs) were cultured with the osteogenic medium to generate MVs. Media MVs (MMVs) were isolated from culture supernatant, and collagenase-released MVs (CRMVs) were acquired from collagenase-digested cell suspension. We compared the biological features of the two MVs and investigated their induction of cell proliferation, migration, mineralization, and the modulation of osteogenic genes expression. Furthermore, we investigated the long-term regenerative capacity of MMVs and CRMVs in an alveolar bone defect rat model.ResultsWe found that both DFC-derived MMVs and CRMVs effectively improved the proliferation, migration, and osteogenic differentiation of DFCs. Notably, CRMVs showed better bone regeneration capabilities. Compared to MMVs, CRMVs-induced DFCs exhibited increased synthesis of osteogenic marker proteins including ALP, OCN, OPN, and MMP-2. In the treatment of murine alveolar bone defects, CRMV-loaded collagen scaffold brought more significant therapeutic outcomes with less unhealing areas and more mature bone tissues in comparison with MMVs and acquired the effects resembling DFCs-based treatment. Furthermore, the western blotting results demonstrated the activation of the PLC/PKC/MAPK pathway in CRMVs-induced DFCs, while this cascade was inhibited by MMVs.ConclusionsIn summary, our findings revealed a novel cell-free regenerative therapy for repairing alveolar bone defects by specific MV subtypes and suggest that PLC/PKC/MAPK pathways contribute to MVs-mediated alveolar bone regeneration.

Exosomes derived from platelet-rich plasma administration in site mediate cartilage protection in subtalar osteoarthritis

Subtalar osteoarthritis (STOA) is often secondary to chronic ankle sprains, which seriously affects the quality of life of patients. Due to its etiology and pathogenesis was not studied equivocally yet, there is currently a lack of effective conservative treatments. Although they have been used for tissue repair, platelet-rich plasma-derived exosomes (PRP-Exo) have the disadvantage of low retention and short-lived therapeutic effects. This study aimed to determine whether incorporation of PRP-Exo in thermosensitive hydrogel (Gel) increased their retention in the joint and thereby playing a therapeutic role on STOA due to chronic mechanical instability established by transecting lateral ligaments (anterior talofibular ligament (ATFL)/calcaneal fibular ligament (CFL)). PRP-Exo incorporated Gel (Exo-Gel) system, composed of Poloxamer-407 and 188 mixture-based thermoresponsive hydrogel matrix in an optimal ratio, was determined by its release ability of Exo and rheology of Gel response to different temperature. The biological activity of Exo-Gel was evaluated in vitro, and the therapeutic effect of Exo-Gel on STOA was evaluated in vivo. Exo released from Exo-Gel continuously for 28 days could promote the proliferation and migration of mouse bone mesenchymal stem cells (mBMSCs) and chondrocytes, at the same time enhance the chondrogenic differentiation of mBMSCs, and inhibit inflammation-induced chondrocyte degeneration. In vivo experiments confirmed that Exo-Gel increased the local retention of Exo, inhibited the apoptosis and hypertrophy of chondrocytes, enhanced their proliferation, and potentially played the role in stem cell recruitment to delay the development of STOA. Thus, Delivery of PRP-Exo incorporated in thermosensitive Gel provides a novel approach of cell-free therapy and has therapeutic effect on STOA.Graphical