Mesenchymal stem cell-derived small extracellular vesicles in the treatment of human diseases:Progress and prospect

Mesenchymal stem cells(MSCs)are self-renewing,multipotent cells that could differentiate into multiple tissues.MSC-based therapy has become an attractive and promising strategy for treating human diseases through immune regulation and tissue repair.However,accumulating data have indicated that MSC-based therapeutic effects are mainly attributed to the properties of the MSC-sourced secretome,especially small extracellular vesicles(sEVs).sEVs are signaling vehicles in intercellular communication in normal or pathological conditions.sEVs contain natural contents,such as proteins,mRNA,and microRNAs,and transfer these functional contents to adjacent cells or distant cells through the circulatory system.MSC-sEVs have drawn much attention as attractive agents for treating multiple diseases.The properties of MSC-sEVs include stability in circulation,good biocompatibility,and low toxicity and immunogenicity.Moreover,emerging evidence has shown that MSC-sEVs have equal or even better treatment efficacies than MSCs in many kinds of disease.This review summarizes the current research efforts on the use of MSC-sEVs in the treatment of human diseases and the existing challenges in their application from lab to clinical practice that need to be considered.     

Phospholipids in small extracellular vesicles: emerging regulators of neurodegenerative diseases and cancer

Small extracellular vesicles (sEVs) are generated by almost all cell types. They have a bilayer membrane structure that is similar to cell membranes. Thus, the phospholipids contained in sEVs are the main components of cell membranes and function as structural support elements. However, as in-depth research on sEV membrane components is conducted, some phospholipids have been found to participate in cellular biological processes and function as targets for cell–cell communication. Currently, sEVs are being developed as part of drug delivery systems and diagnostic factors for various diseases, especially neurodegenerative diseases and cancer. An understanding of the physiological and pathological roles of sEV phospholipids in cellular processes is essential for their future medical application. In this review, the authors discuss phospholipid components in sEVs of different origins and summarize the roles of phospholipids in sEV biogenesis. The authors further collect the current knowledge on the functional roles of sEV phospholipids in cell–cell communication and bioactivities as signals regulating neurodegenerative diseases and cancer and the possibility of using sEV phospholipids as biomarkers or in drug delivery systems for cancer diagnosis and treatment. Knowledge of sEV phospholipids is important to help us identify directions for future studies.     

Bone mesenchymal stromal cell-derived small extracellular vesicles inhibit inflammation and ameliorate sepsis via delivery of microRNA-21a-5p

Background aimsSepsis is a potentially life-threatening disease that results from a severe systemic inflammatory response due to infection. Mesenchymal stromal cell-derived small extracellular vesicles (MSC sEVs) are able to transfer bioactive molecules and have been demonstrated to play an important role in the pathophysiological process of sepsis. Herein the authors aimed to investigate the potential role and downstream molecular mechanism of MSC sEVs in sepsis.MethodsMSC sEVs were acquired by ultracentrifugation and then injected into a cecal ligation and puncture mouse model. The efficacy of MSC sEVs in both in vitro and in vivo models of sepsis was evaluated.ResultsMSC sEV therapy improved survival, reduced sepsis-induced inflammation, attenuated pulmonary capillary permeability and improved liver and kidney function in septic mice. In addition, the authors found that microRNA-21a-5p (miR-21a-5p) was highly enriched in MSC sEVs, could be transferred to recipient cells, inhibited inflammation and increased survival in septic mice. Furthermore, the authors demonstrated that MSC sEV miR-21a-5p suppressed inflammation by targeting toll-like receptor 4 and programmed cell death 4. The therapeutic efficacy of MSC sEVs was partially abrogated by transfection with miR-21a-5p inhibitors.ConclusionsCollectively, the authors’ data suggest that miR-21a-5p-bearing MSC sEVs may be a prospective and effective sepsis therapeutic strategy.     

Immune cell-derived small extracellular vesicles in cancer treatment

Small extracellular vesicles (sEVs) secreted by most cells carry bioactive macromolecules including proteins, lipids, and nucleic acids for intercellular communication. Given that some immune cell-derived sEVs exhibit anti-cancer properties, these sEVs have received scientific attention for the development of novel anti-cancer immunotherapeutic agents. In this paper, we reviewed the latest advances concerning the biological roles of immune cell-derived sEVs for cancer therapy. sEVs derived from immune cells including dendritic cells (DCs), T cells, natural-killer (NK) cells, and macrophages are good candidates for sEV-based cancer therapy. Besides their role of cancer vaccines, DC-shed sEVs activated cytotoxic lymphocytes and killed tumor cells. sEVs isolated from NK cells and chimeric antigen receptor (CAR) T cells exhibited cytotoxicity against cancer cells. sEVs derived from CD8⁺ T and CD4⁺ T cells inhibited cancer-associated cells in tumor microenvironment (TME) and activated B cells, respectively. M1-macrophage-derived sEVs induced M2 to M1 repolarization and also created a pro-inflammatory environment. Hence, these sEVs, via mono or combination therapy, could be considered in the treatment of cancer patients in the future. In addition, sEVs derived from cytokine-stimulated immune cells or sEV engineering could improve their anti-tumor potency.     

Polarized human cholangiocytes release distinct populations of apical and basolateral small extracellular vesicles

Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2–interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.     

Macrophage bioassay standardization to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles

Background aimsOwing to the lack of biological assays, determining the biological activity of extracellular vesicles has proven difficult. Here the authors standardized an in vitro assay to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) based on their ability to prevent acquisition of the M1 phenotype in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Induction of tumor necrosis factor alpha, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS) characterizes the M1 phenotype. Nitric oxide released by iNOS turns into nitrite, which can be easily quantitated in culture media by Griess reaction.MethodsThe authors first tested different assay conditions in 96-well plates, including two seeding densities (2 × 10⁴ cells/well and 4 × 10⁴ cells/well), four LPS doses (1 ng/mL, 10 ng/mL, 100 ng/mL and 1000 ng/mL) and two time points (16 h and 24 h), in order to determine the best set-up to accurately measure nitrite concentration as an index of M1 macrophage polarization.ResultsThe authors found that seeding 2 × 10⁴ cells/well and stimulating with 10 ng/mL LPS for 16 h allowed the inhibition of nitrite production by 60% with the use of dexamethasone. Using these established conditions, the authors were able to test different MSC-sEV preparations and generate dose–response curves. Moreover, the authors fully analytically validated assay performance and fulfilled cross-validation against other M1 markers.ConclusionsThe authors standardized a quick, cheap and reproducible in vitro macrophage assay that allows for the evaluation and estimation of the anti-inflammatory activity of MSC-sEVs.     

CD73 activity of mesenchymal stromal cell-derived extracellular vesicle preparations is detergent-resistant and does not correlate with immunomodulatory capabilities

Background aimsExtracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) show immunomodulatory activity in different assays both in vitro and in vivo. In previous work, the authors compared the immunomodulatory potential of independent MSC-EV preparations in a multi-donor mixed lymphocyte reaction (mdMLR) assay and an optimized steroid-refractory acute graft-versus-host disease (aGVHD) mouse model. The authors observed that only a proportion of the MSC-EV preparations showed immunomodulatory capabilities and demonstrated that only MSC-EV preparations with mdMLR immunomodulating activities were able to suppress aGVHD symptoms in vivo and vice versa. Since the mdMLR assay is complex and depends on primary human cells of different donors, the authors sought to establish an assay that is much easier to standardize and fulfills the requirements for becoming qualified as a potency assay.MethodsThe bona fide MSC antigen CD73 possesses ecto-5’-nucleotidase activity that cleaves pro-inflammatory extracellular adenosine monophosphate into anti-inflammatory adenosine and free phosphate. To test whether the ecto-5’-nucleotidase activity of the MSC-EV preparations reflected their immunomodulatory potential, the authors adopted an enzymatic assay that monitors the ecto-5’-nucleotidase activity of CD73 in a quantitative manner and compared the activity of well-characterized MSC-EV preparations containing or lacking mdMLR immunomodulatory activity.ResultsThe authors showed that the ecto-5’-nucleotidase activity of the MSC-EV preparations did not correlate with their ability to modulate T-cell responses in the mdMLR assay and thus with their potency in improving disease symptomatology in the optimized mouse aGVHD model. Furthermore, the ecto-5’-nucleotidase activity was resistant to EV-destroying detergent treatment.ConclusionsEcto-5’-nucleotidase activity neither reflects the potency of the authors’ MSC-EV preparations nor provides any information about the integrity of the respective EVs. Thus, ecto-5’-nucleotidase enzyme activity is not indicative for the immunomodulatory potency of the authors’ MSC-EV products. The development of appropriate potency assays for MSC-EV products remains challenging.     

Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness

Several studies have examined the functions of nucleic acids in small extracellular vesicles (sEVs). However, much less is known about the protein cargos of sEVs and their functions in recipient cells. This study demonstrates the presence of lysine‐specific demethylase 1 (LSD1), which is the first identified histone demethylase, in the culture medium of gastric cancer cells. We show that sEVs derived from gastric cancer cells and the plasma of patients with gastric cancer harbor LSD1. The shuttling of LSD1‐containing sEVs from donor cells to recipient gastric cancer cells promotes cancer cell stemness by positively regulating the expression of Nanog, OCT4, SOX2, and CD44. Additionally, sEV‐delivered LSD1 suppresses oxaliplatin response of recipient cells in vitro and in vivo, whereas LSD1‐depleted sEVs do not. Taken together, we demonstrate that LSD1‐loaded sEVs can promote stemness and chemoresistance to oxaliplatin. These findings suggest that the LSD1 content of sEV could serve as a biomarker to predict oxaliplatin response in gastric cancer patients.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Human mesenchymal stromal cells small extracellular vesicles attenuate sepsis-induced acute lung injury in a mouse model: the role of oxidative stress and the mitogen-activated protein kinase/nuclear factor kappa B pathway

Background aimsAcute lung injury (ALI) secondary to sepsis is a complex disease associated with high morbidity and mortality. Mesenchymal stem cells (MSCs) and their conditioned medium have been demonstrated to reduce alveolar inflammation, improve lung endothelial barrier permeability and modulate oxidative stress in vivo and in vitro. Recently, MSCs have been found to release small extracellular vesicles (sEVs) that can deliver functionally active biomolecules into recipient cells. The authors’ study was designed to determine whether sEVs released by MSCs would be effective in sepsis-induced ALI mice and to identify the potential mechanisms.MethodsA total of 6 h after cercal ligation and puncture, the mice received saline, sEV-depleted conditioned medium (sEVD-CM) or MSC sEVs via the tail vein.ResultsThe administration of MSC sEVs improved pulmonary microvascular permeability and inhibited both histopathological changes and the infiltration of polymorphonuclear neutrophils into lung tissues. In addition, the activities of antioxidant enzymes were significantly increased in the group treated with sEVs compared with the saline and sEVD-CM groups, whereas lipid peroxidation was significantly decreased. Furthermore, sEVs were found to possibly inhibit phosphorylation of the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) pathway and degradation of IκB but increase the activities of nuclear factor erythroid 2-related factor 2 and heme oxygenase 1.ConclusionsThese findings suggest that one of the effective therapeutic mechanisms of sEVs against sepsis-induced ALI may be associated with upregulation of anti-oxidative enzymes and inhibition of MAPK/NF-κB activation.     

Immune regulatory targets of mesenchymal stromal cell exosomes/small extracellular vesicles in tissue regeneration

Mesenchymal stromal cell (MSC) therapies have demonstrated therapeutic efficacy in a wide-ranging array of tissue injury and disease indications. An important aspect of MSC-mediated therapeutic activities is immune modulation. Consistent with the concentration of MSC therapeutic potency in its secretion, a significant proportion of MSC immune potency resides in the small extracellular vesicles (sEVs) secreted by MSCs. These sEVs, which also include exosomes, carry a large cargo enriched in proteins with potent immunomodulatory activities. They have been reported to exert potent effects on humoral and cellular components of the immune system in vitro and in vivo, and may have the potential to support the diametrically opposite pro- and anti-inflammatory functions necessary for tissue repair and regeneration following injury. Following injury, pro-inflammatory activities are necessary to neutralize injury and remove dead or injured tissue, while anti-inflammatory activities to facilitate migration and proliferation of reparative cell types and to increase vascularization and nutrient supply are necessary to repair and regenerate new tissue. Therefore, a critical immunomodulatory requisite of MSC sEVs in tissue regeneration is the capacity to support the appropriate immune activities at the appropriate time. Here, we review how some of the immune regulatory targets of MSC sEVs could support the dynamic immunomodulatory activities during tissue repair and regeneration.     

Mesenchymal stromal cell variables influencing clinical potency: the impact of viability,fitness, route of administration and host predisposition

The International Society for Cell & Gene Therapy mesenchymal stromal cell (MSC) committee has been an interested observer of community interests in all matters related to MSC identity, mechanism of action, potency assessment and etymology, and it has regularly contributed to this conversation through a series of MSC pre-conferences and committee publications dealing with these matters. Arising from these reflections, the authors propose that an overlooked and potentially disruptive perspective is the impact of in vivo persistence on potency that is not predicted by surrogate cellular potency assays performed in vitro and how this translates to in vivo outcomes. Systemic delivery or extravascular implantation at sites removed from the affected organ system seems to be adequate in affecting clinical outcomes in many pre-clinical murine models of acute tissue injury and inflammatory pathology, including the recent European Medicines Agency-approved use of MSCs in Crohn-related fistular disease. The authors further propose that MSC viability and metabolic fitness likely dominate as a potency quality attribute, especially in recipients poised for salutary benefits as defined by emerging predictive biomarkers of response.     

Characterization of a single reporter-gene potency assay for T-cell-dependent bispecific molecules

ABSTRACT

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit patients’ T cells to kill cancer cells. There are many TDBs in clinical trials, demonstrating their widely recognized therapeutic potential. However, their complex, multi-step mechanism of action (MoA), which includes bispecific antigen binding, T-cell activation, and target-cell killing, presents unique challenges for biological characterization and potency assay selection. Here, we describe the development of a single reporter-gene potency assay for a TDB (TDB1) that is MoA reflective and sensitive to binding of both antigens. Our reporter-gene assay measures T-cell activation using Jurkat cells engineered to express luciferase under the control of an NFkB response element. The potencies of select samples were measured both by this assay and by a flow-cytometry-based cell-killing assay using human lymphocytes as effector cells. Correlating the two sets of potency results clearly establishes our reporter-gene assay as MoA reflective. Furthermore, correlating potencies for the same panel of samples against binding data measured by binding assays for each individual arm demonstrates that the reporter-gene potency assay reflects dual-antigen binding and can detect changes in affinity for either arm. This work demonstrates that one reporter-gene assay can be used to measure the potency of TDB1 while capturing key aspects of its MoA, thus serving as a useful case study of selection and justification of reporter-gene potency assays for TDBs. Furthermore, our strategy of correlating reporter-gene potency, target-cell killing, and antigen binding for each individual arm serves as a useful example of a thorough, holistic approach to biological characterization for TDBs that can be applied to other bispecific molecules.     

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell–extracellular vesicle preparations

Background aimsExtracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed.MethodsBy combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia).ResultsThe authors’ analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types.ConclusionsOverall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors’ MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.     

Orthogonal potency analysis of mesenchymal stromal cell function during ex vivo expansion

Adult bone marrow mesenchymal stromal cells (MSCs) have cross-functional, intrinsic potency that is of therapeutic interest. Their ability to regenerate bone, fat, and cartilage, modulate the immune system, and nurture the growth and function of other bone marrow hematopoietic stem/progenitor cells have all been evaluated by transplant applications of MSCs. These applications require the isolation and expansion scaled cell production. To investigate biophysical properties of MSCs that can be feasibly utilized as predictors of bioactivity during biomanufacturing, we used a low-density seeding model to drive MSCs into proliferative stress and exhibit the hallmark characteristics of in vitro aging. A low-density seeding method was used to generate MSCs from passages 1–7 to simulate serial expansion of these cells to maximize yield from a single donor. MSCs were subjected to three bioactivity assays in parallel to ascertain whether patterns in MSC age, size, and shape were associated with the outcomes of the potency assays. MSC age was found to be a predictor of adipogenesis, while cell and nuclear shape was strongly associated to hematopoietic-supportive potency. Together, these data evaluate morphological changes associated with cell potency and highlight new strategies for purification or alternatives to assessing MSC quality.     

Mesenchymal stromal cell therapy: progress in manufacturing and assessments of potency

Mesenchymal stromal cell (MSC) therapies have been pursued for a broad spectrum of indications but mixed reports on clinical efficacy have given rise to some degree of skepticism regarding the effectiveness of this approach. However, recent reports of successful clinical outcomes and regulatory approvals for graft-versus-host disease, Crohn's disease and critical limb ischemia have prompted a shift in this perspective. With hundreds of clinical trials involving MSCs currently underway and an increasing demand for large-scale manufacturing protocols, there is a critical need to develop standards that can be applied to processing methods and to establish consensus assays for both MSC processing control and MSC product release. Reference materials and validated, uniformly applied tests for quality control of MSC products are needed. Here, we review recent developments in MSC manufacturing technologies, release testing and potency assays. We conclude that, although MSCs hold considerable promise clinically, economies of scale have yet to be achieved although numerous bioreactor technologies for scalable production of MSCs exist. Additionally, rigorous disease-specific product testing and comprehensive understanding of mechanisms of action, which are linked to relevant process and product release potency assays, will be required to ensure that these therapies continue to be successful.     

Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources

Background aimsMesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency.MethodsThe authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics.ResultsMSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications.ConclusionsThe authors’ data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.     

Independent human mesenchymal stromal cell–derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model

Background aimsExtracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow–derived MSCs, this study focused on improving the MSC-EV production for clinical application.MethodsIndependent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized.ResultsThe functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers.ConclusionsStandardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.     

DNAJB8 in small extracellular vesicles promotes Oxaliplatin resistance through TP53/MDR1 pathway in colon cancer

Chemotherapy is one of the most frequently used therapies for the treatment of colon cancer (COAD). However, Oxaliplatin (L-OHP) resistance is a major obstacle to the effective treatment of COAD. Here, we investigated whether DNAJB8, a heat shock protein 40 (HSP40) family protein, could be used for the prognosis and therapy of L-OHP resistance in COAD. Treatment with small interfering RNA targeting DNAJB8 could restore the response to L-OHP in vitro and in vivo. On the mechanism, we demonstrated that DNAJB8 could interact with TP53 and inhibit the ubiquitination degradation of TP53, leading to MDR1 upregulation which promotes colon cancer L-OHP resistance. We found that small extracellular vesicle (sEV)-mediated transfer of DNAJB8 from L-OHP-resistant COAD cells to sensitive cells contributed to L-OHP resistance. A prognostic signature based on the DNAJB8 levels in both tissue and serum showed that COAD patients with high-risk scores exhibited significantly worse overall survival and disease-free survival than patients with low-risk scores. These results indicate that DNAJB8 levels in serum sEVs may serve as a biomarker for COAD. DNAJB8 from sEVs might be a promising therapeutic target for L-OHP resistance and a prognostic predictor of clinical response.Subject terms: Mechanisms of disease, Prognostic markers, Colon cancer     

Collaborative study for the calibration of a replacement International Standard for Tetanus Toxoid Adsorbed

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for Tetanus Toxoid Adsorbed. Two candidate preparations were included in the study, one of which was established as the 4th IS for Tetanus Toxoid Adsorbed at the WHO Expert Committee on Biological Standardization meeting in October 2010. This preparation was found to have a unitage of 490 IU/ampoule, based on calibration in guinea pig challenge assays. Results from mouse challenge assays suggest that the relative performance of two candidate preparations may differ significantly between guinea pigs and mice. The authors note that the number of laboratories that performed guinea pig challenge assays, which are used to calibrate and assign IU, is much lower than in previous collaborative studies and this may have implications for calibration of replacement standards in the future. The issue of assigning separate units to the IS for guinea pig and mouse assays is discussed. The study also assessed performance of the replacement standard in serological assays which are used as alternative procedures to challenge assays for tetanus potency testing. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays.