Apoptosis in mesenchymal stromal cells activates an immunosuppressive secretome predicting clinical response in Crohn's disease.

In vivo apoptosis of human mesenchymal stromal cells (MSCs) plays a critical role in delivering immunomodulation. Yet, caspase activity not only mediates the dying process but also death-independent functions that may shape the immunogenicity of apoptotic cells. Therefore, a better characterization of the immunological profile of apoptotic MSCs (ApoMSCs) could shed light on their mechanistic action and therapeutic applications. We analyzed the transcriptomes of MSCs undergoing apoptosis and identified several immunomodulatory factors and chemokines dependent on caspase activation following Fas stimulation. The ApoMSC secretome inhibited human T cell proliferation and activation, and chemoattracted monocytes in vitro. Both immunomodulatory activities were dependent on the cyclooxygenase2 (COX2)/prostaglandin E2 (PGE2) axis. To assess the clinical relevance of ApoMSC signature, we used the peripheral blood mononuclear cells (PBMCs) from a cohort of fistulizing Crohn's disease (CD) patients who had undergone MSC treatment (ADMIRE-CD). Compared with healthy donors, MSCs exposed to patients' PBMCs underwent apoptosis and released PGE2 in a caspase-dependent manner. Both PGE2 and apoptosis were significantly associated with clinical responses to MSCs. Our findings identify a new mechanism whereby caspase activation delivers ApoMSC immunosuppression. Remarkably, such molecular signatures could implicate translational tools for predicting patients' clinical responses to MSC therapy in CD.

Extracellular vesicles from senescent mesenchymal stromal cells are defective and cannot prevent osteoarthritis.

Age is the most important risk factor in degenerative diseases such as osteoarthritis (OA), which is associated with the accumulation of senescent cells in the joints. Here, we aimed to assess the impact of senescence on the therapeutic properties of extracellular vesicles (EVs) from human fat mesenchymal stromal cells (ASCs) in OA. We generated a model of DNA damage-induced senescence in ASCs using etoposide and characterized EVs isolated from their conditioned medium (CM). Senescent ASCs (S-ASCs) produced 3-fold more EVs (S-EVs) with a slightly bigger size and that contain 2-fold less total RNA. Coculture experiments showed that S-ASCs were as efficient as healthy ASCs (H-ASCs) in improving the phenotype of OA chondrocytes cultured in resting conditions but were defective when chondrocytes were proliferating. S-EVs were also impaired in their capacity to polarize synovial macrophages towards an anti-inflammatory phenotype. A differential protein cargo mainly related to inflammation and senescence was detected in S-EVs and H-EVs. Using the collagenase-induced OA model, we found that contrary to H-EVs, S-EVs could not protect mice from cartilage damage and joint calcifications, and were less efficient in protecting subchondral bone degradation. In addition, S-EVs induced a pro-catabolic and pro-inflammatory gene signature in the joints of mice shortly after injection, while H-EVs decreased hypertrophic, catabolic and inflammatory pathways. In conclusion, S-EVs are functionally impaired and cannot protect mice from developing OA.

Therapeutic potential in rheumatic diseases of extracellular vesicles derived from mesenchymal stromal cells.

The incidence of rheumatic diseases such as rheumatoid arthritis and osteoarthritis and injuries to articular cartilage that lead to osteochondral defects is predicted to rise as a result of population ageing and the increase in high-intensity physical activities among young and middle-aged people. Current treatments focus on the management of pain and joint functionality to improve the patient's quality of life, but curative strategies are greatly desired. In the past two decades, the therapeutic value of mesenchymal stromal cells (MSCs) has been evaluated because of their regenerative potential, which is mainly attributed to the secretion of paracrine factors. Many of these factors are enclosed in extracellular vesicles (EVs) that reproduce the main functions of parental cells. MSC-derived EVs have anti-inflammatory, anti-apoptotic as well as pro-regenerative activities. Research on EVs has gained considerable attention as they are a potential cell-free therapy with lower immunogenicity and easier management than whole cells. MSC-derived EVs can rescue the pathogenetic phenotypes of chondrocytes and exert a protective effect in animal models of rheumatic disease. To facilitate the therapeutic use of EVs, appropriate cell sources for the production of EVs with the desired biological effects in each disease should be identified. Production and isolation of EVs should be optimized, and pre-isolation and post-isolation modifications should be considered to maximize the disease-modifying potential of the EVs.

Recent Advances in Extracellular Vesicle-Based Therapies Using Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) are being widely investigated as acellular therapeutics in regenerative medicine applications. EVs isolated from mesenchymal stromal cells (MSCs) are by far the most frequently used in preclinical models for diverse therapeutic applications, including inflammatory, degenerative, or acute diseases. Although they represent promising tools as cell-free therapeutic agents, one limitation to their use is related to the batch-to-batch unreliability that may arise from the heterogeneity between MSC donors. Isolating EVs from MSCs derived from induced pluripotent stem cells (iMSCs) might allow unlimited access to cells with a more stable phenotype and function. In the present review, we first present the latest findings regarding the functional aspects of EVs isolated from iMSCs and their interest in regenerative medicine for the treatment of various diseases. We will then discuss future directions for their translation to clinics with good manufacturing practice implementation.

Mesenchymal Stromal Cells for Graft Versus Host Disease: Mechanism-Based Biomarkers.

The immunosuppressive activity of mesenchymal stromal cells (MSCs) in graft versus host disease (GvHD) is well-documented, but their therapeutic benefit is rather unpredictable. Prospective randomized clinical trials remain the only means to address MSC clinical efficacy. However, the imperfect understanding of MSC biological mechanisms has undermined patients' stratification and the successful design of clinical studies. Furthermore, although MSC efficacy seems to be dependent on patient-associated factors, the role of patients' signature to predict and/or monitor clinical outcomes remains poorly elucidated. The analysis of GvHD patient serum has identified a set of molecules that are associated with high mortality. However, despite their importance in defining GvHD severity, their role in predicting or monitoring response to MSCs has not been confirmed. A new perspective on the use of MSCs for GvHD has been prompted by the recent findings that MSCs are actively induced to undergo apoptosis by recipient cytotoxic cells and that this process is essential to initiate MSC-induced immunosuppression. This discovery has not only reconciled the conundrum between MSC efficacy and their lack of engraftment, but also highlighted the determinant role of the patient in promoting and delivering MSC immunosuppression. In this review we will revisit the extensive use of MSCs for the treatment of GvHD and will elaborate on the need that future clinical trials must depend on mechanistic approaches that facilitate the development of robust and consistent assays to stratify patients and monitor clinical outcomes.

Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry.

Extracellular vesicles (EVs) are responsible for a multitude of physiological functions, including immunomodulation. A heterogenous mixture of small EV (sEV) subsets, including putative exosomes, is derived when commonly used "exosome" isolation techniques are employed. Subset diversity relates in part to their different intracellular origins, and can be associated with distinct functional properties. Recent progress in the EV field has enabled the categorization of such subsets based on their surface composition. For the first time, we combine such emerging subset-specific markers with advanced imaging flow cytometry (iFCM) to perform high-throughput, multiparametric, vesicle-by-vesicle characterization, and functional assessment of specific small EV subsets, and exosomes in particular. The approach allows researchers to address three important applications. First, it is known that different isolation techniques result in the divergent recovery of particular vesicle subsets. Taking three commonly used "exosome" isolation techniques as test cases (ultracentrifugation, size-exclusion chromatography, and polymer-based precipitation), the capacity for convenient and accurate isolate compositional analysis by iFCM is demonstrated. The approach was able to corroborate and to quantify the known skewing of subtype recovery among different isolation approaches. Second, exosomes are a particularly widely studied EV subset. Applying exosome-specific markers to samples collected from an optimal clinical transplantation model, we verify the capacity for iFCM to detect exosomes in circulation, to establish their tissue of origin, and to provide insights as to their functional immunological potential. Finally, we describe a technique for establishing whether the transfer of a molecule of interest to a target cell is exosomally mediated. In so doing, we highlight the approach's utility in assessing the functional impact of circulating exosomes and in identifying their targets. In conclusion, we set out a new methodological approach by which small extracellular vesicle subsets, exosomes in particular, can be conveniently and comprehensively investigated, thereby offering novel phenotypic and functional insights.

Extracellular vesicles secreted by bone marrow- and adipose tissue-derived mesenchymal stromal cells fail to suppress lymphocyte proliferation.

Recently, mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have been suggested as an alternative to MSCs for the treatment of various inflammatory disorders. However, while a first case report observed beneficial therapeutic effects of repeated MSC-EV infusions in a patient with therapy-refractory graft-versus-host disease, in vitro findings revealed that MSC-EVs were significantly less immunosuppressive than their parental cells. In this study, we compared the immunosuppressive potency of MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs), with their secreted EVs in a standardized lymphocyte proliferation assay (LPA). Both BM-MSCs and AT-MSCs exhibited a remarkable inhibition of lymphocyte proliferation (LP) (88.1%±1.5% and 75.5%±1.5%, respectively), while isolated EVs derived from them failed to suppress LP at dose levels up to 100 µg/mL. Thus, our data further substantiate previous reports suggesting that cell-cell contact plays an important role on the immunosuppressive potential mediated by MSCs. Hence, MSC-EVs are still a matter of debate and might not be a reasonable substitute for MSCs with regard to the immunosuppressive function. Collectively, these contrasting findings may also reflect the importance of relevant translational aspects when designing new studies. Standardization of MSC culture conditions before EV collection as well as isolation and characterization methods with regard to EV purity are urged. Moreover, before clinical use, dose-finding studies evaluating MSC-EV preparations in suitable preclinical models are warranted.