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Summary 2018 was the year of the first marketing authorization of an allogeneic stem cell therapy by the European Medicines Agency. The authorization concerns the use of allogeneic adipose tissue‐derived mesenchymal stromal cells (MSCs) for treatment of complex perianal fistulas in Crohn's disease. This is a breakthrough in the field of MSC therapy. The last few years have, furthermore, seen some breakthroughs in the investigations into the mechanisms of action of MSC therapy. Although the therapeutic effects of MSCs have largely been attributed to their secretion of immunomodulatory and regenerative factors, it has now become clear that some of the effects are mediated through host phagocytic cells that clear administered MSCs and in the process adapt an immunoregulatory and regeneration supporting function. The increased interest in therapeutic use of MSCs and the ongoing elucidation of the mechanisms of action of MSCs are promising indicators that 2019 may be the dawn of the therapeutic era of MSCs and that there will be revived interest in research to more efficient, practical, and sustainable MSC‐based therapies. Stem Cells Translational Medicine 2019;8:1126–1134 Overview of the regenerative and immunomodulatory actions of mesenchymal stem cells. Mesenchymal stem cells can be induced to differentiate into cell types of different mesenchymal lineages. They are furthermore active secretors of trophic and immunomodulatory factors. Upon intravenous infusion, mesenchymal stem cells undergo apoptosis in the lung microenvironment and are engulfed by monocytic cells, which subsequently adapt an immune regulatory and tissue repair phenotype.

Mesenchymal stem cells (MSC) are present throughout the body and are thought to play a role in tissue regeneration and control of inflammation. MSC can be easily expanded in vitro and their potential as a therapeutic option for degenerative and inflammatory disease is therefore intensively investigated. Whilst it was initially thought that MSC would replace dysfunctional cells and migrate to sites of injury to interact with inflammatory cells, experimental evidence indicates that the majority of administered MSC get trapped in capillary networks and have a short life span. In this review, we discuss current knowledge on the migratory properties of endogenous and exogenous MSC and confer on how culture-induced modifications of MSC may affect these properties. Finally, we will discuss how, despite their limited survival, administered MSC can bring about their therapeutic effects.

Mesenchymal stromal cells (MSCs) are considered to be promising agents for the treatment of immunological disease. Although originally identified as precursor cells for mesenchymal lineages, in vitro studies have demonstrated that MSCs possess diverse immune regulatory capacities. Pre-clinical models have shown beneficial effects of MSCs in multiple immunological diseases and a number of phase 1/2 clinical trials carried out so far have reported signs of immune modulation after MSC infusion. These data indicate that MSCs play a central role in the immune response. This raises the academic question whether MSCs are immune cells or whether they are tissue precursor cells with immunoregulatory capacity. Correct understanding of the immunological properties and origin of MSCs will aid in the appropriate and safe use of the cells for clinical therapy. In this review the whole spectrum of immunological properties of MSCs is discussed with the aim of determining the position of MSCs in the immune system.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited treatment options in which the telomere shortening is a strong predictive factor of poor prognosis. Mesenchymal stromal cells (MSC) administration is probed in several experimental induced lung pathologies; however, MSC might stimulate fibrotic processes. A therapy that avoids MSC side effects of transformation would be an alternative to the use of living cells. Membranes particles (MP) are nanovesicles artificially generated from the membranes of MSC containing active enzymes involved in ECM regeneration. We aimed to investigate the anti-fibrotic role of MP derived from MSC in an in vitro model of pulmonary fibrosis. Epithelial cells (A549) and lung fibroblasts, from IPF patients with different telomere length, were co-cultured with MP and TGF-β for 48h and gene expression of major pro-fibrotic markers were analyzed. About 90% of both types of cells effectively took up MP without cytotoxic effects. MP decreased the expression of profibrotic proteins such as Col1A1, Fibronectin and PAI-1, in A549 cells. In fibroblasts culture, there was a different response in the inhibitory effect of MP on some pro-fibrotic markers when comparing fibroblast from normal telomere length patients (FN) versus short telomere length (FS), but both types showed an inhibition of Col1A1, Tenascin-c, PAI-1 and MMP-1 gene expression after MP treatment. MP conserve some of the properties attributed to the living MSC. This study shows that MP target lung cells, via which they may have a broad anti-fibrotic effect.

Extracellular vesicles (EVs) are tissue-specific particles containing valuable diagnostic information. However, single EV analysis in blood is challenging due to their physical properties, the molecular complexity of plasma, and a lack of robust data interpretation methods. We assess the applicability of our recently-developed calibrated Imaging Flow Cytometry (IFCM)-based methodology to detect/characterize circulating tissue-specific EV subsets in the clinical setting of kidney transplantation. Platelet-poor plasma was generated from 36 HLA-A3 mismatched donor (HLA-A3 +) and kidney transplant recipients (KTRs; HLA-A3-). Samples taken before transplantation, 3 days, 7 days, and 6 months after transplantation as well as before ‘for-cause’ kidney transplant biopsies were stained with anti-CD9 (plasma EV-marker) and anti-HLA-A3. Before transplantation, no significant differences in total CD9 + EV concentrations were detected between donor and KTR samples. Tissue-specific EVs were identified as CD9 + HLA-A3 + . Serial dilution experiments of HLA-A3 + in HLA-A3- PPP showed that single CD9 + HLA-A3 + EVs were detectable down to ~ 1% above the recipient ‘self-signal’. After transplantation, CD9 + HLA-A3 + EVs were detected above pre-transplantation concentrations in individuals with stable allograft function, but not in individuals with allograft dysfunction. These results demonstrate the applicability of our calibrated IFCM-based methodology in the direct detection of tissue-specific EV subsets in clinical samples. We believe that this EV methodology is applicable in a variety of clinical contexts.

Mesenchymal stromal cells (MSC) are a promising therapy for immunological disorders. However, culture expanded MSC are large and get trapped in the capillary networks of the lungs after intravenous infusion, where they have a short survival time. Hypothetically, living cells are a risk for tumor formation. To reduce risks associated with MSC infusion and improve the distribution in the body, we generated membrane particles (MP) of MSC and MSC stimulated with IFN-γ (MPγ). Tracking analysis and electron microscopy indicated that the average size of MP was 120 nm, and they showed a round shape. MP exhibited ATPase, nucleotidase and esterase activity, indicating they are enzymatically active. MP and MPγ did not physically interact with T cells and had no effect on CD4 + and CD8 + T cells proliferation. However, MP and MPγ selectively bound to monocytes and decreased the frequency of pro-inflammatory CD14 + CD16 + monocytes by induction of selective apoptosis. MP and MPγ increased the percentage of CD90 positive monocytes, and MPγ but not MP increased the percentage of anti-inflammatory PD-L1 monocytes. MPγ increased mRNA expression of PD-L1 in monocytes. These data demonstrate that MP have immunomodulatory properties and have potential as a novel cell-free therapy for treatment of immunological disorders.

The immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells (Bregs). It is, however, unknown how MSC interact with B cells under inflammatory conditions. In this study, adipose tissue-derived MSC were pretreated with 50 ng/ml IFN-γ for 96 h (MSC-IFN-γ) to simulate inflammatory conditions. Mature B cells were obtained from spleens by CD43 selection. B cells were co-cultured with MSC and stimulated with anti-IgM, anti-CD40, and IL-2; and after 7 days, B cell proliferation, phenotype, Immunoglobulin-G (IgG), and IL-10 production were analyzed. MSC did not inhibit B cell proliferation but increased the percentage of CD38 CD24 B cells (Bregs) and IL-10 production, while MSC-IFN-γ significantly reduced B cell proliferation and inhibited IgG production by B cells in a more potent fashion but did not induce Bregs or IL-10 production. Both MSC and MSC-IFN-γ required proximity to target cells and being metabolically active to exert their effects. Indoleamine 2,3 dioxygenase expression was highly induced in MSC-IFN-γ and was responsible of the anti-proliferative and Breg reduction since addition of tryptophan (TRP) restored MSC properties. Immunological conditions dictate the effect of MSC on B cell function. Under immunological quiescent conditions, MSC stimulate Breg induction; whereas, under inflammatory conditions, MSC inhibit B cell proliferation and maturation through depletion of TRP. This knowledge is useful for customizing MSC therapy for specific purposes by appropriate pretreatment of MSC.

Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)–based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions. A method to quantitate and phenotype Extracellular Vesicles (EVs) in unprocessed human plasma using Imaging Flow Cytometry is presented.

Background Kidney injury, typically accompanied by inflammation, is a driver for kidney fibrosis, which contributes to the development of kidney failure. Mesenchymal stromal cells (MSC) have been proposed to have anti-fibrotic potential, but challenges such as their short persistence after infusion and inability to cross the lung capillary system due to their large size hamper their use for treatment of kidney fibrosis. It is hypothesized that the effects of MSC are partially dependent on phagocytosis of fragments of MSC by target cells and inhibiting excessive immune activation response. To exploit this effect of MSC, we developed nanosized membrane particles (MP) from MSC and explored their anti-fibrotic activity and immunomodulation effect in mouse and human kidney fibrosis models. Methods MP were generated from culture-expanded MSC through extrusion of isolated membranes. Unilateral kidney ischemia reperfusion injury (IRI) in male Balb/c mice was used to induce kidney fibrosis. MP generated from 1 × 10 6 MSC were injected in the tail vein immediately after anesthesia recovery. In a second model, human induced pluripotent stem cell-derived kidney organoids were exposed to 1% O 2 for 48 h and 100 ng/mL IL-1β for 96 h to mimic IRI in vitro for inducing fibrosis. MP generated from 0.5 × 10 6 MSC were added to the medium for 4 consecutive days. Fibrosis and immune cell markers were subsequently measured. Results IRI induced the expression of transforming growth factor beta (TGFβ) and collagen type I alpha 1(COL1A1) in mouse kidneys. MP treatment significantly reduced TGF-β mRNA at day 3 while COL1A1 mRNA and protein were downregulated at day 7. We found no evidence for an immunomodulatory effect of MP, as the number and activity of infiltrating T cells and macrophages did not change. In kidney organoids, a rise in COL1A1 and TGF-β demonstrated successful fibrosis induction by hypoxia and IL-1β. MP significantly decreased these fibrosis markers. Additionally, immunohistochemistry revealed a reduction in the myofibroblast marker alpha smooth muscle actin. Conclusions Our results demonstrate that MP have anti-fibrotic properties in mouse kidney IRI and human kidney organoid models. These results indicate that MP have potential for the development of kidney fibrosis-inhibiting therapy.