Agarose spot migration assay to measure the chemoattractant potential of extracellular vesicles: applications in regenerative medicine and cancer metastasis.

BACKGROUND: The recruitment of effector cells is one of the novel functions described for extracellular vesicles (EVs) that needs further study. For instance, cell recruitment by mesenchymal stromal cell derived-EVs (MSC-EVs) is one of the features by which MSC-EVs may induce regeneration and ameliorate tissue injury. On the other hand, increasing evidence suggests that cancer EVs play an important role in the preparation of the pre-metastatic niche (PMN) by recruiting their primary tumour cells. Understanding and measuring the potential of MSC-EVs or cancer-EVs to induce cell migration and recruitment is essential for cell-free therapeutic approaches and/or for a better knowledge of cancer metastasis, respectively. In this context, classical in vitro migration assays do not completely mimic the potential situation by which EVs exert their chemotactic capacity. RESULTS: We adapted an agarose spot migration assay as an in vitro system to evaluate the cell recruitment capacity of locally delivered or localized EVs. Cell migration was tracked for 12 h or 48 h, respectively. Thereafter, endpoint migration images and time-lapse videos were analysed to quantify several parameters aiming to determine the migration of cells to either MSC-EV or pro-metastatic EV. The number of cells contained inside the agarose spots, the migration distance, the area occupied by cells, the directionality of the cell movement, and the Euclidean distance were measured. This multi-parametric evaluation revealed the potential of different MSC-EV preparations to recruit endothelial cells and to detect an enhanced recruitment capacity of highly metastatic PC3-derived EVs (PC3-EVs) compared to low-metastatic LNCaP-EVs in a tumour cell-specific manner. CONCLUSIONS: Overall, this agarose spot migration assay may offer a diversity of measurements and migration settings not provided by classical migration assays and reveal its potential use in the EV field in two different contexts with recruitment in common: regeneration and cancer metastasis.

N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV-Cell Interaction and Functional Activation of Endothelial Cells.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV-cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells' interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton's Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV-cell interactions and associated functions.

In Vitro Characterization of Human CD24hiCD38hi Regulatory B Cells Shows CD9 Is Not a Stable Breg Cell Marker.

Regulatory B (Breg) cells are endowed with immune suppressive functions. Various human and murine Breg subtypes have been reported. While interleukin (IL)-10 intracellular staining remains the most reliable way to identify Breg cells, this technique hinders further essential functional studies. Recent findings suggest that CD9 is an effective surface marker of murine IL-10 competent Breg cells. However, the stability of CD9 and its relevance as a unique marker for human Breg cells, which have been widely characterized as CD24hiCD38hi, have not been investigated. Here, we demonstrate that CD9 expression is sensitive to in vitro B cell stimulations. CD9 expression could either be re-expressed or downregulated in purified CD9-negative B cells and CD9-positive B cells, respectively. We found no significant differences in the Breg differentiation capacity of the CD9-negative and CD9-positive B cells. Furthermore, CD9-positive B cells co-express CD40 and CD86, suggesting their nature as B cell activation or co-stimulatory molecules, rather than regulatory ones. Therefore, we report the relatively unstable CD9 as a distinct surface molecule, indicating the need for further research for a more reliable marker to purify human Breg cells.

The emergence of regenerative medicine in organ transplantation: 1st European Cell Therapy and Organ Regeneration Section meeting.

Regenerative medicine is emerging as a novel field in organ transplantation. In September 2019, the European Cell Therapy and Organ Regeneration Section (ECTORS) of the European Society for Organ Transplantation (ESOT) held its first meeting to discuss the state-of-the-art of regenerative medicine in organ transplantation. The present article highlights the key areas of interest and major advances in this multidisciplinary field in organ regeneration and discusses its implications for the future of organ transplantation.

Proteomic Characterization of Urinary Extracellular Vesicles from Kidney-Transplanted Patients Treated with Calcineurin Inhibitors.

Use of immunosuppressive drugs is still unavoidable in kidney-transplanted patients. Since their discovery, calcineurin inhibitors (CNI) have been considered the first-line immunosuppressive agents, in spite of their known nephrotoxicity. Chronic CNI toxicity (CNIT) may lead to kidney fibrosis, a threatening scenario for graft survival. However, there is still controversy regarding CNIT diagnosis, monitoring and therapeutic management, and their specific effects at the molecular level are not fully known. Aiming to better characterize CNIT patients, in the present study, we collected urine from kidney-transplanted patients treated with CNI who (i) had a normal kidney function, (ii) suffered CNIT, or (iii) presented interstitial fibrosis and tubular atrophy (IFTA). Urinary extracellular vesicles (uEV) were enriched and the proteome was analyzed to get insight into changes happening during CNI. Members of the uroplakin and plakin families were significantly upregulated in the CNIT group, suggesting an important role in CNIT processes. Although biomarkers cannot be asserted from this single pilot study, our results evidence the potential of uEV as a source of non-invasive protein biomarkers for a better detection and monitoring of this renal alteration in kidney-transplanted patients.

Potential of Extracellular Vesicle-Associated TSG-6 from Adipose Mesenchymal Stromal Cells in Traumatic Brain Injury.

Multipotent mesenchymal stromal cells (MSC) represent a promising strategy for a variety of medical applications. Although only a limited number of MSC engraft and survive after in vivo cellular infusion, MSC have shown beneficial effects on immunomodulation and tissue repair. This indicates that the contribution of MSC exists in paracrine signaling, rather than a cell-contact effect of MSC. In this review, we focus on current knowledge about tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) and mechanisms based on extracellular vesicles (EV) that govern long-lasting immunosuppressive and regenerative activity of MSC. In this context, in particular, we discuss the very robust set of findings by Jha and colleagues, and the opportunity to potentially extend their research focus on EV isolated in concentrated conditioned media (CCM) from adipose tissue derived MSC (ASC). Particularly, the authors showed that ASC-CCM mitigated visual deficits after mild traumatic brain injury in mice. TSG-6 knockdown ASC were, then, used to generate TSG-6-depleted CCM that were not able to replicate the alleviation of abnormalities in injured animals. In light of the presented results, we envision that the infusion of much distilled ASC-CCM could enhance the alleviation of visual abnormalities. In terms of EV research, the advantages of using size-exclusion chromatography are also highlighted because of the enrichment of purer and well-defined EV preparations. Taken together, this could further delineate and boost the benefit of using MSC-based regenerative therapies in the context of forthcoming clinical research testing in diseases that disrupt immune system homeostasis.

Mesenchymal Stem Cell-Derived Interleukin 1 Receptor Antagonist Promotes Macrophage Polarization and Inhibits B Cell Differentiation.

The role of interleukin 1 receptor antagonist (IL1RA) in mediating the immunosuppressive effect of mesenchymal stem/stromal cells (MSCs) has been reported in several studies. However, how MSC-derived IL1RA influences the host response has not been clearly investigated. We therefore derived MSCs from the bone marrow of IL1RA knockout mice and evaluated their immunosuppressive effect on different immune cell subsets. IL1RA deficient (IL1RA(-/-) ) or wild type (wt) MSCs inhibited to the same extend the proliferation of T lymphocytes. On the contrary, IL1RA(-/-) MSCs were less effective than wt MSCs to induce in vitro the macrophage polarization from M1 to M2 phenotype secreting IL10 and exerting a suppressive effect on CD4(+) T cells. Moreover compared with wt MSCs, IL1RA(-/-) MSCs did not efficiently support the survival of quiescent B lymphocytes and block their differentiation toward CD19(+) CD138(+) plasmablasts secreting IgG antibodies. The effectiveness of IL1RA secreted by MSCs in controlling inflammation was further shown in vivo using the collagen-induced arthritis murine model. MSCs lacking IL1RA expression were unable to protect mice from arthritic progression and even worsened clinical signs, as shown by higher arthritic score and incidence than control arthritic mice. IL1RA(-/-) MSCs were not able to decrease the percentage of Th17 lymphocytes and increase the percentage of Treg cells as well as decreasing the differentiation of B cells toward plasmablasts. Altogether, our results provide evidence of the key role of IL1RA secreted by MSCs to both control the polarization of macrophages toward a M2 phenotype and inhibit B cell differentiation in vivo.

Allogeneic chondrogenically differentiated human mesenchymal stromal cells do not induce immunogenic responses from T lymphocytes in vitro.

BACKGROUND AIMS: In regenerative medicine, the use of allogeneic cells could enable the development of "off the shelf" therapies for patients with critical size bone defects, reducing limitations observed with the use of autologous cells, such as cost and time to treat the patient. The idea of the use of allogeneic bone marrow mesenchymal stromal cells (BMSCs) has been of interest in tissue engineering studies. However, little is known about the properties of these cells upon differentiation. Chondrogenically differentiated BMSCs have already been shown to form endochondral bone in immunodeficient and immunocompetent animals. The success of this bone formation is dependent on the host's endogenous cells. This study investigates the interactions between allogeneic chondrogenically differentiated human bone marrow mesenchymal stromal cell (hBMSC) pellets and T lymphocytes in vitro. METHODS: Non-chondrogenic (-transforming growth factor (TGF)ß3) and chondrogenic hBMSC (+TGFß3) pellets were directly co-cultured with unstimulated and CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) for 7 days. hBMSC pellets from the co-culture were either fixed for histological analysis or quantitative real time polymerase chain reaction (qRT-PCR). PBMCs were harvested for flow cytometry. RESULTS: Flow cytometic analysis revealed that chondrogenically differentiated hBMSC pellets did not alter the number or proliferation of CD4+, CD8+ T cells or FoxP3+ T regulatory cells (CD4+CD25+CD127-). Chondrogenic hBMSC pellets did not induce immunogenic responses in unstimulated PBMCs. Infiltrating CD3 T cells were found in the matrix of hBMSC pellets. Furthermore, qRT-PCR demonstrated low levels of T-cell activation genes (CD25, CD69, PRF1 and GZMB) in addition to low gene expression levels of the pro-inflammatory gene tumor necrosis factor alpha (TNFα) in chondrogenically differentiated hBMSC pellets cultured with unstimulated PBMCs in comparison with non-chondrogenic hBMSC pellets. CONCLUSIONS: Collectively the results of this study demonstrate that allogeneic chondrogenically differentiated hBMSC pellets are non-immunogenic and do not induce the activation of destructive T-cell responses in vitro.

Tolerance in Kidney Transplantation: What Is on the B Side?

Regulatory B cells (Breg) are in the spotlight for their role in immune homeostasis maintenance and tolerance achievement as in the last years the correlation with functional and increased Breg numbers in autoimmune diseases and transplantation has been extensively proven. Their study is, however, in its infancy with still little knowledge and consensus on their origin, phenotype, and mechanism of action. All this hampers the pursuit of an effective Breg induction method for therapeutic purposes. In this review we aim to summarize the studies on human Breg and their implication in kidney transplantation and to further discuss the issues surrounding therapeutic applications of this cell subset.

Induction of suppressive allogeneic regulatory T cells via rabbit antithymocyte polyclonal globulin during homeostatic proliferation in rat kidney transplantation.

Experimental studies have shown that rabbit antithymocyte polyclonal globulin (ATG) can expand human CD4+CD25++Foxp3+ cells (Tregs). We investigated the major biological effects of a self-manufactured rabbit polyclonal anti-rat thymoglobulin (rATG) in vitro, as well as its effects on different peripheral T-cell subsets. Moreover, we evaluated the allogeneic suppressive capacity of rATG-induced Tregs in an experimental rat renal transplant model. Our results show that rATG has the capacity to induce apoptosis in T lymphocyte lymphocytes as a primary mechanism of T-cell depletion. Our in vivo studies demonstrated a rapid but transient cellular depletion of the main T cell subsets, directly proportional to the rATG dose used, but not of the effector memory T cells, which required significantly higher rATG doses. After rATG administration, we observed a significant proliferation of Tregs in the peripheral blood of transplanted rats, leading to an increase in the Treg/T effector ratio. Importantly, rATG-induced Tregs displayed a strong donor-specific suppressive capacity when assessed in an antigen-specific allogeneic co-culture. All of these results were associated with better renal graft function in rats that received rATG. Our study shows that rATG has the biological capacity immunomodulatory to promote a regulatory alloimmune milieu during post-transplant homeostatic proliferation.

The effect of rabbit antithymocyte globulin on human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess immunomodulatory properties which are of key interest for their application in autoimmunity and transplantation. In transplantation, administration of MSCs has shown promising results in preclinical models and has recently moved to clinical trials. Therefore, it is important to study the interactions between MSCs and immunosuppressive drugs currently used in transplantation. We aimed to analyze the effect of rabbit antithymocyte globulin (rATG) MSCs. MSCs were obtained from perirenal fat of kidney donors and exposed to ranging doses of rATG (Thymoglobulin(®) , Genzyme; 0.5-100 µg/ml). Binding of rATG, effects on viability and susceptibility to be killed by cytotoxic lymphocytes as well as effects on their immunosuppressive potential of MSCs were tested. rATG binds dose-dependently to MSCs. This binding was associated with slightly impaired viability after 48 and 72 h when compared with nonexposed MSCs. In contrast to nontreated MSCs, rATG preexposed MSCs were susceptible to be lysed by cytokine-activated CD8(+) cytotoxic cells and NKT cells. The capacity of MSCs to suppress the proliferation of anti-CD3/CD28 activated CD4 and CD8 T cells were reduced by the presence of rATG in the culture. rATG reduces the viability and antiproliferative capacity of MSCs in a dose-dependent manner and converts them into targets for CD8 T cells and NKT cell lysis.

Kidney regeneration and repair after transplantation.

PURPOSE OF REVIEW: To briefly show which are the mechanisms and cell types involved in kidney regeneration and describe some of the therapies currently under study in regenerative medicine for kidney transplantation. RECENT FINDINGS: The kidney contains cell progenitors that under specific circumstances have the ability to regenerate specific structures. Apart from the knowledge gained in the self-regenerative properties of the kidney, new concepts in regenerative medicine such as organ engineering and the use of mesenchymal stem cell-based therapies are currently the focus of attention in the field. SUMMARY: Overall, kidney regeneration is a reality and the knowledge on how to control it will be one of the main scopes in the present and future.

The impact of mesenchymal stem cell therapy in transplant rejection and tolerance.

PURPOSE OF REVIEW: Treatment with classical immunosuppressive drugs is associated with low specificity leading to toxicity, infections and malignancies. Cell therapy is emerging as an alternative for the pharmacological therapies and among them mesenchymal stem cells (MSCs) appear as a solid choice for the treatment of transplanted patients. The aim of the present review is to analyze the potency of MSCs cell therapy in solid organ transplantation. RECENT FINDINGS: The increasing knowledge on the role of newly identified cell types like the pro-inflammatory Th17 cells, tolerogenic dendritic cells, regulatory macrophages and regulatory B cells among others in alloreactivity widened our understanding of the immune responses in transplantation patients. Recent studies suggest that MSCs interact with those populations as well as with classically studied Th1 cells to reduce alloreactivity and potentially promote graft acceptance. SUMMARY: MSCs are arising as an immunomodulatory and potentially pro-tolerogenic tool in cell therapeutic intervention in solid organ transplantation and could substitute or minimize current immunosuppressive treatments.

Commonly used methods for extracellular vesicles' enrichment: Implications in downstream analyses and use.

Extracellular vesicles (EVs) are becoming promising tools for clinical application, either as sources of disease-specific molecular signatures for the unraveling of disease pathophysiology and establishment of novel biomarkers, or as platforms for cell-free nanotherapy. Yet, an unsolved issue is to define standardized techniques for EV isolation allowing data comparison across laboratories worldwide. Considering the difficulties to find this necessary consensus, it has to be stressed out that the outcome of the downstream analysis might be deeply biased by the isolation method, among other variables. Thus, it is crucial that the researcher is aware of the strengths and weaknesses of each method keeping their intended use in mind, and to sufficiently report the methodology details for the results to be comparable and solid. This review aims to present the most widely used EV isolation methods, from the initial differential ultracentrifugation (dUC) to newest approaches.

Mesenchymal stromal cells induced regulatory B cells are enriched in extracellular matrix genes and IL-10 independent modulators.

Regulatory B cells (Breg) are essential players in tolerance and immune homeostasis. However, lack of specific Breg markers limit their potential in clinical settings. Mesenchymal stromal cells (MSC) modulate B cell responses and are described to induce Breg in vitro. The aim of this work was to characterize MSC induced Breg (iBreg) and identify specific Breg biomarkers by RNAseq. After 7-day coculture with adipose tissue-derived MSC, B cells were enriched in transitional B cell populations, with increased expression and secretion of IL-10 and no TNFα. In addition, iBreg showed potential to modulate T cell proliferation at 2 to 1 cell ratios and their phenotype remained stable for 72h. RNAseq analysis of sorted IL-10 positive and negative iBreg populations identified over 1500 differentially expressed genes (DEG) among both populations. Analysis of biological processes of DEG highlighted an enrichment of immune regulation and extracellular matrix genes in IL-10- iBreg populations, while IL-10+ iBreg DEG were mostly associated with cell activation. This was supported by T cells modulation assays performed in the presence of anti-IL-10 neutralizing antibodies showing the non-essential role of IL-10 in the immunomodulatory capacity of iBregs on T cells. However, based on RNAseq results we explored the role of TGF-ß and found out that it plays a major role on iBreg induction and iBreg immunomodulatory properties. Therefore, we report that MSC induce B cell populations characterized by the generation of extracellular matrix and immune modulation independently of IL-10.

Costimulatory blockade with mTor inhibition abrogates effector T-cell responses allowing regulatory T-cell survival in renal transplantation.

The advent of novel immunosuppressive strategies in renal transplantation, with immunomodulatory properties, might facilitate long-term allograft survival. T-cell depletion, costimulation-blockade and mTor inhibition have been shown to favour anti-donor hyporesponsiveness. Recently, the combination of rATG, belatacept (Bela) and sirolimus (SRL) has been used in kidney transplantation, showing very low incidence of acute rejection and excellent 12-month graft and patient survival. Herein, we have analysed the 1-year evolution of memory/effector and regulatory T cells and assessed the donor-specific T-cell alloimmune response in a group of these patients and compared with others treated with a calcineurin-inhibitor(CNI)-based (rATG/tacrolimus/MMF), and two other Bela-based regimens (rATG/Bela/MMF and basiliximab/Bela/MMF/steroids). During the first year after transplantation, patients receiving rATG/Bela/SRL had significantly higher percentage of Tregs upon the memory T-cell compartment and showed a potent anti-donor suppressive activity. In an in vitro naive and memory/effector T-cell co-culture, the combination of costimulation-blockade and SRL could abrogate both antigen-specific T-cell responses as efficiently as using a CNI drug. The combination of T-cell depletion, costimulation-blockade and mTor inhibition seems to be able to allow Treg survival and inhibit donor-specific alloreactive effector immune responses after kidney transplantation in humans.

Regulatory B Cell Therapy in Kidney Transplantation.

In the context of kidney injury, the role of Bregs is gaining interest. In a number of autoimmune diseases, the number and/or the function of Bregs has been shown to be impaired or downregulated, therefore restoring their balance might be a potential therapeutic tool. Moreover, in the context of kidney transplantation their upregulation has been linked to tolerance. However, a specific marker or set of markers that define Bregs as a unique cell subset has not been found and otherwise multiple phenotypes of Bregs have been studied. A quest on the proper markers and induction mechanisms is now the goal of many researchers. Here we summarize the most recent evidence on the role of Bregs in kidney disease by describing the relevance of in vitro and in vivo Bregs induction as well as the potential use of Bregs as cell therapy agents in kidney transplantation.

Urinary vitronectin identifies patients with high levels of fibrosis in kidney grafts.

BACKGROUND: In kidney transplantation, fibrosis represents the final and irreversible consequence of the pathogenic mechanisms that lead to graft failure, and in the late stages it irremediably precedes the loss of renal function. The invasiveness of kidney biopsy prevents this condition from being frequently monitored, while clinical data are rather unspecific. The objective of this study was to find noninvasive biomarkers of kidney rejection. METHODS: We carried out proteomic analysis of the urinary Extracellular Vesicles (uEVs) from a cohort of kidney transplant recipients (n = 23) classified according to their biopsy-based diagnosis and clinical parameters as interstitial fibrosis and tubular atrophy (IFTA), acute cellular rejection (ACR), calcineurin inhibitors toxicity (CNIT) and normal kidney function (NKF). RESULTS: Shotgun mass spectrometry of uEV-proteins identified differential expression of several proteins among these different groups. Up to 23 of these proteins were re-evaluated using targeted proteomics in a new independent cohort of patients (n = 41) classified in the same diagnostic groups. Among other results, we found a differential expression of vitronectin (VTN) in patients displaying chronic interstitial and tubular lesions (ci and ct mean > 2 according to Banff criteria). These results were further confirmed by a pilot study using enzyme-linked immunosorbent assay (ELISA). CONCLUSION: Urinary vitronectin levels are a potential stand-alone biomarker to monitor fibrotic changes in kidney transplant recipients in a non-invasive fashion.

Corrigendum: Immunomodulatory Effect of MSC on B Cells Is Independent of Secreted Extracellular Vesicles.

[This corrects the article DOI: 10.3389/fimmu.2019.01288.].

Immunomodulatory Effect of MSC on B Cells Is Independent of Secreted Extracellular Vesicles.

Mesenchymal stem or stromal cells (MSC) have proven immunomodulatory properties toward B cell activation and induce regulatory B cells (Breg), through a dual mechanism of action that relies both on cell contact and secreted factors. One of them are MSC-derived extracellular vesicles (EVs), membrane nanovesicles that mediate cell communication and typically reflect the phenotype of the cell of origin. MSC-EVs could resemble MSC functions, and are being contemplated as an improved alternative to the MSC-based immunomodulatory therapy. In the present work, we focused on the factors secreted by MSC and aimed to elucidate the putative role of MSC-EVs in the immunomodulation of B cells. EVs and soluble protein-enriched fractions (PF) were isolated from MSC-conditioned medium (CM) using size-exclusion chromatography (SEC) and their capacity to modulate B cell activation, induction of Breg and B cell proliferation was compared to that of the whole MSCs. Co-culture with MSC or unfractionated CM induced naïve and CD24hiCD38hi, IL-10 producing (Breg) phenotypes on B cells while not affecting proliferation. MSC-PF had a comparable effect to MSCs, inducing a naïve phenotype, and even though they did not induce the shift toward a CD24hiCD38hi population, MSC-PF fostered IL-10 production by B cells. Conversely, MSC-EVs failed to promote naïve B cells and to reduce memory B cells. MSC-EVs induced CD24hiCD38hi B cells to a similar extent of that of MSC, but not bona fide Bregs since they did not produce IL-10. Our results show that B cell modulation by MSC is partially mediated by soluble factors other than EVs.