Brief Report: Elastin Microfibril Interface 1 and Integrin-Linked Protein Kinase Are Novel Markers of Islet Regenerative Function in Human Multipotent Mesenchymal Stromal Cells

Multipotent mesenchymal stromal cell (MSC) transplantation is proposed as a novel therapy for treating diabetes by promoting the regeneration of damaged islets. The clinical promise of such treatments may be hampered by a high degree of donor-related variability in MSC function and a lack of standards for comparing potency. Here, we set out to identify markers of cultured human MSCs directly associated with islet regenerative function. Stromal cultures from nine separate bone marrow donors were demonstrated to have differing capacities to reduce hyperglycemia in the NOD/SCID streptozotocin-induced diabetic model. Regenerative (R) and non-regenerative (NR) MSC cultures were directly compared using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. A total of 1,410 proteins were quantified resulting in the identification of 612 upregulated proteins and 275 downregulated proteins by ± 1.2-fold in R-MSC cultures. Elastin microfibril interface 1 (EMILIN-1), integrin-linked protein kinase (ILK), and hepatoma-derived growth factor (HDGF) were differentially expressed in R-MSCs, and Ingenuity Pathway Analyses revealed each candidate as known regulators of integrin signaling. Western blot validation of EMILIN-1, ILK, and HDGF not only showed significantly higher abundance levels in R-MSCs, as compared with NR-MSCs, but also correlated with passage-induced loss of islet-regenerative potential. Generalized estimating equation modeling was applied to examine the association between each marker and blood glucose reduction. Both EMILIN-1 and ILK were significantly associated with blood glucose lowering function in vivo. Our study is the first to identify EMILIN-1 and ILK as prospective markers of islet regenerative function in human MSCs. Stem Cells 2016;34:2249–2255

Unveiling Integrated Functional Pathways Leading to Enhanced Respiratory Disease Associated With Inactivated Respiratory Syncytial Viral Vaccine

Respiratory syncytial virus (RSV) infection is a severe threat to young children and the elderly. Despite decades of research, no vaccine has been approved. Notably, instead of affording protection, a formalin-inactivated RSV vaccine induced severe respiratory disease including deaths in vaccinated children in a 1960s clinical trial; however, recent studies indicate that other forms of experimental vaccines can also induce pulmonary pathology in pre-clinical studies. These findings suggest that multiple factors/pathways could be involved in the development of enhanced respiratory diseases. Clearly, a better understanding of the mechanisms underlying such adverse reactions is critically important for the development of safe and efficacious vaccines against RSV infection, given the exponential growth of RSV vaccine clinical trials in recent years. By employing an integrated systems biology approach in a pre-clinical cotton rat model, we unraveled a complex network of pulmonary canonical pathways leading to disease development in vaccinated animals upon subsequent RSV infections. Cytokines including IL-1, IL-6 GRO/IL-8, and IL-17 in conjunction with mobilized pulmonary inflammatory cells could play important roles in disease development, which involved a wide range of host responses including exacerbated pulmonary inflammation, oxidative stress, hyperreactivity, and homeostatic imbalance between coagulation and fibrinolysis. Moreover, the observed elevated levels of MyD88 implicate the involvement of this critical signal transduction module as the central node of the inflammatory pathways leading to exacerbated pulmonary pathology. Finally, the immunopathological consequences of inactivated vaccine immunization and subsequent RSV exposure were further substantiated by histological analyses of these key proteins along with inflammatory cytokines, while hypercoagulation was supported by increased pulmonary fibrinogen/fibrin accompanied by reduced levels of plasma D-dimers. Enhanced respiratory disease associated with inactivated RSV vaccine involves a complex network of host responses, resulting in significant pulmonary lesions and clinical manifestations such as tachypnea and airway obstruction. The mechanistic insight into the convergence of different signal pathways and identification of biomarkers could help facilitate the development of safe and effective RSV vaccine and formulation of new targeted interventions

Detailed Characterization of Mesenchymal Stem/Stromal Cells from a Large Cohort of AML Patients Demonstrates a Definitive Link to Treatment Outcomes

Altres ajuts: Health Canada's Genomics Research and Development Initiative Phase VI (H4080-144541-2014-2019); Obra Social La Caixa-Fundació Josep Carreras and the Generalitat de Catalunya (SGR330); Asociación Española Contra el Cáncer (AECC-CI-2015)Bone marrow mesenchymal stem/stromal cells (BM-MSCs) are key components of the hematopoietic niche thought to have a direct role in leukemia pathogenesis. BM-MSCs from patients with acute myeloid leukemia (AML) have been poorly characterized due to disease heterogeneity. We report a functional, genetic, and immunological characterization of BM-MSC cultures from 46 AML patients, stratified by molecular/cytogenetics into low-risk (LR), intermediate-risk (IR), and high-risk (HR) subgroups. Stable MSC cultures were successfully established and characterized from 40 of 46 AML patients irrespective of the risk subgroup. AML-derived BM-MSCs never harbored tumor-specific cytogenetic/molecular alterations present in blasts, but displayed higher clonogenic potential than healthy donor (HD)-derived BM-MSCs. Although HD- and AML-derived BM-MSCs equally provided chemoprotection to AML cells in vitro, AML-derived BM-MSCs were more immunosuppressive/anti-inflammatory, enhanced suppression of lymphocyte proliferation, and diminished secretion of pro-inflammatory cytokines. Multivariate analysis revealed that the level of interleukin-10 produced by AML-derived BM-MSCs as an independent prognostic factor negatively affected overall survival. Collectively our data show that AML-derived BM-MSCs are not tumor related, but display functional differences contributing to therapy resistance and disease evolution. In this article, Díaz de la Guardia and colleagues report a functional, genetic, and immunological characterization of BM-MSC cultures from 46 AML patients, stratified by molecular/cytogenetics into low-risk (LR), intermediate-risk (IR), and high-risk (HR) subgroups. BM-MSCs never harbored tumor-specific cytogenetic/molecular alterations present in blasts, and IL-10 produced by AML-derived BM-MSCs is an independent prognostic factor negatively impacting on overall survival

Applications of Extracellular Vesicles in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is the most aggressive and refractory subtype of breast cancer, often occurring in younger patients with poor clinical prognosis. Given the current lack of specific targets for effective intervention, the development of better treatment strategies remains an unmet medical need. Over the last decade, the field of extracellular vesicles (EVs) has grown tremendously, offering immense potential for clinical diagnosis/prognosis and therapeutic applications. While TNBC-EVs have been shown to play an important role in tumorigenesis, chemoresistance and metastasis, they could be repurposed as potential biomarkers for TNBC diagnosis and prognosis. Furthermore, EVs from various cell types can be utilized as nanoscale drug delivery systems (NDDS) for TNBC treatment. Remarkably, EVs generated from specific immune cell subsets have been shown to delay solid tumour growth and reduce tumour burden, suggesting a new immunotherapy approach for TNBC. Intrinsically, EVs can cross the blood–brain barrier (BBB), which holds great potential to treat the brain metastases diagnosed in one third of TNBC patients that remains a substantial clinical challenge. In this review, we present the most recent applications of EVs in TNBC as diagnostic/prognostic biomarkers, nanoscale drug delivery systems and immunotherapeutic agents, as well as discuss the associated challenges and future directions of EVs in cancer immunotherapy

Efficient Nonviral Transfection of Human Bone Marrow Mesenchymal Stromal Cells Shown Using Placental Growth Factor Overexpression

Background. Human mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity. Methods. In order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. One transfection method using TransIT-2020 was selected and tested with an emphasis on cell quality (viability, identity, and yield), as well as efficacy with a human placental growth factor (PlGF) plasmid. Results. TransIT-2020 yielded the highest fluorescence signal per cell out of the methods that did not decrease cell recovery. Transfecting GFP to 5 hBM-MSC donors using TransIT-2020 yielded 24–36% GFP-expressing cells with a viability of 85–96%. hBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection. When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. Both growth and secretion of PlGF in overexpressing hBM-MSC were sustained over 7 days, confirming the sustainability and applicability of the TransIT-2020 transfection system. Discussion. We report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics

SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

Summary: Extracellular vesicles (EVs) are entering the clinical arena as novel biologics for infectious diseases, potentially serving as the immunogenic components of next generation vaccines. However, relevant human assays to evaluate the immunogenicity of EVs carrying viral antigens are lacking, contributing to challenges in translating rodent studies to human clinical trials. Here, we engineered EVs to carry SARS-CoV-2 Spike to evaluate the immunogenicity of antigen-carrying EVs using human peripheral blood mononuclear cells (PBMCs). Delivery of Spike EVs to PBMCs resulted in specific immune cell activation as assessed through T cell activation marker expression. Further, Spike EVs were taken up largely by antigen-presenting cells (monocytes, dendritic cells and B cells). Taken together, this human PBMC-based system models physiologically relevant pathways of antigen delivery, uptake and presentation. In summary, the current study highlights the suitability of using human PBMCs for evaluating the immunogenicity of EVs engineered to carry antigens for infectious disease therapeutics

Therapeutic effect of the immunomodulatory drug lenalidomide, but not pomalidomide, in experimental models of rheumatoid arthritis and inflammatory bowel disease

Thalidomide is an immunomodulatory drug (IMiD) with proven therapeutic action in several autoimmune/inflammatory diseases; however, its inherent high toxicity has led to the development of more powerful and safer thalidomide analogs, including lenalidomide and pomalidomide. These are new generation IMiDs that exhibit direct antitumor activity as well as anti-inflammatory/immunomodulatory properties, and are FDA-approved for the treatment of several hematological malignances. Here we investigated the potential therapeutic effects of lenalidomide and pomalidomide in several experimental murine models of autoimmune/inflammatory diseases: 2,4,6-trinitrobenzene sulfonic acid- and dextran sulfate sodium-induced inflammatory bowel disease and type II collagen-induced arthritis. Lenalidomide displayed a strong therapeutic effect in all these models of autoimmune/inflammatory diseases, while the effect of pomalidomide was less pronounced. In vitro experiments confirmed the immunosuppressive effect of both IMiDs on the proliferative response of stimulated human lymphocytes and on the balance of secreted cytokines toward an anti-inflammatory profile. We conclude that lenalidomide may offer a therapeutic opportunity against autoimmune/inflammatory diseases.This work was supported by Health Canada (contract: H4080-144541) to PM and MR-M based on funding from Health Canada’s Genomics Research and Development Initiative Phase VI (2014-2019) obtained by MR-M and JRL. PM also acknowledges the financial support from The Obra Social La Caixa-Fundaciò Josep Carreras and The Generalitat de Catalunya (SGR330). PM is investigator of the Spanish Cell Therapy cooperative network (TERCEL). We are indebted to Dominique Patry from Health Canada for his assistance with the Luminex experiments.Peer reviewe

Bone marrow mesenchymal stem/stromal cells from risk-stratified acute myeloid leukemia patients are anti-inflammatory in in vivo preclinical models of hematopoietic reconstitution and severe colitis

financial support for this work was obtained from: Health Canada (H4084-112281) to PM and MR-M. The European Research Council (CoG-2014-646903) to PM. The Spanish Ministry of Economy and Competitiveness (SAF-SAF2013-43065), to PM. The Generalitat de Catalunya (SGR330 and PERIS 2017-2019 Program) to P.M. The Asociacion Espanola Contra el Cancer (AECC-CI-2015) and Fero Foundation to CB. The "Fundacion Hay Esperanza'' to E.A. The Health Institute Carlos III (ISCIII/FEDER/PI14-01191) to CB. PM also acknowledges the institutional support from The Obra Social La Caixa-Fundacio Josep Carreras. PM is investigator of the Spanish Cell Therapy cooperative network (TERCEL).Peer reviewe