Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-derived Extracellular Vesicle-based Cancer Biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands.

Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro.

Multilevel Proteomic Profiling of Colorectal Adenocarcinoma Caco-2 Cell Differentiation to Characterize an Intestinal Epithelial Model.

Emergent advancements on the role of the intestinal microbiome for human health and disease necessitate well-defined intestinal cellular models to study and rapidly assess host, microbiome, and drug interactions. Differentiated Caco-2 cell line is commonly utilized as an epithelial model for drug permeability studies and has more recently been utilized for investigating host-microbiome interactions. However, its suitability to study such interactions remains to be characterized.

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

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).

A clinically relevant large-scale biomanufacturing workflow to produce natural killer cells and natural killer cell-derived extracellular vesicles for cancer immunotherapy.

Natural killer cell-derived extracellular vesicles (NK-EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune-modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical-grade NK-EVs for pre-clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed-loop hollow-fibre bioreactor to continuously produce NK-EVs from the clinically relevant NK92-MI cell line under serum-free, Xeno-free and feeder-free conditions following GMP-compliant conditions.

The Potential of Hormonal Therapies for Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is considered one of the most aggressive forms of breast cancer with poor survival rates compared to other breast cancer subtypes. TNBC is characterized by the absence of the estrogen receptor alpha, progesterone receptor, and the human epidermal growth factor receptor 2, limiting those viable treatment options available to patients with other breast cancer subtypes. Furthermore, due to the particularly high heterogeneity of TNBC, conventional treatments such as chemotherapy are not universally effective, leading to drug resistance and intolerable side effects.

Canadian Regulatory Framework and Regulatory Requirements for Cell and Gene Therapy Products.

Health Canada regulates gene therapy products and many cell therapy products as biological drugs under the Canadian Food and Drugs Act and its attendant regulations. Cellular products that meet certain criteria, including minimal manipulation and homologous use, may be subjected to a standards-based approach under the Safety of Human Cells, Tissues and Organs for Transplantation Regulations. The manufacture and clinical testing of cell and gene therapy products (CGTP) presents many challenges beyond those for protein biologics.

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.

Hollow-fiber bioreactor production of extracellular vesicles from human bone marrow mesenchymal stromal cells yields nanovesicles that mirrors the immuno-modulatory antigenic signature of the producer cell.

Background: Extracellular vesicles (EVs) produced by human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are currently investigated for their clinical effectiveness towards immune-mediated diseases. The large amounts of stem cell-derived EVs required for clinical testing suggest that bioreactor production systems may be a more amenable alternative than conventional EV production methods for manufacturing products for therapeutic use in humans.

Methods: To characterize the potential utility of these systems, EVs from four hBM-MSC donors were produced independently using a hollow-fiber bioreactor system under a cGMP-compliant procedure.

A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles.

Background: Clinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance.

Methods: Small EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation.

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.

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.

Targeting CD40 enhances antibody- and CD8-mediated protection against respiratory syncytial virus infection.

Respiratory Syncytial Virus (RSV) infects almost all children under the age of one and is the leading cause of hospitalization among infants. Despite several decades of research with dozens of candidate vaccines being vigorously evaluated in pre-clinical and clinical studies, there is no licensed vaccine available to date. Here, the RSV fusion protein (F) was fused with CD40 ligand and delivered by an adenoviral vector into BALB/c mice where the CD40 ligand serves two vital functions as a molecular adjuvant and an antigen-targeting molecule.

Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2.

Background: Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified.

Methods: Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 () gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis.

Micro-RNA Profiling of Exosomes from Marrow-Derived Mesenchymal Stromal Cells in Patients with Acute Myeloid Leukemia: Implications in Leukemogenesis.

Gene regulatory networks in AML may be influenced by microRNAs (miRs) contained in exosomes derived from bone marrow mesenchymal stromal cells (MSCs). We sequenced miRs from exosomes isolated from marrow-derived MSCs from patients with AML (n = 3) and from healthy controls (n = 3; not age-matched). Known targets of mIRs that were significantly different in AML-derived MSC exosomes compared to controls were identified.

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

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.

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.

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.

Adipogenic Mesenchymal Stromal Cells from Bone Marrow and Their Hematopoietic Supportive Role: Towards Understanding the Permissive Marrow Microenvironment in Acute Myeloid Leukemia.

Purpose: The role of bone marrow-derived mesenchymal stem/stromal cells (MSCs) in creating a permissive microenvironment that supports the emergence and progression of acute myeloid leukemia (AML) is not well established. We investigated the extent to which adipogenic differentiation in normal MSCs alters hematopoietic supportive capacity and we undertook an in-depth comparative study of human bone marrow MSCs derived from newly diagnosed AML patients and healthy donors, including an assessment of adipogenic differentiation capacity.

Findings: MSCs from healthy controls with partial induction of adipogenic differentiation, in comparison to MSCs undergoing partial osteogenic differentiation, expressed increased levels of hematopoietic factors and induced greater proliferation, decreased quiescence and reduced in vitro hematopoietic colony forming capacity of CD34(+) hematopoietic stem and progenitor cells (HSPCs).

Proteomic analysis implicates translationally controlled tumor protein as a novel mediator of occlusive vascular remodeling in pulmonary arterial hypertension.

Background: Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular endothelial cells (ECs). Hereditary PAH (HPAH) is often caused by mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear.

Glycogen synthase kinase-3β inhibition augments diabetic endothelial progenitor cell abundance and functionality via cathepsin B: a novel therapeutic opportunity for arterial repair.

Progenitor cell therapy is hindered in patients with diabetes mellitus (DM) due to cellular senescence. Glycogen synthase kinase-3β (GSK3β) activity is increased in DM, potentially exacerbating impaired cell-based therapies. Thus, we aimed to determine if and how GSK3β inhibitors (GSKi) can improve therapeutic efficacy of endothelial progenitor cells (EPC) from patients with DM.

Uncovering the secretes of mesenchymal stem cells.

Mesenchymal stem cells (MSC) show great promise in a wide array of therapeutic applications due mainly to their capacity to suppress immune and inflammatory reactions and instigate normal tissue repair processes. The secretion of bioactive factors is thought to play a predominant role in the mechanisms of action for these clinically relevant functions. As such, a large body of MSC research has focussed on characterization of the MSC secretome; including both soluble factors and factors released in extracellular vesicles (e.

Feeder-independent derivation of induced-pluripotent stem cells from peripheral blood endothelial progenitor cells.

Induced-pluripotent stem cells (iPSCs) are a potential alternative cell source in regenerative medicine, which includes the use of differentiated iPSCs for cell therapies to treat coronary artery and/or peripheral arterial diseases. Late-outgrowth endothelial progenitor cells (late-EPCs) are a unique primary cell present in peripheral blood that exhibit high proliferative capacity, are being used in a wide variety of clinical trials, and have the ability to differentiate into mature endothelial cells. The objective of this study was to reprogram peripheral blood-derived late-EPCs to a pluripotent state under feeder-free and defined culture conditions.