Midlands Innovation Extracellular Vesicles Network Annual Conference

Artemis Stamboulis, Sanjana Vig, Era Cansever Mutlu, Maria Helena Fernandes*

*mhfernandes@fmd.up.pt

Extracellular vesicles (EVs), particularly exosomes, play a crucial role in intercellular communication within the bone microenvironment and are increasingly recognised as regulators of osteoblast–osteoclast (OB–OC) crosstalk. Elucidating their role is essential for the development of orthopaedic implant materials with optimised osteogenic and antibacterial properties. This study investigated the functional role of EVs in osteogenic and osteoclastic differentiation and assessed material-dependent effects on EV characteristics and cargo.

Bone marrow–derived mesenchymal stem cells (BMSCs) and blood-derived monocytes were differentiated into osteoblasts and osteoclasts, respectively. EV biogenesis and secretion were modulated using GW4869, an inhibitor of ceramide-dependent exosome formation, and Nexinhib, an inhibitor of Rab27a-mediated vesicle exocytosis. EVs were isolated using ultracentrifugation and characterised by nanoparticle tracking analysis and transmission electron microscopy. The influence of metallic implant materials, including titanium, stainless steel, and TiAl6V4, on EV release was assessed during osteogenic differentiation under EV-depleted conditions.

GW4869 effectively reduced the release of small EVs from BMSCs in a dose-dependent manner and was associated with reduced alkaline phosphatase activity, indicating impaired osteogenic differentiation. In contrast, Nexinhib induced significant cytotoxicity in BMSCs, highlighting the importance of EV secretion for osteoblast viability. During osteoclast differentiation, both inhibitors altered EV size distribution and concentration, while unexpectedly enhancing tartrate-resistant acid phosphatase activity, suggesting a complex regulatory role of EVs in osteoclastogenesis. Material-dependent differences in EV size and concentration were observed, with a progressive reduction in EV output as osteogenic differentiation advanced.

Functional assays demonstrated that osteoclast-derived EVs significantly enhanced osteogenic differentiation of BMSCs in a dose- and stage-dependent manner, even in the absence of osteogenic supplements. Proteomic profiling of osteoclast-derived EVs revealed enrichment of cytoskeletal, metabolic, and signalling proteins associated with bone remodelling pathways. These findings demonstrate the central role of EV-mediated OB–OC communication and highlight the influence of implant materials on EV-mediated signalling.

1. Dr Anto Praveen Rajkumar Rajamani (Clinical Associate Professor in Old Age Psychiatry in the University of Nottingham, and the Honorary consultant old age psychiatrist in the Nottinghamshire healthcare NHS foundation trust)
2. Dr Mattea Finelli (Assistant Professor, University of Nottingham)
3. Dr Anbarasu Loudusamy (Associate Professor, Nottingham Trent University)
4. Dr Anto Praveen Rajkumar Rajamani (Clinical Associate Professor in Old Age Psychiatry in the University of Nottingham, and the Honorary consultant old age psychiatrist in the Nottinghamshire healthcare NHS foundation trust)
5. Dr Mattea Finelli (Assistant Professor, University of Nottingham)
6. Dr Anbarasu Loudusamy (Associate Professor, Nottingham Trent University)

Background:

Distinguishing Dementia with Lewy Bodies (DLB) from Alzheimer’s Disease (AD) accurately is essential because they differ in their prognosis, risk profiles, treatment, and sensitivity to antipsychotic medications. As DLB-specific imaging biomarkers and lumbar puncture are not feasible in many clinical settings in UK, there is an urgent clinical need for identifying blood-based biomarkers for DLB. We have reported DLB-specific RNA that were significantly differentially expressed in both post-mortem DLB brains and blood-based small extracellular vesicles (SEV) in people living with DLB. We aimed to develop a multiplex plasma SEV RNA biomarker assay using these DLB-specific RNA, and to establish its diagnostic accuracy.

Methods:

We designed customised BBDLB (Blood-based biomarker for DLB) miRNA and mRNA assays using the NanoString nCounter^® technology. We recruited people with DLB, AD, and people without cognitive impairment (N=90;n=30/group) from four NHS sites. We separated SEV from platelet-poor plasma using size exclusion chromatography, and characterised SEV using cryo-transmission electron microscopy. BBDLB assays measured DLB-specific plasma SEV RNA levels. Using clinical diagnoses of consultant old age Psychiatrists with DLB expertise as reference standard, we estimated the diagnostic accuracy of our BBDLB assays.

Results:

We demonstrated the feasibility of measuring very low-input plasma SEV RNA levels from clinically collected blood samples. The BBDLB miRNA assay could correctly classify 78.4% of people with DLB from AD with 91.3% sensitivity, 57.1% specificity, 2.13 positive likelihood ratio (PLR) and 0.15 negative likelihood ratio (NLR). The BBDLB mRNA assay could correctly classify 75.0% of DLB from AD with 70.0% sensitivity, 80.0% specificity, 3.50 PLR and 0.38 NLR.

Conclusion:

The BBDLB assay is the first blood-based diagnostic biomarker assay for DLB that has the potential to be adapted for routine use in the NHS settings. Enriching plasma SEV for neuronal and microglial origin by immunoprecipitation can improve the signal-to-noise ratio and diagnostic accuracy.

The BBDLB assay is the first blood-based diagnostic biomarker assay for DLB that has the potential to be adapted for routine use in the NHS settings. Enriching plasma SEV for neuronal and microglial origin by immunoprecipitation can improve the signal-to-noise ratio and diagnostic accuracy.

Melanie Tepus¹, Maria Pia Savoca¹, Elisabetta A. M. Verderio^1,2 

¹ Centre for Systems Health and integrated Metabolic Research (SHiMR), School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom

²Department of Biological, Geological, and Environmental Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy

Introduction

Urinary extracellular vesicles (uEVs) have diagnostic potential as a source for biomarkers, however, their physical properties such as the zeta potential (ZP) are less characterised. ZP of nanoparticles may reflect the properties of the cells from which they originate and could serve as an indicator of underlying pathological changes. We hypothesized that uEVs’ ZP could vary in diabetic kidney disease (DKD) compared with diabetes with no kidney disease, with the possibility of early DKD detection and stratification.

Methods

Cell-free urines, sourced from Sheffield Kidney Institute and Patras University Hospital, (Greece) with informed consent and uniformed standards were grouped in 3 cohorts: DKD (n=36), further stratified based on eGFR loss in stable (<2 ml/min/year) and progressive (>5 ml/min/year); diabetes with no CKD (n=16); healthy (18). Small and large uEVs were obtained by differential centrifugation and differences in ZP, size distribution, and concentration analysed using nanoparticle tracking analysis and TEM. uEVs markers were further characterized by immunoblotting and SP-IRIS/Exoview.

Results

A significant difference in large uEVs ZP was observed between the diabetes and the healthy cohort, and the diabetes and DKD progressive cohort, with a less negative ZP in diabetes. No differences were reported in small uEVs. We hypothesised that alterations in cell surface heparan sulphate (HS) proteoglycans occurring in diabetes (loss of negatively charged glycocalyx) and DKD (HS deposition underlying tissue fibrosis) may be reflected in the EVs surface, resulting in changes in ZP, mirroring HS content. Indeed, uEVs from DKD displayed more HS than uEVs isolated from diabetic subjects when evaluated by SP-IRIS/Exoview, trapping EVs via HS-binding. Comparative uEV proteomics revealed an increased presence of sulfated glycosaminoglycans lumican in DKD EVs compared to diabetes, reported to increase in DKD in early fibrosis.

Conclusions

The zeta potential of large uEVs could be predictive of the evolution of diabetes into DKD.

McCann, Sophie, University of Nottingham (School of Life Sciences)*; Jackson, Hannah, University of Nottingham (Children’s Brain Tumour Research Centre, School of Medicine); Hathway, Gareth, University of Nottingham (School of Life Sciences); Coyle, Beth, University of Nottingham (Children’s Brain Tumour Research Centre, School of Medicine); Dajas-Bailador, Federico, University of Nottingham (School of Life Sciences). *corresponding author: mbysm9@nottingham.ac.uk

Paediatric brain tumour survivors are more likely to experience major psychiatric and neurodevelopmental disorders than their peers. To date, little research has been done on the neurobiological consequences of experiencing a brain tumour during early development.

We examined the ability of medulloblastoma tumour cells (the most common malignant paediatric brain tumour) to affect the development of cortical neurones via extracellular vesicle (EV)-mediated signalling. EVs were isolated from three medulloblastoma cell lines (DAOY, D425 – derived from a primary cerebellar tumour and D458 – derived from the patient-paired recurrent CSF metastasis) and added to E16.5 mouse primary cortical neurones for 24 hours. As indicators of cortical development and network maturation, axon length and total neuronal arborisation length were measured.

D458 EVs increased both axon and total neuronal arborisation length in the cortical neurones. However, neither D425 nor DAOY EVs had any effect on either output. To investigate the mediators of this effect, next-generation sequencing (Illumina, SY-415-1002) and unsupervised analysis (Qiagen) of the D458 EV microRNA (miRNA) cargo were performed, and three highly abundant miRNA candidates were selected. miRNA power inhibitors (100nM) were added to cortical neurones for 24 hours alongside D458 EVs. Inhibiting hsa-miRNA-X significantly attenuated the D458 EV-mediated increase in axon length and total neuronal arborisation length, indicating a potential mechanism for the observed EV-mediated effect. Potential mRNA targets of hsa-miRNA-X were identified in the literature and by using miRDB target prediction. Expression of these mRNA targets was validated in the mouse primary cortical neurones using RT-qPCR.

This study is helping to define how miRNA-containing medulloblastoma-derived EVs can reshape neuronal development. The outputs of this study are highly relevant for improving long-term neurological outcomes in survivors and for identifying novel biomarkers.

Martina Gabrielli^1,2, Giulia D’Arrigo², Giulia Cutugno², Maria Teresa Golia², Francesca Sironi³, Marta Lombardi², Roberto Frigerio⁴, Marina Cretich⁴, Elisabetta Battocchio², Sara Francesca Colombo², Cristiana Barone⁵, Emanuele Azzoni⁵, Roberta Ghidoni⁶, Caterina Bendotti³, Rosa Chiara Paolicelli⁷, Claudia Verderio²

¹ School of Science and Technology, Centre for Systems Health and Integrated Metabolic Research (SHiMR), Nottingham Trent University, Nottingham, United Kingdom

²  Institute of Neuroscience, National Research Council of Italy, Via Raoul Follereau 3, 20854, Vedano al Lambro, Italy.

³ Research Center for ALS, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Mario Negri, 2, 20156 Milano, Italy.

⁴ National Research Council of Italy, Institute of Chemical Science and Technologies (SCITEC-CNR), Via Mario Bianco 9, 20131 Milan, Italy.

⁵ School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy

⁶ Molecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, 25125 Brescia, Italy.

⁷ Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland.

The complement factor C1q is released by microglia, localizes on weak synapses and tags them for microglia-mediated synaptic pruning, a fundamental process for proper circuit refinement across early postnatal life, reactivated in neurodegeneration. However, how C1q tags synapses at specific times remains elusive. We explored the possible involvement of extracellular vesicles (EVs) released by microglia in C1q delivery to synapses designated for removal.

We optimized a protocol to extract large and small EVs from mouse brain tissue. By Western Blot, TRPS, single molecules arrays (SimoA) and confocal imaging, we measured the amount, cell source and complement cargo of EVs extracted from WT postnatal pups and adult mice brains, and from C9orf72KO adult brains displaying enhanced microglia-mediated pruning. Then, we performed in vitro and ex vivo studies to inquire microglial EVs involvement in C1q deposition to the synapse and synaptic engulfment, by increasing or reducing microglial EVs production through EVs supplementation, C9orf72 silencing or pharmacological inhibition (GW4869).

We show that C9orf72KO microglia releases more EVs carrying C1q, providing a link between microglial EVs production and synaptic removal. Moreover, we report that production of C1q carrying microglial EVs peaks during the pruning period in the early postnatal hippocampus, confirming a positive correlation between EVs production and synaptic removal. In neuron-microglia co-cultures, microglial EVs make preferential contacts with synapses, deliver C1q to pre-synapses that externalize phosphatidylserine and promote synaptic removal. Interestingly, C9orf72KO microglia engulf more synaptic terminals and decrease synaptic density to a greater extent compared to WT microglia whereas inhibition of EVs release by GW4869 restores normal pre-synaptic density, providing mechanistic evidence linking EVs release to synaptic remodeling.

This study identifies microglial EVs as delivery vehicles for C1q to synapses targeted for removal and implicates abnormal EVs production from microglia in both neurodevelopmental and age-related disorders characterized by dysregulated synaptic pruning.

Extracellular vesicle (EV) mediated cellular crosstalk has been implicated in the progression of idiopathic pulmonary fibrosis (IPF), however no mechanisms have been elucidated. We investigated the surface phenotype and cargo of EVs derived from IPF patients, and their effect on immune cell function and metabolism. EVs were isolated from platelet-free plasma and broncho-alveolar lavage (BAL) of IPF patients and age-matched healthy controls; phenotype was assessed using Exoview & nanoflow cytometry. EV microRNA cargo was investigated with small RNA sequencing and quantified and validated with RT-qPCR. Monocyte-derived macrophages (MDMs) from healthy volunteers and alveolar macrophages (AMs) from lobectomy patients were treated with EVs for 24 hours prior to metabolic profiling and flow cytometric assessment of efferocytosis & phagocytosis. EPCAM+/CD9+ EVs are enriched in IPF patients compared to age-matched healthy controls (p=0.0273). MDM treatment with IPF patient plasma EVs decreased efferocytosis (fc=0.36, p=0.0078) and increased phagocytosis. Plasma EVs from healthy age-matched controls were enriched with miR-485-5p compared to IPF patients, suggesting a protective role for this novel microRNA. Macrophage dysfunction is associated with a pro-fibrotic phenotype, thus modulation of plasma EV cargo may offer a novel therapeutic strategy for IPF patients.

Katherine White¹, Daniel Scott¹, Federico Dajas-Bailador¹, Sebastien Serres¹, Rob Layfield¹

¹School of Life sciences, University of Nottingham

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the progressive loss of upper and lower motor neurones (MNs). Mutations in Fused in Sarcoma (FUS) are associated with aggressive, juvenile-onset ALS. FUS encodes a predominantly nuclear RNA-binding protein that regulates RNA metabolism, including RNA transport and miRNA processing. Pathogenic FUS mutations can cause cytoplasmic mislocalisation where it forms aggregates, leading to neuronal death. Recent work has identified FUS as a key regulator of extracellular vesicle (EV)-mediated export of selected miRNAs (García-Martín et al., 2022), but this has not been explored in human disease-relevant cell types such as astrocytes and neurones.

In ALS, reactive astrocytes can in some contexts promote neurotoxicity exacerbating MN death. Astrocytes and neurones communicate bidirectionally, with astrocyte-derived EVs being one route thought to deliver neuroprotective molecules including miRNAs to neurones. We hypothesise that FUS dysfunction contributes to ALS pathogenesis through aberrant EV-mediated cell-cell miRNA signalling.

Small RNA sequencing of control human iPSC-derived MNs and their purified EVs revealed an enrichment of miRNAs with known FUS-binding motifs in EVs relative to whole-cell miRNAs. This enrichment was lost in MNs carrying the pathogenic FUS P525L mutation. Similar EV enrichment of FUS-binding motif miRNAs was observed in primary astrocytes, supporting a role for FUS in the selective sorting and loading of EV-associated miRNAs in ALS-relevant cell types. Future work aims to extend these findings to iPSC-derived astrocytes to generate an EV–miRNA atlas defining “inclusion” versus “exclusion” miRNA motifs and identifying how FUS mutations alter neural EV cargo composition.

 ¹*Junpeng Luo, ^1,2,3 Jessica Whelan, ¹Susan McDonnell.

* lead presenter. junpeng.luo@ucdconnect.ie, UCD School of Chemical and Bioprocess Engineering, Ireland

¹ UCD School of Chemical and Bioprocess Engineering, Ireland

² UCD Conway Institute of Biomolecular and Biomedical Research, Ireland

³ Science Foundation Ireland Research Centre for Pharmaceuticals (SSPC), Ireland

Extracellular Vesicles derived from human Mesenchymal Stem Cells (hMSC-EVs) possess promising efficacy in immunomodulation and tissue regeneration. Yet, their clinical translation is bottlenecked by limited process scalability and a lack of robust manufacturing frameworks. Furthermore, current hMSC-EVs production often relies on ill-defined supplements which complicates regulatory approval.

Here, we present the development of a scalable three-dimensional (3D) process for manufacturing hMSC-EVs and compare its performance with that of a monolayer culture process. We also present the development of a chemically-defined medium to enable regulatory-compliant manufacturing of hMSC-EVs. The work highlights the single vesicle characterization using highly sensitive Nano-scale Flow Cytometry (nFCM).

The 3D process achieved a peak viable cell density of 3.25 ± 0.14 ×10⁵ cells mL^-1, a 3.2-fold improvement over the initial process. MSCs preserved the phenotypes defined by the ISCT guideline throughout cultures with 93.4 ± 0.3 % viability at the end of EV production. The 3D process yielded 5.10 ± 0.14 × 10⁹ particles mL^-1 of conditioned medium and a specific productivity of 6.83 ± 0.35 ×10³ particles cell^-1 day^-1, representing 5.2-fold and 5.8-fold increases over the monolayer process, respectively. Both 2D- and 3D-EVs were positive for canonical tetraspanins CD9, CD63 and CD81, and MSC markers CD29, CD44, CD73, and CD90. Interestingly, the phenotypes of MSC-EVs did not always mirror those of their parental cells. Nano-FCM also revealed that MSC markers preferentially associated with EVs of a size over 100 nm, whereas tetraspanins displayed heterogeneous expression on EVs of all size. Growth factor screening indicated that a combination of Insulin-Transferrin-Selenium, bFGF, TGF-β1, albumin, and ascorbic acid collectively supported consistent MSC expansion in the absence of ill-defined supplements.

In conclusion, upstream process optimization effectively boosts MSC-EV titre and productivity, bridging the gap between manufacturing capacity and clinical demand. Transition to chemically-defined production enables MSC-EV manufactured under the Quality by Design (QbD) framework.

Atiyeh Asadpour, PhD in Pharmacology, University of Reading

Glioblastoma is the most aggressive primary brain tumour with a poor clinical outcome, even after surgery, radiotherapy, and chemotherapy. Their proliferation is facilitated by a pro-inflammatory tumour microenvironment. Changing pro-inflammatory environment through the immunoregulatory secretome of adult neural crest-derived stem cells (NCSCs) could be a possible method to make the tumour more susceptible to standard therapies. NCSC secretomes and small extracellular vesicles (sEVs) can provide a clinically scalable, cell-free approach to reprogram tumour inflammatory networks. Methods: Secretomes and sEVs from fully characterised NCSCs (three donors) were applied to human glioma cell lines. Functional effects were assessed via cytokine arrays, soft agar colony formation, temozolomide (TMZ) sensitivity assays, and immunocytochemistry. Results: NCSC-derived secretomes and sEVs interacted with glioma cells and suppressed TNF α-induced proliferation, migration, sphere growth, and viability. Treatment inhibited nuclear p65 translocation, redirected cytokine output toward an anti-tumour profile, and sensitised tumour colonies to TMZ. Conclusions: NCSC secretomes and sEVs synergise with TMZ to overcome Glioblastoma(GBM) chemoresistance through simultaneous NF-κB suppression and immunomodulation. This Good Manufacturing Practice (GMP)-compatible strategy integrates tumour microenvironment reprogramming.