Anders Toftegaard Boysen

Helminths modulate host immunity, and in this respect, the porcine whipworm Trichuris suis has been explored clinically as an immunotherapy for inflammatory diseases. T. suis secretes a complex mixture of excretory/secretory products (ESP), including extracellular vesicles (EVs), nanosized membranous particles with bioactive cargoes of lipids, proteins and nucleic acids that are implicated in intercellular communication within organisms. Here, we characterise the EV protein and miRNA cargo of T. suis to identify potential roles in host–parasite communication. In contrast to EVs from clade III/V helminth species characterised to date, we show that T. suis EVs did not suppress LPS‐induced TNF production by human macrophages under the conditions tested, in contrast to non‐vesicular ESP fractions which did. By generating the first extracellular miRNA complement for T. suis and quantifying EV‐associated versus free miRNAs across larval and adult stages, we identify selective enrichment of two miRNAs in adult EVs with homology to human miR‐22‐3p and miR‐29‐3p, canonical anti‐fibrotic and tumour‐suppressor miRNAs. Functional assays in human colon cancer epithelial cells reveal that these helminth‐derived orthologues downregulate conserved oncogenic targets, indicating evolutionary conservation of their regulatory capacity. Our findings uncover a previously unrecognised axis of whipworm biology in which selective EV‐mediated export of anti‐fibrotic miRNAs has the potential to modulate host tissue remodeling independently of classical immunomodulation, with implications for understanding helminth‐driven epithelial homeostasis and cancer risk.

Parasitic helminths secrete extracellular vesicles (EVs) into their host tissues to modulate immune responses, but the underlying mechanisms are poorly understood. We demonstrate that Ascaris EVs are efficiently internalised by monocytes in human peripheral blood mononuclear cells and increase the percentage of classical monocytes. Furthermore, EV treatment of monocytes induced a novel anti‐inflammatory phenotype characterised by CD14⁺, CD16⁻, CC chemokine receptor 2 (CCR2⁻) and programmed death‐ligand 1 (PD‐L1)⁺ cells. In addition, Ascaris EVs induced T cell anergy in a monocyte‐dependent mechanism. Targeting professional phagocytes to induce both direct and indirect pathways of immune modulation presents a highly novel and efficient mechanism of EV‐mediated host‐parasite communication. Intra‐peritoneal administration of EVs induced protection against gut inflammation in the dextran sodium sulphate model of colitis in mice. Ascaris EVs were shown to affect circulating immune cells and protect against gut inflammation; this highlights their potential as a subject for further investigation in inflammatory conditions driven by dysregulated immune responses. However, their clinical translation would require further studies and careful consideration of ethical implications.

Mesenchymal stromal cell (MSC) therapy holds great potential yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome that includes extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect both yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to enhance potency, increase circulation time or mediate targeting. However, efficient transfection of MSCs is challenging. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production. We isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to genetically engineer USCs using transposon technology. There were no differences between time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the assessed time points, however, male donors had reduced CD73 + cells. Expanded USCs were incubated without growth factors or serum for 72 h without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell cultures were established. Isolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.

Background Mesenchymal stem cell (MSC) therapy holds great potential as a treatment form, yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome, including extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect the yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to; enhance potency, increase circulation time, or mediate targeting. However, efficient transfection of MSCs is challenging, and the process can affect MSC phenotype and chromosomal integrity, which are both imperative to preserve in a therapeutic setting. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production. Methods We isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to engineer USCs using transposon technology genetically. Results There were no observable differences at time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the different time points, but male donors had reduced CD73 + cells. Expanded USCs were incubated without growth factors or serum for 72 hours without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell culture was established. Conclusions Isolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.

Extracellular vesicles (EVs) large‐scale production is a crucial point for the translation of EVs from discovery to application of EV‐based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET‐OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor‐made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV‐based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.

Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminth‐derived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum, were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)‐induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS‐induced levels of tumour necrosis factor (TNF‐α) in a dose‐dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone.

In the last two decades, extracellular vesicles (EVs) from the three domains of life, Archaea, Bacteria and Eukaryotes, have gained increasing scientific attention. As such, the role of EVs in host-pathogen communication and immune modulation are being intensely investigated. Pivotal to EV research is the determination of how and where EVs are taken up by recipient cells and organs in vivo, which requires suitable tracking strategies including labelling. Labelling of EVs is often performed post-isolation which increases risks of non-specific labelling and the introduction of labelling artefacts. Here we exploited the inability of helminths to de novo synthesise fatty acids to enable labelling of EVs by whole organism uptake of fluorescent lipid analogues and the subsequent incorporation in EVs. We showed uptake of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rho) in Anisakis spp. and Trichuris suis larvae. EVs isolated from the supernatant of Anisakis spp. labelled with DOPE-Rho were characterised to assess the effects of labelling on size, structure and fluorescence of EVs. Fluorescent EVs were successfully taken up by the human macrophage cell line THP-1. This study, therefore, presents a novel staining method that can be utilized by the EV field in parasitology and potentially across multiple species.