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2023 ‘ROS’ Workshop

Join four world experts from engineering, biology, and medicine at the 2023 IMGC annual workshop as they share their insights on the challenges and solutions in scaling up, analyzing and navigating regulatory for milk extracellular vesicles and microRNAs.

TITLE: Deciphering the Physiological Roles of Extracellular Vesicles in Milk

ABSTRACT:
In mammals, milk is the first functional food and contains different components playing a role in the development of the gastrointestinal tract and immune system. One of these components are cell-derived extracellular vesicles (EVs), which are used for cell-cell communication. Their roles in developmental processes have been poorly studied. We explored the molecular mechanisms of human mature milk EV-induced modulation of different cell types present in the gastrointestinal tract and found that milk EVs promote the migration of oral epithelial cells. Functional integrative proteomic analyses unveiled that milk EV proteins regulate hotspots in the p38 MAPK pathway. Milk EVs also inhibited innate and adaptive immune responses and milk EV cargo proteins could be linked to hotspots of regulation in crucial signal transduction pathways involved in the modulation of T cell responses. Moreover, also raw bovine milk-derived EVs could inhibit human CD4+ T cell responses, indicating their conserved and cross-species activity. Interestingly, comparison of the T cell modulatory activity of human milk EVs derived from allergic or non-allergic mothers unveiled subtle differences in their modulatory activity and EV-cargo. Furthermore, we found that bovine milk processing before EV isolation could have detrimental effects on the milk EV bioactivities. In conclusion, EVs are conserved bioactive structures in milk that can modulate canonical signal transduction pathways involved in key processes in the development of the epithelial barrier and the immune system of the new-born, but their bioactivities can be affected by the immune status of the donor and the pre-processing of the milk.

Marca Wauben is Full Professor in intercellular communication and an expert in deciphering the role of nano-sized cell-derived vesicles (EVs) in intercellular communication in biological systems with emphasis on the immune system. Besides investigating fundamental immunological and regenerative aspects of EV-mediated communication the ‘Wauben EV Lab’ works on EV-based biomarker discovery, (therapeutic) EV applications and the development of (nano)technology to analyze EVs, e.g. high resolution flow cytometry-based technology enabling single EV-based analysis and sorting. She is author of over n=150 peer reviewed scientific publications and (Co-)recipient of multiple EV-based research grants, e.g. FP7 EU COST Action Microvesicles and Exosomes in Health and Disease (2012; vice-chair), H2020-MSCA-ITN-2016 TRAIN-EV research & training Network (722148) (2017) and H2020-FETOPEN The EV Foundry (801367)(2018). She has been/and is involved in many national and global initiatives to build a scientific community centered around EV-biology; e.g. the founding of the International Society for Extracellular Vesicles (ISEV), the scientific journal “Journal of Extracellular Vesicles” and the founding of the Netherlands Society for Extracellular Vesicles (NLSEV) in 2018 (founding President).

TITLE: When Species and Tissues Matter: Exerting Physiological Functions of Extracellular Vesicles

ABSTRACT:
Historically, milk and saliva have been shown to exert coagulation properties, promote wound healing and reduce the risk of infection. We demonstrated that extracellular vesicles (EVs) derived from milk and saliva trigger coagulation. We conducted several studies to gain insight on species-specific and tissue-specific coagulation functions of EVs. In several comparative studies, human and bovine milk and salivary EVs were isolated using size-exclusion chromatography. Fibrin generation and (coagulation) factor Xa formation assays were used to investigate the ability of EVs to trigger coagulation. EVs were analyzed for the presence of tissue factor (TF), by several methods, including western blot, surface plasmon resonance imaging, electron microscopy, and flow cytometry. We found that EVs derived from human milk and saliva expose TF antigen and activity, as well as coagulation factor VII. Functionally, EVs from human milk and saliva triggered coagulation which was inhibited by antibodies against TF. In contrast, EVs derived from bovine milk did not trigger coagulation, however, EVs from bovine saliva triggered TF-initiated coagulation. To the best of our knowledge, this is the first time that an entirely EV-associated function, i.e. the ability to trigger coagulation, clearly differs between EVs present in the same body fluid of different mammalian species. Additionally, EVs present in different body fluids of the same mammalian species may also exert different functions. These data demonstrate the need for the burgeoning EV field to address factors such as species, tissue type and other proposed characteristics that could influence EV function for human health applications.

Rienk Nieuwland chairs the Amsterdam Vesicle Observation Center of the Amsterdam University Medical Center (Amsterdam, Netherlands). His main interests is isolation, detection and functional characterization of extracellular vesicles (EVs) in human body fluids for biomarker exploration. He chaired the Scientific Standardization Committee on Vascular biology of the International Society on Thrombosis and Haemostasis (ISTH), and currently chairs the Blood EV task force of the International Society for Extracellular Vesicles (ISEV), the ISEV Rigor & Standardization Subcommittee, and he is ISEV board member. He is one of the founders of the ISTH-ISEV-ISAC (International Society for Advancement of Cytometry) working group on EV flow cytometry (www.evflowcytometry.org), chaired multiple European metrology projects aimed to standardize concentration measurements of EVs (www.metves.eu), and he is vice-president of the Dutch EV society (www.nlsev.nl). Rienk Nieuwland chairs the Amsterdam Vesicle Observation Center of the Amsterdam University Medical Center (Amsterdam, Netherlands). His main interests is isolation, detection and functional characterization of extracellular vesicles (EVs) in human body fluids for biomarker exploration. He chaired the Scientific Standardization Committee on Vascular biology of the International Society on Thrombosis and Haemostasis (ISTH), and currently chairs the Blood EV task force of the International Society for Extracellular Vesicles (ISEV), the ISEV Rigor & Standardization Subcommittee, and he is ISEV board member. He is one of the founders of the ISTH-ISEV-ISAC (International Society for Advancement of Cytometry) working group on EV flow cytometry (www.evflowcytometry.org), chaired multiple European metrology projects aimed to standardize concentration measurements of EVs (www.metves.eu), and he is vice-president of the Dutch EV society (www.nlsev.nl).  

TITLE: Advancing an Industrially Scalable Purification Process for mEVs from Bovine Milk

ABSTRACT:
Milk extracellular vesicles (mEVs) consist of greater than 10% by volume of raw milk, yet the industry does not know how to effectively isolate or process them. mEVs contribute to the development of the brain, gastrointestinal tract, and muscle, yet to date, purifications regularly isolate only in the 10^10-11 range. The Tiny Cargo Company seeks to address this problem by advancing an industrially scalable isolation of mEVs for use as a nutraceutical product. mEVs are fragile vesicles held within a lattice of milk proteins- the effective removal of proteins typically will result in the destruction of mEVs, the primary hurdle we faced throughout this process. We solved this problem by incorporating specifically designed chemical treatment regimens with emphases on temperature, time, chemical concentration, and perhaps most importantly, separation regimen. Through careful theoretical modeling combined with extensive experimentation, we identified a very specific regimen that is highly effective in extracting mEVs from milk, resulting in yields exceeding 10^12 mEVs per mL with over 150 mL per Liter input. These yields support continued commercial development of our milk purification technology.

Dr. Spencer Marsh is the Chief Scientific Officer of startup Tiny Cargo Company, born out of his post doctoral research at the Fralin biomedical research institute in Dr. Robert Gourdie’s lab. The primary work in this capacity was in the scaling-up of nanovesicle isolation and loading for use as a novel drug delivery platform. He is an expert in nanovesicle isolation via Tangential Flow Filtration, Ultracentrifugation, Size Exclusion Chromatography and other methods. Dr. Spencer has spent years applying in vitro cell experimentation and downstream protein and nucleic acid isolation and analysis as well as in vivo experimental procedures and ex vivo organ analysis via fluorescent imaging, and protein quantification in scaling current processes to industrial levels.

TITLE: Human Milk MicroRNA: a Novel Mechanism for Sustained Health Benefits in Breastfeeding Infants

ABSTRACT:
Human milk is the ideal source of infant nutrition, potentially reducing the risk for obesity and child allergies. The precise molecular mechanisms that convey these benefits are poorly understood. Our objective is to characterize novel molecules within human milk, called micro-ribonucleic acids (microRNAs), and determine if these molecules convey health benefits to breastfeeding infants. Through longitudinal cohort studies involving hundreds of maternal-infant dyads, we have cultivated a biobank of thousands of human milk samples and a database of long-term child health outcomes. Using high-throughput sequencing we interrogate the small RNA content of milk samples, allowing us to define how microRNAs and other small RNA molecules evolve during the course of breastfeeding. We then examine milk microRNA patterns for relationships with infant health trajectories. Recently, we characterized longitudinal milk microRNA profiles across the first four months of lactation. We identified two specific microRNAs associated with a significant reduction in infant allergies in the first year of life. Our future investigations will determine how milk microRNA profiles are related to cytokines, oligosaccharides, and other traditional nutritional components in human milk. We also aim to determine how milk microRNAs are transferred to the developing infant and impart physiologic changes on immune functioning.   

Dr. Hicks is a physician scientist at the Penn State College of Medicine in Hershey, Pennsylvania. He received his MD/PhD from SUNY Upstate Medical University in 2012, and completed his residency training in pediatrics at Golisano Children’s Hospital, in Syracuse New York. As a general pediatrician, Dr. Hicks provides medical care for families and children from birth through 21 years of age. His clinical expertise includes neurodevelopment, with a focus on autism spectrum disorder. Dr. Hicks’ research examines a multitude of factors that impact child development. In particular, he studies the biologic composition of human milk and its relationship with infant growth, immune functioning, and brain development.