Enrichment of sweat-derived extracellular vesicles of human and bacterial origin for biomarker identification.

Sweat contains biomarkers for real-time non-invasive health monitoring, but only a few relevant analytes are currently used in clinical practice. In the present study, we investigated whether sweat-derived extracellular vesicles (EVs) can be used as a source of potential protein biomarkers of human and bacterial origin. Methods: By using ExoView platform, electron microscopy, nanoparticle tracking analysis and Western blotting we characterized EVs in the sweat of eight volunteers performing rigorous exercise. We compared the presence of EV markers as well as general protein composition of total sweat, EV-enriched sweat and sweat samples collected in alginate skin patches. Results: We identified 1209 unique human proteins in EV-enriched sweat, of which approximately 20% were present in every individual sample investigated. Sweat derived EVs shared 846 human proteins (70%) with total sweat, while 368 proteins (30%) were captured by medical grade alginate skin patch and such EVs contained the typical exosome marker CD63. The majority of identified proteins are known to be carried by EVs found in other biofluids, mostly urine. Besides human proteins, EV-enriched sweat samples contained 1594 proteins of bacterial origin. Bacterial protein profiles in EV-enriched sweat were characterized by high interindividual variability, that reflected differences in total sweat composition. Alginate-based sweat patch accumulated only 5% proteins of bacterial origin. Conclusion: We showed that sweat-derived EVs provide a rich source of potential biomarkers of human and bacterial origin. Use of commercially available alginate skin patches selectively enrich for human derived material with very little microbial material collected.

Generation of novel in vitro flexible kidney organoid model to investigate the role of extracellular vesicles in induction of nephrogenesis.

BACKGROUND: During kidney organogenesis, metanephric mesenchyme (MM) and ureteric bud (UB) interact reciprocally to form nephrons. Signaling stimuli involved in these interactions include Wnts, growth factors and nano/micro particles. How UB and MM are interacting is not completely understood. Our study investigated the signaling and communication via extracellular vesicles (EVs) during nephrogenesis. Embryonic day (E) 11.5 mouse kidney UB and MM produce very low number of primary cells that have limited ability for proliferation in culture. Such limitations obstruct studying the role of EVs in induction of nephrogenesis. These issues necessitate to generate a nephrogenesis model allowing to study the comprehensive role of EVs during nephrogenesis. RESULTS: Our study generated a UB derived cell line-based in vitro flexible model of nephrogenesis allowing expandable cell culturing, in addition to performing characterization, tracking and blocking of EVs. UB cell line aggregation with E11.5 MM cells induced the formation of segmented nephrons. Most efficient nephrogenesis was obtained by the co-culturing of 30,000 cells of UB cell line with 50,000 MM cells. Results revealed that both the UB and the MM secrete EVs during nephrogenesis. UB cell line derived EVs were characterized by their size, morphology and expression of markers (CD63, TSG101, CD9 and CD81). Furthermore, proteomics data of UB cell line-derived EVs revealed large number of proteins involved in nephrogenesis-related signaling pathways. Palmitoylated GFP-tagged EVs from UB cell line were found in the nephron formation zone in the developing kidney organoid. UB cell line derived EVs did not induce nephrogenesis in MM cells but significantly contributed to the survival and nephrogenesis-competency of MM cells. The secretion of EVs was continuously inhibited during the ongoing nephrogenesis by the knockdown of RalA and RalB gene expression using short hairpin RNAs. This inhibition partially impaired the ability of UB cell line to induce nephrogenesis. Moreover, impaired nephrogenesis was partially rescued by the addition of EVs. CONCLUSION: Our study established a novel in vitro flexible model of nephrogenesis that solved the limitations of primary embryonic kidney cells and mouse embryonic stem cell kidney organoids for the EV research. EVs were found to be an integral part of nephrogenesis process. Video Abstract.

Maternal microbiota communicates with the fetus through microbiota-derived extracellular vesicles.

BACKGROUND: Reports regarding the presence of bacteria in the fetal environment remain limited and controversial. Recently, extracellular vesicles secreted by the human gut microbiota have emerged as a novel mechanism for host-microbiota interaction. We aimed to investigate the presence of bacterial extracellular vesicles in the fetal environment during healthy pregnancies and determine whether extracellular vesicles derived from the gut microbiota can cross biological barriers to reach the fetus. RESULTS: Bacterial extracellular vesicles were detectable in the amniotic fluid of healthy pregnant women, exhibiting similarities to extracellular vesicles found in the maternal gut microbiota. In pregnant mice, extracellular vesicles derived from human maternal gut microbiota were found to reach the intra-amniotic space. CONCLUSIONS: Our findings reveal maternal microbiota-derived extracellular vesicles as an interaction mechanism between the maternal microbiota and fetus, potentially playing a pivotal role in priming the prenatal immune system for gut colonization after birth. Video Abstract.

Purification of Bacterial-Enriched Extracellular Vesicle Samples from Feces by Density Gradient Ultracentrifugation.

Commensal microbiota has huge impact on the maintenance of human health, its dysregulation being associated with the development of a plethora of diseases. Release of bacterial extracellular vesicles (BEVs) is a fundamental mechanism of systemic microbiome influence on the host organism. Nevertheless, due to the technical challenges of isolation methods, BEV composition and functions remain poorly characterized. Hereby, we describe the up-to-date protocol for isolation of BEV-enriched samples from human feces. Fecal extracellular vesicles (EVs) are purified through the orthogonal implementation of filtration, size-exclusion chromatography (SEC), and density gradient ultracentrifugation. EVs are first separated from bacteria, flagella, and cell debris by size. In the next steps, BEVs are separated from host-derived EVs by density. The quality of vesicle preparation is estimated via immuno-TEM (transmission electron microscopy) for the presence of vesicle-like structures expressing EV markers and via NTA (nanoparticle tracking analysis) for assaying particle concentration and size. Distribution of EVs of human origin in gradient fractions is estimated using antibodies against human exosomal markers with Western blot and ExoView R100 imaging platform. The enrichment for BEVs in vesicle preparation is estimated by Western blot for the presence of bacterial OMVs (outer membrane vesicles) marker and OmpA (outer membrane protein A). Taken together, our study describes a detailed protocol for EV preparation with enrichment for BEVs from feces with a purity level suitable for bioactivity functional assays.

Biogenesis of Mesoporous Silica Nanoparticles Enclosed in Extracellular Vesicles by Mouse Renal Adenocarcinoma Cells.

Integrating the versatility of synthetic nanoparticles to natural biomaterials, such as cells or cell membranes, has gained considerable attention as promising alternative cargo delivery platforms in recent years. Extracellular vesicles (EVs), natural nanomaterials composed of a protein-rich lipid bilayer secreted by cells, have also shown advantages and great potential as a nano delivery platform in combination with synthetic particles due to their specific natural properties in overcoming several biology hurdles possessed in the recipient cell. Therefore, the preservation of EV's origin properties is critical for their application as nanocarriers. This chapter will describe the encapsulation procedure of MSN encapsulated in EV membrane derived from mouse renal adenocarcinoma (Renca) cells through biogenesis. The FMSN-enclosed EVs produced through this approach still contain preserved EV's natural membrane properties.

Enrichment of bovine milk-derived extracellular vesicles using surface-functionalized cellulose nanofibers.

The isolation of extracellular vesicles (EVs) from milk, a complex mixture of colloidal structures having a comparable size to EVs, is challenging. Although ultracentrifugation (UC) has been widely used for EV isolation, this has significant limitations, including a long processing time at high g-force conditions and large sample volume requirements. We introduced a new approach based on nature nanoentities cellulose nanofibers (CNFs) and short time and low g-force centrifugation to isolate EVs from various milk fractions. The flexible and entangled network of CNFs forms nanoporous, which entraps the EVs. Further, positively charged CNFs interact with anionic EVs through an electrostatic attraction, promoting their isolation with efficiency comparable with UC. The functionality and toxicity of isolated milk EVs were tested in Caco2 cells. Overall, the newly developed approach provides straightforward isolation and biocompatibility and preserves the natural properties of the isolated EVs, enabling further applications.

Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles.

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell-cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols.

Identification of extracellular nanoparticle subsets by nuclear magnetic resonance.

Exosomes are a subset of secreted lipid envelope-encapsulated extracellular vesicles (EVs) of 50-150 nm diameter that can transfer cargo from donor to acceptor cells. In the current purification protocols of exosomes, many smaller and larger nanoparticles such as lipoproteins, exomers and microvesicles are typically co-isolated as well. Particle size distribution is one important characteristics of EV samples, as it reflects the cellular origin of EVs and the purity of the isolation. However, most of the physicochemical analytical methods today cannot illustrate the smallest exosomes and other small particles like the exomers. Here, we demonstrate that diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) method enables the determination of a very broad distribution of extracellular nanoparticles, ranging from 1 to 500 nm. The range covers sizes of all particles included in EV samples after isolation. The method is non-invasive, as it does not require any labelling or other chemical modification. We investigated EVs secreted from milk as well as embryonic kidney and renal carcinoma cells. Western blot analysis and immuno-electron microscopy confirmed expression of exosomal markers such as ALIX, TSG101, CD81, CD9, and CD63 in the EV samples. In addition to the larger particles observed by nanoparticle tracking analysis (NTA) in the range of 70-500 nm, the DOSY distributions include a significant number of smaller particles in the range of 10-70 nm, which are visible also in transmission electron microscopy images but invisible in NTA. Furthermore, we demonstrate that hyperpolarized chemical exchange saturation transfer (Hyper-CEST) with 129Xe NMR indicates also the existence of smaller and larger nanoparticles in the EV samples, providing also additional support for DOSY results. The method implies also that the Xe exchange is significantly faster in the EV pool than in the lipoprotein/exomer pool.

Characterization of nucleic acids from extracellular vesicle-enriched human sweat.

BACKGROUND: The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles. RESULTS: We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7-45%), with 50-60% of those reads mapping to unannotated region of the genome and 30-55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA. CONCLUSIONS: Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.