scholarly journals Interferon-γ and Hypoxia Priming Have Limited Effect on the miRNA Landscape of Human Mesenchymal Stromal Cells-Derived Extracellular Vesicles

2020 ◽  
Vol 8 ◽  
Author(s):  
Juliette Peltzer ◽  
Kyle Lund ◽  
Marie-Emmanuelle Goriot ◽  
Marion Grosbot ◽  
Jean-Jacques Lataillade ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Principal Component ◽  
Interferon Γ ◽  
Biological Action ◽  
Individual Variability ◽  
Substantial Part ◽  
Beneficial Effects ◽  
Healthy Donors ◽  
Micro Rnas ◽  
Serum Free Condition

Mesenchymal stromal cell (MSC)-based cell therapy has received great interest in regenerative medicine. Priming the cells during the culture phase can improve their efficacy and/or survival after injection. The literature suggests that MSC extracellular vesicles (EV) can recapitulate a substantial part of the beneficial effects of the cells they originate from, and that micro-RNAs (miRNAs) are important players in EV biological action. Here, our aim was to determine if two classical priming methods of MSC, interferon-gamma (IFNγ) and hypoxia (HYP), could modify their EV miRNA content. Human bone marrow MSCs (BM-MSCs) from five healthy donors were cultured with IFNγ or in HYP or in control (CONT) conditions. The conditioned media were collected after 48 h in serum-free condition and EV were isolated by ultracentrifugation. Total RNA was isolated, pools of CONT, IFN, and HYP cDNA were prepared, and a miRNA profiling was performed using RT-qPCR. Then, miRNAs were selected based on their detectability and measured on each individual EV sample. Priming had no effect on EV amount or size distribution. A set of 81 miRNAs was detected in at least one of the pools of EVs. They were measured on each individual sample; 41 miRNAs were detected in all samples. The principal component analysis (PCA) failed to discriminate the groups. HYP induced a significant decrease in EV hsa-miR-34a-3p content and IFN induced a significant increase in five miRNAs (hsa-miR-25-3p, hsa-miR-106a-5p, hsa-miR-126-3p, hsa-miR-451a, and hsa-miR-665). Taken together, we found only limited alterations in the miRNA landscape of MSC EV with a high inter-individual variability.

scholarly journals Chronic Lymphocytic Leukemia-Derived Extracellular Vesicles Contain a Distinctive Proteome, As Well As Specific Micro RNAs and Y RNAs

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1968-1968
Author(s):  
Franziska Haderk ◽  
Etienne Moussay ◽  
Jerome Paggetti ◽  
Maria Göbel ◽  
Jan Dürig ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Blood Plasma ◽  
Extracellular Vesicles ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cell Culture Supernatant ◽  
Small Rna Sequencing ◽  
Target Cells ◽  
Healthy Donors ◽  
Micro Rnas

Abstract The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Of note, CLL cells themselves induce changes in surrounding cells, and extracellular vesicles (EVs) released by CLL cells represent a newly discovered mechanism of cell-cell communication. EVs are membrane enclosed nanoparticles 30 to 1000 nm in size and are able to reprogram recipient cells by transferring proteins and RNA molecules from their cell of origin. Thus, we aimed to analyze CLL cell-derived EVs present in blood plasma of CLL patients as well as those released by the CLL cell line MEC-1, in order to understand their role within the microenvironment. EVs were isolated from blood plasma of CLL patients and healthy donors, as well as from MEC-1 cell culture supernatant by serial centrifugation and density-based separation. Characterization of EVs by electron microscopy and immunoblot analysis revealed vesicles 20 to 300 nm in size, which are positive for various EV marker proteins such as Rab5a and Hsp70. Proteome analysis via mass spectrometry indicated differences in the composition of plasma derived EVs and peripheral blood mononuclear cells (PBMCs) from the respective patient, as well as between plasma derived EVs from healthy donors compared to CLL patients. Regarding the later, CLL EVs were specifically enriched for proteins involved in antigen presentation, endocytosis signaling as well as integrin mediated signaling and leukocyte extravasation. RNA analysis by BioAnalyzer profiling indicated an enrichment of small RNAs in EVs compared to cells. Subsequent small RNA sequencing revealed a unique microRNA signature of MEC-1 EVs with the 5 most abundant miRNAs encompassing about 60% of all detected miRNAs. Among them, the CLL-relevant miR-21 and miR-155, as well as miR-146a were selectively enriched in EVs. Moreover, Y RNAs, another class of small non-coding, regulatory RNAs, were highly enriched in MEC-1 EVs and their presence was also observed in plasma EVs of CLL patients. We uncovered a rapid uptake of CLL cell-derived EVs by human monocytes and macrophages. Whether the identified proteins and RNA transcripts shown to be enriched in CLL EVs induce phenotypic changes in targeted cells is being investigated. Further, quantification of plasma EVs in a large cohort of CLL patients is currently conducted and differences in the amount of EVs in correlation to disease outcome are analyzed. Harboring a distinct RNA and protein profile, EVs are potent vehicles for shuttling RNA and proteins to recipient cells and might be involved in the establishment of a supportive microenvironment in CLL. Functional analyses regarding possible effects of CLL EVs on target cells will broaden the knowledge of CLL pathogenesis and might help to identify new therapeutic options for CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Detection of Prostate Cancer via IR Spectroscopic Analysis of Urinary Extracellular Vesicles: A Pilot Study

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 591
Author(s):  
Xin-Le Yap ◽  
Bayden Wood ◽  
Teng-Aik Ong ◽  
Jasmine Lim ◽  
Bey-Hing Goh ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Extracellular Vesicles ◽  
Cancer Progression ◽  
Prostate Cancer Screening ◽  
Principal Component ◽  
Urine Samples ◽  
Cancer Type ◽  
Marker Proteins ◽  
Novel Strategy ◽  
Ir Spectroscopic

Extracellular vesicles (EVs) are membranous nanoparticles naturally released from living cells which can be found in all types of body fluids. Recent studies found that cancer cells secreted EVs containing the unique set of biomolecules, which give rise to a distinctive absorbance spectrum representing its cancer type. In this study, we aimed to detect the medium EVs (200–300 nm) from the urine of prostate cancer patients using Fourier transform infrared (FTIR) spectroscopy and determine their association with cancer progression. EVs extracted from 53 urine samples from patients suspected of prostate cancer were analyzed and their FTIR spectra were preprocessed for analysis. Characterization of morphology, particle size and marker proteins confirmed that EVs were successfully isolated from urine samples. Principal component analysis (PCA) of the EV’s spectra showed the model could discriminate prostate cancer with a sensitivity of 59% and a specificity of 81%. The area under curve (AUC) of FTIR PCA model for prostate cancer detection in the cases with 4–20 ng/mL PSA was 0.7, while the AUC for PSA alone was 0.437, suggesting the analysis of urinary EVs described in this study may offer a novel strategy for the development of a noninvasive additional test for prostate cancer screening.

scholarly journals Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jingjing Zhang ◽  
Luong T. H. Nguyen ◽  
Richard Hickey ◽  
Nicole Walters ◽  
Xinyu Wang ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Metastatic Breast ◽  
Clinical Samples ◽  
Clinical Use ◽  
Immunomagnetic Beads ◽  
Liquid Biopsies ◽  
Cell Culture Media ◽  
Non Invasive ◽  
Healthy Donors

AbstractExtracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

scholarly journals “Empowering” Cardiac Cells via Stem Cell Derived Mitochondrial Transplantation- Does Age Matter?

2021 ◽  
Vol 22 (4) ◽  
pp. 1824
Author(s):  
Matthias Mietsch ◽  
Rabea Hinkel
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Treatment Strategies ◽  
Cardiac Cells ◽  
Aging Effects ◽  
Beneficial Effects ◽  
Micro Rnas ◽  
New Treatment

With cardiovascular diseases affecting millions of patients, new treatment strategies are urgently needed. The use of stem cell based approaches has been investigated during the last decades and promising effects have been achieved. However, the beneficial effect of stem cells has been found to being partly due to paracrine functions by alterations of their microenvironment and so an interesting field of research, the “stem- less” approaches has emerged over the last years using or altering the microenvironment, for example, via deletion of senescent cells, application of micro RNAs or by modifying the cellular energy metabolism via targeting mitochondria. Using autologous muscle-derived mitochondria for transplantations into the affected tissues has resulted in promising reports of improvements of cardiac functions in vitro and in vivo. However, since the targeted treatment group represents mainly elderly or otherwise sick patients, it is unclear whether and to what extent autologous mitochondria would exert their beneficial effects in these cases. Stem cells might represent better sources for mitochondria and could enhance the effect of mitochondrial transplantations. Therefore in this review we aim to provide an overview on aging effects of stem cells and mitochondria which might be important for mitochondrial transplantation and to give an overview on the current state in this field together with considerations worthwhile for further investigations.

scholarly journals Multiplexed profiling of single-cell extracellular vesicles secretion

2019 ◽  
Vol 116 (13) ◽  
pp. 5979-5984 ◽  
Author(s):  
Yahui Ji ◽  
Dongyuan Qi ◽  
Linmei Li ◽  
Haoran Su ◽  
Xiaojie Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Single Cell Analysis ◽  
Comprehensive Evaluation ◽  
Single Cells ◽  
Deep Understanding ◽  
Principal Component ◽  
Cell Heterogeneity ◽  
Indispensable Tool

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g.,CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

scholarly journals Detection of tumor-derived extracellular vesicles in plasma from patients with solid cancer

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Silvia R. Vitale ◽  
Jean A. Helmijr ◽  
Marjolein Gerritsen ◽  
Hicret Coban ◽  
Lisanne F. van Dessel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Extracellular Vesicles ◽  
Digital Pcr ◽  
Circulating Tumor Dna ◽  
Solid Cancer ◽  
Expression Levels ◽  
Healthy Donors ◽  
Spin Column ◽  
High Concordance ◽  
Tumor Dna

Abstract Background Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA). Methods For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR. Results Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue. Conclusions We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients’ blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.

scholarly journals Small Extracellular Vesicles Isolated from Serum May Serve as Signal-Enhancers for the Monitoring of CNS Tumors

2020 ◽  
Vol 21 (15) ◽  
pp. 5359 ◽  
Author(s):  
Gabriella Dobra ◽  
Matyas Bukva ◽  
Zoltan Szabo ◽  
Bella Bruszel ◽  
Maria Harmati ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Serum Proteins ◽  
Lumbar Disc ◽  
Principal Component ◽  
Cns Tumors ◽  
Serum Samples ◽  
Force Microscopy ◽  
Isolation And Characterization ◽  
Atomic Force ◽  
Patient Groups

Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may help to monitor brain tumors. In this proteomics-based study, we aimed to identify a characteristic protein fingerprint associated with central nervous system (CNS) tumors. Overall, 96 human serum samples were obtained from four patient groups, namely glioblastoma multiforme (GBM), non-small-cell lung cancer brain metastasis (BM), meningioma (M) and lumbar disc hernia patients (CTRL). After the isolation and characterization of small extracellular vesicles (sEVs) by nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM), liquid chromatography -mass spectrometry (LC-MS) was performed on two different sample types (whole serum and serum sEVs). Statistical analyses (ratio, Cohen’s d, receiver operating characteristic; ROC) were carried out to compare patient groups. To recognize differences between the two sample types, pairwise comparisons (Welch’s test) and ingenuity pathway analysis (IPA) were performed. According to our knowledge, this is the first study that compares the proteome of whole serum and serum-derived sEVs. From the 311 proteins identified, 10 whole serum proteins and 17 sEV proteins showed the highest intergroup differences. Sixty-five proteins were significantly enriched in sEV samples, while 129 proteins were significantly depleted compared to whole serum. Based on principal component analysis (PCA) analyses, sEVs are more suitable to discriminate between the patient groups. Our results support that sEVs have greater potential to monitor CNS tumors, than whole serum.

scholarly journals Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research

2019 ◽  
Vol 8 (11) ◽  
pp. 1995 ◽  
Author(s):  
Moon ◽  
Shin ◽  
Kim ◽  
Lee ◽  
Mankhong ◽  
...  
Keyword(s):  
Human Plasma ◽  
Extracellular Vesicles ◽  
Proteinase K ◽  
High Yield ◽  
Clinical Samples ◽  
Diagnostic Biomarkers ◽  
Micro Rnas ◽  
Biomarker Research ◽  
Protein Rich ◽  
Yield And Purity

Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%–65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.

scholarly journals The Fatty Acid and Protein Profiles of Circulating CD81-Positive Small Extracellular Vesicles Are Associated with Disease Stage in Melanoma Patients

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4157
Author(s):  
Giovanni Paolino ◽  
Veronica Huber ◽  
Serena Camerini ◽  
Marialuisa Casella ◽  
Alberto Macone ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Extracellular Vesicles ◽  
Ad Hoc ◽  
Early Stage ◽  
Disease Onset ◽  
Protein Composition ◽  
Disease Stage ◽  
Biological Macromolecules ◽  
Healthy Donors ◽  
Melanoma Patients

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II–IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0–I, II and III–IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0–I) from late (III–IV) stages’ CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III–IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV’ FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.

Sign in / Sign up

Export Citation Format

Share Document