Isolation and identification of exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells

Abstract Background Exosomes, internal proteins, lipids, and nucleic acids coated by phospholipid bilayer membranes, are one type of small extracellular vesicles, which can mediate cell-cell communication. In recent years, exosomes have gained considerable scientific interest due to their widely applied prospect in the diagnosis and therapeutics of human and animal diseases. In this study, we describe for the first time a feasible method designed to isolate and characterize exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells. Results Exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells were successfully isolated by differential centrifugation. Quantification and sizing of exosomes were assessed by transmission electron microscopy, flow nano analysis and western blotting. Detected particles showed the normal size (30–100 nm) and morphology described for exosomes, as well as presence of the transmembrane protein (TSG101, CD9, CD63, and CD81) known as exosomal marker. Conclusions The results suggest that differential centrifugation is a feasible method for isolation of exosomes from different types of feline samples. Moreover, these exosomes can be used to further diagnosis and therapeutics in veterinary pre-clinical and clinical studies.

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Isolation and Identification of Exosomes from Feline Plasma, Urine and Adipose-Derived Mesenchymal Stem Cells

10.21203/rs.3.rs-272048/v1 ◽

2021 ◽

Author(s):

Dongsheng Li ◽

Huina Luo ◽

Huimin Ruan ◽

Zhisheng Chen ◽

Shengfeng Chen ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Transmembrane Protein ◽

Cell Communication ◽

Phospholipid Bilayer ◽

Differential Centrifugation ◽

Isolation And Identification ◽

Feasible Method ◽

Transmission Electron ◽

Mediate Cell

Abstract Background: Exosomes, internal proteins, lipids, and nucleic acids coated by phospholipid bilayer membranes, are one type of small extracellular vesicles, which can mediate cell-cell communication. In recent years, exosomes have gained considerable scientific interest due to their widely applied prospect in the diagnosis and therapeutics of human and animal diseases. In this study, we describe for the first time a feasible method designed to isolate and characterize exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells. Results: Exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells were successfully isolated by differential centrifugation. Quantification and sizing of exosomes were assessed by transmission electron microscopy, flow nano analysis and western blotting. Detected particles showed the normal size (30-100 nm) and morphology described for exosomes, as well as presence of the transmembrane protein (TSG101, CD9, CD63, and CD81) known as exosomal marker.Conclusions: The results suggest that differential centrifugation is a feasible method for isolation of exosomes from different types of feline samples. Moreover, these exosomes can be used to further diagnosis and therapeutics in veterinary pre-clinical and clinical studies.

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The Role of Cytokines in Interactions of Mesenchymal Stem Cells and Breast Cancer Cells

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666201022111942 ◽

2020 ◽

Vol 15 ◽

Author(s):

Hariharan Jayaraman ◽

Nalinkanth V. Ghone ◽

Ranjith Kumaran R ◽

Himanshu Dashora

Keyword(s):

Breast Cancer ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Tumor Microenvironment ◽

Cancer Cells ◽

Cell Communication ◽

Extra Cellular Matrix ◽

Cytokine Signaling ◽

Cellular Matrix

: Mesenchymal stem cells because of its high proliferation, differentiation, regenerative capacity, and ease of availability have been a popular choice in cytotherapy. Mesenchymal Stem Cells (MSCs) have a natural tendency to home in a tumor microenvironment and acts against it, owing to the similarity of the latter to an injured tissue environment. Several studies have confirmed the recruitment of MSCs by tumor through various cytokine signaling that brings about phenotypic changes to cancer cells, thereby promoting migration, invasion, and adhesion of cancer cells. The contrasting results on MSCs as a tool for cancer cytotherapy may be due to the complex cell to cell interaction in the tumor microenvironment, which involves various cell types such as cancer cells, immune cells, endothelial cells, and cancer stem cells. Cell to cell communication can be simple or complex and it is transmitted through various cytokines among multiple cell phenotypes, mechano-elasticity of the extra-cellular matrix surrounding the cancer cells, and hypoxic environments. In this article, the role of the extra-cellular matrix proteins and soluble mediators that acts as communicators between mesenchymal stem cells and cancer cells has been reviewed specifically for breast cancer, as it is the leading member of cancer malignancies. The comprehensive information may be beneficial in finding a new combinatorial cytotherapeutic strategy using MSCs by exploiting the cross-talk between mesenchymal stem cells and cancer cells for treating breast cancer.

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A feasible method for the isolation of mesenchymal stem cells from menstrual blood and their exosomes

Tissue and Cell ◽

10.1016/j.tice.2018.09.010 ◽

2018 ◽

Vol 55 ◽

pp. 53-62 ◽

Cited By ~ 3

Author(s):

Razieh Dalirfardouei ◽

Khadijeh Jamialahmadi ◽

Elahe Mahdipour

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Menstrual Blood ◽

Feasible Method

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Exosomes isolation and identification from equine mesenchymal stem cells

BMC Veterinary Research ◽

10.1186/s12917-019-1789-9 ◽

2019 ◽

Vol 15 (1) ◽

Cited By ~ 13

Author(s):

Michele Christian Klymiuk ◽

Natalie Balz ◽

Mohamed I. Elashry ◽

Manuela Heimann ◽

Sabine Wenisch ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Isolation And Identification

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Very Long-Chain C24:1 Ceramide Is Increased in Serum Extracellular Vesicles with Aging and Can Induce Senescence in Bone-Derived Mesenchymal Stem Cells

Cells ◽

10.3390/cells8010037 ◽

2019 ◽

Vol 8 (1) ◽

pp. 37 ◽

Cited By ~ 21

Author(s):

Andrew Khayrullin ◽

Priyanka Krishnan ◽

Luis Martinez-Nater ◽

Bharati Mendhe ◽

Sadanand Fulzele ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Extracellular Vesicles ◽

Cardiovascular Health ◽

Rhesus Macaques ◽

Cell Communication ◽

Size Exclusion ◽

Target Cells ◽

Serum Samples

Extracellular vesicles (EVs), including exosomes and microvesicles, function in cell-to-cell communication through delivery of proteins, lipids and microRNAs to target cells via endocytosis and membrane fusion. These vesicles are enriched in ceramide, a sphingolipid associated with the promotion of cell senescence and apoptosis. We investigated the ceramide profile of serum exosomes from young (24–40 yrs.) and older (75–90 yrs.) women and young (6–10 yrs.) and older (25–30 yrs.) rhesus macaques to define the role of circulating ceramides in the aging process. EVs were isolated using size-exclusion chromatography. Proteomic analysis was used to validate known exosome markers from Exocarta and nanoparticle tracking analysis used to characterize particle size and concentration. Specific ceramide species were identified with lipidomic analysis. Results show a significant increase in the average amount of C24:1 ceramide in EVs from older women (15.4 pmol/sample) compared to those from younger women (3.8 pmol/sample). Results were similar in non-human primate serum samples with increased amounts of C24:1 ceramide (9.3 pmol/sample) in older monkeys compared to the younger monkeys (1.8 pmol/sample). In vitro studies showed that primary bone-derived mesenchymal stem cells (BMSCs) readily endocytose serum EVs, and serum EVs loaded with C24:1 ceramide can induce BMSC senescence. Elevated ceramide levels have been associated with poor cardiovascular health and memory impairment in older adults. Our data suggest that circulating EVs carrying C24:1 ceramide may contribute directly to cell non-autonomous aging.

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MicroRNA-346-5p Regulates Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Inhibiting Transmembrane Protein 9

BioMed Research International ◽

10.1155/2020/8822232 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Yicai Zhang ◽

Yi Sun ◽

Jinlong Liu ◽

Yu Han ◽

Jinglong Yan

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Protein Level ◽

Molecular Mechanisms ◽

Transmembrane Protein ◽

Luciferase Reporter ◽

Alizarin Red ◽

Protein Levels

The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

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Mesenchymal stem cells enhance tumorigenic properties of human glioblastoma through independent cell-cell communication mechanisms

Oncotarget ◽

10.18632/oncotarget.25346 ◽

2018 ◽

Vol 9 (37) ◽

pp. 24766-24777 ◽

Cited By ~ 17

Author(s):

Carolina Oliveira Rodini ◽

Patrícia Benites Gonçalves da Silva ◽

Amanda Faria Assoni ◽

Valdemir Melechco Carvalho ◽

Oswaldo Keith Okamoto

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Communication ◽

Human Glioblastoma ◽

Cell Cell

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Human Cytomegalovirus Can Induce Alteration of β-Actin mRNA and Microfilaments as Well as Induce Apoptosis in Mesenchymal Stem Cells.

Blood ◽

10.1182/blood.v108.11.4261.4261 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4261-4261

Author(s):

Guoqing Wei ◽

Maofang Lin ◽

Zhen Cai ◽

He Huang

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Electron Microscope ◽

Human Cytomegalovirus ◽

Transmission Electron Microscope ◽

Cmv Infection ◽

Transmission Electron ◽

Stem Cells Transplantation ◽

Actin Mrna

Abstract Mesenchymal stem cells (MSCs) is a very important cell system in bone marrow stromal and human cytomegalovirus (CMV) infection is one of the most common complications following stem cells transplantation. Our studies were to investigate the cytoskeleton change as well as the apoptosis induction effects of CMV infection on MSCs. In our study, MSCs were infected by 100,10,1 TCID50 of CMV. After 48h of culture with DMEM supplemented with 10% (v/v) fetal calf serum, MSCs shapes were observed with microscope. CMV particles and ultra structure in the cells were detected with transmission electron microscope. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene,β-actin gene and GAPDH genes. Flow Cytometry was used to detect the apoptotic cells. After the infection of CMV, shape of MSCs changed, turning from thin shuttle shape to round and thick ball shape, even escaping from wall. Cell shape changed more dominantly as the CMV titer increased. With transmission electron microscope, CMV particles and typical cellular apoptotic character as well as the ruptured- Microfilaments could be seen in the cells infected by CMV. MSCs infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer- dependent manner compared with the uninfected cells whose expression of GAPDH mRNA did not change much. With Flow Cytometry, it was found there were more apoptotic and necrosis cells in the cells infected by CMV. These results indicate that CMV can not only infect MSCs and destroy the cytoskeleton in the cells but also can induce apoptosis of the cells, much more attention of CMV infection on MSCs should be paid after stem cells transplantation.

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Morphological and Cytofluorimetric Analysis of Adult Mesenchymal Stem Cells Expanded Ex Vivo from Periodontal Ligament

International Journal of Immunopathology and Pharmacology ◽

10.1177/039463200501800204 ◽

2005 ◽

Vol 18 (2) ◽

pp. 213-221 ◽

Cited By ~ 97

Author(s):

O. Trubiani ◽

R. Di Primio ◽

T. Traini ◽

J. Pizzicannella ◽

A. Scarano ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Periodontal Ligament ◽

Alveolar Bone ◽

Ex Vivo ◽

Human Mesenchymal Stem Cells ◽

Transmission Electron ◽

Adult Mesenchymal Stem Cells ◽

Physiological Structure ◽

Secretory Apparatus

Many adult tissues contain a population of stem cells that have the ability of regeneration after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining physiological structure tissues and their studies have been considered very important and intriguing after having shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone, tooth-associated tissue, cardiomyocytes, but also to differentiate into cells derived from other embryonic layers, including neurons. Currently, different efforts have been focused on the identification of odontogenic progenitors from oral tissues. In this study we isolated and characterized a population of homogeneous human mesenchymal stem cells proliferating in culture with an attached well-spread morphology derived from periodontal ligament, tissue of ectomesenchymal origin, with the ability to form a specialized joint between alveolar bone and tooth. The adherent cells were harvested and expanded ex vivo under specific conditions and analysed by FACScan flow cytometer and morphological analysis was carried out by light, scanning and transmission electron microscopy. Our results displayed highly evident cells with a fibroblast like morphology and a secretory apparatus, probably indicating, that the enhanced function of the secretory apparatus of the mesenchymal stem cells may be associated with the secretion of molecules that are required to survive and proliferate. Moreover, the presence in periodontal ligament of CD90, CD29, CD44, CD166, CD 105, CD13 positive cells, antigens that are also identified as stromal precursors of the bone marrow, indicate that the periodontal ligament may turn out to be a new efficient source of the cells with intrinsic capacity to self-renewal, high ability to proliferate and differentiate, that can be utilized for a new approach to regenerative medicine and tissue engineering.

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