Dataset Information

Glycome analysis of extracellular vesicles derived from human induced pluripotent stem cells using lectin microarray.

ABSTRACT: Glycans are one of the major building blocks of extracellular vesicles (EVs). However, their roles and applications have not been completely explored. Here, we analyzed the glycome of EVs derived from human induced pluripotent stem cells (hiPSCs) using high-density lectin microarray. The glycan profiles of hiPSC-derived EVs were different from those of non-hiPSC-derived EVs. Moreover, rBC2LCN that shows specific binding to hiPSCs, showed strong specificity for hiPSC-derived EVs but not non-hiPSCs-derived EVs. Further, other hiPSC-specific probes, such as anti-TRA-1-60, anti-SSEA4, and anti-R-10G, exhibited specific, but weaker binding to hiPSC-derived EVs than rBC2LCN. We then developed a sandwich assay using rBC2LCN and a phosphatidylserine receptor, Tim4, to specifically detect hiPSC-derived EVs. The Tim4-rBC2LCN sandwich assay allowed for specific detection of hiPSC-derived EVs but not non-hiPSC-derived EVs, indicating that rBC2LCN could also be used for the specific detection of hiPSC-derived EVs. Together, our findings demonstrate that the characteristic glycan signature of hiPSCs are retained by EVs derived from them. The EV glycome could be novel targets for the identification and characterization of stem cells for use in regenerative medicine.

SUBMITTER: Saito S

PROVIDER: S-EPMC5838221 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Glycome analysis of extracellular vesicles derived from human induced pluripotent stem cells using lectin microarray.

Saito Sayoko S Hiemori Keiko K Kiyoi Kayo K Tateno Hiroaki H

Scientific reports 20180305 1

Glycans are one of the major building blocks of extracellular vesicles (EVs). However, their roles and applications have not been completely explored. Here, we analyzed the glycome of EVs derived from human induced pluripotent stem cells (hiPSCs) using high-density lectin microarray. The glycan profiles of hiPSC-derived EVs were different from those of non-hiPSC-derived EVs. Moreover, rBC2LCN that shows specific binding to hiPSCs, showed strong specificity for hiPSC-derived EVs but not non-hiPSC ...[more]

PMID: 29507392

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Project description:Although cell survival post-transplantation is very low, emerging evidence using stem cell therapy for myocardial repair points toward a primary role of paracrine signaling mechanisms as the basis for improved cardiac function, decreased fibrosis, and increased angiogenesis. Recent studies have demonstrated that extracellular vesicles (EVs) such as exosomes secreted by stem cells stimulate angiogenesis, provide cytoprotection, and modulate apoptosis. However, the angiogenic potential of EVs secreted from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), a terminally differentiated cell type, has not been elucidated yet. Therefore, the main objective of this study is to isolate, characterize, and evaluate the in vitro angiogenic potential of EVs collected from hiPSC-CM conditioned media. The hiPSC-CM were cultured for 2 weeks and EVs were isolated from cell culture medium. Isolated EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis, and immunoblotting. Furthermore, the angiogenic potential of these EVs was evaluated by tube formation, wound-healing, and cell-proliferation assays in bovine aortic endothelial cells (BAEC). In addition, gene expression levels of growth factors was evaluated in hiPSC-derived endothelial cells (hiPSC-EC) treated with hiPSC-CM-derived EV (CM-EVs) to assess their role in promoting angiogenesis. TEM imaging of CM-EVs showed a presence of a double-membrane bound structure, which is a characteristic of EV. Nanoparticle tracking analysis further confirmed the size and shape of the secreted particles to be consistent with EVs. Furthermore, EV-specific markers (CD63 and HSP70) were enriched in these particles as illustrated by immunoblotting. Most importantly, BAEC treated with 100 ?g/ml of CM-EVs showed significant increases in tube formation, wound closure, and cell proliferation as compared to control (no-EVs). Finally, treatment of hiPSC-EC with CM-EVs induced increased expression of pro-angiogenic growth factors by the endothelial cells. Overall, our results demonstrated that EVs isolated from hiPSC-CM enhance angiogenesis in endothelial cells. This acellular/cell-free approach constitutes a potential translational therapeutic to induce angiogenesis in patients with myocardial infarction.

Project description:Extracellular vesicles that are derived from stem cells play an important role in the treatment of disease. To obtain high-quality small extracellular vesicles (sEVs), we optimized the culture conditions of human induced pluripotent stem cells (hiPSCs), the supernatant collection time, and sEVs extraction methods. Firstly, hiPSCs were cultured in extracellular vesicles-production medium (EVs-PM) containing different concentrations (0%, 0.25%, 0.5%, 2%, 5%, and 20%) of extracellular vesicle-depleted knockout serum replacement (ED-KSR), and the culture supernatants were collected continuously for 5 days. Then, the sEVs were isolated, followed by an evaluation of their characteristics. The survival rates of the hiPSCs lines that were cultured in EVs-PM containing 0.5% to 20% ED-KSR were not significantly different (P > 0.05). The survival rates of the hiPSCs in 0.5% ED-KSR after the culture supernatants were continuously collected for day 1, day 3, and day 5 were not statistically significant (P > 0.05). After 5 days of continuous collection of the supernatant, the hiPSCs expressed some pluripotent markers, while SSEA4 and TRA-1-60 expression changed gradually. The sEVs that were extracted by the two methods were all 50-200 nm, double-layered and oval or round cellular vesicles and expressed the marker proteins CD63, TSG101, and HSP70. The characteristics of sEVs extracted on day 1, day 3, and day 5 were almost identical on morphology, size and the relative quantity of annexin V-positive subpopulations. The PKH67 staining showed that the sEVs could be endocytosed by HepG2 cells and aggregated in the cytoplasm. The proliferation experiments showed that the sEVs can promote cell proliferation. In Conclusion, the 0.5% ED-KSR is the optimal concentration, and that the hiPSCs culture supernatant can be continuously collected for 5 days while maintaining high cell viability and some pluripotent characteristics. Both of the methods extraction can be used to obtain biologically active sEVs.

Project description:Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFβ (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-β-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.

Project description:RATIONALE:Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs. OBJECTIVE:To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo. METHODS AND RESULTS:Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV-treated mice exhibited improved left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe. CONCLUSIONS:iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.

Dataset Information

Glycome analysis of extracellular vesicles derived from human induced pluripotent stem cells using lectin microarray.

Publications

Glycome analysis of extracellular vesicles derived from human induced pluripotent stem cells using lectin microarray.

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