Journal: Stem Cell Research & Therapy

Article Title: Exosomes derived from human umbilical cord mesenchymal stem cells inhibit vein graft intimal hyperplasia and accelerate reendothelialization by enhancing endothelial function

doi: 10.1186/s13287-020-01639-1

Figure Lengend Snippet: Morphology and characterization of hucMSCs and hucMSC-exosomes. a The fibroblast-like morphology of hucMSCs shown in light microscopy images (× 40). b Surface markers of hucMSCs analysed by flow cytometry. hucMSCs were positive for CD29, CD44, and CD90 and were negative for CD34 and CD45. c MSCs displayed the ability of adipogenic differentiation (× 40). d MSCs displayed the ability of chondrogenic differentiation (× 40). e Morphology of exosomes under transmission electron microscopy. Scale bar, 100 nm. f The size distribution of exosomes measured by Image-Pro Plus software. g Western blotting analyses of the exosome surface markers (CD9, CD81, and CD63)

Article Snippet: Blots were incubated with the appropriate primary antibodies, including CD9 (1:1000, Proteintech), CD63 (1:1000, Proteintech), CD81 (1:1000, BOSTER, Wuhan, China), inducible nitric oxide synthase (iNOS,1: 1000, Abcam), endothelial nitric oxide synthase (eNOS, 1:1000, Abcam), VEGF-A (1: 1000, Abcam), CD31 (1:1000, Abcam), AKT (1:1000,Cell Signaling, Boston, USA), p-AKT(,1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), p-ERK1/2 (1:1000, Cell Signaling), and GAPDH (1:2000, Proteintech).

Techniques: Light Microscopy, Flow Cytometry, Transmission Assay, Electron Microscopy, Software, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: β‐catenin‐controlled tubular cell‐derived exosomes play a key role in fibroblast activation via the OPN‐CD44 axis

doi: 10.1002/jev2.12203

Figure Lengend Snippet: N‐OPN increases in kidney and urine in various clinical nephropathies and associates with CKD progression primarily through encapsulation within exosomes. (a) Representative micrographs showing the abundance and localization of N‐OPN protein in various human CKD: chronic tubulo‐interstitial nephritis (CTIN), IgA nephropathy (IgAN), membranous nephritis (MN), diabetic nephropathy (DN), focal segmental glomerulosclerosis (FSGS) and lupus nephritis (LN). (b) Normal: nontumor kidney tissue from patients with renal cell carcinoma were used as healthy subjects. Arrow indicate positive staining (scale bar: 50 μm). (c) Quantitative analysis of immunohistochemical staining of N‐OPN in patients with CKD and healthy subjects. (d) Correlation between N‐OPN and fibrosis score. (e–f) Western blot analyses show urinary N‐OPN protein in healthy subjects and patients with CKD. Representative western blot (e) and quantitative data (f) are shown. Numbers (1–10) indicate urine samples from each subject. ** p < 0.01 versus healthy subjects. (g) Pie chart shows the composition of human urine samples. (h) Diagram shows the experimental plan. Morning urine was collected, centrifuged at 3000 rpm for 5 min. The supernatant was collected and tested by ELISA; in addition, after differential centrifugation, the exosomes were extracted by ultracentrifugation. (i) Graphic presentation shows urinary N‐OPN protein levels in cohorts of patients with CKD ( n = 183) and healthy subjects ( n = 30). Urinary N‐OPN levels are presented as pmol/μmol urinary creatinine (Ucr). *** p < 0.001 versus healthy subjects. (j) Graphic presentation shows urinary N‐OPN protein levels in different stages of CKD. There was no statistical difference among different CKD stages. (k) Linear regression shows a negative correlation between urinary N‐OPN protein and kidney function (estimated glomerular filtration rate [eGFR]). (l) Linear regression shows a significant correlation between urinary N‐OPN levels and urinary albumin to creatinine ratio (ACR). (m–t) Analyses of exosomes isolated from the urine of patients with CKD and healthy subjects. (m) Transmission electron microscopy (TEM) image showing the exosomes isolated from urine from patients with CKD. (n) Representative images of Coomassie blue staining of exosomes from the urine of healthy subjects and patients with CKD. (o) Gene ontology (GO) enrichment analysis of the specific proteins from urinary exosomes of patients with CKD. (p) Representative micrographs showing two specific peptides of OPN from urinary exosomes isolated from CKD patients were identified by mass spectrometry. (q) An interaction network of OPN protein with other proteins was identified using the STRING database. (r) GO enrichment analysis of OPN‐interacted proteins shows the potential function of OPN. (s) Exosomes were prepared from the same amounts of urine from healthy subjects or patients with CKD, and were lysed and immunoblotted with antibodies against Alix, CD81, TSG101, CD63, OPN and N‐OPN, respectively. (t) Western blot analyses show N‐OPN protein expression in urine and exosome‐removed urine from patients with CKD. (u) Colloidal gold electron microscopy analysis demonstrates that N‐OPN was encapsulated in urinary exosomes from patients with CKD. N‐OPN was labelled with 10 nm colloidal gold particles. Arrows indicate positive staining; Scale bar: 100 nm

Article Snippet: The following primary antibodies were used: anti‐N‐OPN (Abcam, Cat. ab181440, 1:1000), anti‐CD63 (Abcam, Cat. ab59479, 1:1000), anti‐OPN (Boster Biotechnology, Cat. PB0589, 1:1000), anti‐CD44 (Boster Biotechnology, Cat. A00052, 1:1000), anti‐α‐tubulin (Beijing Ray Antibody Biotech, Cat. RM2007, 1:5000), anti‐fibronectin (Sigma, Cat. F3648, 1:50000), anti‐α‐SMA (Abcam, Cat. ab5648, 1:1000), anti‐PDGFR‐β (Santa Cruz, Cat. sc‐374573, 1:1000), anti‐Collagen I (Boster Biotechnology, Cat. BA0325, 1:1000), anti‐Vimentin (Abcam, Cat. ab8978, 1:1000), anti‐PCNA (Abcam, Cat. ab29; 1:1000), anti‐active‐β‐catenin (Cell Signaling, Cat. #4270s, 1:1000), anti‐c‐Myc (Cell Signaling, Cat. #5605s, 1:1000), and anti‐Foxo4 (Cell Signaling, Cat. #9472s, 1:1000), anti‐TSG101 (Abcam, Cat. Ab83; 1:1000), anti‐CD81 (Boster Biotechnology, Cat. A01281‐2, 1:1000), anti‐Alix (Boster Biotechnology, Cat. BM5496, 1:1000), anti‐Flag (Boster Biotechnology, Cat. M30971, 1:1000).

Techniques: Staining, Immunohistochemical staining, Western Blot, Enzyme-linked Immunosorbent Assay, Centrifugation, Filtration, Isolation, Transmission Assay, Electron Microscopy, Mass Spectrometry, Expressing

Journal: Bioscience Reports

Article Title: Exosomes derived from circRNA Rtn4-modified BMSCs attenuate TNF-α-induced cytotoxicity and apoptosis in murine MC3T3-E1 cells by sponging miR-146a

doi: 10.1042/BSR20193436

Figure Lengend Snippet: ( A ) Western blot analysis showed that BMSCs-Exos were positive for CD63, CD9, CD81, and Alix. ( B ) The exosome uptake assay was performed to assess the uptake of PKH26-labeled exosomes into recipient MC3T3-E1 cells. Red: PKH26-labeled BMSCs-Exos. Blue: nuclei. Scale bar = 20 μm. ( C ) MC3T3-E1 cells were treated with TNF-α (5 ng/ml) and BMSCs-Exos (0, 25, 50 and 100 μg/ml) and then subjected to cell viability testing. Results showed that BMSCs-Exos dose-dependently blocked TNF-α-induced inhibition of cell viability. ( D ) Flow cytometry analysis of MC3T3-E1 cells treated with TNF-α and BMSCs-Exos. The results showed that BMSCs-Exos dose-dependently mitigated TNF-α-induced increase in cell apoptosis. ( E ) qRT-PCR analysis showed that TNF-α-induced increase in miR-146a expression was blocked when MC3T3-E1 cells were co-cultured with BMSCs-Exos. ( F ) Western blot analysis showed that BMSCs-Exos dose-dependently blocked TNF-α-induced cleaved caspase-3 and Bax expression. ( G ) ELISA data showed that BMSCs-Exos inhibited TNF-α-induced caspase-3 activity. All experiments were independently repeated three times. The caspase-3 activity and MTT assays were performed in triplicate. The differences among multiple groups were determined using one-way ANOVA test. n =3. * P <0.05.

Article Snippet: After blocking with 5% skim milk, the membranes were probed with primary antibodies against caspase-3 (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), Bcl-2-associated X protein (Bax; Cell Signaling Technology), CD9 (Boster, Wuhan, China), CD81 (Boster), CD9 (Boster), Alix (Boster), and β-actin (Cell Signaling Technology), followed by incubation with horseradish peroxidase–conjugated secondary antibody (Boster).

Techniques: Western Blot, Labeling, Inhibition, Flow Cytometry, Quantitative RT-PCR, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Bioscience Reports

Article Title: Exosomes derived from circRNA Rtn4-modified BMSCs attenuate TNF-α-induced cytotoxicity and apoptosis in murine MC3T3-E1 cells by sponging miR-146a

doi: 10.1042/BSR20193436

Figure Lengend Snippet: BMSCs were transfected with NC or pcDNA-circ-Rtn4, and their exosomes were isolated. ( A,B ) The expression of circ-Rtn4 was measured in NC- or pcDNA-circ-Rtn4-transfected BMSCs and their exosomes using qRT-PCR. ( C ) MC3T3-E1 cells were co-cultured with Rtn4-Exos or NC-Exos, and tested for circ-Rtn4 expression using qRT-PCR. ( D,E ) Evaluation of miR-146a expression in NC- or pcDNA-circ-Rtn4-transfected BMSCs and their exosomes using qRT-PCR. ( F ) qRT-PCR analysis of miR-146a expression in MC3T3-E1 cells treated with Rtn4-Exos or NC-Exos. ( G ) MC3T3-E1 cells were treated with TNF-α, followed by co-culture with Rtn4-Exos or NC-Exos. The viability of MC3T3-E1 cells was evaluated using MTT assay. ( H ) Flow cytometry analysis to evaluate cell apoptosis in MC3T3-E1 cells treated with TNF-α and exosomes from different sources. ( I ) The protein expression levels of caspase-3, cleaved caspase-3, and Bax were determined using Western blotting. ( J ) Caspase-3 activity was measured in MC3T3-E1 cells treated with TNF-α and exosomes from different sources using ELISA. ( K ) Western blot analysis of surface markers (CD63, CD81, CD9, and Alix) in exosomes. All experiments were independently repeated three times. The caspase-3 activity and MTT assays were performed in triplicate. The differences among multiple groups were determined using one-way ANOVA test. n =3. * P <0.05.

Article Snippet: After blocking with 5% skim milk, the membranes were probed with primary antibodies against caspase-3 (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), Bcl-2-associated X protein (Bax; Cell Signaling Technology), CD9 (Boster, Wuhan, China), CD81 (Boster), CD9 (Boster), Alix (Boster), and β-actin (Cell Signaling Technology), followed by incubation with horseradish peroxidase–conjugated secondary antibody (Boster).

Techniques: Transfection, Isolation, Expressing, Quantitative RT-PCR, Cell Culture, Co-Culture Assay, MTT Assay, Flow Cytometry, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay