Journal: Journal of Extracellular Vesicles

Article Title: Proteomic analysis of MSC‐derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions

doi: 10.1002/jev2.12240

Figure Lengend Snippet: Characteristics of apoptotic vesicles (apoVs) derived from mesenchymal stem cells (MSCs). (a) Schematic diagram indicating the procedures of isolating MSC‐derived apoVs. STS, staurosporine; apoMSCs, apoptotic MSCs. (b) Representative cryo‐electron microscopy (Cryo‐EM) images showing the morphology of apoVs. Scale bars, 200 nm. (c) Nanoparticle tracking analysis by Zetaview exhibiting the size distribution (upper panel) and membrane potential (lower panel) of apoVs. (d) Flow cytometric analysis revealing surface marker expression of apoVs. hBMSCs, human bone marrow MSCs; hASCs, human adipose MSCs

Article Snippet: Primary human bone marrow mesenchymal stem cells (hBMSCs) and human adipose mesenchymal stem cells (hASCs) were obtained from ScienCell (7500 and 7510, ScienCell, USA).

Techniques: Derivative Assay, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Membrane, Marker, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: Proteomic analysis of MSC‐derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions

doi: 10.1002/jev2.12240

Figure Lengend Snippet: Comparison of proteomic profile between apoVs and exosomes from MSCs. (a) Venn diagram representing the numbers of unique and overlapping proteins between two groups. Upper panel showing detected proteins in apoVs from hBMSCs and hASCs. Lower panel showing detected proteins in apoVs and exosomes from hBMSCs or hASCs. hB‐apoVs, apoVs derived from hBMSCs; hA‐apoVs, apoVs derived from hASCs; Exos, exosomes. (b) Clustering heatmap of differentially expressed proteins (DEPs) in apoVs compared to exosomes. The horizontal axis represents the differential measure log 2 (fold change), and the vertical axis represents proteins. Enrichment is depicted in red and depletion in blue. (c) Volcano plots showing significantly upregulated (red dots) and downregulated (green dots) proteins in apoVs compared to exosomes. Fold change > = 2 and adjusted p ‐value < 0.05 were used to obtain DEPs. (d) Subcellular localizations of DEPs in apoVs compared to exosomes. Cyto, cytosol; Nucl, nucleus; Mito, mitochondria; Plas, plasma membrane; Extr, extracellular; Cyto_nucl, cytosol‐nucleus; E.R., endoplasmic reticulum; Cysk, cytoskeleton; Pero, peroxisome; Golg, golgi apparatus; Cyto_mito, cytosol‐mitochondria; E.R._mito, endoplasmic reticulum‐mitochondria; Extr_plas, extracellular‐plasma membrane; Cyto_pero, cytosol‐peroxisome. (e) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of significantly upregulated proteins in apoVs compared to exosomes from hBMSCs and hASCs. Upper panel is KEGG level 1, and lower panel is KEGG level 2. hBMSCs, human bone marrow MSCs; hASCs, human adipose MSCs

Article Snippet: Primary human bone marrow mesenchymal stem cells (hBMSCs) and human adipose mesenchymal stem cells (hASCs) were obtained from ScienCell (7500 and 7510, ScienCell, USA).

Techniques: Comparison, Derivative Assay, Clinical Proteomics, Membrane

Journal: Journal of Extracellular Vesicles

Article Title: Proteomic analysis of MSC‐derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions

doi: 10.1002/jev2.12240

Figure Lengend Snippet: Validation of ubiquitous and specific markers of MSC‐derived apoVs. (a, b) Heatmaps showing the differentially expressed proteins (DEPs) of the four categories (a) and apoptotic markers (b) in hBMSC‐derived apoVs compared to exosomes. Category 1: Transmembrane or GPI‐anchored proteins associated to plasma membrane and/or endosomes; Category 2: Cytosolic proteins recovered in EVs; Category 3: Ribosomal proteins; Category 4: Transmembrane, lipid‐bound and soluble proteins associated to other intracellular compartments than PM/endosomes. Rows represent proteins and columns represent individual replicates. (c) Multiple reaction monitoring (MRM) analysis of candidate proteins for hBMSC‐derived apoV biomarkers. (d) Western blotting analysis showing the inclusive (13 proteins) and exclusive (Syntenin‐1) biomarkers of apoVs from hBMSCs and hASCs. (e) Protein‐protein interaction (PPI) network analysis of the ubiquitous and specific apoV proteins. (f) Representation of the putative apoV markers identified in MSC‐derived apoVs. Exos, exosomes. hBMSCs, human bone marrow MSCs; hASCs, human adipose MSCs

Article Snippet: Primary human bone marrow mesenchymal stem cells (hBMSCs) and human adipose mesenchymal stem cells (hASCs) were obtained from ScienCell (7500 and 7510, ScienCell, USA).

Techniques: Biomarker Discovery, Derivative Assay, Clinical Proteomics, Membrane, Targeted Proteomics, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Proteomic analysis of MSC‐derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions

doi: 10.1002/jev2.12240

Figure Lengend Snippet: Human bone marrow MSC (hBMSC)‐derived apoVs activate human platelet functions in vitro. (a) KEGG pathway enrichment analysis of significantly upregulated proteins in apoVs compared to exosomes. The enriched KEGG pathways are presented as a bar chart. The Y‐axis represents KEGG pathways and the X‐axes represent the number of significantly upregulated proteins (top) and enrichment significance (bottom), respectively. hBMSCs, human bone marrow MSCs. hASCs, human adipose MSCs. mBMSCs, mouse bone marrow MSCs. (b) Representative confocal microscopy images showing binding of apoVs (red) to the surface of platelets (green). After co‐culture with PKH26‐labeled apoVs at 37°C for 30 min, platelets were stained with Alexa Fluor 488‐conjugated WGA. Scale bar, 1 μm. (c) Flow cytometric analysis and the corresponding quantification of apoV binding to the surface of platelets. After incubation with PKH26‐labeled apoVs at 37°C for 30 min, platelets were stained with CD41 and CD62P. N = 5 per group. (d) Aggregation analysis showing the aggregation of platelets when treated with apoVs or exosomes. The maximal aggregation ratio (MAR) was calculated. PBS was used as the negative control, whereas EPI was used as the positive control. Notably, MAR values lower than the detection range of the analyzer were recorded as “0”. EPI, epinephrine. N = 3–4 per group. (e, f) Representative scanning electron microscopy (SEM) images (e) and confocal microscopy images (f) showing the morphological change of platelets after incubating with apoVs or exosomes. For confocal microscopy, platelets were stained with WGA (green). PBS was used as the negative control, whereas ADP was used as the positive control. ADP, Adenosine diphosphate. Scale bar, 1 μm. (g) Flow cytometric analysis and the corresponding quantification of the percentages of CD41 + and CD62P + platelets. The platelets were incubated with apoVs or exosomes at 37°C for 30 min, and stained with CD41 and CD62P. N = 3 per group. (h) Representative platelet spreading and the corresponding quantification of fold changes relative to the negative control group. After incubation with apoVs or exosomes at 37°C for 30 min, platelets were placed on fibrinogen‐coated glass coverslips at 37°C for 90 min followed by staining with WGA (green). Scale bar, 10 μm. N = 3 per group. apoVs, apoVs derived from hBMSCs; Exos, exosomes derived from hBMSCs. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed by Student's t ‐test (two‐tailed) for two group comparisons and one‐way ANOVA with Tukey's post hoc test for multiple group comparisons. NS, not significant; ** p < 0.01; *** p < 0.001

Article Snippet: Primary human bone marrow mesenchymal stem cells (hBMSCs) and human adipose mesenchymal stem cells (hASCs) were obtained from ScienCell (7500 and 7510, ScienCell, USA).

Techniques: Derivative Assay, In Vitro, Confocal Microscopy, Binding Assay, Co-Culture Assay, Labeling, Staining, Incubation, Negative Control, Positive Control, Electron Microscopy, Standard Deviation, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Proteomic analysis of MSC‐derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions

doi: 10.1002/jev2.12240

Figure Lengend Snippet: Fas is responsible for apoV‐mediated upregulation of HA platelet functions in vitro. (a) Flow cytometric analysis and the corresponding quantification of the percentages of CD41 + and CD62P + platelets. The HA platelets were incubated with si‐NC‐apoVs or si‐ Fas ‐apoVs, and subsequently stained with CD41 and CD62P. PBS was used as negative control. si‐NC‐apoVs, apoVs derived from mBMSCs (mouse bone marrow MSCs) treated with siRNA‐negative control; si‐ Fas ‐apoVs, apoVs derived from mBMSCs treated with siRNA‐ Fas . N = 3 per group. (b) Flow cytometric analysis and the corresponding quantification of the percentages of CD41 + and CD62P + platelets. The HA platelets were incubated with apoVs or Fas (‐) ‐apoVs, and subsequently stained with CD41 and CD62P. apoVs, apoVs derived from WT mBMSCs; Fas (‐) ‐apoVs, Fas‐negative apoV subpopulation after magnetic beads sorting. N = 3 per group. (c) Flow cytometric analysis and the corresponding quantification of the percentages of CD41 + and CD62P + platelets. The HA platelets were incubated with apoVs or apoVs + Fas‐nAb, and subsequently stained with CD41 and CD62P. apoVs + Fas‐nAb, apoVs pre‐treated with Fas neutralizing antibody. N = 3 per group. (d) Flow cytometric analysis and the corresponding quantification of the percentages of CD41 + and CD62P + platelets. The HA platelets were incubated with apoVs or apoVs + hDcR3, and subsequently stained with CD41 and CD62P. apoVs + hDcR3, platelets incubated with apoVs in the presence of hDcR3. hDcR3, human decoy receptor 3. N = 3 per group. (e) The quantitative real time polymerase chain reaction (qRT‐PCR) analysis of Fas gene expression levels in apoVs indicating overexpression of Fas . WT, apoVs derived from WT mBMSCs; Fas mut , apoVs derived from Fas mut mBMSCs; Fas mut rescue, apoVs derived from Fas mut mBMSCs overexpressing Fas . (f) Western blotting analysis of Fas protein levels in apoVs showing successful overexpression of Fas protein. (g) Flow cytometric analysis and the corresponding quantification of the percentages of CD41 + and CD62P + platelets. The HA platelets were incubated with apoVs, Fas mut ‐apoVs, or Fas rescue ‐apoVs, and subsequently stained with CD41 and CD62P. apoVs, apoVs derived from WT mBMSCs; Fas mut , apoVs derived from Fas mut mBMSCs; Fas mut rescue, apoVs derived from Fas mut mBMSCs overexpressing Fas . N = 3 per group. (h) Flow cytometric analysis and the corresponding quantification of the percentages of CD41 + and CD62P + platelets. The HA platelets or Fasl mut platelets were incubated with apoVs, respectively, and subsequently stained with CD41 and CD62P. HA Plts, platelets derived from HA mice; Fasl mut Plts, platelets derived from Fasl mut mice. N = 3 per group. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed by one‐way ANOVA with Tukey's post hoc test for multiple group comparisons. NS, not significant; ** p < 0.01; *** p < 0.001

Article Snippet: Primary human bone marrow mesenchymal stem cells (hBMSCs) and human adipose mesenchymal stem cells (hASCs) were obtained from ScienCell (7500 and 7510, ScienCell, USA).

Techniques: In Vitro, Incubation, Staining, Negative Control, Derivative Assay, Magnetic Beads, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Over Expression, Western Blot, Standard Deviation