71d09e8eea25c63414efdac8ee2c536b pmc_9455930.pdf 3ab7be685397ae1d53e0eb4c46b82901d5eabbbe pmc_9455930.pdf d128477bf3283fa374b36cc93b0928642b02c2513f20c8d17379c1d6e6c36468 pmc_9455930.pdf Title: N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV–Cell Interaction and Functional Activation of Endothelial Cells Subject: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV–cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells’ interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton’s Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV–cell interactions and associated functions. Keywords: mesenchymal stem/stromal cells; extracellular vesicles; exosomes; glycans; glycosylation; lectins Author: Marta Clos-Sansalvador, Sergio G. Garcia, Miriam Morón-Font, Charles Williams, Niels-Christian Reichardt, Juan M. Falcón-Pérez, Antoni Bayes-Genis, Santiago Roura, Marcella Franquesa, Marta Monguió-Tortajada and Francesc E. Borràs Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Aug 24 17:05:51 2022 CEST ModDate: Wed Aug 24 17:08:46 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2916380 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- VJAUGW+VnURWPalladioL Type 1 Custom yes yes no 10 0 VFBWPZ+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 NZWUPB+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 SQZOQT+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 PURWBA+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 46 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 CMGFHP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 77 0 CMGFIB+PalatinoLinotype TrueType WinAnsi yes yes no 80 0 CMGFID+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 83 0 VORBJF+CMSY10 Type 1 Builtin yes yes no 91 0 CMGFHP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 98 0 CMGFIB+PalatinoLinotype TrueType WinAnsi yes yes no 101 0 CMGFID+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 104 0 CMGFHP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 117 0 CMGFIB+PalatinoLinotype TrueType WinAnsi yes yes no 120 0 CMGFID+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 123 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 132 0 CMGFHP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 142 0 CMGFIB+PalatinoLinotype TrueType WinAnsi yes yes no 145 0 CMGFID+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 148 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-06-30 02:36:19 CEST RepresentationInformation: pmc_9455930.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-09-23 02:14:56 CEST Size: 2916380 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 386 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Isolation and Characterization of iWJ-MSC-EV Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: N-Glycosylation Is Critical to Uptake iWJ-MSC-EV by HUVEC Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: PNGase-F Treatment Abrogates HUVEC Recruitment by iWJ-MSC-EV Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Tube-like Structures Promoted by iWJ-MSC-EV Are Dependent on N-Glycosylation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: iWJ-MSC-EV and PNGase-F-Treated iWJ-MSC-EV Exhibit Distinct Surface Glycosylation Profile Patterns Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: EV Isolation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: PNGase-F Treatment of iWJ-MSC-EV Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: EV Staining Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Bead-Based Flow Cytometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Dotblot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Cryo-Electron Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: iWJ-MSC-EV Capture Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Agarose Spot Migration Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Matrigel-Based Tube Formation Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Glycoarray Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Statistics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Info: Title: N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV Cell Interaction and Functional Activation of Endothelial Cells Author: Marta Clos-Sansalvador, Sergio G. Garcia, Miriam Morón-Font, Charles Williams, Niels-Christian Reichardt, Juan M. Falcón-Pérez, Antoni Bayes-Genis, Santiago Roura, Marcella Franquesa, Marta Monguió-Tortajada and Francesc E. Borràs Subject: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells’ interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton’s Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV cell interactions and associated functions. 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