title: Glycoengineering of E-Selectin Ligands by Intracellular versus Extracellular Fucosylation Differentially Affects Osteotropism of Human Mesenchymal Stem Cells.
abstract: Human mesenchymal stem cells (MSCs) hold great promise in cellular therapeutics for skeletal diseases but lack expression of E-selectin ligands that direct homing of blood-borne cells to bone marrow. Previously, we described a method to engineer E-selectin ligands on the MSC surface by exofucosylating cells with fucosyltransferase VI (FTVI) and its donor sugar, GDP-Fucose, enforcing transient surface expression of the potent E-selectin ligand HCELL with resultant enhanced osteotropism of intravenously administered cells. Here, we sought to determine whether E-selectin ligands created via FTVI-exofucosylation are distinct in identity and function to those created by FTVI expressed intracellularly. To this end, we introduced synthetic modified mRNA encoding FTVI (FUT6-modRNA) into human MSCs. FTVI-exofucosylation (i.e., extracellular fucosylation) and FUT6-modRNA transfection (i.e., intracellular fucosylation) produced similar peak increases in cell surface E-selectin ligand levels, and shear-based functional assays showed comparable increases in tethering/rolling on human endothelial cells expressing E-selectin. However, biochemical analyses revealed that intracellular fucosylation induced expression of both intracellular and cell surface E-selectin ligands and also induced a more sustained expression of E-selectin ligands compared to extracellular fucosylation. Notably, live imaging studies to assess homing of human MSC to mouse calvarium revealed more osteotropism following intravenous administration of intracellularly-fucosylated cells compared to extracellularly-fucosylated cells. This study represents the first direct analysis of E-selectin ligand expression programmed on human MSCs by FTVI-mediated intracellular versus extracellular fucosylation. The observed differential biologic effects of FTVI activity in these two contexts may yield new strategies for improving the efficacy of human MSCs in clinical applications. Stem Cells 2016;34:2501-2511.
authors.affiliation: Department of Dermatology and Harvard Skin Disease Research Center, Brigham and Women's Hospital, Harvard University, Cambridge, Massachusetts, USA.|Program of Excellence in Glycosciences, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Brad
authors.initials: B
authors.lastname: Dykstra
authors.affiliation: Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Harvard University, Cambridge, Massachusetts, USA.|Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Jungmin
authors.initials: J
authors.lastname: Lee
authors.affiliation: Regenerative Bioscience Center, Rhodes Center for Animal and Dairy Science and College of Engineering, University of Georgia, Athens, Georgia, USA.
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Mortensen
authors.affiliation: Bio-Techne, R&D Systems, Inc, Minneapolis, Massachusetts, USA.
authors.forename: Haixiao
authors.initials: H
authors.lastname: Yu
authors.affiliation: Bio-Techne, R&D Systems, Inc, Minneapolis, Massachusetts, USA.
authors.forename: Zhengliang L
authors.initials: ZL
authors.lastname: Wu
authors.affiliation: Advanced Microscopy Program, Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Charles P
authors.initials: CP
authors.lastname: Lin
authors.affiliation: Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Harvard University, Cambridge, Massachusetts, USA.|Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Derrick J
authors.initials: DJ
authors.lastname: Rossi
authors.affiliation: Department of Dermatology and Harvard Skin Disease Research Center, Brigham and Women's Hospital, Harvard University, Cambridge, Massachusetts, USA. rsackstein@partners.org.|Program of Excellence in Glycosciences, Harvard University, Cambridge, Massachusetts, USA. rsackstein@partners.org.|Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. rsackstein@partners.org.
authors.forename: Robert
authors.initials: R
authors.lastname: Sackstein
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000378:metabolism; D001842:Bone and Bones / Q000166:cytology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D019040:E-Selectin / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005119:Extravasation of Diagnostic and Therapeutic Materials / Q000473:pathology; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D051379:Mice; D012886:Skull / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous
chemical_list: D019040:E-Selectin; D006023:Glycoproteins; D008024:Ligands; D005643:Fucose; D005647:Fucosyltransferases; C515956:FUT6 protein, human
keywords: E-selectin; Exofucosylation; Fucosyltransferase; GPS; HCELL; Intravital microscopy; Mesenchymal stromal cell; Modified mRNA; sialyl Lewis X