Glycoengineering of E-Selectin Ligands by Intracellular versus Extracellular Fucosylation Differentially affects Osteotropism of Human Mesenchymal Stem Cells

Dykstra B, Lee J, Mortensen LJ, Yu H, Wu ZL, Lin CP, Rossi DJ, Sackstein R.

Stem Cells. 2016 Oct;34(10):2501-2511. doi: 10.1002/stem.2435. Epub 2016 Jul 17.


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.