Gene expression profile of human bone marrow-derived mesenchymal stem cells (BMSC) treated with fucosyltransferase 6 (FTVI) or fucosyltransferase 7 (FTVII)

Background. The regenerative and immunomodulatory properties of human mesenchymal stem cells (hMSC) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α-(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin, and at all sites of inflammation. Here we describe how this modification on BM-derived hMSC (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. Methods. BM-hMSC were expanded using xenogenic-free media and exofucosylated using α-(1,3)-fucosyltransferases VI (FTVI) or VII (FTVII). Enforced fucosylation converted CD44 into HCELL, and HCELL formation was assessed by western blot, flow cytometry, and cell binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSC were each characterized by immunophenotype , differentiation potential and cell viability and fucosylation stability were assessed at room temperature and at 4°C. Safety was assessed by microbiological testing, karyotype, and c-Myc mRNA expression, and potential effects on genetic reprogramming and in cell signaling were analyzed by gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. Results. Our protocol efficiently generates HCELL on clinical-scale batches of BM-hMSC. Exofucosylation yields stable HCELL expression for 48 hours at 4°C. Exofucosylation preserved full cell viability and identity, without changing gene expression or RTK phosphorylation. Discussion. The described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSC endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production. Human bone marrow-derived mesenchymal stromal cells (BMSC) from 2 healthy donors, 2 retinitis pigmentosa (RP) patients, and 3 osteoporotic (OSP) patients were treated in vitro with fucosyltransferase 6 (FTVI) or fucosyltransferase 7 (FTVII) during 1 hr, or mock-treated, then reseeded in culture flasks and cultured for 24 hr. Equimolar RNA amounts from 5 untreated BMSC were pooled, labelled with cy3, and used as reference sample. Each of the mock-treated, FTVI-treated, or FTVII-treated samples was labelled with cy5 and used as a test sample. The reference sample and the test samples were hybridized together onto Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarrays (technology 39494), scanned in an Agilent G2565CA DNA Microarray Scanner using the Agilent Scan Control software, processed with the Agilent Feature Extraction software, and analyzed using the GeneSpring GX software.