Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications

• Published in: Cytotherapy (Oxford, England) Vol. 20; no. 9; pp. 1110 - 1123
• Main Authors: LÓPEZ-LUCAS, MARÍA DOLORES, PACHÓN-PEÑA, GISELA, GARCÍA-HERNÁNDEZ, ANA MARÍA, PARRADO, ANTONIO, SÁNCHEZ-SALINAS, DARÍO, GARCÍA-BERNAL, DAVID, ALGUERÓ, MARIA DEL CARMEN, MARTINEZ, FRANCISCA INIESTA, BLANQUER, MIGUEL, CABAÑAS-PERIANES, VALENTÍN, MOLINA-MOLINA, MAR, ASÍN-AGUILAR, CIRA, MORALEDA, JOSÉ M, SACKSTEIN, ROBERT
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.09.2018
• Subjects: Advanced Basic Science; E-selectin ligand; exofucosylation; fucosyltransferase; glycosyltransferase programmed stereosubstitution; good manufacturing practice; HCELL; hematopoietic cell E-/L-selectin ligand; mesenchymal stem cell; Other; sialyl Lewis X; mesenchymal stem cell; sialyl Lewis X; E-selectin ligand; good manufacturing practice; glycosyltransferase programmed stereosubstitution; hematopoietic cell E-/L-selectin ligand; fucosyltransferase; HCELL; exofucosylation
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2018.07.001

Abstract Background The regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) 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 hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. Methods BM-hMSCs 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 using Western blot, flow cytometry and cell-binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSCs were each analyzed for cell viability, immunophenotype and differentiation potential, and E-selectin binding stability was assessed at room temperature, at 4°C, and after cryopreservation. Cell product safety was evaluated using microbiological testing, karyotype analysis, and c-Myc messenger RNA (mRNA) expression, and potential effects on genetic reprogramming and in cell signaling were analyzed using gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. Results Our protocol efficiently generates HCELL on clinical-scale batches of BM-hMSCs. Exofucosylation yields stable HCELL expression for 48 h at 4°C, with retained expression after cell cryopreservation. Cell viability and identity are unaffected by exofucosylation, without changes in gene expression or RTK phosphorylation. Discussion The described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSCs endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production.