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MARÍA DOLORES LÓPEZ-LUCAS GISELA PACHÓN-PEÑA ANA MARÍA GARCÍA-HERNÁNDEZ ANTONIO PARRADO DARÍO SÁNCHEZ-SALINAS DAVID GARCÍA-BERNAL MARIA DEL CARMEN ALGUERÓ FRANCISCA INIESTA MARTINEZ MIGUEL BLANQUER VALENTÍN CABAÑAS-PERIANES MAR MOLINA-MOLINA CIRA ASÍN-AGUILAR JOSÉ M MORALEDA ROBERT SACKSTEIN 《Cytotherapy》2018,20(9):1110-1123
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. 相似文献2.
DEMETRIO BOLTOVSKOY FRANCISCO SYLVESTER ALEJANDRO OTAEGUI VALENTÍN LEITES DANIEL H. CATALDO 《Austral ecology》2009,34(7):719-730
Limnoperna fortunei (Dunker, 1857) (Bivalvia) invaded Argentina through the Río de la Plata estuary around 1990 and is presently established throughout five South American countries as a dominant component of the benthic fauna and a major nuisance for industry and power plants. Between 1997 and 2006 we monitored the reproductive activity of L. fortunei through weekly measurements of planktonic larvae in six South American water bodies: Río de la Plata estuary, Paraná and Carapachay rivers, Salto Grande, Itaipú and Embalse de Río Tercero reservoirs. Mean larval densities varied between 4000 and 7000 individuals m?3; except in the reservoirs of Itaipú (450 ind. m?3) and Salto Grande (869 ind. m?3), where the mussel was first recorded shortly before our surveys, and upstream dispersal is limited (Salto Grande). In all cases, reproductive output decreases during the winter. At four of the six sites surveyed larval densities were comparatively high for 8.8–10.2 months per year. A lower food supply is possibly responsible for the shorter reproductive period of 5.9 months at Embalse. At Salto Grande, there is a well‐defined mid‐summer drop in larval numbers, coinciding with blooms of cyanobacteria. We propose that, in addition to temperature, two major factors may regulate the reproductive activity of L. fortunei: (i) the availability of food; and (ii) blooms of toxic cyanobacteria, significantly shortening the otherwise very long reproductive period. This information is important for the design of antifouling programmes involving the use of molluscicides, and has potential for reduced biocide use. These results provide supporting evidence for some fundamental ecological theories of invasions discussed here. 相似文献
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