Stromal Cells Regulate Sialylations…

Stromal Cells Regulate Sialylations…

The goal of this study was to determine the role of sialylated N-glycans in MSCs. We show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model.

Materials and Methods – MSC Isolation and Expansion MSCs were isolated from commercially available human bone marrow. Bone marrow aspirates were passed through 90 µm pore strainer for isolation of bone spicules. Then, bone marrow aspirates were diluted with an equal volume of phosphate-buffered saline (PBS) and centrifuged over Ficoll at 700 g for 30 min. Mononuclear cells from the Buffy Coat and bone spicules (if any) were plated in plastic culture flasks, using Minimum Essential Medium α plus 10% Fetal Bovine Serum (FBS-Phoenix Scientific, San Marcos, CA, USA) (standard culture media). After two days, non-adherent cells were removed by washing 2–3 times with PBS. MSCs between passage 3–6 were used for experimentation to ensure pure MSC cultures. Additionally, higher passage cells may become senescent, which lead to alteration of gene expression, arrest in cell proliferation, and resistance to apoptosis.

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