Abstract
In 10-20% of the six million cases of bone fractures in the United States there is an impaired healing process due to severe trauma or metabolic diseases that reduce tissue regeneration. Mesenchymal Stromal Cells (MSCs) have been widely studied for their ability to differentiate into various cell types, including osteoblasts. Osteoblasts are cells that develop and mineralize the bone. Cellular therapies using MSCs to promote tissue regeneration have been proposed, but many have been ineffective due to MSCs inability to reach the target site as well as the poor retention/survival of cells after transplantation. Therefore, novel research approaches are needed to investigate MSC survival pathways, in order to develop effective methods of maintaining viable MSCs at transplantation site, thereby expanding the therapeutic window exerted by the cells.Post-translational modifications affect protein function, and therefore cellular functions, such as cell survival. N-linked glycosylations (N-glycans) are post-translation modifications that are known to affect cell signaling, adhesion, and migration. N-glycan modification occurs in the endoplasmic reticulum and Golgi apparatus, where sugars are attached to asparagine in N-X-S/T peptides, where X is any amino acid, except proline. Sialyltransferases are enzymes that synthesize the addition of sialic acid onto N-glycans. ST3Gal1 and ST6Gal1 are sialyltransferases that catalyze the addition of sialic acid onto galactose in an α-2,3 and α-2,6 conformation, respectively. Previous research, especially in the cancer field has linked sialylations on N-glycans to cell survival. The primary goal of the work herein was to determine if sialylation influences MSC survival and to explore the underlying mechanism.
MSCs cultured in serum-free media and 1% oxygen have reduced cell proliferation and survival, these conditions mimic an ischemic environment in vitro. MSCs pretreated with 3F-Neu5Ac, a novel sialyltransferase inhibitor, showed an increase cell survival when incubated under in vitro ischemic conditions. Thus, the decrease in sialylation on MSCs led to an increase in cell survival under serum-free and hypoxic conditions. This increase in MSC survival was associated to an increase in catalase activity, protecting MSCs from oxidative stress and apoptosis. In contrast, there was no association between 3F-Neu5Ac supplementation and MSC proliferation. Altogether, the 3F-Neu5Ac inhibitor led to an increase in MSC survival under ischemic conditions, which could be attributed to apoptosis, but not cell proliferation.
During bone fracture there are many cytokines present for the regulation of bone fracture repair, including interferon γ (IFNγ) and platelet-derived growth factor (PDGF). Previous research showed that IFNγ led to an increase in sialylated N-glycans on MSCs, which could be attributed to the Akt pathway. In the present study, we used PDGF-BB, known to activate the Akt pathway in MSCs, to analyze sialylated N-glycans on MSCs. PDGF-BB causes a reduction of sialylation of MSCs after 48 hours. Then, we explored the effects on MSC survival in the presence of these cytokines (IFNγ and PDGF-BB) or low serum, which mimics nutrient deprivation observed during bone fractures and is known to increase sialylation on MSCs. After two days pre-treatment, MSCs treated with IFNγ showed no significant different in cell number. While PDGF-BB treated cells had an increase in cell number and MSCs cultured in low serum (0.5% FBS) had a decrease in cell number. After nine days in nutrient and oxygen deprivation, IFNγ and PDGF-BB treated MSCs showed no significant difference in cell survival. In contrast, MSCs cultured in low serum had a significant decrease in MSC survival. Although, IFNγ and PDGF-BB treatment affects sialylated N-glycan expression on MSCs, they do not necessarily influence cell survival.