Effect of glucose levels on high versus low molecular weight hyaluronan and transfection of bone marrow derived human Mesenchymal Stromal Cells

摘要

Hyaluronan (HA) is a non-sulfonated linear glycosaminoglycan that is ubiquitously found in the extracellular matrix (ECM) and whose biomechanical properties have been thoroughly characterized. More recently, high molecular weight HA has been implicated in regulation of various physiological processes including embryonic development, inflammation, tissue regeneration, cell proliferation and migration. High molecular weight (HMW) HA is produced by a variety of cell types including mesenchymal stromal cells and functions at a multitude of levels in the regulation of cell behavior. Mesenchymal stromal cells (MSCs) have become an ideal candidate for cellular therapy since they are easily isolated and propagated in vitro, can differentiate into multiple lineages and have immunosuppressive properties. HA has been shown to regulate the potency of MSCs and may play a significant role in their behavior. Here we investigated the effect of different glucose concentrations in minimal media on HA levels produced by the MSCs in the ECM and their effect on MSC transfection with endothelial NO synthase (eNOS). Bone marrow derived MSCs were grown in alpha minimal essential media (aMEM) containing either low (1 mg/mL) or high (4.5 mg/mL) levels of glucose. MSCs were also transfected with pVax-eNOS plasmid using JetPrime (Polyplus Transfection) in the presence or absence of hyaluronidase. HA levels were detected with biotin-conjugated HA binding protein (HABP) followed by FITC-conjugated streptavidin. Cells grown in high glucose media displayed large intricate networks of HMW HA in the ECM as shown by HABP binding and fluorescence microscopy. In comparison MSCs grown in low glucose media displayed disperse networks of HA. Additionally, there was a 3-fold increase observed in levels of HA in the ECM when MSCs were cultured in high glucose media versus cells grown in low glucose media. These MSCs also displayed enhanced levels of CD44, one of the receptors of HA, as determined by flow cytometry. MSCs transfected with pVax-eNOS using JetPrime (Polyplus Transfection) showed no significant loss in viability in comparison to the controls. Furthermore, significantly enhanced levels of eNOS were observed in transfected cells grown in both media containing low and high levels of glucose in comparison with pVax transfected cells. However, hyaluronidase pre-treatment of MSCs resulted in no significant change in eNOS transfection in cells grown in low glucose media. In comparison, there was a significant decrease in eNOS accumulation observed in hyaluronidase treated cells grown in high glucose media in comparison to non-treated cells (1.74±0.09 fold eNOS accumulation with respect to beta-actin in treated vs.2.2±0.12 in non-treated; P ＜ 0.01). Furthermore, Hyaluronidase treated cells grown in high glucose media displayed a lack of network formation of high molecular HA in the ECM. These results suggest that glucose concentration in the media may have significant impact on HA levels in the ECM. Additionally, the balance between HMW and LMW HA as observed through the presence or absence of intricate networks of HA in the ECM could play a role in transfection ability of MSCs as well as their potency.