Effect of glucose depletion and fructose administration during chondrogenic commitment in human bone marrow-derived stem cells.

ABSTRACT

BACKGROUND: Bone marrow mesenchymal stromal cells (BMSCs) are promising for therapeutic use in cartilage repair, because of their capacity to differentiate into chondrocytes. Often, in vitro differentiation protocols employ the use of high amount of glucose, which does not reflect cartilage physiology. For this reason, we investigated how different concentrations of glucose can affect the chondrogenic differentiation of BMSCs in cell culture pellets. Additionally, we investigated how fructose could influence the chondrogenic differentiation in vitro. METHODS: BMSC were isolated from six donors and cultured in DMEM containing glucose at either 25 mM (HG), 5.5 mM (LG) or 1 mM (LLG), and 1% non-essential amino acids, 1% ITS+, in the presence of 100 nM dexamethasone, 50 µg/ml ascorbic acid-2 phosphate and 10 ng/ml TGF-ß1. To investigate the effect of different metabolic substrates, other groups were exposed to additional 25 mM fructose. The media were replaced every second day until day 21 when all the pellets were harvested for further analyses. Biochemical analysis for glycosaminoglycans into pellets and released in medium was performed using the DMMB method. Expression of GLUT3 and GLUT5 was assayed by qPCR and validated using FACS analysis and immunofluorescence in monolayer cultures. Chondrogenic differentiation was further confirmed by qPCR analysis of COL2A1, COL1A1, COL10A1, ACAN, RUNX2, SOX9, SP7, MMP13, and PPARG, normalized on RPLP0. Type 2 collagen expression was subsequently validated by immunofluorescence analysis. RESULTS: We show for the first time the presence of fructose transporter GLUT5 in BMSC and its regulation during chondrogenic commitment. Additionally, decreasing glucose concentration during chondrogenesis dramatically decreased the yield of differentiation. However, the use of fructose alone or together with low glucose concentrations does not limit cell differentiation, but on the contrary it might help in maintaining a stable chondrogenic phenotype comparable with the standard culture conditions (high glucose). CONCLUSION: This study provides evidence that BMSC express GLUT5 and differentially regulate GLUT3 in the presence of glucose variation. This study gives a better comprehension of BMSCs sugar use during chondrogenesis.