De novo synthesis of glycosaminoglycans by equine multipotent mesenchymal stromal cells in vitro – Studied by stable isotopic labeling and matrix-assisted laser desorption ionization mass spectrometry

Glycosaminoglycans (GAGs) play an important role in extracellular matrix (ECM) homeostasis and are crucial for maintaining the specific biomechanical and functional properties of musculoskeletal tissues. Aiming at regenerating these tissues, multipotent mesenchymal stromal cells (MSCs) are frequently used, their targeted differentiation and ECM synthesis being a part of their complex mechanism of action. To achieve a better understanding of these processes and to improve the targeted use of the cells for the development of regenerative therapies, reliable quantification of GAGs synthesized by MSCs represents an important step. The aim of this study was to develop a novel technique to specifically assess the de novo synthesis of GAGs, particularly chondroitin sulfate (CS), by MSCs.

Adipose tissue-derived equine MSCs were cultured in vitro for 2, 7, 14, and partially 28 d. Harvested cell populations were enzymatically digested with chondroitinase ABC from Proteus vulgaris and afterwards subjected to CS analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

The herein chosen detection method of MALDI-TOF MS combined with enzymatic digestion represents a reliable technique to monitor the culture time-dependent GAG biosynthesis of MSCs cultured in vitro. Furthermore, the addition of ¹³C-labeled glucose as cell culture medium supplement is a useful approach to obtain information regarding cellular GAG and in particular CS synthesis.