HOW DOES CHONDROGENIC DIFFERENTIATION AND STIMULATION WITH INTERLEUKIN-1BETA AFFECT THE SECRETOME FROM BONE MARROW DERIVED MESENCHYMAL STEM CELLS

Abstract

Purpose: Similar to humans, the horse is a long-lived, athletic species. Osteoarthritis (OA) is one of the leading causes of lameness in horses, and cell-based therapies are seen as the next generation treatment for OA. Treatment with mesenchymal stem cells (MSC) is already used in the veterinary clinic, and it is believed that MSC exert their therapeutic effects through secreted trophic biomolecules, but little is known about the exact action of MSC in inflamed articular joints. The aim of this study was to compare the in vitro secreted protein profile (SPP) after 48 hours and 10 days of bone marrow derived (BM)-MSC stimulated with the pro-inflammatory cytokine IL-1beta and cultured in either expansion medium (EM) or chondrogenic differentiation medium (CM) containing transforming growth factor beta 3 (TGF-beta3). Methods: BM-MSC derived from equine sternal bone marrow were expanded to 70% confluence in serum-enriched medium followed by a 24h wash in serum-reduced medium. The cells were then cultured with serum-reduced EM alone (EMn), serum-reduced EM supplemented with IL-1beta (10 ng/ml) (EMIL), or serum-reduced CM alone (CMn) or supplemented with IL-1beta (10 ng/ml) (CMIL). Medium for analysis was added day 0 and collected after 48h and medium added day 8 was collected day 10. Samples were analysed by mass spectrometry. A two sample test was used to compare the SPP group-wise: 48hEMn vs 48hCMn, 48hEMIL vs 48hCMIL, 10dEMn vs 10dCMn, 10dEMIL vs 10dCMIL. Results: A total of 823 proteins were identified. The concentration of proteins involved in chondrogenesis and formation of extracellular matrix (ECM) including stimulator of chondrogenesis 1, cartilage oligomeric matrix protein, decorin, osteomodulin, fibronectin 1, and connective tissue growth factor 1 were significantly higher in the SPP from chondrogenic differentiated MSCs in all groups. Proteins with a significantly higher concentration in the SPP from the 48hEMIL and 10dEMIL groups compared to the 48hCMIL and 10dCMIL groups included proteins involved in the inflammatory response and breakdown of extracellular matrix (e.g., IL8, CXCL1, CXCL6, MMP1, and MMP3). Proteins with a significantly higher concentration in the SPP from the 48hCMIL and 10dCMIL groups compared to the 48hEMIL and 10dEMIL groups included proteins involved in formation of ECM and inhibition of the inflammatory response (e.g., chondroitin sulfate synthase 3, extracellular matrix protein 2, and SERPINB1. Conclusions: In conclusion, the results indicate that BM-MSC undergoing chondrogenic differentiation suppress the influence of IL-1beta and promote formation of ECM.