Abstract
Purpose: A major limited factor for the application of human bone marrow mesenchymal stem cells (hBMSCs) in cartilage tissue engineering is our understanding of the process by which cartilage is developed, termed chondrogenesis. This multi-step process is regulated by a variety of growth factors, including bone morphogenic proteins and transforming growth factor β (TGF-β) proteins. In addition, chondrogenic differentiation is also modulated by extracellular matrix (ECM) proteins. Considering that the functional role for the majority of these secreted proteins during chondrogenesis process is not fully established, we have applied SILAC (Isotope Labeling by Amino Acids in Cell Culture) technique to analyze the extracellular protein expression profile of hBMSCs undergoing chondrogenic differentiation. Methods: hBMSCs isolated from 3 osteoarthritic (OA) patients were grown in SILAC DMEM with different isotope variants of lysine and arginine between 4-6 weeks. The fully labelled cell populations were then subjected to chondrogenesis under a home-made medium containing 10 ng/ml TGF-β3 and other factors for 14 days. 24 h before collecting the secretome, the micromasses were carefully washed and changed to serum-free labeling medium containing also chondrogenic inducers. Expression of cartilage specific genes such as type I and II collagen and proteoglycans were used to verify the chondrogenicity of hBMSCs. Proteins in the CM harvested on day 2 and 14 of differentiation were precipitated and combined at a 1:1 ratio. Each mixture was then separated by 1D-SDS-PAGE and subjected to in-gel trypsin digestion using an automatic digester. The resulting peptide mixtures were analyzed by nanoscale liquid chromatography coupled on-line to an LTQ-Orbitrap XL mass spectrometer and quantified using the MaxQuant software. Results: A progressive increase of type II collagen, agreccan and chondroitin-6-sulfate during the course of 14 days of differentiation was detected by immunohistochemical assays, suggesting that hMSCs maintain their chondrogenic capacity in SILAC medium. Using the metabolic labeling strategy, we compared the secretomes at two different time points of chondrogenesis. More than 1000 proteins were identified with high confidence parameters. 20% of them were previously known ECM-related proteins. Among these, 34 exhibited a significant modulation of their levels during the process of chondrogenesis, including cartilage ECM proteins such as COMP, lumican, prolargin, fibromodulin and matrix gla protein (MGP), which could be involved in the organization and/or stabilization of cartilage matrix. All these proteins were increased at day 14 of chondrogenesis. Finally, pentraxin-related protein (PTX3), which plays a role in the regulation of inflammatory response, resulted decreased at day 14. Conclusions: This study was focused on characterizing the specific modulations in the secretome of hBMSCs undergoing chondrogenesis using the SILAC method. The identification and quantification of these secreted proteins provide information about the changes in the ECM synthesis during the differentiation process. Moreover, these findings enhance our knowledge of extracellular regulation of this process and allow the identification of extracellular markers of chondrogenesis, which might have potential value in cartilage regeneration strategies.
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