Background Mesenchymal stem cells (MSCs) have been identified in bone marrow, as well as in other tissues of the joint. These cells have the ability to differentiate into chondrocytes and also to modulate immune responses, improve angiogenesis and prevent fibrosis. These properties confer them the potential to be used for therapeutic applications in rheumatic diseases, including osteoarthritis (OA).
Objectives To identify possible differences between both types of cells, we have performed a quantitative proteomic analysis of bone marrow MSCs undergoing chondrogenic differentiation, which were obtained from OA patients and control donors using a de novo protein labeling approach (SILAC).
Methods Bone marrow MSCs from control (n=3) and OA (n=3) patients were expanded in a DMEM medium lacking arginine and lysine. Regular Lys and Arg were added to the control cell population media, and isotope-labeled Lys (Lys6) and Arg (Arg10) to the OA cell population media. Once a complete incorporation of the isotopes was achieved in the newly synthesized proteins, cells were grown in a three-dimensional environment (micromasses) during a period of 14 days in a commercial chondrogenic medium to promote chondrogenesis. Expression of cartilage specific genes and histological assays were performed to explore the chondrogenicity of the MSCs. For the proteomic analysis, protein samples from OA (heavy) and normal (light) micromasses were combined 1:1 and separated by SDS-PAGE before in-gel digestion with trypsin. Analysis of the resulting peptides was performed by nanoscale LC-MS/MS. Identification and quantification of the proteins was done with Protein Pilot 3.0 software.
Results A significant difference in the gene expression of COL2 was observed in the normal donors when compared to the OA patients. Moreover, histology assays confirmed that both types of cells exhibited aggrecan and sulfated glycosaminoglycans formation after 14 days in chondrogenesis. Using the proteomic approach, we compared the intracellular protein profiles of OA and normal MSCs at the same time of differentiation. Among the 417 quantified proteins, 77 had significantly altered levels. 46 proteins displayed consistently higher levels in the OA samples compared to normal donors. Among them, we found some matrix-regulatory agents like Serpin H1 and Galectin-3. On the other hand, 31 proteins displayed a significantly reduced abundance in OA patients when compared to controls. Interestingly, we detected some proteins that are involved in the regulation of cell morphology and cytoskeletal organization, like Destrin or Fascin, pointing to a lower actin turnover in the MSCs from OA patients.
Conclusions Using the SILAC technology, we have identified a number of novel specific modulations in the proteomic profiles of bone marrow MSCs from OA and healthy patients cultured in an in vitro model of chondrogenesis. The increased presence of some of them in OA patients (like Serpin H1), suggest their putative role in the abnormal cell differentiation that might contribute to the development of the disease. All this information can help us to clarify the underlying mechanisms of OA, and would be useful to improve putative cell therapy strategies.
Disclosure of Interest None Declared
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