Chemotaxis of cultured human mesenchymal stem cells to osteoarthritic cartilage and synovium

Abstract

Purpose: Mesenchymal stem cells (MSCs) are promising candidates for cell therapy in osteoarthritis (OA). MSCs can differentiate to generate repair cartilage tissue and they have potential immunomodulatory and anti-inflammatory effects on synovium and cartilage. To be therapeutic in OA, MSCs need to survive after intra-articular injection, probably by migration towards or into affected tissues. Chemotactic factors secreted by OA tissues and expression of chemotactic factor receptors by MSCs could be involved in this migration. We studied the effect of the OA environment on the migration of MSCs in culture and we studied whether we could prime MSCs with inflammatory factors to influence the expression of chemotactic factor receptors and thereby improve the migration capacity. Methods: MSCs were cultured in monolayer in serum-free medium with 1% ITS at 20% O2 with(out) 50ng/ml, 20ng/ml or 1 ng/ml IFNγ and TNFα for 24 hours or with(out) 50% synovial fluid (SF) of OA patients or healthy donors for 48 hours. The expression of chemotactic factor receptors CXCR1, CXCR3, CXCR4, CCR1, CCR4, CCR5, PDGFRa, PDGFRb and CX3CR1 by MSCs was analysed by RT-PCR (related to HPRT as housekeeper) and FACS. Chemotactic factors fractalkine, MCP-3, PDGF-BB, MIP-1a, MIP-1b, MDC, PDGF-AA, IP10, IL-8 and MCP-1 secreted in the medium by OA cartilage and synovium of six donors were measured with a 10-plex milliplex assay. For the chemotaxis assay 6,000 MSCs were seeded in transwell inserts (8.0um pores) and placed in 24-well plates with OA synovium or cartilage conditioned medium (CM) for 18 hours. Migrated MSCs on the outside of the transwell membrane were stained and counted. Results: All chemotactic factor receptor genes were expressed in cultured MSCs except for CXCR1. MSCs exposed to OA SF lead to an increased mRNA expression of CCR1 and PDGFRa and a decreased expression of PDGFRb (figure 1). The chemotactic factors that bind on these receptors were present in OA synovium. They were secreted by both OA cartilage and synovium; in particular high amounts of MDC, IL-8 and MCP-1 were detected in the CM. Additionally, differences in the composition of the chemokines secreted by synovium and cartilage were obvious: synovium CM contained higher amounts of MIP-1a and MIP-1b and cartilage CM contained higher amounts of PDGF-AA and IP10. The chemotaxis assay then demonstrated that significantly more MSCs migrated towards OA synovium and cartilage CM compared to control medium (figure 2). Priming the MSCs with inflammatory factors IFNγ and TNFα increased the mRNA expression of CCR1 and CCR4 and decreased PDGFRa and PDGFRb with a clear dose response effect on the expression of CCR1 and PDGFRa. The decreased expression of PDGFRa could be confirmed with FACS analyses. OA CM still had an chemotactic effect on primed MSCs, however the chemotactic effect of OA CM was stronger on non-primed MSCs compared to primed MSCs. http://files.abstractsonline.com/CTRL/51/E/C61/29B/95C/475/69C/E31/591/FAC/899/02/g833_1.jpg Fig. 2MSCs migrate to OA synovium and cartilage CM. *** p < 0.001; mean ± SD; (syn CM) synovium conditioned medium, (cart CM) cartilage conditioned medium.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusion: MSCs expressed mRNA for chemotactic factor receptors and migrated towards chemotactic factors secreted by OA synovium and cartilage. Based on our in-vitro data, OA SF will probably not have a negative effect on the expression of the chemotactic factor receptors of intra-articularly injected MSCs. Priming MSCs in culture with inflammatory factors before intra-articular injection with the goal to improve the migration potential, influenced the expression of different chemotactic factor receptor in MSCs, however did not improve chemotaxis. These data suggest that we should be careful with priming MSCs (with inflammatory factors) to activate their capacities or with injecting them into an inflammatory environment and encourage further research to improve and optimize the therapeutic capacities of MSCs for OA.