Synovial stem cell biology

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My NSERC program is focused on understanding how the behaviour of mesenchymal stem cells (MSCs) is regulated. My lab has previously demonstrated that inflammatory changes within the joint alter the chondrogenic differentiation potential of synovial-derived MSCs. During the initial cycle of my NSERC program, we found that synovial macrophages are crucial for the regulation of these inflammatory factors. Further, by depleting macrophages there is a dramatic change in the inflammatory micro-environment within the synovium, following which we observe alterations in the differentiation capacity of synovial MSCs. These results strongly suggest that synovial macrophages have the potential to act as a stem cell niche by regulating the behaviour of synovial MSCs. To complement this finding, we are also investigating if there is reciprocal regulation of macrophages by MSCs. We have undertaken in vitro omics (transcript and protein) based approaches to determine which proteins are secreted by MSCs when exposed to macrophage-secreted cytokines/chemokines. We have found that MSCs express high levels of the proteoglycans Aggrecan (ACAN) and Proteoglycan 4 (PRG4) in response to pro-inflammatory mediators (in vitro). Therefore, we hypothesize that MSCs may regulate macrophages and by extension, their microenvironment (niche) through the expression of proteoglycans (specifically ACAN and PRG4); however, the mechanism by which ACAN and/or PRG4 can regulate the behaviour of macrophages remains unclear. My research objective in this grant cycle is to elucidate the regulatory feedback mechanisms that exist between synovial MSCs and macrophages and understand how these pathways regulate MSC fate in vitro and in vivo. This will be achieved by undertaking the following aims: Aim 1 (in vitro): Elucidate the signaling mechanism between synovial MSCs and macrophages through the use of knockdown/out (human) and transgenic (mouse) approaches. Specifically, we will employ co-culturing techniques (MSC with macrophage) and disrupt signaling in each cell type (e.g. Ccl2 in macrophages; Acan/Prg4 in MSCs). This will allow us to determine how these factors regulate synovial MSC behaviour in terms of self-renewal and differentiation potential and macrophage behavior in terms of polarization state and cytokine expression. Aim 2 (in vivo): Assay for MSC function and proteoglycan expression (Acan/Prg4) within the synovium when macrophage niche regulatory factors (e.g. Ccl2) are conditionally deleted using a transgenic mouse approach. Aim 3 (in vivo): Examine the inverse relationship (MSCs on macrophages) by conditionally deleting proteoglycan (Acan/Prg4) expression in MSCs and assaying for macrophage polarization state and secreted factors. This program of research will result in significant advances to our knowledge of how macrophages control MSC behaviour; and will also provide a framework to understand how MSCs regulate their micro-environment in synovial joints.