Synovial stem cell biology

我的NSERC项目专注于了解间充质干细胞(MSCs)的行为是如何受到调控的。我的实验室以前已经证明，关节内的炎性变化改变了滑膜来源的MSCs的软骨分化潜能。在我的NSERC项目的最初周期中，我们发现滑膜巨噬细胞对这些炎症因子的调节至关重要。此外，通过消耗巨噬细胞，滑膜内的炎性微环境发生了戏剧性的变化，随后我们观察到滑膜间充质干细胞分化能力的变化。这些结果有力地表明，滑膜巨噬细胞有可能通过调节滑膜间充质干细胞的行为而发挥干细胞的作用。为了补充这一发现，我们还在调查MSCs对巨噬细胞是否存在相互调节。我们已经采用了基于体外组学(转录本和蛋白质)的方法来确定当MSCs暴露于巨噬细胞分泌的细胞因子/趋化因子时，哪些蛋白质被分泌。我们发现MSCs在体外表达高水平的蛋白多糖聚集素(Acan)和蛋白多糖4(PRG4)，以响应促炎介质。因此，我们假设MSCs可能通过蛋白多糖(特别是ACAN和PRG4)的表达来调节巨噬细胞及其微环境(生态位)；然而，ACAN和/或PRG4调节巨噬细胞行为的机制尚不清楚。我在这个资助周期的研究目标是阐明滑膜MSCs和巨噬细胞之间存在的调节反馈机制，并了解这些途径如何在体外和体内调节MSC的命运。这将通过以下目标来实现：目标1(体外)：通过基因敲除(人)和转基因(小鼠)方法阐明滑膜间充质干细胞和巨噬细胞之间的信号机制。具体地说，我们将使用共培养技术(MSC与巨噬细胞)，并干扰每种细胞类型的信号(例如，巨噬细胞中的CCL2；MSCs中的Acan/Prg4)。这将使我们能够确定这些因素如何调节滑膜MSC的自我更新和分化潜能，以及巨噬细胞的极化状态和细胞因子表达。目的2(体内)：使用转基因小鼠方法，有条件地删除巨噬细胞生态位调节因子(如CCL2)后，测定滑膜中MSC的功能和蛋白多糖的表达(Acan/Prg4)。目的3(体内)：通过有条件地删除MSCs中蛋白多糖(Acan/Prg4)的表达，并检测巨噬细胞的极化状态和分泌因子，来检验MSCs对巨噬细胞的反向关系。这项研究计划将使我们对巨噬细胞如何控制MSC行为的了解取得重大进展；并将提供一个框架，以了解MSCs如何调节其滑膜关节的微环境。

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.