ARTHRITIS & RHEUMATISM Vol. 63, No. 5, May 2011, pp 1289–1300 DOI 10.1002/art.30234 © 2011, American College of Rheumatology Functional Mesenchymal Stem Cell Niches in Adult Mouse Knee Joint Synovium In Vivo Tobias B. Kurth, 1 Francesco Dell’Accio, 2 Vicki Crouch, 1 Andrea Augello, 1 Paul T. Sharpe, 3 and Cosimo De Bari 1 Objective. We previously reported that human synovium contains cells that, after culture expansion, display properties of mesenchymal stem cells (MSCs). The objective of this study was to identify MSCs in native synovium in vivo. Methods. To identify stem cells in the synovium in vivo, a double nucleoside analog cell-labeling scheme was used in a mouse model of joint-surface injury. For labeling of slow-cycling cells, mice received iododeoxyu- ridine (IdU) for 30 days, followed by a 40-day washout period. For labeling of cells that proliferate after injury, mice underwent knee surgery to produce an articular cartilage defect and received chlorodeoxyuridine (CIdU) for 4 days, starting at multiple time points after surgery. Unoperated and sham-operated joints served as con- trols. Knee joint paraffin sections were analyzed by double and triple immunostaining to detect nucleoside analogs, conventional MSC markers, and chondrocyte- lineage markers. Results. Long-term–retaining, slow-cycling IdU- positive cells were detected in the synovium. At 4 days and 8 days after injury, there was marked proliferation of IdU-positive cells, which costained for CIdU. IdU- positive cells were nonhematopoietic, nonendothelial stromal cells, were distinct from pericytes, and stained positive for MSC markers. MSCs were phenotypically heterogeneous and located in topographically distinct niches in the lining layer and the subsynovial tissue. Twelve days after injury, double nucleoside–labeled cells within synovium were embedded in cartilage- specific metachromatic extracellular matrix and costained positive for the chondrocyte-lineage markers Sox9 and type II collagen. Conclusion. Our findings provide the first evi- dence of the existence of resident MSCs in the knee joint synovium that undergo proliferation and chondrogenic differentiation following injury in vivo. The synovial membrane is a tissue that lines the joint cavity of synovial joints and consists of a lining layer of macrophage-like (type A) and fibroblast-like (type B) synoviocytes and a loose sublining tissue. In the healthy joint, type A synoviocytes act in innate immunologic defense and support adaptive immunity, while type B synoviocytes function to regulate the release of nutrients and molecules, including hyaluronan, into the synovial fluid (1). In response to injury of various types, including trauma, the synovial membrane rapidly becomes hyper- plastic (2,3). It is commonly believed that synovial hyperplasia is sustained mainly by stromal cells including type B (fibroblast-like) synoviocytes, also called synovial fibroblasts, with infiltration of blood-borne inflamma- tory and immune cells particularly in inflammatory joint diseases such as rheumatoid arthritis (4). The biologic function of synovial stromal cell proliferation is likely to depend on the nature of the injury, but it is believed to have a pivotal role in joint homeostasis and disease. Deregulation of this process is thought to contribute to the formation of pannus, which in rheumatoid arthritis causes destruction of cartilage and bone (4). Despite the high frequency and remarkable biologic and clinical relevance of synovial hyperplasia, very little is known Supported by MRC grant G108/620. Dr. De Bari is a Fellow of the Medical Research Council, UK. 1 Tobias B. Kurth, PhD, Vicki Crouch, BSc, Andrea Augello, PhD, Cosimo De Bari, MD, PhD: University of Aberdeen, Aberdeen, UK; 2 Francesco Dell’Accio, MD, PhD: Queen Mary University of London, London, UK; 3 Paul T. Sharpe, PhD: King’s College London and Biomedical Research Centre, Guy’s and St. Thomas National Health Service Foundation Trust, London, UK. Drs. Kurth and Dell’Accio contributed equally to this work. Address correspondence to Cosimo De Bari, MD, PhD, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK. E-mail: c.debari@abdn.ac.uk. Submitted for publication July 28, 2010; accepted in revised form December 30, 2010. 1289