After washing three times, secondary antibodies (Chemmate Envision HRP-polymer, Dako) or anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (Dako) were added, followed by incubation for 30?min at room temparature. were sorted. Proliferation, surface marker expression, chondrogenesis, calcification and adipogenesis potentials were compared in synovial MSCs derived from the three regions. Results We selected CD55+ CD271? for synovial cells in the surface region, CD55? CD271? in the stromal region, and CD55? CD271+ in the perivascular region. The ratio of the sorted cells to non-hematopoietic lineage cells was 5% in the surface region, 70% in the stromal region and 15% in the perivascular region. Synovial cells in the perivascular fraction had the greatest proliferation potential. After expansion, surface marker expression profiles and adipogenesis potentials were similar but chondrogenic and calcification potentials were higher in synovial MSCs derived from the perivascular region than in those derived from the surface and stromal regions. Conclusions We identified specific markers to isolate synovial cells from the surface, stromal, and perivascular regions of the synovium. Synovial MSCs in the perivascular region had the highest proliferative and chondrogenic potentials among the three regions. Background Mesenchymal stem cells (MSCs) are an attractive cell source for cell therapies. These cells participate in tissue homoeostasis, remodeling, and repair by ensuring replacement of mature cells that are lost during the course of physiological turnover, senescence, injury, or disease . Along with preclinical studies, a large number of clinical trials have been conducted for cardiovascular diseases, osteoarthritis, liver disorders, graft versus host disease (GvHD), respiratory disorders, spinal cord injury, and others . MSCs are found not only in bone marrow but multiple adult tissues [3C5]. MSCs are defined as non-hematopoietic-lineage, plastic-adherent, self-renewing cells that can differentiate into chondrocytes, adipocytes and osteoblasts in vitro [6, 7]. Traditionally, the IDF-11774 isolation of MSCs has relied on their adherence to plastic dishes and colony-forming ability in an unfractionated cell population. This technique may give rise to heterogeneous cell populations in MSCs. To better characterize this heterogeneity, surface markers have been investigated for bone marrow MSCs from the osteoblast region , endosteum region , and perivascular region . Synovial MSCs have a higher chondrogenic potential than bone marrow MSCs . Transplantation of synovial MSCs regenerated cartilage  and meniscus . Synovial MSCs are clinically used for cartilage regeneration . To prepare synovial MSCs, synovium is digested, and unfractionated synovial cells are expanded to form cell colonies of synovial MSCs INPP5K antibody [15, 16]. Synovial tissue can be histologically classified into three regions; surface, stromal, and perivascular regions . If synovial cells can be obtained and synovial MSCs can be prepared from each region separately, more attractive synovial MSCs can be used in clinical therapies. This also provides important information on the physiological roles of cells in the synovium. The purpose of the present study was to identify specific markers for the isolation of synovial cells in the surface, stromal, and perivascular regions, and to compare properties of MSCs sorted by the specific markers. Methods Human synovium This study was approved by the Medical Research Ethics Committee of Tokyo Medical and Dental University and all human study subjects provided informed consent. Human synovium was harvested from the knees of ten donors (59C85?years) with osteoarthritis during total knee arthroplasty. Transmission electron microscopy (TEM) The specimens of synovial tissues were rapidly fixed in 2.5% glutaraldehyde in 0.1?M phosphate buffer for 2?h. The samples were washed IDF-11774 with 0.1?M phosphate buffer, post-fixed in 1% OsO4 buffered with 0.1?M phosphate buffer for 2?h, dehydrated in IDF-11774 a graded series of ethanol and embedded in Epon 812. Ultrathin IDF-11774 sections at 90?nm were collected on copper grids, double-stained with uranyl acetate and lead citrate, and then examined by transmission electron microscopy (H-7100, Hitachi, Tokyo, Japan) . Immunostaining Synovial tissues were rapidly embedded in OCT compound (Sakura Finetec Japan, Tokyo, Japan) and 4% carboxymethyl cellulose and were washed with 0.1% Tween-TBS. After blocking with Protein Block Serum-Free (Dako, Glostrup, Denmark), sections (5?m thick) were incubated with 19 antibodies; CD90 (Becton, Dickinson and Company; BD, Franklin.