replenishment from the synovium or synovial fluid. Should the cells of the synovium or synovial fluid be a reservoir of stem cells for normal articular cartilage maintenance and repair, these endogenous sources of chondro-biased cells would be a funda- mental and new strategy for treating OA and cartilage injury if this loss of aggregation & differentiation phenotype can be overcome. Methods Ethics Statements Human: Informed consent to participate was obtained by written agreement. The study protocol was approved by the University of Calgary Research Ethics Board (Application number: 21987). Canine: All procedures received approval by the institutional ethics board (University of Calgary, Animal Care Committee) and were carried out under the supervision of a veterinarian according to the guidelines of the Canadian Council on Animal Care. Human Subjects Patients with clinical and radiographic OA [20] with no other co-morbidities consented to arthrocentesis and had synovial fluid aspirated from their knees (mean volume 63.4 ml+/212.6 ml) during a visit to the University of Calgary Foothills Medical Clinic (N = 5, 3 males age: 47, 50, 52; 2 females age 52, 54; Ethics ID #21987). Synovial fluid from macroscopically normal knees was aspirated (mean volume 5.6 ml+/22.2 ml) from cadavers less than 4 hrs after death (N = 5, 2 males age: 47, 54; 3 female age 46, 51, 52 Ethics ID #21987). Tissue donors were received by the Southern Alberta Organ and Tissue Donation Program (SAOTDP), which obtains the medical history of every donor, including current medication, previous history of joint diseases, and other co-morbidities (e.g., cancer, diabetes, inflammatory diseases). All donor knees received x-ray and macroscopic examination of the joint surfaces. Any abnormalities (cracking, blistering, darkening, abnormal wear) prompted exclusion from the study. In total seven cadaveric joints were examined and two were excluded from the study. Animals Animals were adult, outbred, skeletally mature canines ranging in age from 18–54 months. Briefly, unilateral cruciate ligament transection was performed through a lateral peripatellar athrot- Table 1. Primary antibodies used for analyses. Chondrogenic Potential of CD90+ Progenitors omy (N = 2, 1 male, 1 female). Eight weeks after surgery, animals were euthanized, and sterile samples of joint fluid were withdrawn with a syringe. The volume of joint fluid from un-operated control knee joints ranged from 150–300 micro-liters; the volume of joint fluid from knees with cruciate transection ranged from 5–8 ml. Joint fluid was kept at 4uC and transported to the laboratory within 2 hours of necropsy. All knee and hip joints were inspected for morphological abnormalities, which were not observed in any of the ipsilateral or contralateral hips, nor in any of the ipsilateral (control) knee joints. sMPC derivation and purification Human: Fresh synovial fluid was plated in untreated culture dishes and after 1–2 hrs at 37uC/5%CO2 culture media was added. sMPC culture media consisted of DMEM (Invitrogen # 11965), 10% FBS, 1% Pen/Strep, 1% Non-essential amino acids (NEAA), 0.2% Beta-mercaptoethanol (BME) (all Invitrogen, Carlsbad, CA). Once cells had adhered to the plastic and reached 30–40% confluence, the media was changed and the cells were allowed to reach 60–70% confluence. At this point the cells were dissociated and resuspended in Dulbecco’s PBS (DPBS) at 1 million cells/ml. Primary labeled FACS antibodies (Table 1) to CD105, CD73, CD44, CD45, CD11b (All Becton, Dickinson and Company (BD), Franklin Lakes, NJ) were added to the suspension and the cells were sterilely sorted (University of Calgary, Flow Cytometry Core Facility). This CD105+, CD73+, CD44+, CD452, CD11b2 population accounted for 47.4%(+/25.4%) of the total cells isolated from normal joints, and 67.2%(+/23.1%) from joints with OA. Both normal and OA CD105+, CD73+, CD44+, CD452, CD11b2 cell populations were over 80% positive for CD90 before magnetic purification. The CD105+, CD73+, CD44+, CD452, CD11b2 cell population was returned to culture for 1 passage and then prepared for magnetic enrichment (an aliquot of this population was tested for multi- potency, with differentiation into fat, bone and cartilage assayed with oil red, alizarin red and Alcian blue respectively). Purified sfMPCs were dissociated and treated with anti-CD90 (BD) and then a magnetically labeled secondary (BD), and subsequently exposed to a magnetic field for enrichment following the manufacturer’s instructions. CD90-positive and CD90-negative fractions were cultured for one additional passage and then induced to differentiate. An aliquot of the cells at this passage were Marker CD105 CD73 CD44 CD45 CD11b CD90 CD34 CD45 Collagen 2 Aggrecan doi:10.1371/journal.pone.0043616.t001 Source Becton Dickinson Becton Dickinson Becton Dickinson Becton Dickinson Becton Dickinson Becton Dickinson Becton Dickinson Aviva Systems Biology Hybridoma Bank, Iowa Santa Cruz Identifies Endoglin 59-nucleotidase CD44 Protein tyrosine phosphatase, receptor type, C Integrin alpha M Protein tyrosine phosphatase, receptor type, C Thy-1 CD34 Collagen 2 Aggrecan Species Human Human Human Human Human Canine Canine Human/Canine Human/Canine Human/Canine Cell target MPC MPC MPC MPC hematopoietic cells hematopoietic cells hematopoietic cells hematopoietic cells Chondrocytes Chondrocytes PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43616