We first determined and isolated cellular subpopulations with characteristics of mesenchymal

We first determined and isolated cellular subpopulations with characteristics of mesenchymal progenitor cells (MPCs) in osteoarthritic cartilage using fluorescence-activated cell sorting (FACS). found that various triple combinations of CD9, 1129669-05-1 supplier CD44, CD54, CD90 and CD166 positive cells within osteoarthritic cartilage account for 2C12% of the total population. After adhesion and cultivation their relative amount was markedly higher, with levels between 24% and 48%. Culture expanded cells combined and the initially sorted CD9/CD90/CD166 triple positive subpopulation had multipotency for chondrogenic, osteogenic and adipogenic differentiation. In conclusion, human osteoarthritic cartilage contains cells with characteristics of MPCs. Their relative enrichment during in vitro cultivation and the ability of cell sorting to obtain more homogeneous populations offer interesting perspectives for future studies on the activation of regenerative processes within osteoarthritic joints. Keywords: cartilage, mesenchymal progenitor cell, osteoarthritis Introduction Mesenchymal progenitor cells (MPCs) from bone marrow are able to differentiate in various types of connective tissue, including cartilage, bone and adipose tissue [1-3]. This led to more precise characterization of these cells by analysis of cell surface markers and differentiation related gene expression [4-9]. In parallel, it was recognized that MPCs not only reside in bone marrow but also in various other connective tissues, such as periost, and adipose and muscle tissue [5,6,10-14]. Cells within the joint that are capable of differentiating into chondrocytes, osteoblasts and adipocytes were recently described in synovia, patellar fat pad and articular cartilage [4,5,15-18]. In the present study we purified progenitor-like cells from the cartilage of human osteoarthritic joints and showed that these cells are capable of proliferation and osteogenic, adipogenic and chondrogenic lineage progression. Those cells could be distinguished from articular chondrocytes by simultaneous staining with several triple combinations of cell surface antigens [4-6]. We used these marker sets for quantification of MPCs by flow cytometric analysis in the original 1129669-05-1 supplier cell population and after in vitro cultivation. Finally, we sorted these cells according to the expression of triplicate surface markers and demonstrated that this subpopulation is capable of osteogenic, adipogenic and chondrogenic differentiation. These findings should provide a basis for identification of MPCs in articular cartilage and for studies of their roles in 1129669-05-1 supplier joint physiology and disease, as well as in induction of regenerative processes within osteoarthritic joints. Methods Patient characteristics Human osteoarthritic cartilage (OC) was obtained during routine surgical procedures with informed consent from seven patients with end-stage osteoarthritis, in accordance with the terms of the Ethics Committee of the University of Ulm. The age of the donors ranged from 55 to 89 years (mean 74 years). The diagnosis was based on clinical and radiological criteria. None of the donors had received corticosteroids or cytostatic drugs during the previous few months. Patients with systemic inflammatory diseases such as rheumatoid arthritis or spondyloarthropathies were excluded. Cell isolation, expansion and cryopreservation For cell culture samples, pure cartilage from regions with macroscopically mild-to-moderate osteoarthritic changes was extracted and then subjected to the following: two rinses with phosphate-buffered saline (PBS; Invitrogen, Karlsruhe, Germany) supplemented with antibiotic solution (100 units/ml penicillin, 100 g/ml streptomycin; Biochrom, Berlin, Germany); fine mincing and digestion with 0.2% pronase (Roche, Mannheim, Germany) for 45 min at 37C; and two further washes followed by enzymatic digestion overnight at 37C in 0.025% collagenase (Roche). After filtration through a 40 m pore membrane, the cells were washed twice in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing 10% fetal calf serum (FCS; Biochrom) and antibiotic solution (100 units/ml penicillin, 100 g/ml streptomycin), and counted and plated at low density (5 104 isolated cells/cm2). DMEM supplemented with 10% FCS was used as a medium during the proliferation phase. The cultures were incubated at 37C in a humidified 5% carbon dioxide atmosphere, and media were changed three times a week. FEN1 Cultures were split by trypsin treatment (0.05% trypsin, 0.02% EDTA; Biochrom) at 75% confluence. Flow cytometry analysis of cells Either isolated cells from OC were directly used for flow cytometric analysis or cells were used after adherence and cultivation, as described above. Cells were washed twice with PBS containing 1% FCS and 0.02% sodium azide (Sigma, Taufkirchen, Germany). The cells were incubated with 1 g/106 cells for each mouse anti-human monoclonal antibody that had been directly conjugated to a fluorochrome or biotinylated in the dark for 20 min on ice. The antibodies used are listed in Table ?Table1.1. After a washing step, second staining for biotin-conjugated monoclonal antibodies was done with streptavidin peridinin chlorophyll protein conjugate in a working titre of 1 1:100. After 30 min in the dark on ice, cells were washed again twice with PBS buffer before flow cytometric analysis. MPCs were characterized by three-colour immunoflourescence and 2 104 cells per sample were analyzed on a Becton Dickinson FACScalibur system using CELLQuest software (Becton Dickinson, Heidelberg, Germany). Dead cells were excluded by.