A senescence associated -galactosidase assay was used to assess the degree of senescence within the stem cell lines from OA cartilage. were consistently below 0.1%. Clonal cell lines were expanded beyond 40 population doublings but disparities RPH-2823 were observed in the number of population doublings per day. Clonally derived cell lines also demonstrated multilineage potential via successful differentiation into chondrogenic, osteogenic, and adipogenic lineages. However, variation in the degree of differentiation was observed between these clonal cell lines. Conclusions A viable pool of cells with stem cell characteristics have been identified within human osteoarthritic cartilage. Variation in the degree of differentiation suggests the possibility of further subpopulations of cells. The identification of this stem cell population highlights the reparative potential of these cells in osteoarthritic cartilage, which could be further exploited to aid the field of regenerative medicine. culturing methods to isolate and expand cells in order to initiate new cartilage formation, methods have progressed from using chondrocytes to mesenchymal stem cells (MSCs) as a means of eliminating hurdles such as limited expansion potential, chondrocyte dedifferentiation = 9). South East Wales Research Ethics Committee safety and ethical guidelines were followed. Cells were released from their matrix by sequential enzyme digestion using 70 U mL?1 pronase followed by 300 U RPH-2823 mL?1 collagenase (type I) in supplemented Dulbeccos Modified Eagles Medium F12 (DMEM/F12) plus Glutamax (DMEM/F12 + Glutamax with 100 mg mL?1 gentamicin, 50 g mL?1 l-ascorbic acid 2-phosphate, 1 mg mL?1 glucose, 2 mM l-glutamine, and 5% fetal calf RGS17 serum [FCS]), at 37C. Following digestion, the cells were filtered through a 40-m mesh cell strainer. The remaining cell suspension was centrifuged, supernatant removed, and the pellet was resuspended in supplemented DMEM/F12 to be counted using a hemocytometer. Open in a separate window Figure 1. A tibial plateau removed from a patient with osteoarthritis at the time of total knee replacement. Characteristic features can be seen including osteophytes and subchondral bone. Fibronectin Adhesion Assay to Isolate Cartilage Stem Cells RPH-2823 A differential adhesion assay RPH-2823 onto fibronectin-coated plates was used to specifically isolate cartilage stem cells from the cell population (developed from Jones and Watt12). Cells were resuspended at a concentration of 4,000 cells mL?1 in supplemented DMEM/F12 and seeded onto 6-well plates that had been pretreated with fibronectin (10 g mL?1 in phosphate-buffered saline containing 1 mM MgCl2 and 1 mM CaCl2) for 24 hours at 4C. Cells were incubated for 20 minutes at 37C, after which the media and nonadherent cells were removed. Fresh media (DMEM/F12 + 10% FCS) was then added to the dish and the cells were incubated and maintained in culture in a humidified chamber containing 5% CO2 at 37C. Colony Forming Efficiencies (CFEs) Twenty-four hours after cell selection via fibronectin adhesion, the number of cells were counted. Between 8 and 14 days after the initial seeding day, clusters of 32 cells (defined as a colony) were counted, as this number represents a population of cells derived from more than 5 population doublings (PDs) of a single cell, thereby discounting a transient amplifying cell cohort. CFEs were then calculated based on (a) the initial seeding density and the number of colonies formed and (b) the number of cells that initially adhered and the number of colonies formed. Colony Isolation Colonies were isolated using polystyrene cloning cylinders. One hundred microliters of trypsin-EDTA was used to lift the cells, allowing them to be transferred into 12-well plates containing supplemented DMEM/F12 + 10% FCS, 1 ng mL?1 TGF-2 and 5 ng mL?1 FGF-2. A minimum of eight clones were isolated from each OA specimen. Expansion in Monolayer Culture Clonal cell lines were cultured until confluent and passaged accordingly. PDs could be monitored, using the following formula: PD =?[log(is the number of cells recovered at the end of the passage and value of 0.05 was considered significant. Results RPH-2823 Cartilage Stem Cell Immunodetection, Cell Isolation, and Expansion Cartilage stem cells were successfully isolated from osteoarthritic tibial.