analyzed using FACS to confirm the presence or absence of CD105, CD90, CD73, CD44, CD45, CD11b (Figure 6). Canine: Fresh synovial fluid was plated in untreated culture dishes and after 1–2 hrs at 37uC/5%CO2 culture media was added. sMPC media (as above) was used. 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. The total cell population was negatively purified using biotin-labeled antibodies to canine CD45 (Aviva Systems Biology, San Diego, CA) and CD34 (BD) following the manufacturer’s instructions. This cell population was then selected for the CD90+ (BD) fraction using magnetic separation. CD90-positive and CD90-negative fractions were cultured for one additional passage and then induced to differentiate. Adipogenic Differentiation sfMPCs were plated in triplicate (100,000 cells/well/24 well dish) and exposed to adipogenic differentiation media for 20 days. Differentiation media consisted of sMPC culture media with 0.5 mM isobutylmethylxanthine, 1 mM dexamethasone, 10 mM insulin, 200 mM indomethacin (all Sigma). Media was changed every three days during the 20 day differentiation period. Figure 6. FACS characterization of human sfMPCs. Normal and OA CD90+ and CD902 subpopulations were assayed for expression of CD105, CD90, CD73, CD45 and CD11b prior to chondrogenic differentiation. doi:10.1371/journal.pone.0043616.g006 Chondrogenic Potential of CD90+ Progenitors Osteogenic Differentiation sfMPCs were plated in triplicate (100,000 cells/well/24 well dish) and exposed to osteogenic differentiation media for 20 days. Differentiation media consisted of sMPC culture media with 0.1 mM dexamethasone and 50 mM ascorbate-2-phosphate. Me- dia was changed every three days during the 20 day differentiation period. Chondrogenic Differentiation sfMPCs were plated in triplicate (100,000 cells/well/24 well dish) and exposed to chondrogenic media for 14 days with or without micro-mass aggregation. Aggregation was achieved by placing 100,000 cells in a 1.5 ml sterile tube at 37uC overnight. Differentiation media consisted of sMPC culture media with 500 ng/mL BMP-2 (Peprotech, Rocky Hill, NJ), 10 ng/mL TGF- b3 (Peprotech), 1028 M dexamethasone (Sigma, St. Louis, MO), 50 mg/mL ascorbic acid (Sigma), 40 mg/mL proline (Invitrogen), 100 mg/mL pyruvate (Sigma) and supplemented with insulin, transferrin, and selenium (Sigma). Media was changed every three days during the 14-day differentiation period. Analysis of Differentiation Differentiation was assessed using quantitative RT-PCR (qRT- PCR) and immunofluorescence (IF). qRT-PCR: RNA was collected using Trizol (Invitrogen) and converted to cDNA using a High Capacity cDNA kit (Applied Biosystems (ABS), Carlsbad, CA). The cDNA was probed using pre-validated TaqmanTM primer-sets for human Sox9, Collagen 2, Aggrecan, Osterix (SP7), Osteocalcin, PPAR-gamma, Adipo- nectin, and canine Sox9, Collagen 2 and Aggrecan (Table 2) on an ABI 7900HT using 18S as the internal control and the ddCT method included within the ABI software to analyze results. IF: Aggregates/tissues generated from sfMPCs were washed in PBS and fixed in 4% para-formaldehyde (PFA) in PBS at 4uC overnight. Aggregates were then permeabilized in 0.5% saponin (Sigma) in PBS at 4uC overnight, rinsed once in PBS, then blocked in 3% BSA at 4uC overnight. Primary antibodies (Collagen 2: Developmental Studies Hybridoma Bank, Iowa. Aggrecan: Santa Cruz, Santa Cruz, CA) (Table 1) were diluted 1:50 in 3% BSA, added to the cell samples and incubated overnight at 4uC. The aggregates were then washed 3 times with PBS and blocked again overnight at 4uC. Following the block, the aggregates were incubated with an appropriate Alexa-fluor 488 secondary antibody (Molecular Probes, Carlsbad, CA) and Toto-3TM (Molecular Probes) overnight at 4uC. After incubation, the aggregates were washed thrice with PBS and mounted on slides with mountant (9:1 glycerol:PBS). Slides were analyzed using a Zeiss 510 confocal Table 2. qRT-PCR probes used for analysis. Marker Sox 9 Collagen 2 Aggrecan Osterix (SP7) Osteocalcin PPAR-gamma Adiponectin Source ABI (human and canine probes) ABI (human and canine probes) ABI (human and canine probes) ABI (human) ABI (human) ABI (human) ABI (human) doi:10.1371/journal.pone.0043616.t002 Cell Target Chondrocytes Chondrocytes Chondrocytes Osteoblasts Osteoblasts Adipocytes Adipocytes PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43616