human sfMPCs have been transformed in vitro into scaffolds termed Tissue Engineering Constructs (TECs) that can be used to repair cartilaginous defects (in pigs) in vivo [6,7]. Additionally, another group has recently demonstrated that smMPCs have the ability to bind directly to cartilage ex vivo in minutes, and are able to contribute to cartilage repair in a defect model [11]. Human sfMPCs are typically characterized using cluster of differentiation (CD) antigens [15]: CD105 (Endoglin), CD90 (Thy-1), CD73 (Ecto-59-nucleotidase) and CD44 are present on the surface of MPCs/MSCs, while CD45 (Protein tyrosine phosphatase, recep- tor type, C) and CD11b (Integrin alpha M) are not expressed by this cell population [15]. The present study focuses on CD90 (Thy- 1), which has been shown to interact with Integrins, tyrosine kinases, growth factors, and cytokines thereby promoting down- stream cellular events including: adhesion, apoptosis, proliferation, and migration [16]. CD90 is commonly used as a marker of MPCs/MSCs, though it is also expressed by many other cell types including neurons, endothelial cells, T-cells, and other immune/ non-immune cell types [16]. More recently, CD90 has been utilized as a selection marker of multi-potent progenitors from bone marrow, synovial tissues, fat, amnion and other tissues [17]. However, the exact role of CD90 on the surface of this class of cells remains unknown. A number of recent studies have begun to explore the role of sfMPCs in diseases, including arthritis. Initial reports suggested that there was no difference in the chondrogenic potential of sfMPCs derived from healthy joints and joints with osteoarthritis (OA) or rheumatoid arthritis (RA) [2], notwithstanding the increase in number of sfMPCs in the OA knees [2]. A more recent study by the same group reported that the inflammatory intra-articular environment in RA joints is responsible for the reduced chondrogenic potential of sfMPCs [18]. As OA is generally viewed primarily as a degenerative rather than an inflammatory joint disease, it seems that the milieu of RA and OA joints has a fundamentally different influence on the capacity of sfMPCs to proliferate and differentiate. If, as has been speculated, sfMPCs participate in processes of joint maintenance or repair after injury [9,10], a fuller understanding of sfMPCs is warranted as they are potential therapeutic targets for these common and debilitating joint diseases. In a recent study where synovial membrane stem cells were obtained from OA patients and differentiated using a micro-mass tissue culture, a significant positive correlation was observed between CD90 expression and chondrogenic differentiation [19]. Therefore, the aim of the present study is a comparison of the chondrogenic potential of sfMPCs (human and canine) isolated from normal and osteoarthritic synovial fluid. Results Differentiation potential of normal and OA derived sfMPCs To evaluate the chondrogenic potential of human sfMPCs (CD105+, CD73+, CD44+, CD452. CD11b2) and canine sfMPCs (CD452, CD342) derived from normal and OA synovial fluid, the cells were differentiated into chondrocytes with media supplements over a 14 day period with a prior micro-mass aggregation step. At days 0, 3, 5, 8 and 14, mRNA was collected and probed using qRT-PCR for Sox9, Collagen 2, and Aggrecan (Figure 1 A,E,H,L). By day 14, Sox9, Collagen 2 and Aggrecan were significantly elevated compared to day 0 controls in normal (Figure 1 A,E) and OA (Figure 1 H,L) sfMPCs derived from human and canine synovial fluid. Immunofluorescence confirmed the qRT-PCR data using a Collagen 2 antibody on day 14. Chondrogenic Potential of CD90+ Progenitors sfMPC-derived chondrogenic masses from normal (Figure 1 B,F) and OA (Figure 1 I,M) fluid expressed Collagen 2 protein on day 14. Secondary antibody alone controls demonstrated minimal non-specific staining in human (Figure 1 C) and canine (Figure 1 J) sfMPCs. Furthermore, the micro-masses generated from all conditions stained with Alcian blue (Figure 1 D,G,K,N). Enhanced Chondrogenic Differentiation of CD90+ sfMPCs Since the human and canine sfMPCs contained CD90 positive and negative cells, the chondrogenic potential of the CD90- positive and CD90-negative fractions within the sfMPC popula- tion were studied. Previously isolated sfMPCs (Figure 2) were further enriched for CD90 using immuno-magnetic separation, with the resultant CD90-positive and CD90-negative fractions induced to differentiate into chondrocytes utilizing a micro-mass step (Figure 2). CD90+ sfMPCs from normal individuals (human and canine) behaved in a similar fashion to the total sfMPC population, displaying significantly increased levels of Sox9, type II Collagen, and Aggrecan mRNA (Figure 2 A,K) and demonstrated positive staining for Alcian blue (Figure 2 B,L) and Collagen 2 (Figure 2 C,M) by day 14. The CD90-negative fraction displayed reduced levels of Sox9, Collagen 2 and Aggrecan mRNA (although significantly increased compared to day 0)(Figure 2 A,K), as well as less intense alcian blue (Figure 2 D,N) and Collagen 2 (Figure 2 E, O) staining at day 14 of differentiation compared to the CD90+ population. Similarly, human and canine CD90+ sfMPCs from OA fluid demonstrated increased Sox9, Collagen 2 and Aggrecan mRNA expression (Figure 2 F,P), as well as staining with alcian blue (Figure 2 G,Q) and Collagen 2 (Figure 2 H,R) compared to the CD902 sfMPCs. Specifically, the mRNA level (Figure 2 F,P) and staining with alcian blue (Figure 2 I,S) and Collagen 2 (Figure 2 J,T) were decreased in the CD902 sfMPCs compared to the CD90+ sfMPCs. To examine if there were any differences in the osteogenic or adipogenic capacity of CD90+/2 human sfMPCs (normal & OA), the cells were placed in adipogenic or osteogenic conditions and allowed to differentiate for 20 days (Figure 3). After 20 days of differentiation, the cells were stained for Alkaline phosphatase (Figure 3 A) or Oil Red-O (Figure 3 B), and assayed using qPCR for Adiponectin, PPAR-gamma, Osteonectin and Osterix (Sp7) (Figure 3 C–F). Using these outcome measures, no differences in osteogenic or adipogenic differentiation capacity were observed between normal and OA sfMPCs, or CD90+/2 sub-populations of either group. Chondrogenesis of normal CD90-positive sfMPCs without a prior micro-mass step Based on the foregoing results, the chondrogenic potential of normal and OA sfMPSc were studied without micro-mass culture. Without a prior micro-mass step, normal sfMPCs from human (Figure 4 A) and canine (Figure 4 D) up-regulated Sox 9, Collagen 2 and Aggrecan mRNA during differentiation. In contrast, the Sox 9, Collagen 2 and Aggrecan mNRA levels in OA sfMPCs were significantly lower (Figure 4 A,D). Normal (human and canine) sfMPCs aggregated into small tissue-like structures during differentiation (Figure 4 B,G); OA-derived sfMPCs (human and canine) did not aggregate (Figure 4 C,F). CD90-positive Cells Form a Tissue-like Structure The tissue-like structures formed by normal CD90+ sfMPCs stained positively with antibodies against Aggrecan (Figure 5 A) and Collagen 2 (Figure 5 C). When visualized with TEM, these PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43616